transforming-growth-factor-beta and Nephrosis

Different complex mechanisms control the morphology of podocyte foot processes and their interactions with the underlying basement membrane. Injuries to this system often cause glomerular dysfunction and albuminuria. The present study aimed at identifying early markers of glomerular damage in diabetic nephropathy. For this purpose, we performed a microarray analysis on kidneys of 3-wk-old peroxisome proliferator-activated receptor-γ (PPARγ)-null and AZIP/F1 mice, which are two models of diabetic nephropathy due to lipodystrophy. This was followed by functional annotation of the enriched clusters of genes. One of the significant changes in the early stages of glomerular damage was the increase of hemicentin 1 (HMCN1). Its expression and distribution were then studied by real-time PCR and immunofluorescence in various models of glomerular damage and on podocyte cell cultures. HMCN1 progressively increased in the glomeruli of diabetic mice, according to disease severity, as well as in puromycin aminonucleoside (PA)-treated rats. Studies on murine and human podocytes showed an increased HMCN1 deposition upon different pathological stimuli, such as hyperglycemia, transforming growth factor-β (TGF-β), and PA. In vitro silencing studies showed that HMCN1 mediated the rearrangements of podocyte cytoskeleton induced by TGF-β. Finally, we demonstrated an increased expression of HMCN1 in the kidneys of patients with proteinuric nephropathies. In summary, our studies identified HMCN1 as a new molecule involved in the dynamic changes of podocyte foot processes. Its increased expression associated with podocyte dysfunction points to HMCN1 as a possible marker for the early glomerular damage occurring in different proteinuric nephropathies.

Topics: Animals; Calcium-Binding Proteins; Cells, Cultured; Cytoskeleton; Diabetic Nephropathies; Disease Models, Animal; Extracellular Matrix Proteins; Female; Glucose; Humans; Immunoglobulins; Male; Mice, Inbred C57BL; Mice, Knockout; Nephrosis; Podocytes; PPAR gamma; Proteinuria; Rats, Sprague-Dawley; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

The olfactomedin domain proteins Olfm-1 and myocilin are expressed in podocytes. Myocilin stimulates the formation of focal contacts and actin stress fibres in podocytes and other cell types, effects that are mediated through the Wnt signalling pathway. Here, we tested if the expression of both proteins is modified during puromycin aminonucleoside (PAN) nephrosis, which leads to structural changes in the actin cytoskeleton of podocytes.. Rats were treated with PAN, and the effectiveness of treatment was analysed by electron microscopy of podocytes and protein detection in the urine. The expression of Olfm-1 and myocilin was studied by immunohistochemistry, western blot analysis of glomerular proteins and real-time RT-PCR of glomerular proteins. In parallel experiments, the expression of Olfm-1 was studied in cultured podocytes treated with dexamethasone, TGF-β, TNF-α and PAN.. Between Days 5 and 22 after treatment, the amounts of the BMZ and BMY splice variants of Olfm-1 and their mRNA were markedly elevated in proteins and mRNA from isolated glomeruli. Immunohistochemistry showed that the expression of Olfm-1 was confined to podocytes. Essentially, comparable results were obtained for myocilin. The BMZ variant of Olfm-1 appeared to be secreted from podocytes and was found in high amounts in urine of treated animals. Treatment of cultured podocytes with dexamethasone and PAN caused an increase in Olfm-1 expression, while treatment with recombinant Olfm-1 increased the formation of actin stress fibres.. Olfm-1 and myocilin are markedly induced in podocytes during PAN nephrosis and appear to be involved in the processes that govern the reorganization of the actin cytoskeleton during podocyte repair.

Topics: Animals; Blotting, Western; Cells, Cultured; Cytoskeletal Proteins; Dexamethasone; Extracellular Matrix Proteins; Eye Proteins; Glycoproteins; Humans; Immunoenzyme Techniques; Male; Nephrosis; Podocytes; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

IL-10/TGF-beta-modified macrophages, a subset of activated macrophages, produce anti-inflammatory cytokines, suggesting that they may protect against inflammation-mediated injury. Here, macrophages modified ex vivo by IL-10/TGF-beta (IL-10/TGF-beta Mu2) significantly attenuated renal inflammation, structural injury, and functional decline in murine adriamycin nephrosis (AN). These cells deactivated effector macrophages and inhibited CD4+ T cell proliferation. IL-10/TGF-beta Mu2 expressed high levels of the regulatory co-stimulatory molecule B7-H4, induced regulatory T cells from CD4+CD25- T cells in vitro, and increased the number of regulatory T cells in lymph nodes draining the kidneys in AN. The phenotype of IL-10/TGF-beta Mu2 did not switch to that of effector macrophages in the inflamed kidney, and these cells did not promote fibrosis. Taken together, these data demonstrate that IL-10/TGF-beta-modified macrophages effectively protect against renal injury in AN and may become part of a therapeutic strategy for chronic inflammatory disease.

Topics: Animals; B7-1 Antigen; CD4-Positive T-Lymphocytes; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Doxorubicin; Interleukin-10; Kidney; Macrophages; Male; Mice; Mice, Inbred BALB C; Nephrosis; Nitric Oxide Synthase Type II; Phenotype; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; V-Set Domain-Containing T-Cell Activation Inhibitor 1

Chronic allograft nephropathy (CAN) belongs to the major causes of long-term kidney allograft failure. One of the histologic hallmarks of CAN is interstitial fibrosis, influenced by matrix metalloproteinases (MMPs) that are controlling extracellular matrix (ECM) degradation. Whether MMPs affect the development and progression of CAN is not clear so far. To analyze the role of MMPs in CAN, we investigated the effects of an early and a late application of BAY 12-9566, an inhibitor of MMP-2, -3, and -9 on the development and progression of CAN in a rat kidney-transplantation model.. Fisher kidneys were orthotopically transplanted into Lewis recipients that were treated with BAY 12-9566 (15 mg/kg per day) or vehicle either for the first 10 days after transplantation (early treatment) or from week 12 to week 20 after transplantation (late treatment). Proteinuria was analyzed every 4 weeks up to week 20 after transplantation when kidney grafts were removed for further analysis.. Early MMP-inhibition resulted in a significantly reduced 24-hour protein excretion that was paralleled by a lower grade of CAN after 20 weeks. However, late MMP inhibition starting at week 12 after transplantation resulted in significantly higher proteinuria and a higher grade of CAN as compared with controls. Furthermore, transforming growth factor-beta and platelet-derived growth factor-B chain mRNA levels were significantly increased in these animals.. Inhibition of MMPs early after transplantation reduced the development and progression of CAN but promoted CAN if initiated at later stages. Thus, MMPs are involved in the development and progression of CAN.

Topics: Animals; Biphenyl Compounds; Blood Pressure; Body Weight; Chronic Disease; Creatine; Glomerulosclerosis, Focal Segmental; Graft Rejection; Kidney Transplantation; Macrophages; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Nephrosis; Organic Chemicals; Phenylbutyrates; Proto-Oncogene Proteins c-sis; Rats; Rats, Inbred F344; RNA, Messenger; Time Factors; Transforming Growth Factor beta; Transplantation, Homologous

Non-esterified fatty acids (NEFA) carried on albumin may have a causal role in the development of chronic proteinuria-induced nephropathy. To investigate whether NEFA aggravate renal structural damage, we studied the effects of NEFA addition to delipidated bovine serum albumin (BSA) in protein-overload nephropathy.. Three groups of Wistar rats received daily intraperitoneal injections (3 weeks) of either 1 g NEFA-free BSA (BSA-0), or NEFA-free BSA with three (BSA-3) or six (BSA-6) molecules oleic acid added per BSA molecule. An additional group received saline injections only (SAL). Renal damage was evaluated by immunohistochemistry and RT-PCR.. Interstitial and glomerular alpha-smooth muscle actin (alpha-SMA, marker of myofibroblast transformation) expression were higher in BSA-3/6 than in saline-injected controls (P < 0.001). Glomerular macrophage influx and desmin (marker of glomerular epithelial cell damage) expression were higher in all BSA-injected rats than SAL (P < 0.001). Interstitial macrophage influx was elevated in BSA-0/3 (P < 0.05) and BSA-6 (P < 0.001) compared to SAL. Addition of six molecules of oleic acid to BSA revealed higher interstitial and glomerular alpha-SMA expression (P < 0.001), increased interstitial macrophage numbers (P < 0.001) and enhanced glomerular desmin expression (P < 0.05) compared to BSA-0. RT-PCR revealed higher glomerular alpha-SMA mRNA expression in BSA-3/6 than SAL (P < 0.001 and 0.05, respectively), interstitial alpha-SMA mRNA was elevated in BSA-6 (P < 0.05). Interstitial TGF-beta1 mRNA expression was significantly higher in BSA-3 than SAL (P < 0.05).. These data show that addition of oleic acid to NEFA-free BSA aggravates renal damage, suggesting a role for NEFA in the pathogenesis of proteinuric nephropathies.

Topics: Actins; Animals; Biomarkers; Disease Models, Animal; Gene Expression; Immunohistochemistry; Kidney Glomerulus; Male; Nephrosis; Oleic Acid; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serum Albumin, Bovine; Transforming Growth Factor beta

Terminal stage renal failure is the final common fate of chronic nephropathies independent of the type of initial insult. Abnormally filtered proteins have an intrinsic renal toxicity linked to their over-reabsorption by proximal tubular cells and activation of tubular-dependent pathways of interstitial inflammation and fibrosis. The functional importance of tubulointerstitial events in progressive renal disease is supported by evidence that the severity of tubular interstitial damage strongly correlates with the risk of renal failure. The present study aimed to investigate the expressive tendency of some pro-fibrosis genetic factors mRNA, including thrombospondin-1 (TSP-1), transforming growth factor-beta1 (TGF-beta1), connective tissue growth factor (CTGF) and the major component of extracellular matrix-fibronectin (FN) in renal tissues at different time points during early stage of renal lesions caused by proteinuria in bovine serum albumin (BSA) injection-induced proteinuria overload nephrotic young rats and the significance of these factors on tubulointerstitial fibrosis development.. Female young Wistar rats aged 3-4 weeks with proteinuria overload nephrosis induced by BSA (1.0 g/d) injected intraperitoneally were used as experimental models. The 80 young rats were divided into control group (n = 40) and BSA injected group (n = 40). At different time points (weeks 1, 2, 3 and 4), the urinary protein was measured by Coomassie brilliant blue colorimetric assay; the renal tissues morphologic changes were evaluated after HE staining; the P(65)/Rel-A, TSP-1, TGF-beta1 and CTGF mRNA expression in renal tissues was determined by in situ hybridization method and the FN mRNA expression was detected by Northern blot. The experimental data were evaluated by statistics software SPSS10.0.. (1) Three to four weeks after BSA injection, heavy proteinuria was observed in the rats of BSA group (week 3: 104.3 +/- 21.8 mg/24 h; week 4: 131.1 +/- 18.3 mg/24 h). The proteinuria deteriorated progressively afterwards. Histopathological examination revealed that inflammatory cells infiltrated into tubulointerstitial areas extensively, protein casts were seen in tubules and edema occurred in tubulointerstitial areas. (2) In situ hybridization showed that NF-kappaB (P(65)/Rel-A) mRNA expression was up-regulated progressively in nuclei of tubular epithelial cells, the semi-quantitative scores (at 1, 2, 3 and 4 weeks) were 2.33 +/- 0.20, 2.76 +/- 0.12, 2.96 +/- 0.19, and 3.76 +/- 0.18, respectively (F = 37.34, P < 0.01). (3) At 1 week after BSA injection, the TSP-1 mRNA expression appeared in glomeruli and increased, but was light in tubulointerstitial areas, its expressive peak was observed at week 2, and declined to mild after weeks 3 and 4. The semi-quantitative scores at different time points suggested that TSP-1 mRNA was expressed mainly in early stage of lesion in this model, then, this tendency turned to a flat roof smoothly. (4) TGF-beta1 and CTGF mRNA was up-regulated simultaneously in tubular epithelial cells (F = 8.80, P < 0.01 F = 19.41, P < 0.01). (5) Northern blotting showed that FN mRNA was considerably up-regulated at second week in the kidneys of rats in BSA group, 2.7-fold higher at week 4 than that at week 1 in BSA group rats, and was 3.6-fold higher than that of control group.. The present study suggested that NF-kappaB (P(65)/Rel-A) mRNA expression and its activity was enhanced significantly by proteinuria-loading and synchronized with high expression of TSP-1, TGF-beta1, and CTGF mRNA in the kidney, at the same time, FN mRNA was up-regulated in renal tissues and an aggravating tendency in tubulointerstitial lesions was observed in nephrotic young rats with heavy proteinuria.

Topics: Animals; Connective Tissue Growth Factor; Female; Fibronectins; Kidney; Nephrosis; NF-kappa B; Proteinuria; Rats; Rats, Wistar; RNA, Messenger; Thrombospondin 1; Transcription Factor RelA; Transforming Growth Factor beta

Nephrotic syndrome has long been treated in China with two herbs, Astragalus mongholicus and Angelica sinensis, which may have antifibrotic effects.. Rats with chronic puromycin-induced nephrosis were treated with Astragalus and Angelica 3 mL/d (n = 7) or enalapril 10 mg/kg/d (n = 7). Normal control rats (n = 7) received saline rather than puromycin, and an untreated control group (n = 7) received puromycin but no treatment. After 12 weeks, stained sections of the glomerulus and tubulointerstitium were evaluated for injury. Immunohistochemistry staining measured extracellular matrix components, transforming growth factor-beta1 (TGFbeta1), osteopontin, ED-1-positive cells, and alpha-actin. TGFbeta1 mRNA was assessed by in situ hybridization. Renin, ACE activity, angiotensin, and aldosterone were measured by radioimmunoassay or colorimetry. In the untreated rats, chronic renal injury progressed to marked fibrosis at 12 weeks. Astragalus and Angelica significantly reduced deterioration of renal function and histologic damage. Expressions of type III and IV collagen, fibronectin, and laminin also decreased significantly. This anti-fibrotic effect was similar to that of enalapril. The herbs had no effect on the renin-angiotensin system but did reduce the number of ED-1-positive, and alpha-actin positive cells and expression of osteopontin compared to untreated controls. The combination of Astragalus and Angelica retarded the progression of renal fibrosis and deterioration of renal function with comparable effects of enalapril. These effects were not caused by blocking the intrarenal renin-angiotensin system, but associated with suppression of the overexpression of TGFbeta1 and osteopontin, reduction of infiltrating macrophages, and less activation of renal intrinsic cells [corrected].

Topics: Angelica sinensis; Angiotensin-Converting Enzyme Inhibitors; Animals; Antibiotics, Antineoplastic; Astragalus Plant; China; Drugs, Chinese Herbal; Enalapril; Fibrosis; Humans; Kidney; Male; Medicine, Chinese Traditional; Nephrosis; Phytotherapy; Plant Preparations; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley; Regression Analysis; Transforming Growth Factor beta; Transforming Growth Factor beta1

The TGFbeta superfamily plays diverse and essential roles in kidney development. Gdf11 and Bmp4 are essential for outgrowth and positioning of the ureteric bud, the inducer of metanephric mesenchyme. During nephrogenesis, Bmp7 is required for renewal of the mesenchyme progenitor population. Additionally, in vitro studies demonstrate inhibitory effects of BMPs and TGFbetas on collecting duct branching and growth. Here, we explore the predicted models of TGFbeta superfamily function by cell-specific inactivation of Smad4, a key mediator of TGFbeta signaling. Using a HoxB7cre transgene expressed in ureteric bud and collecting duct, we find that development of the collecting duct is Smad4 independent. By contrast, removal of Smad4 in nephrogenic mesenchyme using the Bmp7(cre/+) allele leads to disorganization of the nephrogenic mesenchyme and impairment of mesenchyme induction. Smad4-deficient metanephric mesenchyme does not display defects in inducibility in LiCl or spinal cord induction assays. However, in situ hybridization and lineage analysis of Smad4 null mesenchyme cells at E11.5 show that the nephrogenic mesenchyme does not aggregate tightly around the ureteric bud tips, but remains loosely associated, embedded within a population of cells expressing markers of both nephrogenic mesenchyme and peripheral stroma. We conclude that the failure of recruitment of nephrogenic mesenchyme leaves a primitive population of mesenchyme at the periphery of the kidney. This population is gradually depleted, and by E16.5 the periphery is composed of cells of stromal phenotype. This study uncovers a novel role for TGFbeta superfamily signaling in the recruitment and/or organization of the nephrogenic mesenchyme at early time-points of kidney development. Additionally, we present conclusive genetic lineage mapping of the collecting duct and nephrogenic mesenchyme.

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cell Division; Cell Lineage; DNA-Binding Proteins; Gene Deletion; Homeodomain Proteins; Kidney; Mesoderm; Mice; Mice, Transgenic; Morphogenesis; Multigene Family; Nephrosis; Signal Transduction; Smad4 Protein; Stem Cells; Trans-Activators; Transforming Growth Factor beta

To explore the mechanism of protective effect of Compound Salvia Injection (CSI) on experimental cyclosporin A induced nephrotoxicity.. Rats were on low-salt diet and cyclosporin A (CsA) was administered once a day through gastrogavage at dosage of 30 mg/kg.d for 28 days. Expression of the mRNA for intrarenal transforming growth factor-beta 1 (TGF-beta 1) and renin was measured by reverse transcription-polymerase chain reaction (RT-PCR). Intrarenal expression of TGF-beta 1 and Collagen IV was determined by immunohistochemical assays. The effects of CSI on these changes were also evaluated.. Chronic CsA-induced nephropathy might be correlated to TGF-beta 1 and renin mRNA up-regulation as well as matric proteins accumulation in interstitium. CSI could reduce these changes.. Decreased CsA-related TGF-beta 1 and renin upregulation expression and accumulation of matrix proteins in the kidney might be related to the protective mechanism of CSI on CsA-induced chronic nephrotoxicity.

Topics: Animals; Chronic Disease; Collagen Type IV; Cyclosporine; Drugs, Chinese Herbal; Male; Nephrosis; Phytotherapy; Plant Extracts; Random Allocation; Rats; Rats, Sprague-Dawley; Renin; RNA, Messenger; Salvia miltiorrhiza; Transforming Growth Factor beta

Tubulointerstitial fibrosis is one of the most important histologic features that predicts progression in kidney disease. Thrombospondin 1 is an extracellular matrix protein that can activate latent TGF-beta, a cytokine implicated in the pathogenesis of tubulointerstitial fibrosis. We examined the expression of thrombospondin 1 in several animal models of glomerulonephritis (anti-Thy1 model, aminonucleoside nephrosis, passive Heymann nephritis) that are associated with tubulointerstitial disease. Thrombospondin 1 mRNA and protein were transiently increased in tubular cells, myofibroblasts and some macrophages in areas of tubulointerstitial injury. Thrombospondin 1 expression always preceded the development of tubulointerstitial fibrosis, and correlated quantitatively and spatially with the later development of interstitial fibrosis. Thrombospondin 1 expression predicted the severity of tubulointerstitial fibrosis better than the degree of macrophage or myofibroblast accumulation. Thrombospondin 1 expression was associated with increased expression and activation of TGF-beta1 and decreased expression of LAP-TGF-beta in areas of tubulointerstitial injury. We conclude that thrombospondin 1 is an early marker predicting the development of tubulointerstitial kidney disease. De novo expression of thrombospondin 1 is associated and colocalized with increased expression of TGF-beta1 and decreased expression of LAP-TGF-beta during the development of tubulointerstitial disease in vivo. These data are consistent with the possibility that thrombospondin 1 may be an endogenous activator of TGF-beta.

Topics: Animals; Anticoagulants; Becaplermin; Biopsy; Extracellular Matrix Proteins; Fibroblasts; Fibrosis; Gene Expression; Glomerular Mesangium; Glomerulonephritis, Membranoproliferative; Kidney Tubules; Macrophages; Male; Nephritis, Interstitial; Nephrosis; Platelet-Derived Growth Factor; Predictive Value of Tests; Proto-Oncogene Proteins c-sis; Rats; Rats, Wistar; RNA, Messenger; Thrombospondin 1; Transforming Growth Factor beta

Chronic renal disease (CRD) is generally thought to be incurable, except through renal transplantation, and the number of patients with CRD is on the increase. Glomerulosclerosis and tubulointerstitial fibrosis represent the morphological equivalent of end-stage CRD. In this study, we demonstrated the preventive effect of hepatocyte growth factor (HGF) on the progression of renal dysfunction and fibrosis, using a spontaneous mouse model for CRD (ICGN strain). The mice progressively developed glomerular sclerotic injury, tubular atrophy, and renal dysfunction until they were 17 wk of age. When recombinant HGF was injected into these mice during a 4-wk-period (from weeks 14-17 after birth), DNA synthesis of tubular epithelial cells was found to be 4.4-fold higher than in mice without HGF injection, thereby suggesting tubular parenchymal expansion promoted by HGF. Notably, HGF suppressed the expression of transforming growth factor-beta and of platelet-derived growth factor as well as myofibroblast formation in the affected kidney. Consequently, the onset of tubulointerstitial fibrosis was almost completely inhibited by HGF, while HGF attenuated the progression of glomerulosclerosis, both leading to preventing manifestation of renal dysfunction. From our results, supplement therapy with HGF may be taken into consideration as a novel option for prevention and treatment of CRD.

Topics: Animals; Disease Models, Animal; Fibrosis; Glomerulonephritis; Hepatocyte Growth Factor; Kidney; Kidney Failure, Chronic; Kidney Glomerulus; Kidney Tubules; Mice; Nephrosis; Platelet-Derived Growth Factor; Recombinant Proteins; Transforming Growth Factor beta

Macrophage infiltration into the glomerular mesangium is a prominent feature of various glomerulopathies. Recent evidence suggests that infiltrating macrophages may play a role in propagating initial glomerular injury to the development of glomerulosclerosis via transforming growth factor-beta (TGF-beta)-stimulating matrix accumulation. Rats with the acute puromycin aminonucleoside (PA) nephrosis exhibit an elevated gene expression of glomerular TGF-beta 1; however, the cellular origin of this upregulation is unknown. Using polymerase chain reaction (PCR), we detected that the TGF-beta 1 isoform is expressed in glomerular macrophages isolated from experimental rats made hypercholesterolemic by either diet or by induction of PA nephrosis. Peritoneal macrophages from nephrotic or dietary-hypercholesterolemic animals also exhibited a significant increment in the expression of TGF-beta 1 mRNA on Northern analysis, in contrast to similar cells obtained from normal control rats. PCR analysis of glomerular RNA also detected the expression of the TGF-beta 2 mRNA isoform. TGF-beta 2 mRNA expression was not observed in isolated glomerular macrophages from either glomeruli of PA-nephrotic rats or from glomeruli of animals with dietary hypercholesterolemia. Expression of the TGF-beta 3 mRNA isoform was only observed by PCR in J774 A.1 cells. Thus the as a cellular source for the enhanced expression of TGF-beta 1 during the acute nephrotic phase of our toxic, progressive glomerulopathy model and within several days of inducing only hypercholesterolemia by dietary means.

Topics: Albuminuria; Animals; Base Sequence; Cholesterol, Dietary; Gene Expression; Hypercholesterolemia; Immunohistochemistry; Kidney Glomerulus; Lipids; Macrophages; Male; Molecular Sequence Data; Nephrosis; Polymerase Chain Reaction; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor beta

Hypercholesterolemia aggravates experimental progressive glomerular injury. Evidence suggests the infiltrating glomerular macrophage (M phi) is a potential effector mechanism for the noxious effects of hypercholesterolemia. Because transforming growth factor (TGF)-beta 1 is secreted by activated M phi s and also stimulates fibronectin production by glomerular cells, we evaluated the kinetics of gene expression for these moieties in glomeruli isolated from nephrotic rats at 3, 7, 11, and 42 days after the delivery of puromycin aminonucleoside (PA). We also assessed whether cholesterol feeding, which raises the glomerular M phi number, alters the glomerular mRNA levels for TGF-beta 1 and fibronectin. Glomerular mRNA levels for TGF-beta 1 and fibronectin in nephrotic rats exhibited a biphasic temporal pattern, decreasing significantly below control at 3 and 7 days after PA but increasing significantly at 11 and 42 days after PA. The upregulated gene expression for TGF-beta 1 and fibronectin at 11 days after PA temporally corresponded to the phase of mesangial M phi infiltration in this model. Cholesterol feeding to both normal and nephrotic rats significantly increased glomerular TGF-beta 1 and fibronectin mRNA levels at 11 days after PA. Immunohistochemical labeling for M phi s and intracellular TGF-beta 1 demonstrated both mesangial and cortical interstitial localization with the TGF-beta1-positive cells possessing M phi nuclear morphology. These findings identify a novel interaction between hypercholesterolemia, augmented glomerular M phi accumulation, and upregulated glomerular TGF-beta 1 and fibronectin gene expression. These perturbations within the acutely injured glomerulus constitute an early pathobiological determinant for the later development of mesangial matrix expansion and glomerulosclerosis.

Topics: Albuminuria; Animals; Cholesterol; Cholic Acids; Extracellular Matrix; Fibronectins; Food, Fortified; Gene Expression; Hypercholesterolemia; Immunohistochemistry; Kidney Glomerulus; Lipids; Macrophages; Male; Nephrosis; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor beta; Up-Regulation

Progressive renal fibrosis is considered to be the final common pathway leading to chronic renal insufficiency. In this study, the authors examined some of the cellular and molecular mechanisms regulating the renal accumulation of extracellular matrix (ECM) proteins using rats with puromycin amino-nucleoside (PAN) nephrosis as an acute model system. Puromycin aminonucleoside rats developed reversible nephrotic syndrome accompanied by an interstitial infiltrate of monocytes. The number of interstitial fibroblasts expressing ST4 antigen did not increase. During the first 4 days, steady-state mRNA levels for all genes examined remained at or below control levels. At 1 week, nephrotic syndrome and interstitial inflammation were established, and a period of renal cell proliferation occurred, identified by increased histone mRNA levels and localized by tritiated thymine autoradiography to tubular epithelial cells and occasional interstitial cells. Transforming growth factor-beta (TGF-beta) steady-state mRNA levels were increased eightfold, but returned to control levels by 3 weeks. At week 1, there was a 10- to 20-fold increase in kidney steady-state mRNA levels for genes encoding interstitial matrix proteins collagen I and fibronectin and basement membrane collagen IV. By in situ hybridization, alpha 1(I) procollagen mRNA was localized to interstitial cells. Immunofluorescence microscopy demonstrated focal accumulation of ECM proteins in the tubulointerstitial compartment at 2 and 3 weeks, but by 6 weeks, kidney immunohistology was normal again. Steady-state mRNA levels for the matrix degrading metalloproteinase stromelysin remained at control values, whereas the levels for interstitial collagenase were normal at week 1 and increased twofold to threefold at 2 and 3 weeks. Steady-state mRNA levels for the tissue inhibitor of metalloproteinases (TIMP) increased fivefold at 1 week and returned to baseline values over the next 2 weeks. The results of this study suggest that tubulointerstitial ECM accumulation occurs in rats with acute PAN nephrosis because of the activation of genes encoding several matrix proteins and inhibition of matrix degradation mediated by TIMP. These events are reversed during the phase of recovery from nephrotic syndrome. Increased mRNA levels for TGF-beta, possibly originating from inflammatory interstitial monocytes, are likely to be one of the mediators of the molecular events observed.

Topics: Animals; Basement Membrane; Blotting, Northern; Cell Division; Collagen; Disease Models, Animal; DNA; Extracellular Matrix; Extracellular Matrix Proteins; Female; Fibronectins; Fluorescent Antibody Technique; Gene Expression; In Situ Hybridization; Injections, Intraperitoneal; Kidney; Metalloendopeptidases; Nephrosis; Puromycin Aminonucleoside; Rats; Rats, Inbred Lew; RNA; RNA, Messenger; Thymidine; Transforming Growth Factor beta

A cellular and molecular approach was used to gain new insight into the pathogenesis of interstitial fibrosis in chronic purine aminonucleoside nephrosis (PAN) nephrosis. Thirty experimental rats (PAN rats) were given 15 mg/100 g body wt of i.p. PAN at time 0, followed by 4.3 mg/100 g body wt i.p. on days 20, 27 and 34; 25 control rats received i.p. saline at the same time intervals. All rats had a right unilateral nephrectomy within the first four days. Groups of control and PAN rats were killed at 21, 37, 52, 72 and 91 days. Renal sections were studied by immunofluorescence to quantitate interstitial macrophages, T lymphocytes and fibroblasts, and to characterize the deposition of the extracellular matrix (ECM) proteins (collagens I, III and IV, fibronectin and laminin) and the tissue inhibitor of the metalloproteinases (TIMP). Steady state concentrations of mRNA from the whole kidney for these ECM proteins, the metalloproteinases, TIMP, and transforming growth factor beta (TGF-beta 1) were quantitated by Northern blot analysis. Significant increases in the number of interstitial macrophages and T lymphocytes were found in the PAN rat groups compared to that in controls. All ECM proteins examined were quantitatively increased in the tubulo-interstitium of PAN rats. The pattern of distribution of some ECM proteins was also modified in experimental animals. TIMP was increased in the interstitium of PAN rats; at later times, TIMP was most prominent in sclerotic regions of the glomeruli and in tubular protein droplets. Northern blot analysis revealed increased steady-state mRNA levels for components of each of the ECM proteins, no change for the metalloproteinases--stromelysin or collagenase--and a marked increase for TIMP and TGF-beta 1 in PAN animals. The results of this study suggest that the diffuse interstitial fibrosis found in chronic PAN nephrosis results from both increased production of ECM proteins and decreased matrix degradation.

Topics: Albuminuria; Animals; Chronic Disease; Creatinine; Disease Models, Animal; Extracellular Matrix Proteins; Female; Fibrosis; Gene Expression; Glycoproteins; Macrophages; Metalloendopeptidases; Nephrosis; Puromycin Aminonucleoside; Rats; Rats, Inbred Lew; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta