transforming-growth-factor-beta and Nephritis

SUMOylation of proteins is an important regulatory element in modulating protein function and has been implicated in the pathogenesis of numerous human diseases such as cancers, neurodegenerative diseases, brain injuries, diabetes, and familial dilated cardiomyopathy. Growing evidence has pointed to a significant role of SUMO in kidney diseases such as DN, RCC, nephritis, AKI, hypertonic stress and nephrolithiasis. Recently, emerging studies in podocytes demonstrated that SUMO might have a protective role against podocyte apoptosis. However, the SUMO code responsible for beneficial outcome in the kidney remains to be decrypted. Our recent experiments have revealed that the expression of both SUMO and SUMOylated proteins is appreciably elevated in hypoxia-induced tubular epithelial cells (TECs) as well as in the unilateral ureteric obstruction (UUO) mouse model, suggesting a role of SUMO in TECs injury and renal fibrosis. In this review, we attempt to decipher the SUMO code in the development of kidney diseases by summarizing the defined function of SUMO and looking forward to the potential role of SUMO in kidney diseases, especially in the pathology of renal fibrosis and CKD, with the goal of developing strategies that maximize correct interpretation in clinical therapy and prognosis.

Topics: Acute Kidney Injury; Apoptosis; Carcinoma, Renal Cell; Diabetic Nephropathies; Fibrosis; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney Neoplasms; Nephritis; Nephrolithiasis; Podocytes; Protein Processing, Post-Translational; Smad Proteins; Small Ubiquitin-Related Modifier Proteins; Sumoylation; Transforming Growth Factor beta; Ureteral Obstruction

To provide a perspective by investigating the potential cross-talk between the adipose tissue and the kidney during obesity.. It is well established that excessive caloric intake contributes to organ injury. The associated increased adiposity initiates a cascade of cellular events that leads to progressive obesity-associated diseases such as kidney disease. Recent evidence has indicated that adipose tissue produces bioactive substances that contribute to obesity-related kidney disease, altering the renal function and structure. In parallel, proinflammatory processes within the adipose tissue can also lead to pathophysiological changes in the kidney during the obese state.. Despite considerable efforts to better characterize the pathophysiology of obesity-related metabolic disease, there are still a lack of efficient therapeutic strategies. New strategies focused on regulating adipose function with respect to AMP-activated protein kinase activation, NADPH oxidase function, and TGF-β may contribute to reducing adipose inflammation that may also provide renoprotection.

Topics: Adipose Tissue; AMP-Activated Protein Kinases; Cytokines; Fibrosis; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; Insulin Resistance; Kidney; NADPH Oxidases; Nephritis; Obesity; Receptors, G-Protein-Coupled; Transforming Growth Factor beta

Topics: Angiotensin II; Animals; Anti-Inflammatory Agents, Non-Steroidal; Fibrosis; Humans; Inflammation; Inflammation Mediators; Kidney Diseases; Models, Biological; Mycophenolic Acid; Nephritis; Renin-Angiotensin System; Transforming Growth Factor beta

Recent insights into the etiopathogenesis of nephritogenic immune responses are derived primarily from experimental models of systemic and organ-specific autoimmunity. Genetic analyses and immune-related gene ablation studies indicate that multiple independent mechanisms determine disease susceptibility. However, full characterization of proximal immunologic events in many diseases awaits identification of the renal antigens recognized by nephritogenic lymphocytes. Advances in characterization of effector mechanisms include epitope mapping of several putative pathogenic glomerular antigens and identification of novel pathways of immune-mediated tissue injury, including those involved in glomerular-tubulointerstitial communication and tubulointerstitial fibrosis. Finally, successful interruption of signal transduction pathways and transforming growth factor-beta 1 blockade by gene therapy suggest novel approaches to therapeutic intervention in immunologic renal injury.

Topics: Animals; Antibodies, Antineutrophil Cytoplasmic; Autoantigens; Autoimmune Diseases; Autoimmunity; Complement System Proteins; Humans; Inflammation Mediators; Integrins; Kidney; Kidney Diseases; Lymphocyte Activation; Nephritis; T-Lymphocytes; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) is a cytokine that is important in embryogenesis, development, carcinogenesis, and tissue repair. TGF-beta is unique among cytokines in its widespread actions on the regulation of extracellular matrix. In a model of acute mesangial proliferative glomerulonephritis, it was shown that overproduction of TGF-beta is the cause of pathologic matrix accumulation in the nephritic glomeruli. TGF-beta acted to increase matrix production, inhibit matrix degradation, and modulate matrix receptors in the glomerulonephritic rats. Elevated expression of TGF-beta was also found in other experimental glomerular diseases, including diabetic nephropathy. Studies of humans with glomerulonephritis and diabetic nephropathy also strongly implicated TGF-beta in the pathogenesis of glomerular matrix build-up. Recently, the proteoglycan decorin was shown to neutralize TGF-beta. When injected into glomerulonephritic rats, decorin markedly suppressed pathologic matrix deposition in the glomeruli. Thus, decorin offers hope as a treatment for progressive kidney diseases caused by the overproduction of TGF-beta.

Topics: Animals; Cells, Cultured; Diabetic Nephropathies; Disease Models, Animal; Extracellular Matrix; Glomerulonephritis; Glomerulonephritis, Membranoproliferative; Humans; Kidney Glomerulus; Nephritis; Transforming Growth Factor beta

One of the central features of many human glomerular diseases is the proliferation of the smooth muscle cell-like mesangial cells. While a multitude of mitogens for mesangial cells has been proposed on the basis of in vitro experiments, the factors involved in the regulation of mesangial cell proliferation in vivo remain largely undefined. To investigate the regulation of mesangial cell proliferation in vivo we have studied the mesangioproliferative glomerulonephritis that is induced by injection of antibody directed against the Thy 1.1 antigen on the mesangial cell surface in rats. In this review, we discuss the role of three cytokines in the mesangioproliferative response, namely platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta (TGF-beta). All three cytokines are present in various inflammatory cells as well as in mesangial cells themselves, thereby allowing these factors to exert both paracrine and autocrine regulatory functions on mesangial cells. In vivo studies show that PDGF, bFGF and TGF-beta participate in either the mesangial cell proliferation or the mesangial matrix expansion that follows mesangial cell injury with anti-Thy 1.1 antibody. Based on currently available data we propose that bFGF may participate in the initiation, PDGF in the maintenance, and TGF-beta in the resolution of mesangial cell proliferation in vivo. Further analysis of the mitogens operative in vivo may ultimately result in the design of new therapeutic strategies to treat progressive glomerular mesangioproliferative diseases.

Topics: Animals; Cell Division; Fibroblast Growth Factor 2; Glomerular Mesangium; Humans; In Vitro Techniques; Kidney Diseases; Mitogens; Nephritis; Platelet-Derived Growth Factor; Transforming Growth Factor beta

Hypertensive nephropathy is a chronic kidney disease caused by hypertension. Eicosapentaenoic acid (EPA) has been reported to possess an antihypertensive effect, and our previous study suggested that EPA-enriched phospholipid (EPA-PL) had more significant bioactivities compared with traditional EPA. However, the effect of dietary EPA-PL on hypertensive nephropathy has not been studied. The current study was designed to examine the protection of EPA-PL against kidney damage in spontaneously hypertensive rats (SHRs). Treatment with EPA-PL for three weeks significantly reduced blood pressure through regulating the renin-angiotensin system in SHRs. Moreover, dietary EPA-PL distinctly alleviated kidney dysfunction in SHRs, evidenced by reduced plasma creatinine, blood urea nitrogen, and 24 h proteinuria. Histology results revealed that treatment of SHRs with EPA-PL alleviated renal injury and reduced tubulointerstitial fibrosis. Further mechanistic studies indicated that dietary EPA-PL remarkably inhibited the activation of TGF-β and Smad 3, elevated the phosphorylation level of PI3K/AKT, suppressed the activation of NF-κB, reduced the expression of pro-inflammatory cytokines, including IL-1β and IL-6, and repressed the oxidative stress and the mitochondria-mediated apoptotic signaling pathway in the kidney. These results indicate that EPA-PL has potential value in the prevention and alleviation of hypertensive nephropathy.

Topics: Animals; Antihypertensive Agents; Blood Pressure; Disease Models, Animal; Eicosapentaenoic Acid; Fibrosis; Hypertension, Renal; Male; Nephritis; NF-kappa B; Oxidative Stress; Phosphatidylinositol 3-Kinase; Phospholipids; Rats; Rats, Inbred SHR; Rats, Sprague-Dawley; Renin-Angiotensin System; Signal Transduction; Transforming Growth Factor beta

The Hippo/YAP (yes-associated protein) pathway is an important signaling pathway to control organ development and tissue homeostasis. YAP is a downstream effector of the Hippo pathway and a critical mediator of mechanic stress. Hypertensive nephropathy is characterized with glomerular sclerosis stiffness and renal fibrosis. The present study investigated the role of YAP pathway in angiotensin (Ang) II hypertensive renal injury by using YAP activation inhibitor verteporfin. Ang II increased the protein expression of YAP in renal nucleus fraction, decreased phospho-YAP, and phospho-LATS1/2 (large tumor suppressors 1 and 2) expressions in renal cytoplasmic fraction, suggesting Ang II activation of renal YAP. Ang II significantly increased systolic blood pressure (SBP), proteinuria, glomerular sclerosis, and fibrosis; treatment with verteporfin attenuated Ang II-induced proteinuria and renal injury with a mild reduction in SBP. Moreover, Ang II increased the protein expressions of inflammatory factors including tumor necrosis factor α, interleukin 1β, and monocyte chemoattractant protein-1, and profibrotic factors including transforming growth factor β, phospho-Smad3 and fibronectin. Verteporfin reversed abovementioned Ang II-induced molecule expressions. Our results for the first time demonstrate that the activation of the YAP pathway promotes hypertensive renal inflammation and fibrosis, which may promote hypertensive renal injury. YAP may be a new target for prevention and treatment of hypertensive renal diseases.

Topics: Acute Kidney Injury; Angiotensin II; Animals; Blood Pressure; Chemokine CCL2; Fibrosis; Hypertension; Hypertension, Renal; Male; Mice; Mice, Inbred C57BL; Nephritis; Photosensitizing Agents; Protein Serine-Threonine Kinases; Signal Transduction; Transforming Growth Factor beta; Vasoconstrictor Agents; Verteporfin; YAP-Signaling Proteins

Innate lymphoid cells are a recently recognized group of immune cells with critical roles in tissue homeostasis and inflammation. Regulatory innate lymphoid cells are a newly identified subset of innate lymphoid cells, which play a suppressive role in the innate immune response, favoring the resolution of intestinal inflammation. However, the expression and role of regulatory innate lymphoid cells in kidney has not been reported. Here, we show that regulatory innate lymphoid cells are present in both human and mouse kidney, express similar surface markers and form a similar proportion of total kidney innate lymphoid cells. Regulatory innate lymphoid cells from kidney were expanded in vitro with a combination of IL-2, IL-7 and transforming growth factor-β. These cells exhibited immunosuppressive effects on innate immune cells via secretion of IL-10 and transforming growth factor-β. Moreover, treatment with IL-2/IL-2 antibody complexes (IL-2C) promoted expansion of regulatory innate lymphoid cells in vivo, and prevent renal ischemia/reperfusion injury in Rag-/- mice that lack adaptive immune cells including Tregs. Depletion of regulatory innate lymphoid cells with anti-CD25 antibody abolished the beneficial effects of IL-2C in the Rag-/- mice. Adoptive transfer of ex vivo expanded regulatory innate lymphoid cells improved renal function and attenuated histologic damage when given before or after induction of ischemia/reperfusion injury in association with reduction of neutrophil infiltration and induction of reparative M2 macrophages in kidney. Thus, our study shows that regulatory innate lymphoid cells suppress innate renal inflammation and ischemia/reperfusion injury.

Topics: Adoptive Transfer; Animals; Cell Separation; Cells, Cultured; Coculture Techniques; Disease Models, Animal; Flow Cytometry; Homeodomain Proteins; Humans; Immunity, Innate; Interleukin-10; Interleukin-2; Kidney; Lymphocyte Subsets; Macrophages; Male; Mice; Mice, Knockout; Nephritis; Primary Cell Culture; Reperfusion Injury; Transforming Growth Factor beta

Hypertensive nephropathy, clinically characterized by progressive renal fibrosis and inflammation, is a severe complication of hypertension. The objectives of this study were to investigate the roles of miR-101a in relieving angiotensin II (Ang II)-mediated hypertensive nephropathy and uncover the possible underlying mechanisms. A hypertensive mouse model was established via continuous 28-day AngII infusion. Systolic blood pressure (SBP), ratio of urine albumin to creatinine, blood urea nitrogen (BUN), serum creatinine (Scr) and glomerular filtration rate (GFR) were evaluated. Dual luciferase reporter assay was used to explore the target of miR-101a. mRNA levels of miR-101a, TGFβRI, fibrotic markers (Collagen I and α-SMA) and pro-inflammatory cytokines (IL-1β and TNF-α) were determined by real-time PCR. Protein levels of TGFβRI, Collagen I, α-SMA, IL-1β, TNF-α, t-p65, P-p65, t-Smad3, P-Smad3, t-IκBα and P-IκBα were detected by western blot. MiR-101a mimics significantly improved GFR and inhibited AngII-induced increase in the ratio of urine albumin to creatinine, BUN and Scr. MiR-101a mimics partially abolished AngII-induced increase in the mRNA and protein level of fibrotic markers by targeting TGFβRI and inhibiting TGFβ/Smad3 pathway. Moreover, TGFβRI inhibitor galunisertib inhibited AngII-mediated renal injury in mice with hypertensive nephropathy. Additionally, miR-101a overexpression blocked AngII-induced up-regulation of pro-inflammatory markers via suppressing NF-κB pathway. MiR-101a exhibited protective effects against hypertensive nephropathy via inhibiting TGFβ/Smad3 and NF-κB signalling pathways.

Topics: Angiotensin II; Animals; Base Sequence; Cell Line; Disease Models, Animal; Fibrosis; Hypertension, Renal; Kidney; Male; Mice; MicroRNAs; Nephritis; NF-kappa B; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Angiotensin-converting enzyme-2 (ACE2) is downregulated in hypertensive nephropathy. The present study investigated the mechanisms whereby loss of ACE2 promoted angiotensin II-induced hypertensive nephropathy in ACE2 gene knockout mice. We found that compared with wild-type animals, mice lacking ACE2 developed much more severe hypertensive nephropathy in response to chronic angiotensin II infusion, including higher levels of blood pressure, urinary protein excretion, serum creatinine, and progressive renal fibrosis and inflammation. Mechanistic studies revealed that worsening kidney injury in ACE2 knockout mice was associated with an increase in Smurf2 (Smad-specific E3 ubiquitin protein ligase 2), a decrease in renal Smad7, and marked activation of TGF-β (transforming growth factor β)/Smad3 and NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells) signaling, suggesting that Smurf2-dependent Smad7 ubiquitin degradation may be a key mechanism whereby loss of ACE2 promotes angiotensin II-induced TGF-β/Smad3 and NF-κB-mediated hypertensive nephropathy. This was validated by restoring Smad7 locally in the kidneys of ACE2 knockout mice to block angiotensin II-induced TGF-β/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation. Moreover, we found that angiotensin II could induce microRNA-21 in the mouse kidney and in cultured mesangial cells via a Smad3-dependent mechanism, which was enhanced by deleting ACE2 but inhibited by overexpressing renal Smad7. In conclusion, loss of ACE2 promotes angiotensin II-induced renal injury by targeting Smad7 for degradation via a Smurf2-dependent mechanism. Overexpression of renal Smad7 protects against hypertensive nephropathy by inactivating angiotensin II-induced TGF-β/Smad3 and NF-κB pathways and by targeting the Smad3-dependent microRNA-21 axis.

Topics: Angiotensin-Converting Enzyme 2; Animals; Blood Pressure; Down-Regulation; Fibrosis; Hypertension; Hypertension, Renal; Inflammation; Kidney; Mice; Mice, Knockout; Nephritis; NF-kappa B; Peptidyl-Dipeptidase A; Signal Transduction; Smad1 Protein; Smad7 Protein; Transforming Growth Factor beta; Ubiquitin

Blockade of the renin-angiotensin II (Ang II) system by AT1 blockers (ARBs) and angiotensin-converting enzyme inhibitors retards the progression of chronic kidney disease (CKD) by reducing albuminuria/proteinuria. However, many patients with CKD suffer from residual albuminuria/proteinuria, which is an independent risk factor for CKD progression. The aim of the current study is to investigate the effect of pitavastatin, one of the adjunctive agents to ARBs, on the reduction of albuminuria/proteinuria and further renoprotection mediated by telmisartan in spontaneously hypertensive rats.. Forty-two-week-old spontaneously hypertensive rats were grouped randomly and received 8 weeks of treatments with vehicle, telmisartan, pitavastatin or a combination of telmisartan and pitavastatin. Both albuminuria and proteinuria were inhibited significantly in the telmisartan-treated group, but an obviously residual albuminuria was maintained. The combination treatment with telmisartan and pitavastatin displayed a more effective decrease in albuminuria and proteinuria, even to the normal level. Enhanced nephroprotection was also observed in this combination group, which was independent of the cholesterol-lowering effects. Further mechanistic studies revealed that the combination therapy greatly attenuated the expression of intrarenal Ang II and AT1, thereby decreasing the activation of TGF-β-Smad and NF-κB and inhibiting fibrosis and inflammation.. Adjunctive therapy with pitavastatin dramatically reduced residual albuminuria/proteinuria and enhanced nephroprotection, likely by downregulating the expression of intrarenal Ang II and AT1. It could be concluded that statins might be a promising adjunctive therapeutic agent to conventional ARB treatment in hypertensive renal damage.

Topics: Albuminuria; Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Benzimidazoles; Benzoates; Down-Regulation; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypertension, Renal; Kidney; Male; Nephritis; NF-kappa B; Proteinuria; Rats; Rats, Inbred SHR; Receptor, Angiotensin, Type 1; Renin-Angiotensin System; Signal Transduction; Telmisartan; Transforming Growth Factor beta

Renal interstitial fibrosis is characterized by excessive accumulation of extracellular matrix, which leads to end-stage renal failure.. The aim of this study was to explore the effect of Elsholtzia ciliata (Thunb.) Hylander ethanol extract (ECE) on renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO).. After quantitative analysis of ECE using the high performance liquid chromatography-photodiode array (HPLC-PDA) method, an in vitro study was performed to assess the anti-inflammatory and anti-fibrotic effects of ECE, using lipopolysaccharide (LPS) and transforming growth factor-ß (TGF-ß), respectively.. For in vivo study, all male Sprague Dawley (SD) rats (n=10/group), except for those in the control group, underwent UUO. The rats were orally treated with water (control), captopril (positive control, 200 mg/kg), and ECE (300 and 500 mg/kg) for 14 days.. In ECE, luteolin and rosmarinic acid were relatively abundant among the other flavonoids and phenolic acids. ECE treatment ameliorated LPS-induced overexpression of nuclear factor-κB, tumor necrosis factor (TNF-α), and interleukin-6 and improved oxidative stress in RAW 264.7 cells. Furthermore, ECE treatment suppressed TGF-ß-induced α-smooth muscle actin and matrix metalloproteinase 9 expression in human renal mesangial cells. In the UUO model, 14 consecutive days of ECE treatment improved UUO-induced renal damage and attenuated histopathological alterations and interstitial fibrosis. Moreover, the renal expression of TNF-α, TGF-ß, and Smad 3 were inhibited by ECE treatment.. Taken together, the effects of ECE may be mediated by blocking the activation of TGF-ß and inflammatory cytokines, leading subsequently to degradation of the ECM accumulation pathway. Based on these findings, ECE might serve as an improved treatment strategy for renal fibrotic disease.

Topics: Animals; Cytokines; Fibrosis; Humans; Inflammation; Kidney; Kidney Diseases; Lamiaceae; Male; Matrix Metalloproteinase 9; Mesangial Cells; Mice; Nephritis; Oxidative Stress; Phytotherapy; Plant Extracts; Polyphenols; Rats; Rats, Sprague-Dawley; RAW 264.7 Cells; Smad3 Protein; Transforming Growth Factor beta; Ureteral Obstruction

Transforming growth factor beta 1 (TGF-β1) is one of the most studied cytokines involved in renal tubulo-interstitial fibrosis, which is characterized by myofibroblast abundance and proliferation, and high buildup of extracellular matrix in the tubular interstitium leading to organ failure. Endoglin (Eng) is a 180-kDa homodimeric transmembrane protein that regulates a great number of TGF-β1 actions in different biological processes, including ECM synthesis. High levels of Eng have been observed in experimental models of renal fibrosis or in biopsies from patients with chronic kidney disease. In humans and mice, two Eng isoforms are generated by alternative splicing, L-Eng and S-Eng that differ in the length and composition of their cytoplasmic domains. We have previously described that L-Eng overexpression promotes renal fibrosis after unilateral ureteral obstruction (UUO). However, the role of S-Eng in renal fibrosis is unknown and its study would let us analyze the possible function of the cytoplasmic domain of Eng in this process. For this purpose, we have generated a mice strain that overexpresses S-Eng (S-ENG(+)) and we have performed an UUO in S-ENG(+) and their wild type (WT) control mice. Our results indicate that obstructed kidney of S-ENG(+) mice shows lower levels of tubulo-interstitial fibrosis, less inflammation and less interstitial cell proliferation than WT littermates. Moreover, S-ENG(+) mice show less activation of Smad1 and Smad2/3 pathways. Thus, S-Eng overexpression reduces UUO-induced renal fibrosis and some associated mechanisms. As L-Eng overexpression provokes renal fibrosis we conclude that Eng-mediated induction of renal fibrosis in this model is dependent on its cytoplasmic domain.

Topics: Animals; Cell Proliferation; Collagen Type I; Disease Models, Animal; Endoglin; Fibronectins; Fibrosis; Humans; Kidney; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Transgenic; Models, Biological; Myofibroblasts; Nephritis; Protein Isoforms; Recombinant Proteins; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Up-Regulation; Ureteral Obstruction

Conversion from calcineurin inhibitor (CNI) to mTOR inhibitors may reduce and even halt the progression of chronic allograft dysfunction (CAD) which is the most important cause of renal allograft loss. We aimed to investigate the effects of conversion from CNI to everolimus on parameters of fibrosis, inflammation, glomerulotubular damage and vascular functions in renal transplant recipients.. Fifteen stable renal transplant recipients who were under CNI treatment (male/female 13/2, mean age 41 ± 10 years) were enrolled and switched to everolimus. Serum and urinary transforming growth factor-β (TGF-β), urinary neutrophil gelatinase-associated lipocalin (NGAL) and monocyte chemoattractant protein-1 (MCP-1) were measured as markers of fibrosis, tubular damage and inflammation. As parameters of vascular functions, pulse wave velocity (PWV), augmentation index (AIx), serum asymmetric dimethyl-arginine and fibroblast growth factor-23 (FGF-23) were measured. All these measurements were repeated at the 3rd month of conversion.. Estimated GFR (52 ± 7-57 ± 11 ml/min/l.73 m(2), p = 0.02) (was increased after conversion to everolimus. However, serum uric acid levels were significantly decreased (6.21 ± 1.21-5.50 ± 1.39 mg/dL, p = 0.01). Serum TGF-β levels (8727 ± 2897-1943 ± 365 pg/mL, p = 0.03) and urinary NGAL levels (26 ± 10-12 ± 2 ng/mg creatinine, p = 0.05) were significantly decreased. However, urinary MCP-1, FGF-23, PWV and AIx did not change. Urinary TGF-β was associated with urinary NGAL (r = 0.62, p = 0.01), urinary MCP-1 (r = 0.68, p = 0.005) and proteinuria (r = 0.50, p = 0.05).. Conversion from CNI to everolimus resulted in significant decreases of serum TGF-β and urinary NGAL which may represent less fibrosis and tubular damage. Association of urinary TGF-β with NGAL and MCP-1 suggests that tubular damage, fibrosis and inflammation may act together for progression of CAD.

Topics: Acute-Phase Proteins; Adult; Calcineurin Inhibitors; Chemokine CCL2; Everolimus; Female; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Fibrosis; Graft Rejection; Humans; Immunosuppressive Agents; Kidney Transplantation; Kidney Tubules; Lipocalin-2; Lipocalins; Male; Middle Aged; Nephritis; Proto-Oncogene Proteins; Pulse Wave Analysis; Renal Artery; Risk Factors; Sirolimus; Transforming Growth Factor beta; Transplant Recipients

Increased renal expression of periostin, a protein normally involved in embryonic and dental development, correlates with the decline of renal function in experimental models and patient biopsies. Because periostin has been reported to induce cell differentiation, we investigated whether it is also involved in the development of renal disease and whether blocking its abnormal expression improves renal function and/or structure. After unilateral ureteral obstruction in wild-type mice, we observed a progressive increase in the expression and synthesis of periostin in the obstructed kidney that associated with the progression of renal lesions. In contrast, mice lacking the periostin gene showed less injury-induced interstitial fibrosis and inflammation and were protected against structural alterations. This protection was associated with a preservation of the renal epithelial phenotype. In vitro, administration of TGF-β to renal epithelial cells increased the expression of periostin several-fold, leading to subsequent loss of the epithelial phenotype. Furthermore, treatment of these cells with periostin increased the expression of collagen I and stimulated the phosphorylation of FAK, p38, and ERK 42/44. In vivo delivery of antisense oligonucleotides to inhibit periostin expression protected animals from L-NAME-induced renal injury. These data strongly suggest that periostin mediates renal disease in response to TGF-β and that blocking periostin may be a promising therapeutic strategy against the development of CKD.

Topics: Animals; Cell Adhesion Molecules; Cell Culture Techniques; Disease Models, Animal; Female; Gene Silencing; Male; Mice, Inbred C57BL; Nephritis; Nephrosclerosis; Podocytes; Rats, Sprague-Dawley; Transforming Growth Factor beta; Ureteral Obstruction

Emerging data have suggested that acute kidney injury (AKI) is often incompletely repaired and can lead to chronic kidney disease (CKD), which is characterized by tubulointerstitial inflammation and fibrosis. However, the underlying mechanisms linking AKI to CKD remain obscure. The present study aimed to investigate the role of cysteine-rich protein 61 (Cyr61) after unilateral kidney ischemia-reperfusion injury (IRI) in mice. After IRI, increased expression of Cyr61 was detected, predominately in the proximal tubular epithelium. This was confirmed by in vitro experiments, which showed that hypoxia stimulated Cyr61 expression in cultured proximal tubular epithelial cells. The proinflammatory property of Cyr61 was indicated by its ability to upregulate monocyte chemoattractant protein-1 and IL-6. Additionally, we found elevated urinary Cyr61 excretion in patients with AKI. Notably, treatment of mice with an anti-Cyr61 antibody attenuated the upregulation of kidney monocyte chemoattractant protein-1, IL-6, IL-1β, and macrophage inflammatory protein-2 and reduced the infiltration of F4/80-positive macrophages on days 7 and 14 after IRI. In addition, blockade of Cyr61 reduced the mRNA expression of collagen, transforming growth factor-β, and plasminogen activator inhibitor-I as well as the degree of collagen fibril accumulation, as evaluated by picrosirius red staining, and levels of α-smooth muscle actin proteins by day 14. Concurrently, in the treated group, peritubular microvascular density was more preserved on day 14. We conclude that Cyr61 blockade inhibits the triad of inflammation, interstitial fibrosis, and capillary rarefaction after severe ischemic AKI. The results of this study expand the knowledge of the mechanisms underlying the AKI-to-CKD transition and suggest that Cyr61 is a potential therapeutic target.

Topics: Acute Kidney Injury; Animals; Antibodies, Anti-Idiotypic; Cells, Cultured; Chemokine CCL2; Cysteine-Rich Protein 61; Disease Models, Animal; Fibrosis; Hypoxia; In Vitro Techniques; Interleukin-6; Kidney; Kidney Tubules, Proximal; Male; Mice; Mice, Inbred ICR; Nephritis; Reperfusion Injury; Serpin E2; Transforming Growth Factor beta

We examined the effects of increased expression of proximal tubule peroxisome proliferator-activated receptor (PPAR)α in a mouse model of renal fibrosis. After 5 days of unilateral ureteral obstruction (UUO), PPARα expression was significantly reduced in kidney tissue of wild-type mice but this downregulation was attenuated in proximal tubules of PPARα transgenic (Tg) mice. When compared with wild-type mice subjected to UUO, PPARα Tg mice had reduced mRNA and protein expression of proximal tubule transforming growth factor (TGF)-β1, with reduced production of extracellular matrix proteins including collagen 1, fibronectin, α-smooth muscle actin, and reduced tubulointerstitial fibrosis. UUO-mediated increased expression of microRNA 21 in kidney tissue was also reduced in PPARα Tg mice. Overexpression of PPARα in cultured proximal tubular cells by adenoviral transduction reduced aristolochic acid-mediated increased production of TGF-β, demonstrating PPARα signaling reduces epithelial TGF-β production. Flow cytometry studies of dissociated whole kidneys demonstrated reduced macrophage infiltration to kidney tissue in PPARα Tg mice after UUO. Increased expression of proinflammatory cytokines including IL-1β, IL-6, and TNF-α in wild-type mice was also significantly reduced in kidney tissue of PPARα Tg mice. In contrast, the expression of anti-inflammatory cytokines IL-10 and arginase-1 was significantly increased in kidney tissue of PPARα Tg mice when compared with wild-type mice subjected to UUO. Our studies demonstrate several mechanisms by which preserved expression of proximal tubule PPARα reduces tubulointerstitial fibrosis and inflammation associated with obstructive uropathy.

Topics: Animals; Arginase; Aristolochic Acids; B7-2 Antigen; Collagen Type I; Collagen Type IV; Down-Regulation; Fibrosis; Interleukin-10; Kidney Diseases; Kidney Tubules, Proximal; Laminin; Mice; Mice, Transgenic; MicroRNAs; Nephritis; PPAR alpha; Transforming Growth Factor beta; Ureteral Obstruction

Obesity is prevalent worldwide and is a major risk factor for many diseases including renal complications. Thrombospondin 1 (TSP1), a multifunctional extracellular matrix protein, plays an important role in diabetic kidney diseases. However, whether TSP1 plays a role in obesity-related kidney disease is unknown. In the present studies, the role of TSP1 in obesity-induced renal dysfunction was determined by using a diet-induced obese mouse model. The results demonstrated that TSP1 was significantly upregulated in the kidney from obese mice. The increased TSP1 was localized in the glomerular mesangium as well as in the tubular system from obese wild-type mice. Obese wild-type mice developed renal hypertrophy and albuminuria, which was associated with increased kidney macrophage infiltration, augmented kidney inflammation, and activated transforming growth factor (TGF)-β signaling and renal fibrosis. In contrast, obese TSP1-deficient mice did not develop these kidney damages. Furthermore, in vitro studies demonstrated that leptin treatment stimulated the expression of TSP1, TGF-β1, fibronectin, and collagen type IV in mesangial cells isolated from wild-type mice. These leptin-stimulated effects were abolished in TSP1-deficient mesangial cells. Taken together, these data suggest that TSP1 is an important mediator for obesity- or hyperleptinemia-induced kidney dysfunction.

Topics: Animals; Cells, Cultured; Collagen Type IV; Diabetic Nephropathies; Diet, High-Fat; Disease Models, Animal; Fibronectins; Fibrosis; Kidney; Leptin; Male; Mice; Nephritis; Obesity; Thrombospondin 1; Transforming Growth Factor beta

Hypertension is a risk factor for chronic kidney disease, particularly when associated with impaired renal autoregulation and thereby increased intraglomerular pressure (Pgc). Elevated Pgc can be modeled in vitro by exposing glomerular mesangial cells to mechanical strain. We previously showed that RhoA mediates strain-induced matrix production. Here, we show that RhoA activation is dependent on an intact microtubule network. Upregulation of the profibrotic cytokine connective tissue growth factor (CTGF) by mechanical strain is dependent on RhoA activation and inhibited by microtubule disruption. We tested the effects of the microtubule depolymerizing agent colchicine in 5/6 nephrectomized rats, a model of chronic kidney disease driven by elevated Pgc. Colchicine inhibited glomerular RhoA activation and attenuated both glomerular sclerosis and interstitial fibrosis without affecting systemic blood pressure. Upregulation of the matrix proteins collagen I and fibronectin, as well as CTGF, was attenuated by colchicine. Activity of the profibrotic cytokine TGF-β, as assessed by Smad3 phosphorylation, was also inhibited by colchicine. Microtubule disruption significantly decreased renal infiltration of lymphocytes and macrophages. Our studies thus indicate that colchicine modifies hypertensive renal fibrosis. Its protective effects are likely mediated by inhibition of RhoA signaling and renal infiltration of inflammatory cells. Already well-established in clinical practice for other indications, prevention of hypertension-associated renal fibrosis may represent a new potential use for colchicine.

Topics: Animals; Cells, Cultured; Colchicine; Collagen Type I; Connective Tissue Growth Factor; Cytoprotection; Disease Models, Animal; Enzyme Activation; Fibronectins; Fibrosis; Hypertension, Renal; Kidney; Male; Microtubules; Nephrectomy; Nephritis; Phosphorylation; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; Renal Insufficiency, Chronic; rhoA GTP-Binding Protein; Smad3 Protein; Stress, Mechanical; Transfection; Transforming Growth Factor beta

Iron is the most abundant metal in mammalian cells, and plays a pivotal role in many metabolic processes. Dysregulated iron homeostasis is involved in the cause of a number of pathological processes including renal diseases.. Longitudinal MRI scans of salt-loaded spontaneously hypertensive stroke-prone rats (SHRSP), an animal model that spontaneously develops hypertensive nephropathy, showed a decrease in renal and hepatic T2 SI (a sign of iron accumulation) of, respectively, 42.3 ± 2.5% (P < 0.01) and 60.4 ± 15.1% (P < 0.01) in comparison with SHRSP fed a standard diet. This was accompanied by the development of renal inflammation and oxidative stress (as evaluated by immunohistochemical and proteomic analyses), mitochondrial dysfunction, massive proteinuria and sustained intravascular hemolysis with the subsequent depletion of plasma haptoglobin, which was responsible for the renal uptake of hemoglobin and iron accumulation. In order to investigate the role of iron in these pathological processes, we subcutaneously treated the salt-loaded rats with the iron chelator deferoxamine (200 mg/kg per day). The pharmacological treatment prevented iron tissue accumulation, as indicated by the increase in renal and hepatic T2 SI of, respectively, 120.0 ± 10.1% (P < 0.01) and 73.9 ± 4.4% (P < 0.01) in comparison with salt-loaded rats treated with vehicle alone. Deferoxamine also preserved renal morphology and function, the renal infiltration of ED-1-positive macrophages/monocytes, and the expression of MCP-1 and TGF-β mRNA, reduced the level of reactive oxygen species, and improved the activity of mitochondrial cytochrome c oxidase.. These findings suggest that iron dysmetabolism is involved in the development of hypertensive nephropathy in SHRSP.

Topics: Animals; Blood Pressure; Deferoxamine; Disease Models, Animal; Hemolysis; Homeostasis; Hypertension, Renal; Iron; Kidney; Male; Models, Animal; Nephritis; Oxidative Stress; Proteinuria; Proteomics; Rats; Rats, Inbred SHR; Reactive Oxygen Species; Sodium Chloride, Dietary; Stroke; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) implicated in the pathogenesis of diabetic nephropathy. Hence, developing agents that antagonize fibrogenic signals is a critical issue facing researchers.. Fifty rats were allocated to five groups: 1=control rats, 2=diabetic hypertensive rats 3=diabetic hypertensive rats treated with spironolactone, 4=diabetic hypertensive rats treated with moexpril, 5=diabetic hypertensive rats treated with both spironolactone and moexpril. Measurement of TGF-β, aldosterone, creatinine and ACE. Degree of fibrosis was calculated.. Serum creatinine, mean arterial blood pressure (MAP), aldosterone, ACE, TGF-β and renal fibrosis increased significantly in untreated diabetic hypertensive rats compared with control rats. Administration of spironolactone, moexpril, or both decreased these changes.. Addition of the spironolactone to moexpril was more effective in reducing fibrosis and improvement of renal function than monotherapy with either drug, possibly due to a dual inhibitory effect on the RAS, and thus suppression of TGF-β.

Topics: Animals; Drug Therapy, Combination; Hypertension, Renal; Kidney; Kidney Function Tests; Male; Nephritis; Random Allocation; ras Proteins; Rats; Rats, Wistar; Spironolactone; Tetrahydroisoquinolines; Transforming Growth Factor beta

Recent evidence indicates that bone marrow-derived mesenchymal stem cells (BM-MSCs) possess immunosuppressive properties both in vitro and in vivo. We have previously demonstrated that transplantation of human MSCs can significantly improve the autoimmune conditions in MRL/lpr mice. The current study aimed to determine the mechanisms by which murine BM-MSC transplantation (MSCT) ameliorates nephritis in MRL/lpr mice. In this study, we found that MSCT can significantly prolong the survival of MRL/lpr mice. Eight weeks after transplantation, MSCT-treated mice showed significantly smaller spleens than control animals, with fewer marginal zones (MZs), T1, T2, activated B-cells, and plasma cells. Moreover, serum levels of B-cell activating factor (BAFF) and IL-10 in MSCT-treated mice decreased significantly compared to those in the control group, while levels of serum TGF-β were increased. Notably, decreased BAFF expression in both spleen and kidney was accompanied by decreased production of anti-dsDNA autoantibodies and proteinuria in MSCT-treated mice. Since BAFF is mainly expressed by T-cells and dendritic cells, we incubated BM-MSCs and DCs together and found that the production of BAFF by DCs was suppressed by MSCs. Thus, our findings suggest that MSCT may suppress the excessive activation of B-cells via inhibition of BAFF production in MRL/lpr mice.

Topics: Animals; Autoantibodies; B-Cell Activating Factor; B-Lymphocytes; Bone Marrow Cells; Cells, Cultured; Female; Interleukin-10; Kidney; Lymphocyte Activation; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred BALB C; Mice, Inbred MRL lpr; Nephritis; Proteinuria; Spleen; T-Lymphocytes; Transforming Growth Factor beta; Transplantation, Homologous

The mechanism by which TGF-β regulates renal inflammation and fibrosis is largely unclear; however, it is well accepted that its biological effects are mediated through Smad2 and Smad3 phosphorylation. Following activation, these Smads form heteromeric complex with Smad4 and translocate into the nucleus to bind and regulate the expression of target genes. Here we studied the roles of Smad4 to regulate TGF-β signaling in a mouse model of unilateral ureteral obstruction using conditional Smad4 knockout mice and in isolated Smad4 mutant macrophages and fibroblasts. Disruption of Smad4 significantly enhanced renal inflammation as evidenced by a greater CD45(+) leukocyte and F4/80(+) macrophage infiltration and upregulation of IL-1β, TNF-α, MCP-1, and ICAM-1 in the obstructed kidney and in IL-1β-stimulated macrophages. In contrast, deletion of Smad4 inhibited renal fibrosis and TGF-β1-induced collagen I expression by fibroblasts. Further studies showed that the loss of Smad4 repressed Smad7 transcription, leading to a loss of functional protein. This, in turn, inhibited IκBα expression but enhanced NF-κB activation, thereby promoting renal inflammation. Interestingly, deletion of Smad4 influenced Smad3-mediated promoter activities and the binding of Smad3 to the COL1A2 promoter, but not Smad3 phosphorylation and nuclear translocation, thereby inhibiting the fibrotic response. Thus, Smad4 may be a key regulator for the diverse roles of TGF-β1 in inflammation and fibrogenesis by interacting with Smad7 and Smad3 to influence their transcriptional activities in renal inflammation and fibrosis.

Topics: Animals; Extracellular Signal-Regulated MAP Kinases; Fibrosis; Gene Expression Regulation; Interleukin-1beta; Kidney; Mice; Mice, Inbred C57BL; Nephritis; NF-kappa B; Smad3 Protein; Smad4 Protein; Smad7 Protein; Transcription, Genetic; Transforming Growth Factor beta

It is known that angiotensin (Ang)-converting enzyme (ACE) 2 catalyzes Ang II to Ang 1-7 to prevent the detrimental effect of Ang II on blood pressure, renal fibrosis, and inflammation. However, mechanisms of renoprotective role of Ace2 remain largely unclear. The present study tested the hypothesis that deficiency of Ace2 may accelerate intrarenal Ang II-mediated fibrosis and inflammation independent of blood pressure in a model of unilateral ureteral obstructive (UUO) nephropathy induced in Ace2(+/y) and Ace2(-/y) mice. Results showed that both Ace2(+/y) and Ace2(-/y) mice had normal levels of blood pressure and plasma Ang II/Ang 1-7. In contrast, deletion of ACE2 resulted in a fourfold increase in the ratio of intrarenal Ang II/Ang 1-7 in the UUO nephropathy. These changes were associated with the development of more intensive tubulointerstitial fibrosis (α-SMA, collagen I) and inflammation (TNF-α, IL-1β, MCP-1, F4/80(+) cells, and CD3(+)T cells) in Ace2(-/y) mice at day 3 (all P<0.05) after UUO, becoming more profound at day 7 (all P<0.01). Enhanced renal fibrosis and inflammation in the UUO kidney of Ace2(-/y) mice were largely attributed to a marked increase in the intrarenal Ang II signaling (AT1-ERK1/2 mitogen-activated protein kinase), TGF-β/Smad2/3, and NF-κB signaling pathways. Further studies revealed that enhanced TGF-β/Smad and NF-κB signaling in the UUO kidney of Ace2(-/y) mice was associated with upregulation of an E3 ligase Smurf2 and a loss of renal Smad7. In conclusion, enhanced Ang II-mediated TGF-β/Smad and NF-κB signaling may be the mechanisms by which loss of Ace2 enhances renal fibrosis and inflammation. Smad7 ubiquitin degradation mediated by Smurf2 may be a central mechanism by which Ace2(-/y) mice promote TGF-β/Smad2/3-mediated renal fibrosis and NF-κB-driven renal inflammation in a mouse model of UUO nephropathy.

Topics: Analysis of Variance; Angiotensin II; Angiotensin-Converting Enzyme 2; Animals; Blood Pressure; Cytokines; Disease Models, Animal; Fibrosis; Kidney; Mice; Mice, Inbred C57BL; Mice, Knockout; Nephritis; NF-kappa B; Peptidyl-Dipeptidase A; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad7 Protein; Transforming Growth Factor beta; Ubiquitin-Protein Ligases; Ureteral Obstruction

Elevated blood level of C-reactive protein (CRP) is associated with increased risk of chronic kidney disease. However, whether this association reflects functional importance of CRP in the pathogenesis of kidney disease remains unclear. In this study, we examined the biological role of CRP in a well-characterized model of progressive kidney disease, unilateral ureteral obstruction (UUO), in mice that express the human CRP gene (CRPtg). Compared with wild-type (Wt) mice at 3 days after UUO, CRPtg mice developed more severe renal inflammation with a significant increase in tubulointerstitial T cells and macrophages, upregulation of proinflammatory cytokines (IL-1β and TNF-α), chemokines (MCP-1), and adhesion molecules (ICAM-1). Renal fibrosis was also significantly enhanced in CRPtg mice as demonstrated by increased expression of tubulointerstitial α-smooth muscle actin and collagen types I and III compared with Wt mice. Interestingly, on days 7 and 14 after UUO, an equal severity of renal inflammation and fibrosis were observed in CRPtg and Wt mice. These findings suggested that CRP may have a role in the initiation of renal inflammation and fibrosis. Further study revealed that enhanced early renal inflammation and fibrosis on day 3 in CRPtg mice was associated with a significant upregulation of endogenous mouse CRP and FcγRI mRNA and increased activation of both NF-κB/p65 and TGF-β/Smad2/3 signaling, while equal severity of progressive renal injury at day 7 and day 14 between CRPtg and Wt mice were attributed to equivalent levels of CRP, FcγRI, phospho-NF-κB/p65, and TGF-β/Smad2/3 signaling. Based on these findings, we conclude that CRP may not only be a biomarker, but also a mediator in the early development of renal inflammation and fibrosis in a mouse model of UUO. Enhanced activation of both NF-κB and TGF-β/Smad signaling pathways may be mechanisms by which CRP promotes early renal inflammation and fibrosis.

Topics: Actins; Analysis of Variance; Animals; C-Reactive Protein; Cell Adhesion Molecules; Chemokines; Collagen Type I; Collagen Type II; Cytokines; Fibrosis; Humans; Immunohistochemistry; Kidney Diseases; Macrophages; Mice; Mice, Transgenic; Nephritis; NF-kappa B; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; T-Lymphocytes; Transforming Growth Factor beta; Up-Regulation; Ureteral Obstruction

Therapeutic agents currently in use to treat systemic lupus erythematosus (SLE) are predominantly immunosuppressive agents with limited specificities. Multiple groups, including ours, have illustrated that inducing tolerance in SLE animal models ameliorates disease symptoms and increases survival. We examined if oral administration of a tolerogenic peptide could affect SLE disease progression. The pConsensus (pCons) peptide, based on protein sequences of anti-double stranded (anti-ds)DNA antibodies, induces tolerance through upregulation of regulatory T cells when administered intravenously. Six different forms of pCons, including multiple antigenic peptides (MAP) and cyclic peptides made up of L- and D-amino acids, at three different concentrations, were fed to BWF1 SLE-susceptible mice for 30 weeks. Mice fed 100 µg of L-MAP or D-MAP had less cumulative proteinuria and serum anti-dsDNA antibody levels than controls. In addition, animals in these groups also survived significantly longer than controls with a corresponding increase in serum transforming growth factor beta (TGFβ, implying a protective role for pCons-induced regulatory T cells. Oral administration of a tolerogenic peptide is a safe, effective method for ameliorating SLE disease manifestations and prolonging survival in SLE-prone mice. Induction of oral tolerance using modified pCons peptides could lead to a novel targeted therapy for human SLE.

Topics: Administration, Oral; Animals; Antibodies, Antinuclear; Humans; Immune Tolerance; Immunosuppressive Agents; Lupus Erythematosus, Systemic; Mice; Nephritis; Organic Chemicals; Peptides; Survival Rate; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Selectins play an essential role in most inflammatory reactions, mediating the initial leukocyte-rolling event on activated endothelium. Heparin and dermatan sulfate (DS) bind and block P- and L-selectin function in vitro. Recently, we reported that subcutaneous administration of DS inhibits colon inflammation in rats by reducing macrophage and T-cell recruitment and macrophage activation. In the present study, we examined the effect of porcine intestinal mucosa DS on renal inflammation and fibrosis in mice after unilateral ureteral obstruction (UUO). Twenty-four adult male Swiss mice weighing 20-25 g were divided into 4 groups: group C (N = 6) was not subjected to any surgical manipulation; group SH (N = 6) was subjected to surgical manipulation but without ureter ligation; group UUO (N = 6) was subjected to unilateral ureteral obstruction and received no treatment; group UUO plus DS (N = 6) was subjected to UUO and received DS (4 mg/kg) subcutaneously daily for 14 days. An immunoblot study was also performed for TGF-β. Collagen (stained area ~3700 µm(2)), MCP-1 (stained area ~1700 µm(2)), TGF-β (stained area ~13% of total area), macrophage (number of cells ~40), and myofibroblast (stained area ~1900 µm(2)) levels were significantly (P < 0.05) higher in the UUO group compared to control. DS treatment significantly (P < 0.05) reduced the content of collagen (stained area ~700 µm(2)), MCP-1 (stained area ~160 µm(2)) and TGF-β (stained area ~5% of total area), in addition to myofibroblast (stained area ~190 µm(2)) and macrophage (number of cells ~32) accumulation in the obstructed kidney. Overall, these results indicate that DS attenuates kidney inflammation by reducing macrophage recruitment, myofibroblast population and fibrosis in mice submitted to UUO.

Topics: Animals; Anti-Inflammatory Agents; Chemokine CCL2; Dermatan Sulfate; Disease Models, Animal; Fibrosis; Injections, Subcutaneous; Kidney; Macrophage Activation; Macrophages; Male; Mice; Myofibroblasts; Nephritis; Transforming Growth Factor beta; Ureteral Obstruction

Significant reduction of renal mass initiates a series of hemodynamic and nonhemodynamic events which lead to proteinuria, glomerulosclerosis, tubulointerstitial injury, and end-stage renal failure. Lipid mediators derived from fatty acids participate in regulation of renal hemodynamic and nonhemodynamic processes that influence progression of renal disease. Composition of cellular fatty acids and hence related signaling responses are influenced by their dietary contents. Consumption of omega-3 fatty acids (O-3FA) has proven effective in mitigating atherosclerosis. We tested the hypothesis that O-3FA supplementation may retard progression and attenuate upregulation of pathways involved in oxidative stress, inflammation, and fibrosis in rats with renal mass reduction. Sprague-Dawley rats were subjected to 5/6 nephrectomy [chronic renal failure (CRF)] and randomly assigned to the untreated and O-3FA-treated (0.3 g.kg(-1).day(-1) by gastric gavage for 12 wk) groups. Sham-operated rats served as controls. The untreated CRF rats exhibited proteinuria, hypertension, azotemia, upregulations of renal tissue NAD(P)H oxidase, MCP-1, COX-2, PAI-1, TGF-beta, Smad2, alpha-smooth muscle actin, fibronectin, and hepatocyte growth factor, activation of ERK1/2 and NF-kappaB, downregulation of Smad7, intense mononuclear leukocyte infiltration, tubulointerstitial fibrosis, and glomerulosclerosis. O-3FA supplementation significantly lowered COX-2, NAD(P)H oxidase (NOX-4, gp91(phox), p47(phox), p22(phox)), PAI-1, TGF-beta, connective tissue growth factor, alpha-smooth muscle actin, fibronectin, Smad2, and MCP-1, raised Smad7, and attenuated ERK1/2 and NF-kappaB activation, tubulointerstitial fibrosis, and inflammation. Thus, long-term O-3FA supplementation can reduce or reverse upregulation of prooxidant, proinflammatory, and profibrotic pathways and attenuate tubulointerstitial fibrosis in the remnant kidney.

Topics: Actins; Animals; Chemokine CCL2; Connective Tissue Growth Factor; Cyclooxygenase 2; Dietary Supplements; Extracellular Signal-Regulated MAP Kinases; Fatty Acids, Omega-3; Fibronectins; Fibrosis; Hepatocyte Growth Factor; Inflammation; Kidney; Male; NADPH Oxidases; Nephritis; NF-kappa B; Oxidative Stress; Plasminogen Activator Inhibitor 1; Rats; Rats, Sprague-Dawley; Renal Insufficiency, Chronic; Smad2 Protein; Smad7 Protein; Transforming Growth Factor beta

End-stage renal disease has been associated with an inflammatory state. TGF-beta plays a critical role in antiinflammation counteracting inflammatory cytokines, wound healing, and tissue repair. We, therefore, speculated the protective role of TGF-beta in renal inflammation rather than inducing fibrosis. Three polymorphisms of TGF-beta (713-8delC), i.e., C deletion in intron sequence 8 base prior to exon-5 by PCR-RFLP and codon-10, Leu/Pro, and codon-25, Arg/Pro by Amplification Refractory Mutation System (ARMS-PCR) techniques were genotyped in 228 end-stage renal disease (ESRD) patients and 180 controls. Linkage disequilibrium (LD) and haplotype analysis was performed by Arlequin software. Our data showed positive association between codon-10 polymorphism and ESRD risk (P < 0.001; OR 4.845, 95% CI 2.57-9.11 for Pro/Pro). However, genotype frequencies were comparable in patients and controls for 713-8delC, while in the case of codon-25, a trend of higher frequency of Pro/Pro genotype (16.2% versus 10.0%) was observed but the P-value did not reach significant (P = 0.187). Significant association of codon-10 Pro/Pro was observed in patients with glomerulonephritis (P = 0.001; OR 4.138, 95%CI 2.1-8.13). LD was found significant between codon-10 and 25 (P = 0.021). Haplotype "Pro-Pro" showed 1.8-fold higher risk for ESRD (p = 0.003; OR = 1.867, 95%CI = 1.229-2.838). A combined analysis of the effect of TGF-beta (codon-10) with C-deletion and codon-25 showed significant difference for TGF-beta(10)-TGF-beta(C-del) (P = 0.010). In conclusion, the present study suggests that low-producing genotype (Pro/Pro) of TGF-beta (codon-10) polymorphism is associated with ESRD. Haplotype analysis further suggested that "Pro-Pro" (low producer) is associated with higher risk for ESRD. Thus, high-producing genotype of TGF-beta may be beneficial and may play a potential role in the resolution of renal inflammation.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Alleles; Codon; Female; Genotype; Haplotypes; Humans; Kidney Failure, Chronic; Linkage Disequilibrium; Male; Middle Aged; Nephritis; Polymorphism, Genetic; Proline; Risk; Transforming Growth Factor beta

High-potassium diets have been shown to be beneficial in cardiovascular disease partly because of a blood pressure-lowering effect. The effect of potassium on inflammation has not been studied. We investigated the influence of potassium supplementation on the degree of renal inflammation and the intracellular signaling mechanisms that could mediate inflammation in chronic kidney disease (CKD). CKD was created in male Sprague-Dawley rats by subtotal nephrectomy. Two groups of CKD rats were pair fed with diets containing 2.1% potassium (potassium-supplemented diet) or 0.4% potassium (basal diet). Body weight, blood pressure, and blood and urine electrolytes were measured biweekly. The animals were euthanized at week 8, and the remnant kidneys were analyzed by histology, immunohistochemistry, Western blotting, and real-time quantitative PCR. In the CKD pair-fed groups, blood potassium concentration did not differ significantly, but blood pressure was lower in the potassium-supplemented group. Compared with the basal diet, potassium supplementation decreased renal tubulointerstitial injury and suppressed renal inflammation as evidenced by decreased macrophage infiltration, lower expression of inflammatory cytokines, and decreased NF-kappaB activation. These renoprotective effects were associated with downregulation of renal transforming growth facto-beta, upregulation of renal Smad7, and lower blood pressure. Our results show that potassium supplementation can reduce renal inflammation and hence, could modulate the progression of kidney injury in CKD.

Topics: Aldosterone; Angiotensin II; Animals; Blood Pressure; Chronic Disease; Creatinine; Disease Models, Animal; Kidney; Kidney Diseases; Male; Nephritis; NF-kappa B; Potassium; Potassium, Dietary; Rats; Rats, Sprague-Dawley; Smad7 Protein; Sodium; Transforming Growth Factor beta

We previously showed that supplementation with 17beta-estradiol (E2) from the onset of diabetes attenuates the development of diabetic renal disease. The aim of the present study was to examine whether E2 can also attenuate the disease process once it has developed. The present study was performed in nondiabetic and streptozotocin-induced diabetic Sprague-Dawley rats. E2 supplementation began after 9 wk of diabetes and continued for 8 wk. Diabetes was associated with an increase in urine albumin excretion, glomerulosclerosis, tubulointerstitial fibrosis, renal cortical collagen type I and IV, laminin, plasminogen activator inhibitor-1, tissue inhibitors of metalloproteinase-1 and -2, transforming growth factor (TGF)-beta, TGF-beta receptor type I and II, Smad2/3, phosphorylated Smad2/3, and Smad4 protein expression, and CD68-positive cell abundance. Decreases in matrix metalloproteinase (MMP)-2 protein expression and activity and decreases in Smad6 and Smad7 protein expression were also associated with diabetes. E2 supplementation completely or partially attenuated all these changes, except Smad4 and fibronectin, on which E2 supplementation had no effect. These data suggest that E2 attenuates the progression of diabetic renal disease once it has developed by regulating extracellular matrix, TGF-beta, and expression of its downstream regulatory proteins. These findings support the notion that sex hormones in general, and E2 in particular, are important regulators of renal function and may be novel targets for the treatment and prevention of diabetic renal disease.

Topics: Albuminuria; Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Progression; Estradiol; Extracellular Matrix; Extracellular Matrix Proteins; Female; Fibrosis; Glomerulosclerosis, Focal Segmental; Kidney Tubules; Matrix Metalloproteinases; Nephritis; Plasminogen Activator Inhibitor 1; Rats; Rats, Sprague-Dawley; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta

Chronic allograft nephropathy is a sclerotic process characterized by an increased extracellular matrix (ECM) protein deposition. Fibronectin (FN) is a major component of ECM. FN has been reported to undergo alternative splicing and produce several isoforms including the extra domain-B (ED-B) containing embryonic isoform. In the present study, we investigated ED-B FN expression in chronic allograft nephropathy and its relationship with endothelins (ET).. To establish chronic allograft nephropathy, allografts were performed between Fisher 344 --> Lewis rats. Allograft recipients were then randomly divided into two groups, allografts and allografts treated with ET receptor antagonist bosentan. Lewis --> Lewis recipients were used as isograft controls. Grafts were harvested at 30, 90 and 140 days for histological and gene expression analyses with respect to ED-B FN, ET-1 and transforming growth factor-beta1 (TGF-beta1) mRNA. ED-B FN protein levels were assessed by immunohistochemical analysis. Additionally, we analyzed human renal allograft biopsies.. Our data demonstrates that rat chronic allograft nephropathy is associated with progressive upregulation of ED-B FN mRNA and protein. ET-1 and TGF-beta1 mRNA were also upregulated. Treatment of allograft recipient rats with bosentan prevented upregulation of ED-B FN and TGF-beta1. We further show that total FN, ED-B FN, ET-1 and TGF-beta1 mRNA expression were upregulated in human chronic allograft nephropathy specimens.. Results obtained from both human and rat renal allograft tissues suggest that expression of ED-B FN is upregulated in chronic allograft nephropathy and such upregulation is mediated via ET-1 and its interaction with TGF-beta1.

Topics: Adolescent; Adult; Aged; Animals; Biopsy; Child; Chronic Disease; Endothelin-1; Female; Fibronectins; Gene Expression Regulation; Graft Rejection; Humans; Kidney; Kidney Transplantation; Male; Middle Aged; Nephritis; Protein Isoforms; Rats; RNA, Messenger; Transforming Growth Factor beta; Transplantation, Homologous

In the kidney, a unique plasticity exists between epithelial and mesenchymal cells. During kidney development, the metanephric mesenchyme contributes to emerging epithelium of the nephron via mesenchymal to epithelial transition (MET). In the injured adult kidney, renal epithelia contribute to the generation of fibroblasts via epithelial-mesenchymal transition, facilitating renal fibrosis. Recombinant human bone morphogenic protein (BMP)-7, a morphogen that is essential for the conversion of epithelia from condensing mesenchyme during kidney development, enhances the repair of tubular structures in the kidney. In this setting, BMP-7 inhibits epithelial-mesenchymal transition involving adult renal epithelial tubular cells and decreases secretion of type I collagen by adult renal fibroblasts. In search of a mechanism behind the ability of BMP-7 to repair damaged renal tubules, we hypothesized that systemic treatment with BMP-7 might induce MET involving adult renal fibroblasts in the injured kidney, generating functional epithelial cells. Here we report that BMP-7 induces formation of epithelial cell aggregates in adult renal fibroblasts associated with reacquisition of E-cadherin expression and decreased motility, mimicking the effect of BMP-7 on embryonic metanephric mesenchyme to generate epithelium. In addition, we provide evidence that BMP-7-mediated repair of renal injury is associated with MET involving adult renal interstitial fibroblasts in mouse models for renal fibrosis. Collectively, these findings suggest that adult renal fibroblasts might retain parts of their original embryonic imprint and plasticity, which can be re-engaged by systemic administration of BMP-7 to mediate repair of tubular injury in a fibrotic kidney.

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cadherins; Cell Movement; Cell Proliferation; Collagen Type I; Epithelial Cells; Fibroblasts; Humans; Immunohistochemistry; Kidney; Mesoderm; Mice; Nephritis; Promoter Regions, Genetic; Protein Structure, Tertiary; Recombinant Proteins; Regeneration; Sepharose; Time Factors; Transforming Growth Factor beta

Treating patients with kidney failure by organ transplantation has been extraordinarily successful. Although, current immunosuppressants have improved short-term allograft survival, most transplants are eventually lost due to chronic allograft nephropathy (CAN). The molecular mechanisms underlying CAN are poorly understood. Smooth muscle cells (SMC) play a major role in the pathogenesis of CAN by contributing to the thickening of the intima and narrowing of the lumen of blood vessels. We show that selenium-binding protein-1 (SBP-1), a protein implicated in protein trafficking and secretion, is localized primarily to SMC in vivo. SBP-1 was heavily tyrosine-phosphorylated in vivo. Remarkably, SBP-1 was absent or strongly downregulated in vascular SMC in monkey kidney allografts with CAN. In contrast, the SMC alpha-actin was strongly expressed in the vascular SMC of the same allografts, indicating that the decrease in SBP-1 was not due to a global decrease in SMC proteins. Out of four growth factors implicated in the pathogenesis of CAN, only TGF-beta blocked the expression of SBP-1; thus, TGF-beta could regulate the expression of SBP-1 in CAN. These results show that SBP-1 localizes primarily to SMC in vivo and implicate this phosphoprotein in the effects of TGF-beta on SMC and in the process of CAN.

Topics: Actins; Animals; Carrier Proteins; Cell Line; Coronary Vessels; Detergents; Disease Models, Animal; Down-Regulation; Electrophoresis, Gel, Two-Dimensional; Female; Humans; Immunohistochemistry; Immunosuppressive Agents; Kidney; Kidney Diseases; Macaca mulatta; Mass Spectrometry; Muscle, Smooth; Nephritis; Phosphoproteins; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; Selenium-Binding Proteins; Transforming Growth Factor beta; Tyrosine; Uterus

Transforming growth factor-beta (TGF-beta) in renal fibrosis has been well studied, but little attention has been paid to the potential role of TGF-beta in the resolution of renal inflammation. We hypothesize that TGF-beta exerts its anti-inflammation properties by stimulating its negative signaling pathway involving Smad7.. A rat remnant kidney model was treated with a doxycycline-regulated Smad7 gene or control empty vector using an ultrasound-microbubble (Optison)-mediated system. Smad7 transgene expression within the kidney was tightly controlled by the addition of doxycycline in the daily drinking water. All animals were euthanized at week 4 for examination of inflammatory responses.. Real-time polymerase chain reaction (PCR) and immunohistochemistry revealed that gene transfer of Smad7 resulted in a substantial inhibition of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) expression (all P < 0.01 vs. control). This was associated with the attenuation of histology damage, proteinuria, serum creatinine, and an increase in creatinine clearance (all P < 0.05). In addition, overexpression of Smad7 significantly inhibited renal inflammation, including ICAM-1, iNOS, and accumulation of macrophages and T cells in both glomeruli and tubulointerstitium. Furthermore, gene transfer of Smad7 also substantially blocked nuclear factor kappa B (NFkappaB) activation in the rat remnant kidney (P < 0.01).. TGF-beta/Smad7 signaling plays a critical role in the resolution of renal inflammation in rat remnant kidney model. Inhibition of NFkappaB activation is a key mechanism by which Smad7 suppresses renal inflammation, which suggests a crosstalk pathway between NFkappaB and Smad7. The ability of Smad7 to inhibit renal inflammation indicates that ultrasound-microbubble-mediated Smad7 gene therapy may represents a new therapeutic strategy for glomerulonephritis.

Topics: Animals; Blood Pressure; DNA-Binding Proteins; Genetic Therapy; Intercellular Adhesion Molecule-1; Interleukin-1; Kidney Function Tests; Leukocytes; Male; Nephrectomy; Nephritis; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley; Signal Transduction; Smad7 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Ultrasonography, Interventional

SB-525334 (6-[2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl]-quinoxaline) has been characterized as a potent and selective inhibitor of the transforming growth factor-beta1 (TGF-beta1) receptor, activin receptor-like kinase (ALK5). The compound inhibited ALK5 kinase activity with an IC(50) of 14.3 nM and was approximately 4-fold less potent as an inhibitor of ALK4 (IC(50) = 58.5 nM). SB-525334 was inactive as an inhibitor of ALK2, ALK3, and ALK6 (IC(50) > 10,000 nM). In cell-based assays, SB-525334 (1 microM) blocked TGF-beta1-induced phosphorylation and nuclear translocation of Smad2/3 in renal proximal tubule cells and inhibited TGF-beta1-induced increases in plasminogen activator inhibitor-1 (PAI-1) and procollagen alpha1(I) mRNA expression in A498 renal epithelial carcinoma cells. In view of this profile, SB-525334 was used to investigate the role of TGF-beta1 in the acute puromycin aminonucleoside (PAN) rat model of renal disease, a model of nephritis-induced renal fibrosis. Orally administered doses of 1, 3, or 10 mg/kg/day SB-525334 for 11 days produced statistically significant reductions in renal PAI-1 mRNA. Also, the compound produced dose-dependent decreases in renal procollagen alpha1(I) and procollagen alpha1(III) mRNA, which reached statistical significance at the 10-mg/kg/day dose when compared with vehicle-treated PAN controls. Furthermore, PAN-induced proteinuria was significantly inhibited at the 10-mg/kg/day dose level. These results provide further evidence for the involvement of TGF-beta1 in the profibrotic changes that occur in the PAN model and for the first time, demonstrate the ability of a small molecule inhibitor of ALK5 to block several of the markers that are predictive of fibrosis and renal injury in this model.

Topics: Activin Receptors; Animals; Biomarkers; Cell Line, Tumor; Collagen Type I; Dose-Response Relationship, Drug; Fibrosis; Humans; Kidney; Nephritis; Phosphorylation; Plasminogen Activator Inhibitor 1; Protein Serine-Threonine Kinases; Puromycin; Rats; Rats, Sprague-Dawley; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

We investigated the effects of pravastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, on interstitial inflammation and fibrosis, using an animal model of chronic cyclosporine A (CsA)-induced nephropathy. Sprague-Dawley rats were maintained on a low-salt diet (0.05% sodium) and treated daily for 1 or 4 wk with vehicle (olive oil; 1 ml/kg sc), CsA (15 mg/kg sc), or both CsA and pravastatin (5 or 20 mg/kg in the drinking water). Anti-inflammatory and antifibrotic effects of pravastatin were studied by evaluating the concentrations of the inflammatory mediators osteopontin (OPN) and C-reactive protein (CRP), of fibrotic cytokine-transforming growth factor (TGF)-beta1, and the presence of ED-1-positive cells (macrophages). In addition, renal function, serum lipid levels, histopathology (arteriolopathy and tubulointerstitial fibrosis), and the expression of the vasoactive factors endothelial nitric oxide synthase (eNOS) and renin protein were also compared for different treatment groups. Pravastatin induced dose-dependent decreases in the expression of OPN, intrarenal CRP, and TGF-beta1, and in the numbers of ED-1-positive cells at 1 and 4 wk. These were accompanied by a significant attenuation of tubulointerstitial fibrosis at 4 wk. The downregulation of eNOS protein expression in CsA-treated rat kidney was markedly upregulated by pravastatin treatment, although intrarenal renin expression was unaffected. Renal dysfunction induced by CsA significantly improved with administration of pravastatin at a dose of 20 mg/kg. Neither CsA nor pravastatin influenced serum lipid or high-sensitivity CRP levels in the treatment groups. Thus in chronic CsA nephropathy, pravastatin effectively abrogates the progression of tubulointerstitial inflammation and fibrosis. This may support the clinical use of pravastatin.

Topics: Animals; Anticholesteremic Agents; C-Reactive Protein; Cyclosporine; Fibrosis; Immunosuppressive Agents; Inflammation Mediators; Kidney; Macrophages; Male; Nephritis; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Osteopontin; Pravastatin; Rats; Rats, Sprague-Dawley; Renin; RNA, Messenger; Sialoglycoproteins; Transforming Growth Factor beta; Transforming Growth Factor beta1

The relative roles of angiotensin II (Ang II) type 1 receptor (AT(1)R) and Ang II type 2 receptor (AT(2)R) in immune-mediated nephritis are unknown, and the effect of the blockade of AT(1)R and its indirect counter-activation of AT(2)R relative to the anti-fibrotic action in this disease is unclear. To address this question, we studied the role of AT(1)R and AT(2)R in anti-glomerular basement membrane nephritis in SJL mice. Groups of mice were treated with either an AT(1)R antagonist (CGP-48933; CGP group), an AT(2)R antagonist (PD-123319; PD group), both (CGP/PD group), or a vehicle (PCt group) from Day 29 to 56. At Day 56 post-treatment, fibrosis-related parameters such as interstitial matrix deposition, and the expression of genes of TGF-beta1, plasminogen activator inhibitor-1, and type I collagen were significantly reduced in the kidney in the CGP group. There were no significant effects on these parameters in the PD group. However, this anti-fibrotic action by CGP-48933 was totally abolished by co-treatment with PD-123319 in the CGP/PD group. The gene expression of renin was significantly increased in the kidneys in the CGP and CGP/PD groups, suggesting that CGP-48933 had increased Ang II generation in those groups. In conclusion, counter-activation of AT(2)R by increased Ang II under AT(1)R blockade likely conferred an anti-fibrotic protection in this model.

Topics: Angiotensin II; Animals; Basement Membrane; Collagen; Creatinine; Kidney; Kidney Glomerulus; Male; Mice; Nephritis; Plasminogen Activator Inhibitor 1; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Renin; Ribonucleases; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

Bone morphogenic protein (BMP)-7 is a 35-kDa homodimeric protein and a member of the transforming growth factor (TGF)-beta superfamily. BMP-7 expression is highest in the kidney, and its genetic deletion in mice leads to severe impairment of eye, skeletal and kidney development. Here we report that BMP-7 reverses TGF-beta1-induced epithelial-to-mesenchymal transition (EMT) by reinduction of E-cadherin, a key epithelial cell adhesion molecule. Additionally, we provide molecular evidence for Smad-dependent reversal of TGF-beta1-induced EMT by BMP-7 in renal tubular epithelial cells and mammary ductal epithelial cells. In the kidney, EMT-induced accumulation of myofibroblasts and subsequent tubular atrophy are considered key determinants of renal fibrosis during chronic renal injury. We therefore tested the potential of BMP-7 to reverse TGF-beta1-induced de novo EMT in a mouse model of chronic renal injury. Our results show that systemic administration of recombinant human BMP-7 leads to repair of severely damaged renal tubular epithelial cells, in association with reversal of chronic renal injury. Collectively, these results provide evidence of cross talk between BMP-7 and TGF-beta1 in the regulation of EMT in health and disease.

Topics: Activin Receptors, Type I; Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cadherins; Cell Differentiation; Cells, Cultured; Chronic Disease; DNA-Binding Proteins; Epithelial Cells; Kidney Tubules; Mesoderm; Mice; Mice, Inbred Strains; Nephritis; Phosphoproteins; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Signal Transduction; Smad3 Protein; Smad5 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1

The overexpression of transforming growth factor (TGF)-beta and Smad-mediated intracellular TGF-beta signaling in the kidney underlies the development of renal scarring from pathological matrix accumulation. However, changes in the Smad proteins during the progression of kidney disease are unclear. In this study, we investigated the regulation of Smad proteins in the glomeruli of rats with anti-thymocyte serum nephritis. We found that Smad2 protein decreased markedly in nephritic glomeruli, whereas no significant changes were observed in the levels of Smad3 and Smad4 proteins. In contrast, the level of Smad2 mRNA in nephritic glomeruli did not differ significantly from that in control glomeruli. Based on recent reports of the ubiquitin-mediated degradation of Smad2, we investigated the degradation and ubiquitination activity directed against Smad2 in glomerular extracts. Both the degradation and ubiquitination of Smad2 were markedly increased in glomerular extracts from rats with nephritis. We also found that Smurf2, a ubiquitin ligase for Smad2, was increased in the nephritic glomerular extracts. These data suggest that the decrease in Smad2 resulted from enhanced ubiquitin-dependent degradation of Smad2 mediated by Smurf2, and is involved in the regulation of Smad2-mediated TGF-beta signaling in nephritic glomeruli.

Topics: Animals; Antilymphocyte Serum; DNA-Binding Proteins; Glutathione Transferase; In Vitro Techniques; Kidney Glomerulus; Ligases; Male; Nephritis; Phosphoproteins; Proteinuria; Rats; Rats, Wistar; Recombinant Fusion Proteins; Sheep; Smad2 Protein; Smad3 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Ubiquitin; Ubiquitin-Protein Ligases

The present study was performed to clarify the mechanism underlying the beneficial effects of lisinopril on chronic glomerulonephritis. Chronic glomerulonephritis was induced by a single injection of E30 monoclonal antibody (E30) recognizing Thy-1.1 antigen to unilaterally nephrectomized rats. E30 injection resulted in persistent massive proteinuria with a decrease in anionic charge sites on the glomerular basement membrane (GBM) at 8 weeks. Also, renal tissue from rats treated with E30 showed typical glomerulosclerosis and tubulointerstitial fibrosis. Lisinopril exerted a potent antiproteinuric effect and suppressed the progression of both glomerulosclerosis and tubulointerstitial fibrosis. Lisinopril recovered the reduced number of anionic charge sites on GBM, accounting for the positive action against massive proteinuria. Immunostaining for desmin revealed that lisinopril treatment prevented the injury of glomerular epithelial cells (GECs) occurring in the chronic nephritic stage. Also, the level of gene expression of transforming growth factor-beta (TGF-beta) and plasminogen activator inhibitor-1 (PAI-1) in the renal cortex were reduced, suggesting that lisinopril improved extracellular matrix (ECM) metabolism. These results indicated that proteinuria in Thy-1.1 antibody-induced chronic nephritis is associated with a decrease in anionic charge sites on GBM, and that the antiproteinuric effect of lisinopril is attributable to protection against GEC damage. Suppression of TGF-beta and PAI-1 expression contributed to the preventive effect of lisinopril on ECM deposition in renal tissue.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Base Sequence; DNA Primers; Immunohistochemistry; Kidney Glomerulus; Lisinopril; Male; Microscopy, Electron; Nephritis; Plasminogen Activator Inhibitor 1; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

We have previously shown that renovascular hypertension does not inhibit healing of the acute Thy-1 nephritis. To test whether a chronic model of the Thy-1 nephritis is more susceptible to high blood pressure, the repetitive hit model was evaluated in rats with 2-kidney, 1-clip Goldblatt hypertension.. Six weeks after initiation of 2-kidney, 1-clip hypertension, chronic Thy-1 glomerulonephritis was induced in hypertensive rats by four consecutive injections of rabbit antiserum in weekly intervals. Renal structure and function were examined two weeks after the last injection. Glomerular binding of rabbit IgG as well as expression of transforming growth factor-beta (TGF-beta), alpha-smooth muscle actin (alpha-SMA) and cyclooxygenase (COX)-1 and -2 were evaluated by Western blotting.. Similar glomerular deposition of rabbit IgG was detected in normotensive rats and in both kidneys of Goldblatt hypertensive rats indicating similar delivery and binding of the heterologous antibody. Induction of the repetitive Thy-1 model significantly enhanced glomerular damage in the nonclipped kidney and increased albuminuria. Surprisingly, no glomerular damage developed in the clipped kidney of nephritic hypertensive rats. In contrast, increased glomerular volume and increased expression of TGF-beta, alpha-SMA as well as COX-1 and COX-2 were found in normotensive nephritic rats and in both kidneys of nephritic hypertensive rats.. Glomerular and tubulointerstitial damage of the chronic Thy-1 model is dramatically enhanced in the nonclipped kidneys of Goldblatt hypertensive rats. In contrast, the clipped kidney is completely protected from this immunological injury despite similar activation of glomerular cells, induction of TGF-beta, COX-1 and COX-2 and glomerular hypertrophy.

Topics: Albuminuria; Animals; Blood Pressure; Chronic Disease; Cyclooxygenase 1; Cyclooxygenase 2; Hypertension, Renovascular; Immune Sera; Isoantibodies; Isoenzymes; Kidney; Kidney Glomerulus; Kidney Tubules; Male; Membrane Proteins; Nephritis; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

Tubulitis is a defining feature for the diagnosis and management of acute renal allograft rejection. Lymphocytes extracted from rejecting renal tissue are known to express the alphaEbeta7-integrin (CD103), a receptor for E-cadherin expressed on epithelial cells. In this study, expression of CD103 was examined in situ in tubulitis associated with acute rejection.. Immuno-labeling detected CD8+ and CD103+ lymphocytes and E-cadherin on epithelial cells in cryostat sections from 34 diagnostic biopsy specimens and a limited number of transplant nephrectomies. CD8+ and CD103+ intratubular cells were enumerated as mean numbers per tubular crosssection and median values were compared between rejection grades as were median ratios of CD103+ to CD8+ cells. Active transforming growth factor (TGF) beta1 was quantified in paraffin sections by immunofluorescence and confocal microscopical analysis. A parallel in vitro study quantified CD103+ T cells after allospecific activation with and without exogenous TGFbeta1.. CD8+ T cells were present in tubules and tubular interstitium in acute rejection. CD103+ T cells were restricted exclusively to the tubules. The numbers of intratubular CD8+ and CD103+ cells and the ratio of intratubular CD103+ to CD8+ cells increased significantly with tubulitis score (P values 0.005, 0.009, and 0.02, respectively). TGFbeta1 expression was wide-spread in tubules also increasing significantly with tubulitis score (P=0.034). In chronic rejection, CD103+ T cells and TGFbeta1 were present within both tubules and interstitial cell populations. The in vitro study demonstrated that addition of TGFbeta1 to activated, alloantigen-specific T cells increased the proportion of CD8+ cells that also expressed CD103.. These data indicate that specific upregulation of the alphaEbeta7-integrin by activated, intratubular T cells in acute renal allograft rejection could be a consequence of exposure to high local concentrations of TGFbeta1. The capacity of CD103+ T cells to bind E-cadherin on tubular epithelial cells may be an important factor in the pathogenesis of specific tissue damage observed in acute renal allograft rejection.

Topics: Antigens, CD; Biopsy; Cadherins; Graft Rejection; Humans; Integrin alpha Chains; Kidney Transplantation; Kidney Tubules; Nephritis; T-Lymphocytes; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor-beta has three main isoforms (TGF-beta1, TGF-beta2, and TGF-beta3) that have distinct but overlapping functions in immunity, inflammation, and tissue repair. TGF-beta1 has been implicated in progressive renal scarring, but the roles of TGF-beta2 and TGF-beta3 are less clear. The purpose of this study was to characterize the expression of all three isoforms in nephrotoxic nephritis (NTN) in rats and to determine the effect of TGF-beta3 infusions on injury because of its reported combined anti-inflammatory and antifibrotic effects.. TGF-beta1, TGF-beta2, and TGF-beta3 expression was analyzed by immunohistochemistry and RNase protection assays. TGF-beta3 was administered by osmotic minipumps at 2 microg/day, a dose shown to alter glomerular macrophage function in vivo. Injury was assessed morphologically and functionally.. The three TGF-beta isoforms showed a different distribution in normal rats and after the induction of nephritis. TGF-beta1 was only detected in glomeruli of the most severely nephritic rats. TGF-beta2 was found in glomerular neutrophils, whereas damaged podocytes expressed TGF-beta3. Infusions of TGF-beta3 did not reduce proteinuria over seven days after the induction of nephritis. They did, however, have a profound effect on glomerular macrophage number (7.76 +/- 4.1 in treated rats vs. 14.4 +/- 4.7 in controls, P < 0.02). The numbers of class II-positive macrophages were similar in the two groups, whereas class II-negative macrophages infiltrating glomeruli were significantly decreased (4.06 +/- 3.1 vs. 9.1 +/- 4.4, P < 0.02). TGF-beta did not influence the amount of glomerular matrix.. TGF-beta isoforms have different expressions and presumptively different roles in NTN. The infusion of pharmacological doses of TGF-beta3 has profound effects on macrophages infiltrating nephritic glomeruli and reveals marked heterogeneity of infiltrating macrophages.

Topics: Animals; Infusion Pumps; Kidney; Nephritis; Neutrophils; Protein Isoforms; Rats; Rats, Sprague-Dawley; Reference Values; Tissue Distribution; Transforming Growth Factor beta

Under normal conditions, kidney expresses Smad6 and Smad7 most abundantly among the organs of the body. To understand the physiological roles of these Smad expressions in the kidney, we first identified the sites of Smad6 and Smad7 expression in the rat kidney by in situ hybridization. The expression of Smad7 in the rat kidney was only observed in the glomeruli, while Smad6 was expressed in both the glomeruli and thick ascending limb of Henle's loop. In order to investigate whether Smad6 and 7 are also involved in the negative feedback loop of TGF-beta signaling in vivo, we examined the changes of mRNA levels of these Smads in the glomeruli of rat anti-Thy1 (1-22-3) nephritis, a model where the expression of TGF-beta in the glomeruli has been shown to be most up-regulated from day 4 to 14 after the antibody injection. Unexpectedly, 7 days after injection, the levels of Smad6 and Smad7 did not increase but rather decreased to approximately 70% of the levels on day 0. During that period, Smad7 immunostaining was observed in the glomerular endothelial cells (GEN) where Smad3 immunostaining was also observed. This suggested that Smad7 expression was not augmented by the TGF-beta signal in GEN in vivo in anti-Thy-1 nephritis. The absence of up-regulation of these inhibitory Smads may be involved in the pathogenesis of anti-Thy-1 nephritis.

Topics: Animals; Cloning, Molecular; DNA Primers; DNA-Binding Proteins; Immunoenzyme Techniques; In Situ Hybridization; Isoantibodies; Kidney; Kidney Glomerulus; Macrophages; Male; Nephritis; Polymerase Chain Reaction; Rats; Rats, Wistar; RNA, Messenger; Smad6 Protein; Smad7 Protein; Trans-Activators; Transforming Growth Factor beta

Topics: Animals; Cell Line; Chimera; Exons; Feasibility Studies; Fibronectins; Gene Transfer Techniques; Glomerulosclerosis, Focal Segmental; Growth Inhibitors; Immunoglobulin Fc Fragments; Nephritis; Protein Serine-Threonine Kinases; Rats; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Fusion Proteins; RNA, Messenger; Transforming Growth Factor beta

Previous studies have demonstrated that enalapril and verapamil seem to attenuate the cyclosporine nephrotoxicity. However, the mechanisms have not been completely understood, especially on molecular events. The aim of this study was to examine the effect of individual or combined treatment on osteopontin, TGF-beta, endothelin-1 and procollagen alpha 1(I) mRNA expressions. Enalapril (50 mg/L in drinking water) and verapamil (0.5 mg/kg/day, subcutaneously), alone or in combination, were administered to rats with chronic cyclosporine nephrotoxicity (cyclosporine, 25 mg/kg/day, subcutaneously) (n = 5 each). Five rats treated with olive oil vehicle were used as control. After 4 weeks, biochemical parameters were measured, and renal cortical mRNA levels were evaluated by Northern blot analysis. Cyclosporine reduced renal creatinine clearance significantly and induced renal cortical osteopontin, TGF-beta, endothelin-1 and procollagen alpha 1(I) gene expressions around 13.5 +/- 1.3, 2.4 +/- 0.2, 1.5 +/- 0.1, 1.9 +/- 0.1 folds, respectively. Individual treatment with enalapril or verapamil significantly suppressed the osteopontin and TGF-beta mRNA expression, but not endothelin-1 and procollagen alpha 1(I). Combined treatment also inhibited the osteopontin and TGF-beta mRNA expression but there was no difference between combined and individual treatment. In conclusion, enalapril or verapamil significantly blunted the cyclosporine-induced osteopontin and TGF-beta gene expressions. However, combined treatment did not show any additive effect.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Calcium Channel Blockers; Cyclosporine; Drug Therapy, Combination; Enalapril; Endothelin-1; Gene Expression Regulation; Immunosuppressive Agents; Kidney Cortex; Male; Nephritis; Osteopontin; Procollagen; Rats; Rats, Wistar; RNA, Messenger; Sialoglycoproteins; Transforming Growth Factor beta; Verapamil

MRL/lpr mice suffer from a systemic lupus erythematosus-like autoimmune disease. We studied the effects of oral treatments with Ninjin-youei-to (NYT, Ren-shen-yang-rong-tang, 1000 mg/kg/day), a suboptimal dose (2 mg/kg/day) of prednisolone(PSL) and their combination on nephritis in MRL/lpr mice. Treatments with NYT or PSL alone inhibited the development of proteinuria and prolonged survival. The combined treatment reduced the incidence of proteinuria and prolonged survival. In histological analysis, NYT treatments decreased the degree of mesangial proliferative glomerulonephritis and infiltration of mononuclear cells in the kidneys. PSL treatment was effective in reducing periglomerular nephritis and vasculitis in addition to such effects as NYT and NYT plus PSL treatment was more effective than PSL alone. The active form of TGF-beta was reduced in NYT and PSL-treated mouse serum, and the combined treatments further suppressed it. However, the treatment with NYT alone did not induce a decrease in the latent form of TGF-beta. The effect of NYT can be assumed to be different from an immunosuppressive effect of PSL. Therefore, the combined treatment with NYT and PSL can be expected to be more useful for the therapy of autoimmune disease such as nephritis, compared with NYT or PSL alone treatments.

Topics: Animals; Anti-Inflammatory Agents; Antigen-Antibody Complex; Autoimmune Diseases; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Female; Kidney; Mice; Mice, Inbred C3H; Mice, Inbred MRL lpr; Nephritis; Prednisolone; Proteinuria; Transforming Growth Factor beta

The effects of a low-casein diet fortified with methionine and threonine on renal cortical and glomerular transforming growth factor (TGF)-beta activity were studied in rats with nephritis induced by anti-rat kidney glomerular basement membrane antiserum. Both normal and nephritic rats were fed experimental diets for 10 days. An injection of nephrotoxic serum increased urinary protein excretion and renal TGF-beta activity. A methionine-threonine-supplemented 8.5% casein diet, compared with a basal 20% casein diet, decreased these two measurements without aggravating growth retardation in nephritic rats. These results suggest that aggravation and alleviation of symptoms incident to anti-GBM nephritis are relevant to elevation and reduction of TGF-beta activity, respectively. The results also suggest that amino acid-balanced low-protein diets would have beneficial effects on glomerulonephritis without causing severe protein malnutrition.

Topics: Animals; Caseins; Cell Line; Dietary Proteins; Eating; Food, Fortified; Growth Disorders; Humans; Kidney; Kidney Glomerulus; Male; Methionine; Mink; Nephritis; Rats; Rats, Wistar; Threonine; Transforming Growth Factor beta

Rats with significant proteinuria induced by daily injections of bovine serum albumin develop interstitial inflammation and fibrosis. The present study was designed to investigate the molecular basis of interstitial monocyte (Mø) recruitment and early interstitial fibrosis. Groups of rats were sacrificed after one, two and three weeks. Despite an increase in interstitial Mø at week 1, whole kidney mRNA levels were not elevated for monocyte chemoattractant protein-1 (MCP-1), osteopontin or vascular cell adhesion molecule-1 (VCAM-1). Only osteopontin mRNA levels were significantly elevated in the renal cortex at four days. At two and three weeks, MCP-1 and osteopontin mRNA levels were increased and the proteins showed distinct tubular patterns of distribution. By immunostaining increased expression of VCAM-1 and intercellular adhesion molecule-1 (ICAM-1) was restricted to their presence or the surface of the interstitial inflammatory cells. TGF-beta 1 mRNA levels were increased at weeks 1, 2 and 3 (2.1, 2.9, 3.6x); interstitial and occasional cortical tubular cells expressed TGF-beta 1 mRNA and protein. There was a progressive rise in the number of cortical interstitial fields with increased staining for collagen (col) 1 (18, 29, 44%), col III (39, 61, 63%), col IV (7, 13, 29%), laminin (4, 10, 30%), fibronectin (14, 28, 37%), tenascin (19, 22, 14%) and in total renal col measured biochemically (1.1, 1.4, 2.0x) at weeks 1, 2 and 3, respectively. Renal matrix protein mRNA levels were variable and not always predictive of fibrosis. Only col I and tenascin levels were increased at week 1; all matrix protein mRNA levels except col IV were increased at week 2; but only tenascin, laminin and col IV mRNA levels remained elevated at three weeks. Plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metallo-proteinases (TIMP)-1 mRNA levels were significantly increased at two weeks. During the three weeks there was no change in urokinase, stromelysin or TIMP-3 mRNA levels. These results suggest that both increased matrix protein synthesis and altered matrix remodeling/degradation contribute to the final interstitial fibrogenic process in rats with protein-overload proteinuria. Mø, one of the sources of TGF-beta 1, infiltrate the interstitium by complex recruitment mechanisms which may depend in part on osteopontin, ICAM-1 and VCAM-1 expression.

Topics: Animals; Cell Movement; Extracellular Matrix Proteins; Female; Fibrosis; Gene Expression; Glycoproteins; Kidney; Kidney Diseases; Monocytes; Nephritis; Protease Inhibitors; Proteins; Proteinuria; Rats; Rats, Inbred Lew; Reference Values; RNA, Messenger; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta

Nephrotic syndrome induced by puromycin aminonucleoside (PAN) is characterized by tubulointerstitial (TI) inflammation, foci of TI fibrosis, and increased renal mRNA levels for matrix genes, the tissue inhibitor of metalloproteinases (TIMP), and the transforming growth factor-beta 1 (TGF-beta 1). To investigate the ability of a low-protein diet known to decrease TI inflammation to alter the degree of renal fibrosis, we studied four groups of rats: 27% protein PAN, 27% protein control, 8% protein PAN, and 8% protein control. Renal TGF-beta 1 mRNA levels correlated with the number of interstitial macrophages (r = 0.76) and were significantly reduced by dietary protein restriction. On day 10, Northern blot analysis showed that the elevated renal mRNA levels for procollagens alpha 1 (I), alpha 1(III), and alpha 2(IV) and fibronectin in the PAN-treated rats were significantly reduced by 8% dietary protein. In contrast, genes regulating matrix degradation (stromelysin and TIMP) were relatively unchanged by the low-protein diet. The number of foci of interstitial fibrosis and total renal collagen were greater in the PAN + 27% protein group than in the control groups. Both parameters of fibrosis were partially normalized in the PAN + 8% protein group. The results of this study suggest that dietary protein restriction attenuates TI fibrosis in PAN-induced nephrosis by partially reversing the increase in renal matrix synthesis. This effect was associated with decreased renal expression of the fibrogenic cytokine TGF-beta 1, which may be partially mediated by the concomitant reduction in the number of interstitial inflammatory macrophages.

Topics: Animals; Cholesterol; Dietary Proteins; Extracellular Matrix Proteins; Female; Fibrosis; Kidney; Nephritis; Nephrotic Syndrome; Proteinuria; Rats; Rats, Inbred Lew; RNA, Messenger; Transforming Growth Factor beta

Transforming growth factor-beta 1 (TGF-beta 1) is generally considered to exert positive effects on the accumulation of extracellular matrices. These occur as the net result of enhanced matrix protein synthesis, diminished matrix metalloproteinase (MMP) synthesis, and augmented production of specific inhibitors, including the tissue inhibitor of metalloproteinases (TIMP-1). Given that glomerular TGF-beta 1 synthesis is induced by inflammation, the effects of this cytokine on synthesis of the 72-kd type IV collagenase and TIMP-1 by cultured human mesangial cells were evaluated. Concentrations of TGF-beta 1 of 5 ng/ml and above specifically stimulated the synthesis of the 72-kd type IV collagenase. This effect was independent of the stimulatory effect of TGF-beta 1 on TIMP-1 synthesis, which was maximal in a lower concentration range (0.1 to 1 ng/ml). Most significantly, the net effect at the higher concentrations of TGF-beta 1 was an excess of enzyme over the TIMP-1 inhibitor. Northern blot analysis of TGF-beta 1-stimulated human mesangial cells demonstrated a specific increase in the abundance of the 3.1 kb mRNA transcript encoding the 72-kd type IV collagenase, presumably mediated by a direct stimulation of 72-kd type IV collagenase mRNA transcription observed as early as 3 hours after exposure to TGF-beta 1. These studies were extended to an analysis of the expression of TGF-beta 1 and 72-kd type IV collagenase mRNAs in normal and nephritic rats. In normal animals, basal TGF-beta 1 and 72-kd type IV collagenase mRNA expression was observed in a strictly mesangial distribution. After induction of acute immune complex-mediated glomerulonephritis, there was a major increase in TGF-beta 1 and 72-kd type IV collagenase mRNA expression, which was strictly limited to the expanded, hypercellular mesangial compartment. Enhanced synthesis of the mesangial type IV collagenase in response to TGF-beta 1 released during glomerular inflammatory processes could have an important role in the extensive glomerular matrix remodeling that accompanies these disorders.

Topics: Animals; Base Sequence; Blotting, Northern; Cells, Cultured; Collagenases; Enzyme-Linked Immunosorbent Assay; Glomerular Mesangium; Humans; Immunohistochemistry; Matrix Metalloproteinase 9; Molecular Probes; Molecular Sequence Data; Nephritis; Rats; Rats, Wistar; Transcription, Genetic; Transforming Growth Factor beta

Crescentic nephritis was induced in rabbits by injection of antirabbit glomerular basement membrane (GBM) antibodies. Urine samples were obtained by catheterization and assayed for transforming growth factor-beta (TGF-beta) activity. On day 31, all animals were sacrificed for evaluation of renal cortical histopathology and collagen content. The results show that control rabbit urine contains measurable amounts of TGF-beta. Urine TGF-beta activity was expressed in relation to urine creatinine concentration to correct for variation in urine concentration. When expressed in this manner, urine TGF-beta activity increased from day 2 onwards, peaked on day 7 and returned to normal levels after day 14. This time course is identical to that previously seen for the cortical and glomerular production of TGF-beta in the same model. Furthermore, when the normalized TGF-beta values for each animal were compared to their respective fibrosis parameters on day 31, significant correlations were observed for the values of urine TGF-beta activities on day 7 and all indices of fibrosis. These results suggest that measurements of urine TGF-beta activity at certain critical stages of disease could be useful in predicting the progression to end-stage renal disease with fibrosis and might serve as a helpful noninvasive adjunct in monitoring response to therapy.

Topics: Animals; Basement Membrane; Cicatrix; Collagen; Creatinine; Immunoglobulin G; Kidney; Male; Nephritis; Prognosis; Rabbits; Time Factors; Transforming Growth Factor beta

The purpose of this study was to assay for the presence of collagen synthesis stimulatory activity in the kidney during immune-induced renal injury that results in severe fibrosis in both glomerular and interstitial compartments. A model of antiglomerular basement (anti-GBM) disease in the rabbit was induced on day 0 by the injection of anti-GBM antibody and renal cortex tissues were then sampled at various time points. Only conditioned media prepared from diseased renal cortical samples showed collagen synthesis stimulatory activity when tested on rabbit mesangial cells. The activity had an estimated molecular weight range of 16 to 25 kd and was neutralized by antibody to transforming growth factor-beta (TGF-beta). A standard assay for TGF-beta using a mink lung epithelial cell line confirmed the increase in TGF-beta activity in conditioned media of diseased cortex from day 7 and day 14 animals, which was not significantly activated by previous acidification. This suggests that most of the TGF-beta present in renal conditioned media was in the active form. The increase in renal cortical secretion of active TGF-beta was accompanied by increases in renal cortical TGF-beta mRNA content on days 4 and 7 after induction, with subsequent return to control levels. A similar increase in TGF-beta activity was present in nonacidified conditioned media of purified glomeruli from diseased days 7 and 14 animals, which was also accompanied by significant increases in TGF-beta mRNA. However with acidification no significant differences were noted between control and diseased samples, suggesting the presence of substantial latent TGF-beta activity in control glomerular conditioned media. These same control-conditioned media contained inhibitor activity for added exogenous TGF-beta. These results support the conclusion that the association between increased TGF-beta secretion and increased renal cortical collagen synthesis in this model is consistent with a role for this cytokine in directing fibrogenesis in the kidney.

Topics: Animals; Antibodies; Basement Membrane; Chromatography, Gel; Collagen; Culture Media; Kidney Cortex; Kidney Glomerulus; Nephritis; Rabbits; RNA, Messenger; Transforming Growth Factor beta