nephrin and Glomerulonephritis

Podocyte is one of the main components of glomerular filtration barrier in the kidney; the loss or dysfunction of podocyte could impair the functions of glomerular filtration barrier, leading to development of various renal diseases. Podocyte is a terminally differentiated cell, and thus does not possess any proliferative properties. Accordingly, its number and contribution to renal function are initially determined by its normal development. Information from the literature and results of our research indicate that genetic factors or prenatal adverse environment could cause developmental retardation of podocytes, thereby suggesting the potential fetal developmental origin(s) of kidney diseases, and involvement of epigenetic mechanisms in the regulation of key genes in podocyte development. In this review, we provide a brief overview on the podocyte normal development; discussion on the potential pathogenic mechanisms for developmental disorders of podocytes; as well as renal diseases associated with podocyte developmental retardation. We aim to provide some insights in articulating the strategies for diagnosis and treatments of renal diseases associated with podocyte developmental abnormalities.

Topics: Animals; Glomerulonephritis; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Membrane Proteins; Mutation; Nephrotic Syndrome; Podocytes; Wnt Signaling Pathway

Mycophenolic acid (MPA) was introduced into clinical practice as immunosuppressive drug therapy to prevent allograft rejection. Since then, its clinical application has widened. Our goal was to review the lessons learned from experimental nontransplant glomerular disease models on the mechanisms of MPA therapy. T and B lymphocytes are preferentially dependent on de novo purine synthesis. By inhibiting the rate-limiting enzyme of de novo purine synthesis, MPA depletes the pool of deoxyguanosine triphosphate (dGTP) and inhibits proliferation of these immune cells. Furthermore, MPA can also induce apoptosis of immune cells and is known to inhibit synthesis of fucose- and mannose-containing membrane glycoproteins altering the surface expression and binding ability of adhesion molecules. However, MPA exerts a direct effect also on nonimmune cells. Mesangial cells are partially dependent on de novo purine biosynthesis and are thus susceptible to MPA treatment. Additionally, MPA can inhibit apoptosis in podocytes and seems to be beneficial in preserving the expression of nephrin and podocin, and by attenuation of urokinase receptor expression leads to decreased foot-process effacement. In summary, our manuscript sheds light on the molecular mechanisms underlying the antiproteinuric effect of MPA. Overall, MPA is an excellent treatment option in many immunologic glomerulopathies because it possesses immunosuppressive properties, has a remarkable effect on nonimmune cells and counteracts the proliferation of mesangial cells, expansion of mesangial matrix, and foot-process effacement of podocytes combined with a low systemic toxicity.

Topics: Animals; Cell Proliferation; Disease Models, Animal; Glomerular Basement Membrane; Glomerulonephritis; Humans; Immunosuppressive Agents; IMP Dehydrogenase; Intracellular Signaling Peptides and Proteins; Lymphocytes; Membrane Proteins; Mesangial Cells; Monocytes; Mycophenolic Acid; Nephrotic Syndrome; Podocytes; Purines; Rats

Topics: Animals; Disease Models, Animal; Glomerulonephritis; Immune Sera; Immunoglobulin A; Immunotoxins; Interleukin-2 Receptor alpha Subunit; Japan; Membrane Proteins; Thy-1 Antigens

Topics: Animals; Antigen-Antibody Complex; Complement Activation; Complement Membrane Attack Complex; Glomerulonephritis; Glomerulonephritis, Membranous; Humans; Immunoglobulin G; Intracellular Signaling Peptides and Proteins; Low Density Lipoprotein Receptor-Related Protein-2; Membrane Proteins; Neprilysin; Proteinuria

Anti-neutrophil cytoplasmic antibody-associated glomerulonephritis (ANCA-GN) is a critical kidney disease that sometimes results in an unfavorable renal outcome. Cellular crescent formation is a hallmark of ANCA-GN and is associated with renal prognosis, response to treatment, and it was reportedly associated with podocyte detachment. Because there is a need to explore non-invasive biomarkers for the evaluation of ANCA-GN activity, we tested whether urinary podocyte mRNA might be a potent non-invasive biomarker.. We measured two different types of urinary podocyte mRNA, including podocin mRNA in relation to urine creatinine concentration (U-PodCR) and urinary podocin mRNA in relation to nephrin mRNA (U-PNR), which were reportedly associated with the activity of various glomerular diseases.. In ANCA-GN patients (n = 19), we discovered that U-PodCR was positively correlated with the percent of crescent formation until 50% crescent was reached because of podocyte depletion; U-PNR was correlated with the percent of crescent formation in all patients. Furthermore, patients with high levels of urinary podocyte mRNA exhibited a favorable renal outcome compared with the outcomes of patients with low levels of urinary podocyte mRNA. The levels of urinary podocyte mRNA were correlated with the rate of improvement in estimated glomerular filtration rate.. U-PodCR, U-PNR or a combination of these parameters might serve as a non-invasive potential biomarker in patients with ANCA-GN to predict the percent of crescent formation and renal prognosis.

Topics: Aged; Antibodies, Antineutrophil Cytoplasmic; Biomarkers; Case-Control Studies; Female; Glomerulonephritis; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Male; Membrane Proteins; Middle Aged; RNA, Messenger

The endocapillary proliferative (EP) lesion is not included in the International Study of Kidney Disease in Children (ISKDC) pathological classification of Henoch-Schönlein purpura nephritis (HSPN). The main objective of the study was to determine the pathological importance of EP in the development of proteinuria in children with Henoch-Schönlein purpura nephritis (HSPN).. The pathological features of 148 HSPN children with nephrotic-range proteinuria were investigated retrospectively. Urinary IgG, transferrin, and albumin levels were measured by immunonephelometry. The correlations between EP lesion and 24-h proteinuria, urinary IgG, urinary transferrin, and urinary albumin were analyzed. Renal biopsy specimens were immunohistochemically stained for nephrin and podocalyxin.. Of the total 581 cases of children with HSPN who underwent renal biopsy, 148 cases (25.5%) presented with nephrotic-range proteinuria. The pathological types of HSPN with nephrotic-range proteinuria were categorized as IIb, IIIa, IIIb, IIIb with diffuse EP, IVb, pure focal EP type, and pure diffuse EP type. Among these types, pure diffuse EP type accounted for 7.4%. The levels of 24-h proteinuria and urinary albumin were the highest in pure diffuse EP type among all pathological types, and the percentage of EP correlated with 24-h proteinuria and urinary albumin levels. 24-h proteinuria was significantly higher in pure diffuse EP type relative to HSPN IIb type, and significantly higher in IIIb with EP, compared with HSPN IIIb. Nephrin, but not podocalyxin, was downregulated in EP segment.. EP is an independent pathogenic factor in HSPN with nephrotic-range proteinuria. Downregulation of nephrin in EP segment is a potential molecular mechanism of nephrotic-range proteinuria. Albumin is the major urinary protein component in HSPN with EP.

Topics: Adolescent; Albuminuria; Capillaries; Child; Female; Glomerulonephritis; Humans; IgA Vasculitis; Immunoglobulin G; Kidney Glomerulus; Male; Membrane Proteins; Neovascularization, Pathologic; Prognosis; Retrospective Studies; Risk Factors; Sialoglycoproteins; Transferrin

The glomerular filtration barrier, has historically only been spatially resolved using electron microscopy due to the nanometer-scale dimensions of these structures. Recently, it was shown that the nanoscale distribution of proteins in the slit diaphragm can be resolved by fluorescence based stimulated emission depletion microscopy, in combination with optical clearing. Fluorescence microscopy has advantages over electron microscopy in terms of multiplex imaging of different epitopes, and also the amount of volumetric data that can be extracted from thicker samples. However, stimulated emission depletion microscopy is still a costly technique commonly not available to most life science researchers. An imaging technique with which the glomerular filtration barrier can be visualized using more standard fluorescence imaging techniques is thus desirable. Recent studies have shown that biological tissue samples can be isotropically expanded, revealing nanoscale localizations of multiple epitopes using confocal microscopy. Here we show that kidney samples can be expanded sufficiently to study the finest elements of the filtration barrier using confocal microscopy. Thus, our result opens up the possibility to study protein distributions and foot process morphology on the effective nanometer-scale.

Topics: Animals; Autoantibodies; Biomarkers; Collagen Type IV; Disease Models, Animal; Fluorescent Antibody Technique, Indirect; Glomerular Filtration Barrier; Glomerulonephritis; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice, Transgenic; Microscopy, Confocal; Microscopy, Fluorescence; Rats; Tissue Expansion

It is accepted that alteration of connexin43 (Cx43) expression in glomeruli is a common pathological response in several forms of kidney diseases. To date, however the change of the Cx43 expression in obesity-related glomerulopathy (ORG) has not been reported. In this study, the alteration of Cx43 expression in the glomeruli of rat with ORG was defined. Five-week-old rats were fed with high-fat diet for 18weeks to establish the ORG model, then the histological change of glomeruli, the foot process effacement of podocyte, the markers for podocyte injury (nephrin,podocin and WT1) and Cx43 expression in glomeruli were examined respectively. The results demonstrated metabolic disorder, hyperinsulinemia, systemic inflammation and microalbuminuria in ORG rats. There was significant hypertrophy, glomerular expansion and inflammatory cell infiltration in the kidney of ORG rats compared to the control group. Significant foot process effacement of the podocyte in the glomeruli, nephrin loss and density reduction were shown in the ORG rats, and Cx43 expression was significant upregulated in glomeruli of ORG rats compared to the control group. The results indicate the correlation of overexpressed Cx43 with the obesity related renal inflammation and suggest that Cx43 might be a potential target in the development of obesity related glomerulopathy.

