nephrin and Glomerulonephritis--Membranoproliferative

Extra-capillary hypercellularity is a common finding in crescentic glomerulonephritis (GN) and focal segmental glomerulosclerosis (FSGS). In diabetic nephropathy (DN), extra-capillary hypercellularity is often observed as a finding of complications such as IgA nephropathy or microscopic polyangiitis superimposed on DN. However, in rare cases, epithelial cell proliferation may accompany DN. We experienced a case of nodular diabetic glomerulosclerosis with marked extra-capillary hypercellularity and revealed the origin of this atypical lesion using immunostainings.. A man in his 50 s was admitted to the hospital with nephrotic syndrome, and a renal biopsy was performed. Diffuse nodular lesions and extra-capillary hypercellularity were observed, but the results of serological examination or immunofluorescent assays did not implicate any other crescentic GN. Immunostaining for claudin-1 and nephrin was performed to identify the origin of the extra-capillary lesions. Given the clinical course and pathological findings, a diagnosis of DN-associated extra-capillary cell proliferation was made.. Extra-capillary hypercellularity, which resembles FSGS or crescentic GN, is a rare finding in DN and should therefore be treated with caution. In such cases, co-staining for claudin-1 and nephrin may facilitate the diagnosis of DN.

Topics: Cell Proliferation; Claudin-1; Diabetes Mellitus; Diabetic Nephropathies; Glomerulonephritis, IGA; Glomerulonephritis, Membranoproliferative; Glomerulosclerosis, Focal Segmental; Humans; Male; Membrane Proteins; Microscopic Polyangiitis; Middle Aged

The Wilms' tumor suppressor WT1 is a key regulator of podocyte function that is mutated in Denys-Drash and Frasier syndromes. Here we have used an integrative approach employing ChIP, exon array, and genetic analyses in mice to address general and isoform-specific functions of WT1 in podocyte differentiation. Analysis of ChIP-Seq data showed that almost half of the podocyte-specific genes are direct targets of WT1. Bioinformatic analysis further identified coactivator FOXC1-binding sites in proximity to WT1-bound regions, thus supporting coordinated action of these transcription factors in regulating podocyte-specific genes. Transcriptional profiling of mice lacking the WT1 alternative splice isoform (+KTS) had a more restrictive set of genes whose expression depends on these alternatively spliced isoforms. One of these genes encodes the membrane-associated guanylate kinase MAGI2, a protein that localizes to the base of the slit diaphragm. Using functional analysis in mice, we further show that MAGI2α is essential for proper localization of nephrin and the assembly of the slit diaphragm complex. Finally, a dramatic reduction of MAGI2 was found in an LPS mouse model of glomerular injury and in genetic cases of human disease. Thus, our study highlights the central role of WT1 in podocyte differentiation, identifies that WT1 has a central role in podocyte differentiation, and identifies MAGI2α as the crucial isoform in slit diaphragm assembly, suggesting a causative role of this gene in the etiology of glomerular disorders.

Topics: Adaptor Proteins, Signal Transducing; Alternative Splicing; Animals; Binding Sites; Cell Differentiation; Down-Regulation; Exons; Female; Forkhead Transcription Factors; Glomerulonephritis, Membranoproliferative; Glomerulosclerosis, Focal Segmental; Guanylate Kinases; Humans; Lipopolysaccharides; Membrane Proteins; Mice; Mutation; Oligonucleotide Array Sequence Analysis; Podocytes; Promoter Regions, Genetic; Protein Isoforms; Repressor Proteins; Transcription, Genetic; WT1 Proteins

Proteinuria is a major feature of lupus nephritis (LN) and reflects podocyte injury. Analysis of podocyte biomarkers was performed attempting to identify if podocyte phenotype is distinct in pure membranous and proliferative LN. Expression of synaptopodin, Wilms tumor protein 1 (WT1), glomerular epithelial protein 1 (GLEPP1) and nephrin was evaluated in 52 LN biopsies by immunohistochemistry. Preserved synaptopodin expression was observed in only 10 (19.2%) of all biopsies while 42 (80.8%) had reduced expression. Both groups had comparable proteinuria at the time of biopsy (p = 0.22); however, in the mean follow-up of four years there was a tendency toward lower mean levels of proteinuria in patients with preserved synaptopodin staining (0.26±0.23 vs. 0.84±0.90 g/24 h, p = 0.05) compared with those with diminished expression. Thirty-nine (75%) biopsies were classified as proliferative and 13 (25%) as pure membranous. Comparison of podocyte biomarkers demonstrated a predominance of preserved staining of synaptopodin (69.2%), WT1 (69.2%), GLEPP1 (53.9%) and nephrin (60%) in the pure membranous group whereas only <10% of the proliferative showed preserved expression. Our data suggest that in proliferative forms there seems to occur structural podocyte damage, whereas in the pure membranous the predominant preserved pattern suggests a dysfunctional podocyte lesion that may account for the better long-term prognosis of proteinuria outcome.

Topics: Adult; Biomarkers; Biopsy; Cell Proliferation; Female; Glomerulonephritis, Membranoproliferative; Glomerulonephritis, Membranous; Humans; Immunohistochemistry; Lupus Nephritis; Male; Membrane Proteins; Microfilament Proteins; Middle Aged; Podocytes; Prognosis; Proteinuria; Receptor-Like Protein Tyrosine Phosphatases, Class 3; Time Factors; WT1 Proteins; Young Adult

Membranous proliferative glomerulonephritis (MPGN) is a major primary cause of chronic kidney disease (CKD). Podocyte injury is crucial in the pathogenesis of glomerular disease with proteinuria, leading to CKD. To assess podocyte injuries in MPGN, the pathological features of spontaneous murine models were analyzed.. The autoimmune-prone mice strains BXSB/MpJ-Yaa and B6.MRL-(D1Mit202-D1Mit403) were used as the MPGN models, and BXSB/MpJ-Yaa(+) and C57BL/6 were used as the respective controls. In addition to clinical parameters and glomerular histopathology, the protein and mRNA levels of podocyte functional markers were evaluated as indices for podocyte injuries. The relation between MPGN pathology and podocyte injuries was analyzed by statistical correlation.. Both models developed MPGN with albuminuria and elevated serum anti-double-strand DNA (dsDNA) antibody levels. BXSB/MpJ-Yaa and B6.MRL showed severe proliferative lesions with T and B cell infiltrations and membranous lesions with T cell infiltrations, respectively. Foot process effacement and microvillus-like structure formation were observed ultrastructurally in the podocytes of both MPGN models. Furthermore, both MPGN models showed a decrease in immune-positive areas of nephrin, podocin and synaptopodin in the glomerulus, and in the mRNA expression of Nphs1, Nphs2, Synpo, Actn4, Cd2ap, and Podxl in the isolated glomerulus. Significant negative correlations were detected between serum anti-dsDNA antibody levels and glomerular Nphs1 expression, and between urinary albumin-to-creatinine ratio and glomerular expression of Nphs1, Synpo, Actn4, Cd2ap, or Podxl.. MPGN models clearly developed podocyte injuries characterized by the decreased expression of podocyte functional markers with altered morphology. These data emphasized the importance of regulation of podocyte injuries in MPGN.

Topics: Adaptor Proteins, Signal Transducing; Animals; CD3 Complex; Cytoskeletal Proteins; Disease Models, Animal; Female; Glomerulonephritis, Membranoproliferative; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Leukocyte Common Antigens; Male; Membrane Proteins; Mice; Microfilament Proteins; Microscopy, Electron; Myosin Heavy Chains; Nonmuscle Myosin Type IIA; Podocytes; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sialoglycoproteins

Glomerular podocyte molecules are involved in the pathogenesis of congenital nephrotic syndrome. However, their role in primary nephrotic syndrome is not clear. This study investigated the expression of nephrin, podocin and synaptopodin in primary nephrotic syndrome.. Eighty-seven patients with primary nephrotic syndrome including minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN) and membranoproliferative glomerulonephritis Type I (MPGN) were included in the study. Glomerular expression of nephrin, podocin and synaptopodin was studied in renal biopsies by immunofluorescence and immunohistochemistry. Correlation of expression with clinical and biochemical parameters was performed.. The pattern of expression for all podocyte proteins in controls was uniform fine granular along the capillary walls towards the visceral epithelial cell aspect. Glomerular expression of nephrin was present in all renal biopsies and was similar to that in controls. Glomerular synaptopodin expression was seen in all MN and MPGN patients, while it was seen in 74 % (17/23) MCD and 93.5 % (29/31) FSGS. Reduced synaptopodin expression showed no correlation with clinical and biochemical factors. Podocin expression was present in 5/23 MCD (22 %), 3/31 FSGS (9.6 %), 13/17 MN (76.4 %) and 13/16 MPGN (81 %) patients. The reduced expression of podocin significantly correlated with the degree of proteinuria (p = 0.032). No correlation with age, gender and serum creatinine level was observed.. Reduction of glomerular podocin expression found in MCD and FSGS is related to the amount of proteinuria. Our findings suggest that alteration in podocyte phenotype may not be a primary event and may reflect the degree of podocyte injury in primary nephrotic syndrome.

Topics: Adolescent; Adult; Creatinine; Glomerulonephritis, Membranoproliferative; Glomerulosclerosis, Focal Segmental; Humans; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Male; Membrane Proteins; Microfilament Proteins; Middle Aged; Nephrosis, Lipoid; Nephrotic Syndrome; Podocytes; Proteinuria

Multi-glycoside of Tripterygium wilfordii Hook. f. (GTW) has been proved clinically effective in reducing proteinuria in chronic kidney disease in China. However, the mechanisms involved are still unclear. In this study we examined the effects of GTW at the different dosages on proteinuria and podocyte slit diaphragm (SD) dysfunction in anti-Thy1.1 glomerulonephritis (GN).. Rats with anti-Thy1.1 GN were divided into 2 groups, a GTW group and a vehicle group, and sacrificed at 30 min, on day 7, and on day 14 in Experiments 1, 2 and 3, respectively. The administration of GTW at the moderate and high doses was started 3 days before or at the same time of antibody injection till sacrifice. Proteinuria was determined in Experiments 1, 2, and 3. After sacrifice, the staining intensity of SD-associated key functional molecules including nephrin and podocin, podocyte structure, mesangial change, macrophage infiltration, and blood biochemical parameters were examined, respectively. Protein and mRNA expressions of nephrin and podocin in glomeruli were also investigated. Besides, liver histological characteristics were analyzed.. In Experiment 1, GTW pretreatment at the medium dose (75 mg/kg body weight) caused no influence on the induction of anti-Thy1.1 GN and the basal nephrin expression. In Experiment 2, the high dosage (100mg/kg body weight) of GTW ameliorated proteinuria, the distribution of nephrin and podocin, mesangial proliferation, and the activated macrophage accumulation, as compared with vehicle group (P<0.05). Additionally, it increased mRNA and protein expressions of nephrin and podocin in glomeruli on day 7, but had no influence on podocyte structure. In Experiment 3, the medium dosage (75 mg/kg body weight) of GTW improved proteinuria, the partial matrix expansion, and the distribution of nephrin and podocin on day 14, as compared with anti-Thy1.1 GN rats (P<0.05). GTW at the high or moderate dose did not affect hepatic function on day 7 and on day 14.. Podocyte SD dysfunction, such as the disordered distribution and down-regulation of nephrin and podocin expression, is critically involved in the pathogenesis of anti-Thy1.1 GN induced by mAb 1-22-3. The restoration of the distribution and expression of nephrin and podocin by GTW could be an important mechanism by which GTW ameliorates proteinuria and podocyte SD dysfunction.

Topics: Animals; Disease Models, Animal; Female; Glomerular Mesangium; Glomerulonephritis, Membranoproliferative; Intracellular Signaling Peptides and Proteins; Macrophage Activation; Membrane Proteins; Phytotherapy; Plant Extracts; Podocytes; Proteinuria; Rats; Rats, Wistar; RNA, Messenger; Thy-1 Antigens; Tripterygium

To examine the effect of multi-glycoside of Tripterygium wilfordii Hook. f. (GTW) on proteinuria and expression of slit diaphragm-associated molecules such as nephrin and podocin in glomerulonephritis induced by anti-Thy1.1 antibody (anti-Thy1 . 1 GN).. Anti-Thy1.1 GN was induced in rats by a single intravenous injection with 500 microg of anti-Thy1.1 mAb 1-22-3. Fourteen rats were randomly divided into 2 groups, the GTW-treated group and vehicle treated group, and sacrificed on day 14 in Experiment 1 or on day 7 in Experiment 2 after induction of Anti-Thy1.1 GN. Daily oral administration of GTW and vehicle as a control was started from 3 days before injection or at the same time of injection to the day of sacrifice in Experiment 1 or 2. Proteinuria was determined during 14 days in Experiment 1 or during 7 days in Experiment 2. From kidneys taken at sacrifice, glomerular morphological changes, glomerular macrophage infiltration, glomerular expression of nephrin and podocin, and its mRNA expression in renal tissue were examined.. In Experiment 1, proteinuria and mesangial matrix expansion were significantly attenuated by GTW treatment. No difference in staining intensity of nephrin and podocin in glomeruli was observed between GTW treated group and vehicle treated group on day 14. In Experiment 2, GTW treatment significantly ameliorated proteinuria, mesangial injury and activated macrophage infiltration in glomerulus. In addition, it significantly increased the expression of nephrin and podocin and its mRNA expression in glomeruli on day 7.. In anti-Thy1.1 GN, the reduced expression of nephrin and podocin may contribute to the development of mesangial injury and proteinuria. The findings suggest that GTW ameliorates not only proteinuria but also mesangial lesions in anti-Thy1 . 1 GN most likely by increasing the expression of nephrin and podocin.

Topics: Animals; Female; Fluorescent Antibody Technique; Glomerulonephritis, Membranoproliferative; Glycosides; Intracellular Signaling Peptides and Proteins; Isoantibodies; Membrane Proteins; Phytotherapy; Podocytes; Proteinuria; Rats; Reverse Transcriptase Polymerase Chain Reaction; Thy-1 Antigens; Tripterygium

Blockade of the renin-angiotensin system has been established as a treatment for heart failure with hypertension and left ventricular hypertrophy, and for progressive kidney diseases. The present study was conducted to examine whether spironolactone, a mineralocorticoid receptor antagonist, alone or in combination with cilazapril, an angiotensin converting enzyme (ACE) inhibitor, ameliorates proteinuria and renal lesions in an immune-initiated progressive nephritis model. Wistar rats were uninephrectomized 7 days before injection of anti-Thy-1 monoclonal antibody 1-22-3 to induce progressive glomerulonephritis. The nephritic rats were untreated or treated with spironolactone (400 mg/kg body weight/day), cilazapril (1 mg/kg body weight/day), or both for 10 weeks. Proteinuria was increased in the untreated rats 1 week after nephritis induction and was maintained throughout the experiment. Compared with the untreated animals (212.9+/-49.2 mg/day), proteinuria was significantly reduced in the spironolactone-treated group (62.0+/-4.0 mg/day, p=0.0046) and the cilazapril-treated group (71.8+/-26.0 mg/day, p=0.0048) on day 70 after antibody injection. Further reduction of proteinuria (42.4+/-4.5 mg/day, p=0.0019 vs. the untreated group) and less renal cortex interstitial fibrotic change (fibrosis score: 142.0+/-18.4 vs. 80.3+/-18.5 in the untreated group, p=0.0123) were detected in the spironolactone plus cilazapril-treated group. Blood pressure did not differ among the three treatment groups. In conclusion, spironolactone ameliorates proteinuria to the same degree as cilazapril, and concomitant use of spironolactone and an ACE inhibitor further suppresses renal disease progression. These data suggest that concomitant treatment with spironolactone and an ACE inhibitor has beneficial effects on immune-initiated progressive kidney disease.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Antibodies, Monoclonal; Blood Pressure; Cilazapril; Drug Therapy, Combination; Fibrosis; Glomerulonephritis, Membranoproliferative; Kidney; Male; Membrane Proteins; Mineralocorticoid Receptor Antagonists; Proteinuria; Rats; Rats, Wistar; Spironolactone; Thy-1 Antigens

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