Topics: Animals; Connexin 43; Diet, High-Fat; Disease Models, Animal; Glomerulonephritis; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Obesity; Podocytes; Rats; Rats, Sprague-Dawley; WT1 Proteins

Obesity-related glomerulopathy (ORG) is morphologically characterized by glomerulomegaly with or without observable focal segmental glomerulosclerosis under light microscope, with decreased podocyte density and number, and with increased foot‑process width observed under electron microscope. The severity of podocyte injury is correlated with the degree of proteinuria and renal dysfunction. However, the pathogenesis of ORG is not well understood. The aim of the present study was to explore the possible pathogenic role of aldosterone (ALDO) in ORG. In the in vivo animal experiments, body weight, Lee's obesity index, abdominal fat index, urinary protein excretion, average glomerular diameter were significantly increased, the mRNA and protein expression of podocyte‑associated molecules including nephrin, podocin, podoplanin and podocalyxin were significantly reduced, and the Wnt/β‑catenin signaling pathway was activated in ORG model mice compared with the Control mice, whereas the administration of spironolactone significantly ameliorated these effects. In the in vitro experiments on cultured podocytes, the mRNA and protein expression levels of the aforementioned podocyte‑associated molecules were significantly downregulated and the Wnt/β‑catenin signaling pathway was activated following ALDO stimulation, whereas eplerenone significantly attenuated all the above effects. Dickkopf‑related protein 1 (DKK1), an inhibitor of Wnt/β‑catenin signaling pathway, also reduced the effects of ALDO exposure on the expression of podocyte‑associated molecules. The present study hypothesized that ALDO may be involved in the pathogenesis of ORG through the activation of Wnt/β‑catenin signaling pathway in podocytes.

Topics: Aldosterone; Animals; beta Catenin; Disease Models, Animal; Down-Regulation; Eplerenone; Glomerulonephritis; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Kidney Cortex; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Obesity; Podocytes; Sialoglycoproteins; Spironolactone; Wnt Proteins; Wnt Signaling Pathway

Preclinical Research Vanillic acid (VA) is a dihydroxybenzoic acid derivative widely used as a flavoring agent. It has chemopreventive effects on experimentally-induced carcinogenesis and in ulcerative colitis. The object of the present study was to investigate the effects of VA, alone and in combination with methylprednisolone (MP), on cationic bovine serum albumin (cBSA induced immune-complex glomerulonephritis in female BALB/c mice. Pre-immunization was carried out with cBSA in BALB/c mice and repeated (cBSA, 13 mg/kg, 3 times/week, i.v.) for 6 weeks to induce glomerulonephritis which was confirmed by the presence of severe proteinuria. The effect of VA (50, 100, and 200 mg/kg, p.o.) and its combination with MP (12.5 mg/kg, p.o.) was assessed in the nephrotic disease model. Treatment with VA decreased inflammatory nephrotic injury as evidenced by decreased proteinuria, serum creatinine, blood urea nitrogen, serum IgG1 and TNF-α levels. Co-administration of VA with MP showed an improvement in the immunohistochemistry of glomerular nephrin and podocin. The present results indicate that VA has a nephroprotective effect in the management of autoimmune nephritis. Drug Dev Res 77 : 171-179, 2016.   © 2016 Wiley Periodicals, Inc.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Therapy, Combination; Female; Glomerulonephritis; Immune Complex Diseases; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Methylprednisolone; Mice; Mice, Inbred BALB C; Serum Albumin, Bovine; Treatment Outcome; Vanillic Acid

Glomerular visceral epithelial cells (podocytes) play a critical role in the maintenance of glomerular permselectivity. Podocyte injury, manifesting as proteinuria, is the cause of many glomerular diseases. We reported previously that calcium-independent phospholipase A2γ (iPLA2γ) is cytoprotective against complement-mediated glomerular epithelial cell injury. Studies in iPLA2γ KO mice have demonstrated an important role for iPLA2γ in mitochondrial lipid turnover, membrane structure, and metabolism. The aim of the present study was to employ iPLA2γ KO mice to better understand the role of iPLA2γ in normal glomerular and podocyte function as well as in glomerular injury. We show that deletion of iPLA2γ did not cause detectable albuminuria; however, it resulted in mitochondrial structural abnormalities and enhanced autophagy in podocytes as well as loss of podocytes in aging KO mice. Moreover, after induction of anti-glomerular basement membrane nephritis in young mice, iPLA2γ KO mice exhibited significantly increased levels of albuminuria, podocyte injury, and loss of podocytes compared with wild type. Thus, iPLA2γ has a protective functional role in the normal glomerulus and in glomerulonephritis. Understanding the role of iPLA2γ in glomerular pathophysiology provides opportunities for the development of novel therapeutic approaches to glomerular injury and proteinuria.

Topics: Aging; Animals; Autophagy; Cells, Cultured; Endoplasmic Reticulum Stress; Glomerulonephritis; Group VI Phospholipases A2; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Phospholipases A2, Calcium-Independent; Podocytes; Proteinuria

The phosphorylation of nephrin plays an important role in maintaining the normal structure and function in podocytes. Dexamethasone (Dex) is usually used to treat glomerular diseases with proteinuria. In this study, we observated the effect of Dex and angiotensin II (AngII) on the change of nephrin phosphorylation in cultured podocytes. In vitro, cultured podocytes were exposed to AngII (10(-6) mol/L) pretreated with or without Dex (100 nM) for different time periods. Nck or Fyn were silenced by small interfering RNA (siRNA), nephrin and its phosphorylation expression were analyzed by Western blotting. In vitro, the phosphorylation of nephrin was significantly reduced after AngII stimulation (P < 0.05). Dex significantly resisted podocyte injury inducted by AngII via increasing the phosphorylation of nephrin (P < 0.05), siRNA silencing Nck can partially inhibited nephrin phosphorylation, siRNA silencing Fyn can completely inhibited nephrin phosphorylation. Phosphorylation of nephrin is important for the survival status of podocytes. Glucocorticoid treatment for human glomerulonephritis may exert its function by regulating Nck and Fyn complex to promote phosphorylation of nephrin. These results elucidate a novel mechanism of glucocorticoid treatment for glomerulonephritis.

Topics: Angiotensin II; Cell Survival; Cells, Cultured; Dexamethasone; Glomerulonephritis; Humans; Kidney Glomerulus; Membrane Proteins; Phosphorylation; Podocytes; RNA, Small Interfering

Cellular crescents are typically inflammatory and associated with rapidly progressive glomerulonephritis. Their pathogenesis involves glomerular basement membrane rupture due to circulating or intrinsic factors. Crescents associated with diabetic glomerulosclerosis are rarely reported. Furthermore, the nature of cells forming crescents in diabetes is unknown. To investigate the nature of crescents in diabetes, we examined renal biopsies from diabetic patients with nodular glomerulosclerosis and crescents (n = 2), diabetes without crescents (n = 5), nondiabetic renal biopsies (n = 3), and crescentic glomerulonephritis with inflammatory crescents (n = 5). Electron microscopy and confocal immunofluorescence analysis with antibodies against nephrin (a podocyte marker) and claudin 1 (parietal epithelial cell marker) were performed. Diabetic glomeruli with crescents contained a mixture of crescentic cells expressing either claudin 1 (11 ± 1.4 cells/glomerulus) or nephrin (5.5 ± 3.0 cells/glomerulus). Rare crescentic cells coexpressed nephrin and claudin 1 (2.5 ± 1.6 cells/glomerulus). In contrast, inflammatory crescents were almost exclusively composed of claudin 1-positive cells (25 ± 5.3 cells/glomerulus). Cells coexpressing claudin 1 and nephrin were absent in inflammatory crescents and all cases without crescents. Electron microscopy showed podocyte bridge formation between the glomerular basement membrane and parietal basement membrane but no glomerular basement membrane rupture as in inflammatory crescents. Crescents in diabetes may occur in diabetes in the absence of a secondary etiology and are composed of a mixture of parietal epithelial cells and visceral podocytes. Cells coexpressing parietal epithelial and podocyte markers suggest that parietal epithelial cells may transdifferentiate into podocytes in response to severe glomerular injury.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Claudin-1; Diabetic Nephropathies; Female; Glomerulonephritis; Humans; Kidney Glomerulus; Male; Membrane Proteins; Middle Aged; Podocytes

To investigate urinary nephrin and podocalyxin standardized by aquaporin (AQP)-2 using the enzyme-linked immunosorbent assay (ELISA) method in adult nephrotic syndrome (NS) patients.. In 107 adult NS patients (27 proliferative nephritis, 77 non-proliferative, and 3 amyloidosis) undergoing renal biopsy, urinary nephrin, podocalyxin and AQP2 were measured by ELISA. Urinary nephrin and podocalyxin were standardized by AQP2 (neph/AQP and PCX/AQP) and values were compared with 11 healthy controls.. Urinary neph/AQP correlated positively to PCX/AQP (r = 0.51, p < 0.001). Urinary neph/AQP and PCX/AQP were lower in controls than NS patients. Both proliferative and non-proliferative NS patients excreted high urinary neph/AQP and PCX/AQP without a significant difference between them (p > 0.05). Patients with focal segmental glomerular sclerosis (FSGS) excreted higher urinary neph/AQP (p = 0.09) and PCX/AQP (p < 0.05) compared to the other patients. Urinary neph/AQP and PCX/AQP were increased in the immunoglobulin M nephropathy patients. Amyloidosis patients excreted lower neph/AQP and PCX/AQP. The sensitivity was 0.87 and specificity 0.37 when the neph/AQP borderline value of 0.16 was adopted [area under the curve (AUC) = 0.61]. The sensitivity was 0.74 and specificity 0.61 when the PCX/AQP borderline value was 3.06 (AUC = 0.69).. Urinary neph/AQP and PCX/AQP are increased in NS patients, with FSGS patients showing the highest levels. To distinguish FSGS from other NS forms, the measurement of urinary PCX/AQP may be a practical method, and superior to neph/AQP.

Topics: Adolescent; Adult; Aged; Amyloidosis; Aquaporin 2; Case-Control Studies; Child; Enzyme-Linked Immunosorbent Assay; Female; Glomerulonephritis; Humans; Male; Membrane Proteins; Middle Aged; Nephrosis, Lipoid; Nephrotic Syndrome; Podocytes; Sensitivity and Specificity; Sialoglycoproteins; Urine; Young Adult

It is not clear how the podocyte damage manifests in different glomerulopathies. This study evaluated the podocyte-associated mRNA profiles in renal tissue and urine of patients with proliferative (PGs) or non-proliferative (NPGs) glomerulopathies.. Messenger RNA levels of nephrin, podocin, podocalyxin, synaptopodin, and alpha-actinin-4 were measured in the kidney tissue and urinary cells by real-time polymerase chain reaction. Podocyte-associated mRNAs were correlated with proteinuria and renal function, and the effect of immunosuppressive treatment of PGs and NPGs on urine mRNAs was assessed up to one year of follow up.. Podocyte-associated mRNAs were expressed consistently less in kidney tissue from patients with NPGs, and urinary podocyte mRNA levels were significantly higher in the PG group. After six months of immunosuppressive therapy, patients with PGs showed a significant reduction in the expression of podocin, podocalyxin, and alpha-actinin-4 compared with baseline (p<0.001). In the NPG group, alpha-actinin-4 levels decreased (p=0.008), and there was also a trend toward reduced podocalyxin mRNA (p=0.08). Urine podocyte-associated mRNAs correlated with the level of proteinuria at baseline and at six months, and there was a trend toward an inverse correlation between urinary mRNAs and kidney function at one year of follow up.. Podocyte-associated mRNAs were inhibited in kidney tissue concomitantly with their increase in urine in these patients with glomerulopathies. Different profiles of mRNA expression were seen, pointing to a higher degree of intra-renal podocytopenia in the NPGs and of podocyturia in the PGs. The immunosuppressive therapy effectively reduced the urinary levels of podocyte-associated mRNAs.

Topics: Actinin; Adult; Aged; Case-Control Studies; Cross-Sectional Studies; Female; Gene Expression Profiling; Genetic Markers; Glomerulonephritis; Humans; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Microfilament Proteins; Middle Aged; Podocytes; Predictive Value of Tests; Prospective Studies; Proteinuria; RNA, Messenger; Sialoglycoproteins; Time Factors; Treatment Outcome; Urinalysis; Young Adult

Although angiotensin (Ang) II blockade has become a standard antifibrotic therapy in kidney disease, the therapeutic efficacy of Ang II blockade is yet to be optimized. Considering the prognostic impact of proteinuria reduction, we hypothesized that titration of Ang II blockade for optimal anti-proteinuric effect would improve renoprotection. One day after induction of Thy 1.1 glomeruonephritis, rats were treated with increasing doses of the Ang II receptor blocker valsartan in drinking water. Six days after disease induction, the therapeutic effect on proteinuria, podocyte injury and glomerular fibrosis was evaluated. Increasing doses of valsartan resulted in increasing reduction of proteinuria. The maximally effective dose of valsartan was determined to be 1000 mg/l, which reduced proteinuria by 80% and maximally reduced glomerular matrix expansion, fibronectin, collagen I and collagen III staining and glomerular mRNAs for TGFß1, PAI-1, FN and collagen I. Notably, valsartan given at this dose prevented podocyte dysfunction by preserving expression of podocin and nephrin and the counter-regulating molecule B7-1 that is involved in podocyte injury. These results support the hypothesis that higher doses of valsartan are required to optimize proteinuria reduction and glomerulosclerosis amelioration. Further, the optimal dose of valsartan also provides an additional therapeutic effect by preventing podocyte dysfunction.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Dose-Response Relationship, Drug; Fibrosis; Glomerulonephritis; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Function Tests; Male; Membrane Proteins; Podocytes; Proteinuria; Rats; Rats, Sprague-Dawley; Tetrazoles; Valine; Valsartan

The role of parietal epithelial cells (PECs) in glomerular disease is unclear because they also express podocyte proteins under pathophysiological conditions. To help resolve this, we established a novel PEC isolation technique in rats and mice to investigate which regulatory mechanisms lead to podocyte protein expression in PECs. This pure pool of naive PECs was then compared with PECs in primary culture and immortalized PECs in permanent culture. The naive PECs expressed low levels of podocyte-specific mRNA. Accordingly, in crescentic glomerulonephritis, single PECs activated the podocin promoter in vivo. In primary culture, PECs expressed a distinct morphology from podocytes but with high transcript and protein levels of PEC markers. In contrast to naive PECs, cultured PECs also expressed podocyte proteins, and this correlated with reduced proteolytic activity but not with increased transcript levels. Activation of autophagy or proteasomal degradation decreased the levels of podocyte proteins in PECs, whereas inhibition of proteasomal degradation led to the stabilization of podocyte proteins in PECs. Thus, naive PECs express podocyte transcripts physiologically and these podocyte proteins are stable under pathological conditions through decreased proteolysis.

Topics: Animals; Autophagy; Cells, Cultured; Desmin; Disease Models, Animal; Female; Glomerulonephritis; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Podocytes; Proteolysis; Rats; Rats, Sprague-Dawley; Rats, Wistar; RNA, Messenger; Sialoglycoproteins; Transcriptome

Tenc1 (also known as tensin2) is an integrin-associated focal adhesion molecule that is broadly expressed in mouse tissues including the liver, muscle, heart and kidney. A mouse strain carrying mutated Tenc1, the ICR-derived glomerulonephritis (ICGN) strain, develops severe nephrotic syndrome.. To elucidate the function of Tenc1 in the kidney, Tenc1(ICGN) was introduced into 2 genetic backgrounds, i.e. DBA/2J (D2) and C57BL/6J (B6), strains that are respectively susceptible and resistant to chronic kidney disease.. Biochemical and histological analysis revealed that homozygous Tenc1(ICGN) mice develop nephrotic syndrome on the D2 background (D2GN) but not on the B6 background (B6GN). Initially, abnormal assembly and maturation of glomerular basement membrane (GBM) were observed, and subsequently effacement of podocyte foot processes was noted in the kidneys of D2GN but not B6GN mice. These defects are likely to be involved in the integrin signaling pathway.. This study suggests that Tenc1 contributes to the maintenance of GBM structures and that the genetic background influences the severity of nephrotic syndrome.

Topics: Adaptor Proteins, Signal Transducing; Animals; Blotting, Western; Collagen Type IV; Cytoskeletal Proteins; Glomerular Basement Membrane; Glomerulonephritis; Integrin alpha3beta1; Kidney Glomerulus; Laminin; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Inbred ICR; Mice, Knockout; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Nephrotic Syndrome; Phosphoprotein Phosphatases; Podocytes; Proteinuria; Species Specificity; Tensins

An early lesion in many kidney diseases is damage to podocytes, which are critical components of the glomerular filtration barrier. A number of proteins are essential for podocyte filtration function, but the signaling events contributing to development of nephrotic syndrome are not well defined. Here we show that class II phosphoinositide 3-kinase C2α (PI3KC2α) is expressed in podocytes and plays a critical role in maintaining normal renal homeostasis. PI3KC2α-deficient mice developed chronic renal failure and exhibited a range of kidney lesions, including glomerular crescent formation and renal tubule defects in early disease, which progressed to diffuse mesangial sclerosis, with reduced podocytes, widespread effacement of foot processes, and modest proteinuria. These findings were associated with altered expression of nephrin, synaptopodin, WT-1, and desmin, indicating that PI3KC2α deficiency specifically impacts podocyte morphology and function. Deposition of glomerular IgA was observed in knockout mice; importantly, however, the development of severe glomerulonephropathy preceded IgA production, indicating that nephropathy was not directly IgA mediated. PI3KC2α deficiency did not affect immune responses, and bone marrow transplantation studies also indicated that the glomerulonephropathy was not the direct consequence of an immune-mediated disease. Thus, PI3KC2α is critical for maintenance of normal glomerular structure and function by supporting normal podocyte function.

Topics: Animals; Antigens, Surface; Bone Marrow Transplantation; Glomerulonephritis; Glomerulonephritis, IGA; Humans; Immunoglobulin A; Immunoglobulin G; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Phosphatidylinositol 3-Kinases; Podocytes; Renal Insufficiency; Transplantation Chimera

Podocytes provide a slit diaphragm to inhibit proteinuria, and nephrin between podocytes functions as a barrier during glomerular filtration. Hepatocyte growth factor (HGF) can improve proteinuria in rodents with various renal injuries, but little is known about the role of HGF in podocyte-based events during glomerulonephritis. In the present study, we examined whether and how nephrin expression is sustained by podocytes during the HGF-mediated attenuation of albuminuria.. Lipopolysaccharide (LPS)-treated mice were used as an animal model of albuminuria. We evaluated the effect of HGF on slit proteins using immunohistochemistry, western blotting and real-time polymerase chain reaction.. Albuminuria occurred 36 h after LPS treatment in mice. This albuminuria did not involve podocyte loss, but was associated with a decrease in nephrin and its key anchor, synaptopodin. In these processes, c-Met tyrosine phosphorylation, which represented HGF signal activation, occurred in glomerular cells including podocytes. When recombinant HGF was administrated to nephritic mice, c-Met tyrosine phosphorylation became evident in podocytes. The enhancement of the HGF-c-Met signal was associated with increases in nephrin and synaptopodin. An electron microscopic examination revealed that LPS induced the foot process effacement of podocytes, while HGF injections suppressed the foot process injury. Overall, albuminuria was attenuated in the LPS-treated mice after HGF administration.. HGF protects podocytes from a loss of nephrin, at least in part, through maintaining synaptopodin. As a result, HGF was shown to sustain foot process structure, and albuminuria was attenuated under inflammation.

Topics: Albuminuria; Animals; Blotting, Western; Disease Models, Animal; Female; Glomerulonephritis; Hepatocyte Growth Factor; Humans; Immunohistochemistry; Lipopolysaccharides; Membrane Proteins; Mice; Mice, Inbred C57BL; Microfilament Proteins; Phosphorylation; Podocytes; Polymerase Chain Reaction; Proto-Oncogene Proteins c-met; Recombinant Proteins; Time Factors

Our previous studies using puromycin aminonucleoside (PAN) established that podocyte damage leads to glomerular growth arrest during development and glomerulosclerosis later in life. This study examined the potential benefit of maintaining podocyte-derived VEGF in podocyte defense and survival after PAN injury using conditional transgenic podocytes and mice, in which human VEGF-A (hVEGF) transgene expression is controlled by tetracycline responsive element (TRE) promoter and reverse tetracycline transactivator (rtTA) in podocytes. In vitro experiments used primary cultured podocytes harvested from mice carrying podocin-rtTA and TRE-hVEGF transgenes, in which hVEGF can be induced selectively. Induction of VEGF in PAN-exposed podocytes resulted in preservation of intrinsic VEGF, α-actinin-4 and synaptopodin, antiapoptotic marker Bcl-xL/Bax, as well as attenuation in apoptotic marker cleaved/total caspase-3. In vivo, compared with genotype controls, PAN-sensitive neonatal mice with physiologically relevant levels of podocyte-derived VEGF showed significantly larger glomeruli. Furthermore, PAN-induced up-regulation of desmin, down-regulation of synaptopodin and nephrin, and disruption of glomerular morphology were significantly attenuated in VEGF-induced transgenic mice. Our data indicate that podocyte-derived VEGF provides self-preservation functions, which can rescue the cell after injury and preempt subsequent deterioration of the glomerulus in developing mice.

Topics: Actinin; Animals; Animals, Newborn; Apoptosis; Autocrine Communication; bcl-2-Associated X Protein; bcl-X Protein; Caspase 3; Cells, Cultured; Desmin; Disease Models, Animal; Doxycycline; Glomerulonephritis; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microfilament Proteins; Podocytes; Puromycin Aminonucleoside; Response Elements; Vascular Endothelial Growth Factor A

Monocyte/ chemoattractant protein-1/chemokine ligand (CCL) 2 and stromal cell-derived factor-1/CXCL12 both contribute to glomerulosclerosis in mice with type 2 diabetes mellitus, through different mechanisms. CCL2 mediates macrophage-related inflammation, whereas CXCL12 contributes to podocyte loss. Therefore, we hypothesized that dual antagonism of these chemokines might have additive protective effects on the progression of diabetic nephropathy. We used chemokine antagonists based on structured l-enantiomeric RNA (so-called Spiegelmers) ie, the CCL2-specific mNOX-E36 and the CXCL12-specific NOX-A12. Male db/db mice, uninephrectomized at the age of 6 weeks, received injections of Spiegelmer, both Spiegelmers, nonfunctional control Spiegelmer, or vehicle from the age of 4 months for 8 weeks. Dual blockade was significantly more effective than monotherapy in preventing glomerulosclerosis. CCL2 blockade reduced glomerular leukocyte counts and renal-inducible nitric oxide synthase or IL-6 mRNA expression. CXCL12 blockade maintained podocyte numbers and renal nephrin and podocin mRNA expression. Consistently, CXCL12 blockade suppressed nephrin mRNA up-regulation in primary cultures of human glomerular progenitors induced to differentiate toward the podocyte lineage. All previously mentioned parameters were significantly improved in the dual-blockade group, which also suppressed proteinuria and was associated with the highest levels of glomerular filtration rate. Blood glucose levels and body weight were identical in all treatment groups. Dual chemokine blockade can have additive effects on the progression of diabetic kidney disease when the respective chemokine targets mediate different pathomechanisms of disease (ie, inflammation and progenitor differentiation toward the podocyte lineage).

Topics: Animals; Blotting, Western; Cells, Cultured; Chemokine CCL2; Chemokine CXCL12; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Glomerular Filtration Rate; Glomerulonephritis; Immunoenzyme Techniques; Interleukin-6; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Obese; Nitric Oxide Synthase Type II; Podocytes; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stem Cells

To evaluate severity of nephrinuria (NU) as a marker of podocyte dysfunction (PD) in patients with proteinuric forms of chronic glomerulonephritis (CGN) and to specify efficacy of this test for assessment of activity and prognosis of CGN.. We examined 74 CGN patients: 18 with inactive nephritis (group 1), 18--with subnephrotic proteinuria (group 2), 38--with nephrotic syndrome--NS (group 3). The control group consisted of 10 healthy subjects. Urinary excretion of nephrin was studied with indirect enzyme immunoassay. A response to immunosuppressive treatment (IST) was studied in 23 NS patients depending on a baseline NU level.. An NU level was higher in patients with proteinuric forms of CGN (groups 2 and 3) than in inactive disease and in healthy subjects, in NS patients significantly higher than in less severe proteinuria. NU was significantly higher in arterial hypertension, in persistent NS. Remission of NS was achieved within 6 months of treatment in 9 of 11 (82%) patients with a baseline NU level < 17 ng/ml. Eight from 12 (67%) patients with high NU did not respond to IST conducted for 9 months to 2 years. ROC-curve construction showed that NU assessment in NS patients has high informative value in assessment of prognosis and efficacy of treatment in 6 months to come.. The NU test in CGN patients is an informative diagnostic test allowing prognosis of a response to IST and assessment of PD severity.

Topics: Adolescent; Adult; Aged; Chronic Disease; Female; Glomerulonephritis; Humans; Male; Membrane Proteins; Middle Aged; Podocytes; Prognosis; Proteinuria; Remission, Spontaneous; Severity of Illness Index; Young Adult

There is much interest in the mechanisms that regulate adult tissue homeostasis and their relationship to processes governing foetal development. Mice deleted for the Wilms' tumour gene, Wt1, lack kidneys, gonads, and spleen and die at mid-gestation due to defective coronary vasculature. Wt1 is vital for maintaining the mesenchymal-epithelial balance in these tissues and is required for the epithelial-to-mesenchyme transition (EMT) that generates coronary vascular progenitors. Although Wt1 is only expressed in rare cell populations in adults including glomerular podocytes, 1% of bone marrow cells, and mesothelium, we hypothesised that this might be important for homeostasis of adult tissues; hence, we deleted the gene ubiquitously in young and adult mice. Within just a few days, the mice suffered glomerulosclerosis, atrophy of the exocrine pancreas and spleen, severe reduction in bone and fat, and failure of erythropoiesis. FACS and culture experiments showed that Wt1 has an intrinsic role in both haematopoietic and mesenchymal stem cell lineages and suggest that defects within these contribute to the phenotypes we observe. We propose that glomerulosclerosis arises in part through down regulation of nephrin, a known Wt1 target gene. Protein profiling in mutant serum showed that there was no systemic inflammatory or nutritional response in the mutant mice. However, there was a dramatic reduction in circulating IGF-1 levels, which is likely to contribute to the bone and fat phenotypes. The reduction of IGF-1 did not result from a decrease in circulating GH, and there is no apparent pathology of the pituitary and adrenal glands. These findings 1) suggest that Wt1 is a major regulator of the homeostasis of some adult tissues, through both local and systemic actions; 2) highlight the differences between foetal and adult tissue regulation; 3) point to the importance of adult mesenchyme in tissue turnover.

Topics: Animals; Atrophy; Cell Lineage; Epithelial-Mesenchymal Transition; Female; Gene Deletion; Gene Expression Regulation; Glomerulonephritis; Gonads; Hematopoiesis; Homeostasis; Insulin-Like Growth Factor I; Kidney Glomerulus; Male; Membrane Proteins; Mesenchymal Stem Cells; Mice; Mice, Transgenic; Multiple Organ Failure; Pancreas, Exocrine; Podocytes; Spleen; Tamoxifen; WT1 Proteins

Adrenomedullin (ADM) has antiproliferative effects on glomerular mesangial cells. The study was performed to determine changes in glomerular gene expression of the ADM system by ADM treatment in anti-Thy1 glomerulonephritis (GN).. GN in rats was induced by injecting anti-Thy-1 antibody. To show the effect of ADM treatment, rats received ADM from day 3 to day 6 of GN. Supplemental rats were sacrificed on day 3, 7 and 14 of GN to show the expression pattern of adrenomedullin and its receptors. Glomeruli were prepared by sieving or laser-assisted microdissection. Expression of ADM, calcitonin receptor-like receptor (CLR), receptor activity-modifying proteins (RAMP) 1-3, CD34, Thy1 and nephrin was analyzed using real-time PCR.. During GN a reduction of CLR and RAMP 2 + 3 expressions was detected on days 3, 7 and 14, while RAMP 1 rose. ADM mRNA decreased on days 3 and 7. Thy1 expression as a surrogate of mesangial cell number was downregulated during GN. A significant reduction of CD34 expression, as a surrogate for endothelial cell number, was detected on day 7. A tendency towards reduction of nephrin gene expression, as a surrogate for number of podocytes, was seen. The administration of ADM during GN did not change the expression on Thy1, CD34 or nephrin. The results were similar for microdissected and sieved glomeruli. In ADM-treated GN animals ADM gene expression rose compared to untreated GN animals on day 6. These effects were detected both in sieved and microdissected glomeruli. ADM administration did not change the expression of the receptors.. The downregulation of adrenomedullin during GN at the gene level can be improved by ADM application.

Topics: Adrenomedullin; Animals; Antigens, CD34; Calcitonin Receptor-Like Protein; Down-Regulation; Glomerulonephritis; Intracellular Signaling Peptides and Proteins; Isoantibodies; Kidney Glomerulus; Male; Membrane Proteins; Rats; Rats, Sprague-Dawley; Receptor Activity-Modifying Proteins; Receptors, Adrenomedullin; Receptors, Calcitonin; Receptors, G-Protein-Coupled

Tissue factor (TF) is a transmembrane protein that is essential for coagulation. TF is expressed on podocytes and its cytoplasmic domain has cell signalling functions in epithelial cells.. Mice lacking the cytoplasmic domain of TF (TF(CT-/-) mice) were used to study its role in physiological albuminuria and pathological proteinuria following induction of glomerulonephritis (GN).. Absence of the cytoplasmic domain of TF was associated with increased albuminuria, podocyte effacement, reduced podocyte numbers and increased spontaneous glomerular tumour necrosis factor α(TNFα) production under physiological conditions. In mice developing GN, absence of the cytoplasmic domain of TF resulted in increased proteinuria and enhanced renal TNFα production without altering other parameters of renal inflammation and injury. Studies in TF(CT-/-) chimeric mice (created by bone marrow transplantation) showed increased proteinuria and renal TNFα mRNA in GN was associated with absence of the cytoplasmic domain of TF in the kidney and was independent of the leucocyte phenotype.. These studies demonstrate that the cytoplasmic domain of TF contributes to renal albumin retention and its renal expression protects against proteinuria in leucocyte-mediated renal inflammation. Increased glomerular production of TNFα in the absence of cytoplasmic domain of TF may contribute to podocyte injury resulting in albuminuria and proteinuria.

Topics: Albuminuria; Animals; Cytoplasm; Glomerulonephritis; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Podocytes; Protein Structure, Tertiary; Proteinuria; Thromboplastin; Tumor Necrosis Factor-alpha

Progressive proteinuria and glomerulosclerosis characterize chronic allograft nephropathy. However, the causes are not fully elucidated. Podocytes function to prevent proteinuria; injury to this glomerular cell leads to glomerulosclerosis. The potential role of podocytes in the failing transplanted kidney is unknown. A rat model of kidney transplantation, characterized by proteinuria and glomerulosclerosis, was utilized to examine the potential role of podocytes.. Archival tissue was examined from allografts (Dark Agouti kidneys transplanted into operationally tolerant Albino Surgery rats), isografts (Dark Agouti) and controls (Dark Agouti: age-matched or after unilateral nephrectomy). The number of podocytes (by WT-1 staining) as well as the podocyte proteins podocin, nephrin and synaptopodin (by immunostaining) were measured at days 0, 2, 6 and at 6 months after transplantation. Changes in these parameters were compared between groups and correlated with urinary protein excretion.. At 6 months, podocyte number was reduced in allografted kidneys, accompanied by a decrease in nephrin and synaptopodin, but not podocin staining. Remnant kidneys in the uninephrectomized rats also showed a decreased podocyte number but no change in podocyte protein staining. Podocyte loss in allografts was established on day 6, whereas a decrease in nephrin and synaptopodin was not evident until 6 months. In contrast, podocyte number and protein staining was decreased but not significantly so in remnant and isografted kidneys.. A decrease in the slit diaphragm proteins, nephrin and synaptopodin, is a component of chronic allograft pathology.

Topics: Animals; Cell Count; Chronic Disease; Creatinine; Disease Models, Animal; Glomerulonephritis; Kidney Transplantation; Male; Membrane Proteins; Microfilament Proteins; Podocytes; Proteinuria; Rats; Rats, Inbred Strains; Transplantation, Homologous

Podocytopenia characterizes many forms of glomerular disease, preceding the development of glomerulosclerosis. While detachment of viable podocytes from the underlying glomerular basement membrane is an important mechanism of podocyte loss, the underlying factors involved remain unclear. Secreted protein acidic and rich in cysteine (SPARC), a matricellular protein with counteradhesive properties, is normally expressed at low levels by the podocyte but is markedly increased following podocyte injury. Accordingly, we elucidate the role of SPARC in mediating experimental crescentic glomerulonephritis by inducing passive nephrotoxic nephritis in SPARC(+/+) and SPARC(-/-) mice. By days 4, 7, and 21 following disease induction, podocyte number is better preserved, glomerulosclerosis is ameliorated, and proteinuria is reduced in SPARC(-/-) mice as compared with SPARC(+/+) littermates. Moreover, the preserved podocyte number in SPARC(-/-) mice correlates with reduced urinary levels of both nephrin and podocin. To establish a causal role for SPARC in mediating detachment, cultured SPARC(+/+) and SPARC(-/-) podocytes were subjected to mechanical strain as well as trypsin digestion, and detachment assays were performed. While podocytes lacking SPARC were more resistant to stretch-induced detachment, stable re-expression of SPARC restored detachment rates to levels comparable with SPARC(+/+) podocytes. Taken together, this study proves that SPARC plays a causal role in mediating podocyte detachment and accelerating glomerulosclerosis in experimental crescentic glomerulonephritis.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Disease Progression; Glomerulonephritis; Immunoblotting; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mice, Knockout; Osteonectin; Podocytes

Within the glomerulus, the scaffolding protein nephrin bridges the actin-rich foot processes that extend from adjacent podocytes to form the slit diaphragm. Mutations affecting a number of slit diaphragm proteins, including nephrin, cause glomerular disease through rearrangement of the actin cytoskeleton and disruption of the filtration barrier. We recently established that the Nck family of Src homology 2 (SH2)/SH3 cytoskeletal adaptor proteins can mediate nephrin-dependent actin reorganization. Formation of foot processes requires expression of Nck in developing podocytes, but it is unknown whether Nck maintains podocyte structure and function throughout life. Here, we used an inducible transgenic strategy to delete Nck expression in adult mouse podocytes and found that loss of Nck expression rapidly led to proteinuria, glomerulosclerosis, and altered morphology of foot processes. We also found that podocyte injury reduced phosphorylation of nephrin in adult kidneys. These data suggest that Nck is required to maintain adult podocytes and that phosphotyrosine-based interactions with nephrin may occur in foot processes of resting, mature podocytes.

Topics: Adaptor Proteins, Signal Transducing; Animals; Anti-Bacterial Agents; Cell Line; Disease Models, Animal; Doxycycline; Glomerular Filtration Rate; Glomerulonephritis; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, Transgenic; Oncogene Proteins; Phosphorylation; Podocytes; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley; Tight Junctions

Increasing evidence implicates activation of NF-kappaB in a variety of glomerular diseases, but the mechanisms involved are unknown. Here, upregulation of NF-kappaB in the podocytes of transgenic mice resulted in glomerulosclerosis and proteinuria. Absence of the podocyte protein nephrin resulted in NF-kappaB activation, suggesting that nephrin negatively regulates the NF-kappaB pathway. Signal transduction assays supported a functional relationship between nephrin and NF-kappaB and suggested the involvement of atypical protein kinase C (aPKCzeta/lambda/iota) as an intermediary. We propose that disruption of the slit diaphragm leads to activation of NF-kappaB; subsequent upregulation of NF-kappaB-driven genes results in glomerular damage mediated by NF-kappaB-dependent pathways. In summary, nephrin may normally limit NF-kappaB activity in the podocyte, suggesting a mechanism by which it might discourage the evolution of glomerular disease.

Topics: Animals; Apoptosis Regulatory Proteins; Cell Line; Cytoplasm; Dogs; Gene Silencing; Glomerulonephritis; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mice, Knockout; NF-kappa B; Podocytes; Protein Kinase C; Proteinuria; Transcription Factor RelA

Induction of heme oxygenase (HO)-1 is a key defense mechanism against oxidative stress. Compared with tubules, glomeruli are refractory to HO-1 upregulation in response to injury. This can be a disadvantage as it may be associated with insufficient production of cytoprotective heme-degradation metabolites. We, therefore, explored whether 1) targeted HO-1 expression can be achieved in glomeruli without altering their physiological integrity and 2) this expression reduces proteinuria in immune injury induced by an anti-glomerular basement membrane (GBM) antibody (Ab). We employed a 4.125-kb fragment of a mouse nephrin promoter downstream to which a FLAG-tagged hHO-1 cDNA sequence was inserted and subsequently generated transgenic mice from the FVB/N parental strain. There was a 16-fold higher transgene expression in the kidney than nonspecific background (liver) while the transprotein immunolocalized in glomerular epithelial cells (GEC). There was no change in urinary protein excretion, indicating that GEC-targeted HO-1 expression had no effect on glomerular protein permeability. Urinary protein excretion in transgenic mice with anti-GBM Ab injury (days 3 and 6) was significantly lower compared with wild-type controls. There was no significant change in renal expression levels of profibrotic (TGF-beta1) or anti-inflammatory (IL-10) cytokines in transgenic mice with anti-GBM Ab injury. These observations indicate that GEC-targeted HO-1 expression does not alter glomerular physiological integrity and reduces proteinuria in glomerular immune injury.

Topics: Animals; Autoantibodies; Disease Models, Animal; Epithelial Cells; Gene Expression Regulation, Enzymologic; Genetic Vectors; Glomerulonephritis; Heme Oxygenase-1; Interleukin-10; Kidney Glomerulus; Macrophages; Membrane Proteins; Mice; Mice, Transgenic; Permeability; Promoter Regions, Genetic; Proteinuria; Transforming Growth Factor beta1

Topics: Animals; Glomerulonephritis; Humans; Membrane Proteins; NF-kappa B; Podocytes

The development of proteinuria and glomerulosclerosis in kidney disease is associated with podocyte damage, including down-regulation of nephrin and podocin. Macrophages are known to induce renal injury, but the mechanisms involved are not fully understood. This study examined macrophage-mediated podocyte damage. Conditioned media (CM) from activated macrophages caused a 50-60% reduction in nephrin and podocin mRNA and protein expression in cultured mouse podocytes and rat glomeruli. This was abolished by a neutralizing anti-TNFalpha antibody. The addition of recombinant TNFalpha to podocytes or glomeruli caused a comparable reduction in podocyte nephrin and podocin expression to that of macrophage CM. Inhibition of c-Jun amino terminal kinase (JNK) or p38 kinase abolished the TNFalpha-induced reduction in nephrin and podocin expression. This study demonstrates that activated macrophages can induce podocyte injury via a TNFalpha-JNK/p38-dependent mechanism. This may explain, in part, the protective effects of JNK and p38 blockade in experimental kidney disease.

Topics: Animals; Cells, Cultured; Culture Media, Conditioned; Down-Regulation; Glomerulonephritis; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Macrophage Activation; Macrophages; Membrane Proteins; Mice; p38 Mitogen-Activated Protein Kinases; Podocytes; Rats; Tumor Necrosis Factor-alpha

Our previous comprehensive analysis of the genes expressed in kidneys with anti-glomerular basement membrane (GBM) nephritis using DNA microarrays showed that SM22alpha was one of the highly expressed genes. SM22alpha is a 22-kDa cytoskeletal protein that is exclusively expressed in smooth muscle cells. We investigated the localization of SM22alpha at mRNA and protein levels, and its pathological significance in anti-GBM nephritis kidneys.. Northern blot analysis, in situ hybridization, immunohistochemistry and double immunofluorescence studies were performed. The specific antibody (Ab) against SM22alpha was obtained by immunization of rabbits with recombinant rat SM22alpha protein.. SM22alpha mRNA expression was upregulated in kidneys and inducibly expressed in the parietal and visceral glomerular epithelial cells in anti-GBM nephritis kidneys. Immunohistochemistry with anti-SM22alpha Ab showed that SM22alpha protein was localized in the same series of cells. Double immunofluorescence with anti-SM22alpha and anti-glomerular cell markers demonstrated that SM22alpha might be expressed in epithelial cells of injured glomeruli. In visceral epithelial cells, SM22alpha might be expressed in cells in which podocyte specific markers, podocalyxin and nephrin were lost.. The injured glomerular epithelial cells in anti-GBM nephritis might undergo structural and functional alterations, including the expression of a smooth muscle marker, SM22alpha.

Topics: Animals; Basement Membrane; Biomarkers; Disease Models, Animal; Epithelial Cells; Gene Expression Regulation; Glomerulonephritis; Immunoglobulin G; Kidney Glomerulus; Male; Membrane Proteins; Microfilament Proteins; Muscle Proteins; Oligonucleotide Array Sequence Analysis; Phenotype; Random Allocation; Rats; Rats, Inbred WKY; RNA, Messenger; Sialoglycoproteins

Kidney podocytes and their slit diaphragms (SDs) form the final barrier to urinary protein loss. There is mounting evidence that SD proteins also participate in intracellular signaling pathways. The SD protein nephrin serves as a component of a signaling complex that directly links podocyte junctional integrity to actin cytoskeletal dynamics. Another SD protein, CD2-associated protein (CD2AP), is an adaptor molecule involved in podocyte homeostasis that can repress proapoptotic TGF-beta signaling in podocytes. Here we show that dendrin, a protein originally identified in telencephalic dendrites, is a constituent of the SD complex, where it directly binds to nephrin and CD2AP. In experimental glomerulonephritis, dendrin relocates from the SD to the nucleus of injured podocytes. High-dose, proapoptotic TGF-beta1 directly promotes the nuclear import of dendrin, and nuclear dendrin enhances both staurosporine- and TGF-beta1-mediated apoptosis. In summary, our results identify dendrin as an SD protein with proapoptotic signaling properties that accumulates in the podocyte nucleus in response to glomerular injury and provides a molecular target to tackle proteinuric kidney diseases. Nuclear relocation of dendrin may provide a mechanism whereby changes in SD integrity could translate into alterations of podocyte survival under pathological conditions.

Topics: Animals; Apoptosis; Cell Line; Cell Nucleus; Cells, Cultured; Fluorescent Antibody Technique, Direct; Glomerulonephritis; Glutathione Transferase; Green Fluorescent Proteins; Humans; Male; Membrane Proteins; Mice; Mice, Inbred Strains; Nerve Tissue Proteins; Podocytes; Recombinant Fusion Proteins

In crescentic glomerulonephritis (GN), monocyte chemoattractant protein-1 (MCP-1) is overexpressed within the glomeruli, and MCP-1 blockade has renoprotective effects. Adult podocytes are in a quiescent state, but acquisition of a migratory/proliferative phenotype has been described in crescentic GN and implicated in crescent formation. The cognate CC chemokine receptor 2 (CCR2), the MCP-1 receptor, is expressed by other cell types besides monocytes and has been implicated in both cell proliferation and migration. We investigated whether MCP-1 binding to CCR2 can induce a migratory/proliferative response in cultured podocytes. MCP-1 binding to CCR2 enhanced podocyte chemotaxis/haptotaxis in a concentration-dependent manner and had a modest effect on cell proliferation. Closure of a wounded podocyte monolayer was delayed by CCR2 blockade, and CCR2 was overexpressed at the wound edge, suggesting a role for CCR2 in driving podocyte migration. Immunohistochemical analysis of kidney biopsies from patients with crescentic GN demonstrated CCR2 expression in both podocytes and cellular crescents, confirming the clinical relevance of our in vitro findings. In conclusion, the MCP-1/CCR2 system is functionally active in podocytes and may be implicated in the migratory events triggered by podocyte injury in crescentic GN and other glomerular diseases.

Topics: Cell Movement; Cells, Cultured; Chemokine CCL2; Chemotaxis; Glomerulonephritis; Humans; Kidney; Membrane Proteins; Podocytes; Receptors, CCR2

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that maintains the glomerular and peritubular capillary (PTC) network in the kidney. The soluble form of the VEGF receptor-1 (soluble fms-like tyrosine kinase 1 (sFlt-1)) is known to regulate VEGF activity by binding VEGF in the circulation. We hypothesized that VEGF may be beneficial for maintaining glomerular filtration barrier and vascular network in rats with progressive glomerulonephritis (GN). For blockade of VEGF activity in vivo, rats were transfected twice with plasmid DNA encoding the murine sFlt-1 gene into femoral muscle 3 days before and 2 weeks after the induction of antiglomerular basement membrane antibody-induced GN. Inhibition of VEGF with sFlt-1 resulted in massive urinary protein excretion, concomitantly with downregulated expression of nephrin in nephritic rats. Further, blockade of VEGF induced mild proteinuria in normal rats. Administration of sFlt-1 affected neither the infiltration of macrophages nor crescentic formation. In contrast, treatment of sFlt-1 accelerated the progression of glomerulosclerosis and interstitial fibrosis accompanied with renal dysfunction and PTC loss at day 56. VEGF may play a role in maintaining the podocyte function as well as renal vasculature, thereby protecting glomeruli and interstitium from progressive renal insults.

Topics: Animals; Disease Progression; Glomerulonephritis; Male; Membrane Proteins; Proteinuria; Rats; Rats, Inbred WKY; Time Factors; Vascular Endothelial Growth Factor A

Synaptic vesicle protein 2B (SV2B) was identified by the subtraction hybridization technique as a molecule of which mRNA expression was decreased in puromycin aminonucleoside (PAN) nephropathy by glomerular cDNA subtraction assay. The expression of SV2B was detected in glomerular lysate with Western blot analysis. Dual-labeling immunofluorescence studies with glomerular cell markers demonstrated that SV2B is expressed in glomerular visceral epithelial cells (podocytes). The expression of SV2B is detected also in cultured podocyte and in human kidney section as podocytic pattern. The decrease of SV2B mRNA was already detected before the onset of proteinuria in PAN nephropathy. The mRNA expression of SV2B clearly is altered not only in PAN nephropathy but also in another proteinuric state that is caused by an antibody against nephrin, a functional molecule of the slit diaphragm. The decreased intensity in SV2B staining was already detected before the peak of proteinuria in both models with immunofluorescence study. A reduced amount of SV2B was detected in both models also with Western blot analysis. CD2AP, another functional molecule of the slit diaphragm, was observed in cytoplasm, including the processes area of the cultured podocyte, and when the podocyte was treated with small interfering RNA for SV2B, CD2AP staining at the process area was not detected. These results suggest that SV2B is a functional molecule of podocyte, and SV2B may play a role in the expression and proper localization of CD2AP.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antibiotics, Antineoplastic; Blotting, Western; Cells, Cultured; Chlorocebus aethiops; COS Cells; Cytoplasm; Cytoskeletal Proteins; DNA, Complementary; Female; Fluorescent Antibody Technique; Glomerulonephritis; Humans; Kidney Glomerulus; Membrane Glycoproteins; Membrane Proteins; Nerve Tissue Proteins; Podocytes; Proteinuria; Puromycin Aminonucleoside; rab3A GTP-Binding Protein; Rats; Rats, Wistar; RNA, Messenger; RNA, Small Interfering; src Homology Domains; Subtraction Technique

Podocytes are terminally differentiated and highly specialized epithelial cells. The factors governing podocyte differentiation are poorly understood. We tested the hypothesis that all-trans retinoic acid (ATRA), a vitamin A derivative, induces podocyte differentiation in vitro and in vivo.. We tested the effects of ATRA on podocytes. Primary rat, primary mouse, and immortalized mouse podocytes were exposed to ATRA (1, 5, 10, 20, 40, 50, 80, 160, and 200 micromol/L) or control (ethanol) for 72 hours. Cell morphology was examined by electron microscopy, the expression of podocyte specific proteins was measured by immunoflourescence and Western blot analysis, cell number and apoptosis were measured by 3-[4,5] dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) assay and Hoechst staining, respectively. To determine if ATRA alters podocyte differentiation in vivo, experimental injury was induced in C57BL6 mice using the antiglomerular antibody. Animals were given either daily intraperitoneal ATRA (16 mg/kg) or vehicle (corn oil). For end points, we measured proteinuria, podocyte-specific protein immunostaining, and proliferation [proliferating cell nuclear antigen (PCNA)] at days 5 and 14 (N= 5/group/time point).. ATRA induced podocyte process formation in vitro, and significantly increased the expression of nephrin and podocin. This coincided with a reduction in proliferation. ATRA also significantly prevented the decrease in staining for synaptopodin, nephrin, and podocin in experimental animals (P < 0.05 vs. control). This was accompanied by reduced proteinuria and decreased podocyte proliferation (P < 0.05 vs. control).. ATRA induces podocyte differentiation in vitro and in vivo and alters the expression of certain podocyte-specific proteins. Further studies are ongoing to delineate the mechanism of this effect.

Topics: Animals; Antineoplastic Agents; Cell Differentiation; Cell Division; Cell Line, Transformed; Glomerulonephritis; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Proteinuria; Rats; Rats, Sprague-Dawley; Tretinoin

Nephrin, a product of the NPHS1 gene, is a component of the slit diaphragms that are found between glomerular foot processes and is a crucial element for glomerular filtration barrier. Recently, nephrin has been focused in a number of studies of proteinuria development including various types of acquired glomerular diseases including minimal change nephrotic syndrome and membranous nephropathy. However, the precise role of nephrin in such acquired glomerular diseases is still unknown. To analyse the role of nephrin further, two kinds of anti-nephrin antibodies were raised in the rabbits and applied to an experimental mouse model of chronic graft-versus-host disease, in which (C57BL/10 x DBA/2) F1 mice developed clinically apparent severe proteinuria with significant glomerular lesions 7 weeks after parental DBA/2 cell transfer. Antibody-sandwich ELISA detected anti-nephrin antibodies during week 2 to week 6, with the peak at week 2 or week 4. Colocalization of nephrin and IgG on week 4, week 6, and week 8 was revealed by confocal microscopic analysis, suggesting that in situ immune complex formation with nephrin in glomerular lesion. Taken together, it seems to be suggested nephrin and its autoantibody have a certain role in the development of glomerular lesion in our model mice.

Topics: Animals; Autoantibodies; Chronic Disease; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Glomerulonephritis; Graft vs Host Disease; Immune Complex Diseases; Lupus Nephritis; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Proteinuria; Rabbits

Nephrin is an important constituent of the glomerular filtration barrier and alteration of its expression is associated with severe proteinuria. In this study we show that injection of an anti-Thy1.1 antibody in rats not only induces a mesangioproliferative glomerulonephritis associated with increased proteinuria, but also leads to a sustained increase of nephrin mRNA and protein expression in renal glomeruli over a time period of 29 days. In contrast, podocin and CD2AP, two proteins shown to interact with nephrin in the slit diaphragm, are acutely downregulated at days 3-7 and, thereafter, recovered again to normal levels after 29 days. Interestingly, immunofluorescence staining of kidney sections at day 10 of the disease shows a highly heterogeneous pattern, in that some podocytes show complete absence of nephrin, whereas others show highly accumulated staining for nephrin compared to control sections, which in total results in an increased level of nephrin per glomerulus. In summary, our data show that in the course of mesangioproliferative glomerulonephritis in rats, an upregulation of nephrin expression occurs with a concomitant transient downregulation of podocin and CD2AP which may account for a highly dysregulated filtration barrier and increased proteinuria.

Topics: Adaptor Proteins, Vesicular Transport; Animals; Antibodies; Cell Shape; Glomerulonephritis; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Proteins; Proteinuria; Rats; Rats, Wistar; RNA, Messenger; Thy-1 Antigens

Nephrin, the molecule responsible for congenital nephrotic syndrome of Finnish type, is crucial in maintaining the glomerular filtration barrier. Recently, its complete gene structure and common gene polymorphisms in its exons have been reported, although the functional and clinical significance of these polymorphisms has not yet been elucidated. We investigated a possible association of the NPHS1 polymorphisms with the development of Ig A nephropathy (IgAN), as well as the clinical and histologic manifestations in IgAN. A total of 464 Japanese subjects, including 267 patients with histologically proven IgAN and 197 healthy controls with normal urinalysis, were genotyped for the NPHS1 G349A, G2289A, and T3315C polymorphisms. The frequencies of the genotypes, alleles, and estimated haplotypes of NPHS1 polymorphisms were no different between patients with IgAN and the controls. Within the IgAN group, patients carrying at least one G allele of G349A tended to present with more proteinuria, lower renal function, and more severe histopathologic injury than those with the AA genotype, although the time from the first urinary abnormality to the renal biopsy was no different between both groups. The logistic regression analysis indicated that even after adjusting for the effect of proteinuria and hypertension the GG genotype of NPHS1 G349A was an independent risk factor for the deteriorated renal function at the time of diagnosis. This study suggests that the NPHS1 G349A polymorphism may be associated with heavy proteinuria and a decline in renal function in patients with IgAN.

Topics: Adolescent; Adult; Aged; Case-Control Studies; Child; DNA Primers; Female; Gene Frequency; Genotype; Glomerulonephritis; Glomerulonephritis, IGA; Humans; Male; Membrane Proteins; Middle Aged; Nephrotic Syndrome; Polymorphism, Genetic; Proteins; Proteinuria

The onset of proteinuria in passive Heymann nephritis, (PHN), a rat model of human membranous nephropathy (MN), is complement-dependent and is associated with altered podocyte slit diaphragm integrity and dissociation of nephrin from the actin cytoskeleton. These studies examined if complement is responsible for these podocyte changes.. PHN was induced with sheep anti-Fx1A. Controls were injected with normal sheep globulin. A third group was injected with anti-Fx1A and depleted of complement with cobra venom factor. Four days later, proteinuria was measured, slit diaphragm integrity was examined by electron microscopy, nephrin distribution was studied by immunofluorescence, and the glomerular content of nephrin and its association with actin were assessed by sequential extraction of isolated glomeruli and Western blotting.. Four days after immunization, seven out of eight PHN rats were proteinuric, whereas none of the complement depleted group had proteinuria despite similar levels of antibody deposition. Complement depletion preserved slit diaphragm morphology. Immunofluorescence microscopy with an antibody to the extracellular domain of nephrin showed a normal staining pattern in the rats depleted of complement and a shift to a more dispersed and clustered pattern in the PHN group. Western blot analysis of the glomerular extracts showed a significant reduction in the total amount of nephrin and in the fraction of actin-associated nephrin in the PHN group, whereas the amounts in the complement-depleted rats were similar to normal controls.. The onset of proteinuria in the PHN model of MN is coincident with complement-dependent alterations in the association of nephrin with the actin cytoskeleton and loss of podocyte slit diaphragm integrity.

Topics: Actins; Animals; Blotting, Western; Complement System Proteins; Disease Models, Animal; Fluorescent Antibody Technique; Glomerulonephritis; Glomerulonephritis, Membranous; Kidney; Membrane Proteins; Microscopy, Electron; Proteins; Proteinuria; Rats; Tissue Distribution

Topics: Animals; Disease Models, Animal; Glomerulonephritis; Glomerulonephritis, Membranous; Membrane Proteins; Proteins; Rats

Nephrin is a recently identified protein, which is synthesized in the podocytes and localized in the slit diaphragm area. Nephrin is a cell adhesion molecule of the immunoglobulin superfamily, and presumably is a part of the zipper-like structure of the slit membrane. As the mutation of the gene coding nephrin induces congenital nephrotic syndrome of Finnish type, which is a prototype of nephrotic syndrome, it has been suggested that nephrin also plays a role in acquired proteinuric kidney disease.. To address the above issue, the expression of nephrin in acquired human glomerular disease was studied by immunoelectron microscopy employing a polyclonal antibody against nephrin. Four normal human kidneys from nephrectomy specimens and eight kidney biopsy specimens from glomerular disease patients (one minimal change disease, one membranous glomerulonephritis (GN), one membranoproliferative GN, four IgA nephropathy, and one lupus nephritis) were studied. Proteinuria of the patients ranged from 448 to 11725 mg/day. Effacement of the foot processes was observed in all patients.. The study demonstrated that the number and distribution of gold particles in the glomerular region, where the podocyte foot process was well preserved, were similar to that found in normal kidneys; however, gold particles were almost always absent in regions where the foot processes were effaced. The number of gold particles per foot process interspace was not different between normal controls and GN patients; however, the number of gold particles per defined length (1000 nm) of the glomerular basement membrane underlying the foot processes was significantly reduced in GN patients.. Using immunoelectron microscopy, we observed that the expression of nephrin in GN was lower in regions where the foot processes were effaced, and comparable with that of normal controls where the foot process interspaces were preserved. The significance of our observation in the context of proteinuria in acquired GN needs further clarification.

Topics: Adult; Case-Control Studies; Female; Gene Expression; Glomerulonephritis; Glomerulonephritis, IGA; Glomerulonephritis, Membranoproliferative; Glomerulonephritis, Membranous; Humans; Kidney Glomerulus; Lupus Nephritis; Male; Membrane Proteins; Microscopy, Fluorescence; Microscopy, Immunoelectron; Middle Aged; Nephrosis, Lipoid; Proteins

Glomerular disease is one of the most common causes of end-stage renal failure. Increasing evidence suggests that these glomerulopathies are frequently caused by primary lesions in the renal podocytes. One of the major consequences of podocyte lesions is the accumulation of mesangial matrix in the glomerular basement membrane, a process called glomerulosclerosis. Mesangial sclerosis is one of the most consistent findings in Denys-Drash patients and can be caused by dominant mutations in the Wilms' tumor 1 gene (WT1). The underlying mechanism, however, is poorly understood. WT1 is expressed in the podocytes throughout life, but its function in this cell type is unknown. Combining Wt1-knockout and inducible yeast artificial chromosome transgenic mouse models, we demonstrate that reduced expression levels of WT1 result in either crescentic glomerulonephritis or mesangial sclerosis depending on the gene dosage. Strikingly, the two podocyte-specific genes nphs1 and podocalyxin are dramatically downregulated in mice with decreased levels of Wt1, suggesting that these two genes act downstream of Wt1. Taken together, our data provide genetic evidence that reduced levels of Wt1 are responsible for the pathogenesis of two distinct renal diseases and offer a molecular explanation for the increased occurrence of glomerulosclerosis in patients with WAGR syndrome.

Topics: Animals; Down-Regulation; Glomerular Mesangium; Glomerulonephritis; Glomerulosclerosis, Focal Segmental; Humans; Kidney Diseases; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Knockout; Proteins; Sclerosis; Sialoglycoproteins; WT1 Proteins

These studies examined the expression of the podocyte slit diaphragm protein nephrin and its association with actin at the onset of proteinuria in passive Heymann nephritis (PHN), a rat model of human membranous nephropathy. Four days after immunization, 58% of PHN rats had mild proteinuria. At that time, most slit diaphragms were still visible on electron microscopy; however, in those locations where the deposits encroached on the filtration slits, the slit diaphragms were either displaced or absent. On day 7, the PHN rats were severely proteinuric, and most slit diaphragms were either absent, displaced, or replaced by occluding-type junctions. Immunofluorescence microscopy with antibodies to the external and cytoplasmic domains of nephrin showed a progressive loss of staining and a change in the distribution of nephrin from an interrupted linear pattern in normal controls to a more dispersed and clustered pattern in PHN. In contrast, the intensity of staining for ZO-1 and CD2-associated protein (CD2AP), two other proteins that are located on the cytoplasmic face of the slit diaphragm, was undiminished. Immunogold electron microscopy confirmed the progressive disappearance of nephrin from podocyte foot processes and retention of CD2AP. Glomeruli and glomerular cell membranes were extracted sequentially with Triton X-100, followed by DNase I or potassium iodide to depolymerize actin. Western blot analysis of the extracts showed a progressive decline of total nephrin on days 4 and 7 of PHN as well as a reduction in the actin-associated fraction. These findings show that nephrin partly dissociates from actin at the onset of podocyte injury in PHN. This is accompanied by a progressive loss of nephrin from the podocyte foot processes and prominent changes in the morphology of the slit diaphragms. These events may underlie the loss of podocyte barrier function in membranous nephropathy.

Topics: Actins; Animals; Fluorescent Antibody Technique; Glomerulonephritis; Kidney Glomerulus; Male; Membrane Proteins; Microscopy, Electron; Microscopy, Immunoelectron; Proteins; Proteinuria; Rats; Rats, Sprague-Dawley; Solubility

Angiotensin-converting enzyme inhibitors restore size-selective dysfunction of the glomerular barrier in experimental animals and humans with proteinuric nephropathies, although the structural and molecular determinants of such an effect are not completely understood. This study used an accelerated model of experimental nephrosis to assess nephrin gene and protein expression in the kidney and the possible modulating effect of drugs that block angiotensin II (AII) synthesis/activity. Passive Heymann nephritis (PHN) and control animals were studied at day 7, month 4, and month 8. Additional PHN rats were treated with lisinopril or AII receptor blocker L-158,809 and studied at 8 mo. Lisinopril and L-158,809 controlled BP, prevented proteinuria, and protected PHN animals from renal injury. An intense signal of nephrin mRNA was detected in glomeruli of control animals mainly restricted to podocytes. In PHN rats, nephrin staining progressively and remarkably decreased with time. Lisinopril and L-158,809 fully prevented the decrease in nephrin transcripts to levels comparable to those of control rats. Consistent with nephrin mRNA expression, immunostaining of the protein showed a progressive decrease in kidneys from PHN rats that was completely abolished by lisinopril and L-158,809. In summary, progressive renal injury was associated with downregulation of nephrin gene that was totally prevented by angiotensin-converting enzyme inhibitor and AII receptor blocker, suggesting that renoprotection afforded by drugs that interfere with AII synthesis/activity was related to an effect on nephrin assembly.

Topics: Angiotensin II; Angiotensin Receptor Antagonists; Angiotensin-Converting Enzyme Inhibitors; Animals; Base Sequence; DNA Primers; Glomerulonephritis; Imidazoles; In Situ Hybridization; Kidney Glomerulus; Lisinopril; Male; Membrane Proteins; Proteins; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tetrazoles

The recently identified gene NPHS1 with its mutations causing congenital nephrotic syndrome of the Finnish type (CNF) is highly promising in providing new understanding of pathophysiology of proteinuria. Earlier we cloned a rat NPHS1 homologue, as well as characterized and raised antibodies to the respective protein product nephrin.. Changes in the expression levels of nephrin-specific mRNA in commonly used experimental models of proteinuria were examined using semiquantitative reverse transcription-polymerase chain reaction, immunofluorescence, and immunoelectron microscopy (IEM) of nephrin.. Notably, a 40% down-regulation of the nephrin-specific mRNA of cortical kidney was seen already at day 3 after induction of the puromycin aminonucleoside nephrosis (PAN), while no major elevation of urinary protein secretion was seen at this stage. A further decrease of 80% of nephrin message was seen at the peak of proteinuria at day 10. A similar decrease of up to 70% from the basal levels was seen in mercuric chloride-treated rats. Changes in the protein expression paralleled those of the mRNA in indirect immunofluorescence. Interestingly, a remarkable plasmalemmal dislocation from the normal expression site at the interpodocyte filtration slits could be observed in IEM.. Nephrin appears to be an important causative molecule of proteinuria and shows a remarkable redistribution from the filtration slits to the podocyte plasma membrane, especially in PAN.

Topics: Albuminuria; Animals; Antimetabolites, Antineoplastic; Disease Models, Animal; Disinfectants; Gene Expression; Glomerulonephritis; Kidney Glomerulus; Lipid Peroxidation; Male; Membrane Proteins; Mercuric Chloride; Microscopy, Immunoelectron; Proteins; Puromycin Aminonucleoside; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic