Pathogenesis Of Focal Segmental Glomerulosclerosis Research Articles

Proteomic Analysis of Idiopathic Nephrotic Syndrome Triggered by Primary Podocytopathies in Adults: Regulatory Mechanisms and Diagnostic Implications.

Idiopathic nephrotic syndrome (NS) is a heterogeneous group of glomerular disorders which includes two major phenotypes: minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS). MCD and FSGS are classic types of primary podocytopathies. We aimed to explore the molecular mechanisms in NS triggered by primary podocytopathies and evaluate diagnostic value of the selected proteomic signatures by analyzing blood proteome profiling. Totally, we recruited 90 participants in two cohorts. The first cohort was analyzed using label-free quantitative (LFQ) proteomics to discover differential expressed proteins and identify enriched biological process in NS which were further studied in relation to clinical markers of kidney injury. The second cohort was analyzed using parallel reaction monitoring-based quantitative proteomics to verify the data of LFQ proteomics and assess the diagnostic performance of the selected proteins using receiver-operating characteristic curve analysis. Several biological processes (such as immune response, cell adhesion, and response to hypoxia) were found to be associated with kidney injury during MCD and FSGS. Moreover, three proteins (CSF1, APOC3, and LDLR) had over 90% sensitivity and specificity in detecting adult NS triggered by primary podocytopathies. The identified biological processes may play a crucial role in MCD and FSGS pathogenesis. The three blood protein markers are promising for diagnosing adult NS triggered by primary podocytopathies.

A Prospective Study on Complement Activation Distinguishes Focal Segmental Glomerulosclerosis from Minimal Change Disease

IntroductionMinimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are related podocytopathies with distinct kidney outcomes. Surprisingly, elevated urinary activation fragments have been found in FSGS despite little complement deposition on immunofluorescence staining. Whether complement activation distinguishes FSGS from MCD, participating in the development of segmental lesions, remains unknown. MethodsWe performed an observational study in subjects with MCD and FSGS, and proteinuria ≥ 1 g/g of creatinine. We included both primary and secondary or unknown causes. We compared urinary fragments of terminal pathway activation, sC5b9 and C5a expressed as creatinine ratios, between MCD and FSGS. ResultsPatients with FSGS (n=41) had a serum albumin of 31±10 g/L and proteinuria of 5.1 (2.6-9.1) g/g at sampling, while those with MCD (n=15) had a lower serum albumin (22±9 g/L, p=0.002), and a proteinuria of 3.8 (1.9-7.7) g/g (p=0.40). Urinary sC5b9 and C5a were 8.7 (1.7-52.3) and 1.26 (0.45-1.84) μg/mmol of creatinine, respectively in FSGS subjects, compared to 0.8 (0.0-1.5) and 0.06 (0.01-0.15) μg/mmol of creatinine in MCD (p<0.001), respectively. We found no association between urinary complement fragments and age, eGFR, or chronic kidney lesions. When analyzing samples with proteinurias ≥ 3 g/g, the c-statistics for urinary sC5b9 and C5a were 0.96 and 1.00, respectively, in differentiating FSGS from MCD. ConclusionWe found no urinary complement activation fragments in MCD, in comparison to FSGS, despite similar levels of proteinuria. This suggests a role for complement activation in the pathogenesis of FSGS and provides an additional tool for distinguishing these two entities.

Urinary SPP1 has potential as a non-invasive diagnostic marker for focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a type of chronic glomerular nephropathy showing characteristic glomerular sclerosis, diagnosed by kidney biopsy. However, it is difficult and expensive to monitor disease progression with repeated renal biopsy in clinical practice, and thus here we explored the feasibility of urine biomarkers as non-invasive diagnostic tools. We downloaded scRNA-seq datasets of 20 urine cell samples and 3 kidney tissues and obtained two gene lists encoding extracellular proteins for bioinformatic analysis; in addition, we identified key EP-Genes by immunohistochemical staining and performed bulk RNA sequencing with 12 urine samples. We report that urine cells and kidney cells were correlated. A total of 64 EP-Genes were acquired by intersecting genes of distal tubular cluster with extracellular proteins. Function enrichment analysis showed that EP-Genes might be involved in the immune response and extracellular components. Six key EP-Genes were identified and correlated with renal function. IMC showed that key EP-Genes were located mainly in tubules. Cross verification and examination of a urine RNAseq dataset showed that SPP1 had diagnostic potential for FSGS. The presence of urine SPP1 was primarily associated with macrophage infiltration in kidney, and the pathogenesis of FSGS may be related to innate immunity. Urinary cells seemed to be strongly similar to kidney cells. In summary, SPP1 levels reflect renal function and may have potential as a biomarker for non-invasive diagnosis of FSGS.

Current understanding of the molecular mechanisms of circulating permeability factor in focal segmental glomerulosclerosis.

The pathogenetic mechanisms underlying the onset and the post-transplant recurrence of primary focal segmental glomerulosclerosis (FSGS) are complex and remain yet to be fully elucidated. However, a growing body of evidence emphasizes the pivotal role of the immune system in both initiating and perpetuating the disease. Extensive investigations, encompassing both experimental models and patient studies, have implicated T cells, B cells, and complement as crucial actors in the pathogenesis of primary FSGS, with various molecules being proposed as potential "circulating factors" contributing to the disease and its recurrence post kidney-transplantation. In this review, we critically assessed the existing literature to identify essential pathways for a comprehensive characterization of the pathogenesis of FSGS. Recent discoveries have shed further light on the intricate interplay between these mechanisms. We present an overview of the current understanding of the engagement of distinct molecules and immune cells in FSGS pathogenesis while highlighting critical knowledge gaps that require attention. A thorough characterization of these intricate immune mechanisms holds the potential to identify noninvasive biomarkers that can accurately identify patients at high risk of post-transplant recurrence. Such knowledge can pave the way for the development of targeted and personalized therapeutic approaches in the management of FSGS.

Asparagine endopeptidase protects podocytes in adriamycin-induced nephropathy by regulating actin dynamics through cleaving transgelin

Focal segmental glomerulosclerosis (FSGS) is the most common glomerular disorder causing end-stage renal diseases worldwide. Central to the pathogenesis of FSGS is podocyte dysfunction, which is induced by diverse insults. However, the mechanism governing podocyte injury and repair remains largely unexplored. Asparagine endopeptidase (AEP), a lysosomal protease, regulates substrates by residue-specific cleavage or degradation. We identified the increased AEP expression in the primary proteinuria model which was induced by adriamycin (ADR) to mimic human FSGS. Invivo, global AEP knockout mice manifested increased injury-susceptibility of podocytes in ADR-induced nephropathy (ADRN). Podocyte-specific AEP knockout mice exhibited much more severe glomerular lesions and podocyte injury after ADR injection. In contrast, podocyte-specific augmentation of AEP in mice protected against ADRN. Invitro, knockdown and overexpression of AEP in human podocytes revealed the cytoprotection of AEP as a cytoskeleton regulator. Furthermore, transgelin, an actin-binding protein regulating actin dynamics, was cleaved by AEP, and, as a result, removed its actin-binding regulatory domain. The truncated transgelin regulated podocyte actin dynamics and repressed podocyte hypermotility, compared to the native full-length transgelin. Together, our data reveal a link between lysosomal protease AEP and podocyte cytoskeletal homeostasis, which suggests a potential therapeutic role for AEP in proteinuria disease.

Targeting ferroptosis attenuates podocytes injury and delays tubulointerstitial fibrosis in focal segmental glomerulosclerosis

Ferroptosis is a non-apoptotic form of cell death, involved in chronic kidney diseases (CKD) and acute kidney injury (AKI), so far, the role of ferroptosis in focal segmental glomerulosclerosis (FSGS) remains largely unknown. We aimed to investigate the role of ferroptosis in FSGS, in this study, we found the reduced expression of GPX4 in podocytes, as well as tubular epithelial cells (TECs), from patients with FSGS. Treatment with ferrostatin-1 (Fer-1), a potent and selective ferroptosis inhibitor, significantly reduced proteinuria, prevented glomerulosclerosis, attenuated podocyte injury in ADR-induced FSGS murine model. As expected, ADR treatment caused downregulation of GPX4 in human podocytes, treatment with Fer-1 greatly blocked the downregulation of GPX4, restored the GSH level and attenuated cell death. Furthermore, Fer-1 treatment greatly delayed the development of tubulointerstitial fibrosis in ADR-induced FSGS murine model. Taken together, ferroptosis is involved in the pathogenesis of FSGS, targeting ferroptosis is a promising therapeutic option for patients with FSGS.

Renal Glomerular Expression of WT-1, TGF-β, VEGF, and ET-1 Immunostains in Murine Models of Focal and Segmental Glomerulosclerosis.

Primary focal segmental glomerulosclerosis (FSGS) is a type of chronic renal disease that commonly progresses to renal failure as the treatments are not particularly effective. Glomerular podocyte injury and loss are pivotal to the pathogenesis of FSGS. This study aims to explore the glomerular immunohistochemistry stain expression of Wilms tumor-1 (WT-1) (podocyte-specific protein), transforming growth factor beta (TGF-β) (cytokine protein), vascular endothelial growth factor (VEGF) (angiogenic protein), and endothelin-1 (ET-1) (profibrotic growth factor), in rats with adriamycin nephropathy, which represents the murine model of human FSGS. By the end of 8 and 12 weeks, the kidneys of adriamycin-treated rats and control rats were harvested and the histomorphology was studied. Both 8- and 12-week test groups developed proteinuria, and hypoalbuminemia and showed FSGS on hematoxylin and eosin-stained slides. The renal tissue samples were also treated with immunostains for WT-1, TGF-β, VEGF, and ET-1. The glomeruli in all the FSGS kidneys showed loss of WT-1 expression with a concomitant notable increased expression of TGF-β, VEGF, and ET-1 immunostains. These results demonstrate that as FSGS evolves, the WT-1-expressing podocytes are lost and it correlates inversely with the overexpression of TGF-β, VEGF, and ET-1, suggesting that during the pathogenesis of FSGS, podocyte damage triggers the activation of these proteins. The findings in the current study echo the theory hypothesized in world literature that TGF-β, VEGF, and ET-1 play an integral part in the evolution of FSGS. More research is needed to further detail the pathogenic role of these proteins as it may open routes to more targeted and effective treatment modalities.

#4635 CIRCULATING NEPHRIN AUTOANTIBODIES IN PATIENTS WITH POST-TRANSPLANT RECURRENCE OF PRIMARY FOCAL SEGMENTAL GLOMERULOSCLEROSIS

Abstract Background and Aims Post-transplant recurrence of primary focal segmental glomerulosclerosis (FSGS) is a major challenge in the field of nephrology and kidney transplantation. Primary FSGS is thought to be caused by circulating factors (CFs) that injure podocytes, but these factors remain unknown despite intensive efforts to identify them. Recently, one post-transplant recurrent FSGS patient with pretransplant nephrin autoantibodies was reported (Watts AJB et al, JASN 33:238, 2022). Furthermore, we reported very early post-transplant recurrence changes of podocytes and possible roles of circulating nephrin autoantibodies in a patient with FSGS recurrence (Hattori M et al, AJT 22:2478, 2022). To confirm these preliminary results, we examined the autoantibodies to nephrin and the punctate deposition of IgG colocalized with nephrin using stocked plasma samples and grafted kidney biopsy specimens of the patients with FSGS recurrence. Method This study was approved by the local Ethics Committee of Tokyo Women's Medical University (approval no. 2021-0184). The clinical and research activities reported here are consistent with the Principles of the Declaration of Istanbul as outlined in the “Declaration of Istanbul on Organ Trafficking and Transplant Tourism”. From 1989 to 2022, a total of 78 patients with FSGS underwent kidney transplantation in our institution, and 16 patients experienced post-transplant recurrence of FSGS. Since the stoked plasma samples were available in 14 out of 16 patients, these 14 patients were included in this study. All 14 patients showed immediate post-transplant recurrence and had no pathogenic variants in FSGS-related genes including NPHS1. Circulating anti-nephrin autoantibodies were measured using the ELISA methods based on the Watts's report with one modification, that is, recombinant extracellular domain of human nephrin was obtained commercially (R&amp;D systems, USA). To validate the ELISA system, circulating anti-nephrin autoantibodies were also measured in 9 genetic FSGS patients, 13 membranous nephropathy (MN) patients, 4 lupus nephritis (LN) patients, and 13 healthy controls. Dual immunofluorescence (IF) staining of nephrin and IgG and image acquisition using the two-dimensional structured illumination microscopy mode with a Nikon microscope were performed as previously reported (Hattori M et al, AJT 22:2478, 2022). Results The cut-off level of anti-nephrin antibody calculated from the results of a total 30 cases including 13 MN patients, 4 LN patients, and 13 healthy controls using Frey's formula (J Immunol Method, 1998) was 172.2 U/mL All 14 recurrent patients enrolled in this study had higher titter than the cut-off level and the mean ± SD of the anti-nephrin antibody level in these patients was 939.9 ± 261.8 U/mL. In contrast, the mean anti-nephrin antibody level in patients with genetic FSGS was 98.6 ± 77.3 U/mL, which was comparable with healthy controls. The IF staining of graft biopsy specimens showed punctate depositions of IgG colocalized with nephrin in all 14 recurrent patients. Conclusion The autoantibodies to nephrin in plasma samples and the punctate deposition of IgG colocalized with nephrin in grafted kidney biopsy specimens were observed in all 14 patients with post-transplant recurrent FSGS. Further studies in larger numbers of patients as well as in patients of other ethnicities are clearly required to confirm this surprising result; however, circulating nephrin autoantibodies could be a possible candidate for CFs in the pathogenesis of recurrent FSGS.

Urine proteome profile in primary podocytopathies

BACKGROUND. Primary focal segmental glomerulosclerosis (FSGS) and membranous nephropathy (MN) are diseases with primary podocyte damage with high proteinuria and nephrotic syndrome. While the mechanisms in primary MN are well understood, the pathogenesis of primary FSGS is still unknown, and therefore, the search for biomarkers that could expand ourunderstanding of its pathogenetic mechanisms. THE AIM: to determine the urine proteomic profile of patients with primary podocytopathies – FSGS in comparison with MN. PATIENTS AND METHODS. The study included 48 patients with a morphologically confirmed diagnosis of CGN occurring with nephrotic syndrome – 32 men and 16 women. In 18 patients, a decrease in glomerular filtration rate &lt; 60 ml/min/1.73 m2 was observed. The histological diagnosis was confirmed by biopsy: 31 patients had FSGS, 17 patients with MN were included as a comparison group. The study of the urinary proteome was carried out by high performance liquid chromatography/mass spectrometry. RESULTS. In patients with FSGS, compared with the MN group, an increased content of 22 different proteins was noted, the most abundant were apolipoprotein A-I, hemopexin, vitronectin, pigment epithelial growth factor, components of the complement system (C3, C4b, factors B and H), retinol – and vitamin D-binding proteins, alpha-2-HS-glycoprotein, histidine-rich glycoprotein, plasma C1 protease inhibitor. In MN, increased urinary excretion of the complement component C2, fibrinogen alpha chain, osteopontin, and the SH3 domain-binding glutamic acid-rich-like protein 3, was detected. CONCLUSION. The proteomic profile of urine in FSGS, compared to MN, reflects the activation of variety of pathological processes – podocyte damage, involvement of parietal epithelial cells, tubulo-interstitial damage, accumulation of extracellular matrix, and complement activation process.

Parietal Epithelial Cell Behavior and Its Modulation by microRNA-193a.

Glomerular parietal epithelial cells (PECs) have been increasingly recognized to have crucial functions. Lineage tracking in animal models showed the expression of a podocyte phenotype by PECs during normal glomerular growth and after acute podocyte injury, suggesting a reparative role of PECs. Conversely, activated PECs are speculated to be pathogenic and comprise extracapillary proliferation in focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CrescGN). The reparative and pathogenic roles of PECs seem to represent two sides of PEC behavior directed by the local milieu and mediators. Recent studies suggest microRNA-193a (miR193a) is involved in the pathogenesis of FSGS and CrescGN. In a mouse model of primary FSGS, the induction of miR193a caused the downregulation of Wilms' tumor protein, leading to the dedifferentiation of podocytes. On the other hand, the inhibition of miR193a resulted in reduced crescent lesions in a mouse model of CrescGN. Interestingly, in vitro studies report that the downregulation of miR193a induces trans-differentiation of PECs into a podocyte phenotype. This narrative review highlights the critical role of PEC behavior in health and during disease and its modulation by miR193a.

Glomerular parietal epithelial expression of CD44 in minimal change nephrotic syndrome and primary focal segmental glomerulosclerosis: A clinico-pathological study.

Minimal change nephrotic syndrome (MCNS) and focal segmental glomerulosclerosis (FSGS) are the two common causes of nephrotic syndrome (NS) in both children and adults with overlapping clinical features, but with distinct prognostic and therapeutic implications. The distinction between these relies entirely on histopathology, which can sometimes be difficult. CD44 is expressed by activated parietal epithelial cells, plays a role in matrix deposition and thus in the pathogenesis of FSGS. To assess the expression of CD44 in MCNS and FSGS and to evaluate its association with the known clinical and histopathological prognostic factors. Thirty cases each of MCNS and FSGS were studied. The clinical, laboratory, histopathological, and CD 44 immunohistochemical data were recorded. The findings were analyzed and correlated. A P value of < 0.05 was considered statistically significant. Statistical association was noted between CD44 positivity and serum creatinine (p = 0.031), estimated glomerular filtration rate (p = 0.040), segmental sclerosis (p < 0.001), tubular atrophy (p = 0.027), interstitial fibrosis (p = 0.027), and histological diagnosis (p < 0.001). The sensitivity, specificity, positive predictive, and negative predictive values were 90%, 76.67%, 79.41% and 88.46%, respectively. CD44 immunostain can effectively distinguish MCNS from FSGS. The congruent results of CD44 positivity with known prognostic factors support the possibility of using the CD44 marker as a predictive tool in selecting high-risk patients and offering appropriate therapeutic measures.

Extracellular vesicles from focal segmental glomerulosclerosis pediatric patients induce STAT3 activation and mesangial cell proliferation.

Primary focal segmental glomerulosclerosis (FSGS), a major cause of end-stage kidney disease (ESKD) in adolescents and young adults, is attributable to recognized genetic mutations in a minority of cases. For the majority with idiopathic primary FSGS, the cause of the disease is unknown. We hypothesize that extracellular vesicle (EVs), that carry information between podocytes and mesangial cells, may play a key role in disease progression. A total of 30 participants (20 primary nephrotic syndrome/ 10 healthy controls) were enrolled in this study. Primary nephrotic syndrome subjects were grouped based on pathologic diagnosis. The FSGS group was compared to healthy control subjects based on demographic and clinical findings. EVs were isolated from the urine of each group before being characterized by Western blotting, transmission electron microscopy, and nanoparticle tracking analysis. The effects of the EVs from each group on normal human mesangial cells and activation of certain pathways were then investigated. Based on demographic and clinical findings, mean serum creatinine was significantly higher in the FSGS group than the normal healthy control group. The mean size of the EVs in the FSGS group was significantly higher than the healthy control group. The mesangial cells that were challenged with EVs isolated from FSGS patients showed significant upregulation of STAT-3, PCNA, Ki67, and cell proliferation. Our data demonstrate that EVs from FSGS patients stimulate mesangial cell proliferation in association with upregulation of the phospho-STAT-3 pathway. Additional studies are planned to identify the molecular cargo within the EVs from FSGS patients that contribute to the pathogenesis of FSGS.

Circulating nephrin autoantibodies and posttransplant recurrence of primary focal segmental glomerulosclerosis

To the Editor: Posttransplant recurrence of primary focal segmental glomerulosclerosis (FSGS) is a major challenge in the field of kidney transplantation. Primary FSGS is thought to be caused by circulating factors (CFs) that injure podocytes,1 but these factors remain unknown despite intensive efforts to identify them.2 Recently, one posttransplant recurrent FSGS patient with pretransplant nephrin autoantibodies was reported; however, the involvement of nephrin autoantibodies in the pathogenesis of recurrence has not been clarified.3 Here, we present very early posttransplant recurrence changes of podocytes and possible roles of circulating nephrin autoantibodies in FSGS recurrence. This study was approved by the local Ethics Committee of Tokyo Women’s Medical University (approval no. 2021–0184). We analyzed plasma samples and kidney tissue specimens of preperfusion (0 h) and postperfusion (1 h) in one patient (patient 1) with and one (patient 2) without FSGS recurrence after first kidney transplantation. Patient 1 had no pathogenic variants in FSGS-related genes including NPHS1. Patient 2 was shown to carry compound heterozygous LAMB2 variants after his kidney transplantation. Plasma samples were obtained before any prophylactic therapies were administered for their kidney transplantation. Patient clinical data are presented in Table S1 and methods are provided in the Supplementary Methods. We identified autoantibodies to nephrin in plasma of the patient showing recurrence (patient 1), but not in plasma of the patient without recurrence (patient 2) (Figure 1A). We further observed that punctate IgG was present and specifically colocalized with nephrin in the 1 h biopsy from the FSGS recurrent patient (Figure 1B and Figure S1), which is in line with the findings of Watts et al. for a subset of minimal change disease biopsies.3 In a previous in vitro experiment, a monoclonal anti-nephrin antibody induced nephrin tyrosine phosphorylation.4 Moreover, Src homology and collagen homology A (ShcA) protein was found to be expressed at low levels in podocytes and to bind tyrosine-phosphorylated nephrin as an adaptor protein.5 Here, dual immunofluorescence (IF) staining for IgG and phosphorylated (p)-nephrinY1176 revealed induced nephrin tyrosine phosphorylation colocalizing with IgG in the 1 h biopsy from the recurrent patient (Figure 1C and Figure S2). We further observed increased expression of ShcA in the 1 h biopsy from the recurrent patient and observed it to colocalize with nephrin (Figure 1D and Figure S3). These findings indicate that nephrin tyrosine phosphorylation was induced very early after exposure of circulating nephrin autoantibodies. Nephrin tyrosine phosphorylation is known to influence signal transduction pathways which control several podocyte functions including nephrin trafficking and cytoskeletal organization.5 In this study, IF staining for nephrin showed linear expression along the glomerular capillary in the 0 h biopsy from the recurrent patient, but nephrin expression took on a granular pattern in the 1 h biopsy (Figure 1E). Moreover, glomeruli appeared normal under light microscopy in the 1 h biopsy (Figure 1F), but podocyte foot process effacement (FPE) was segmentally observed under electron microscopy in the 1 h biopsy from the recurrent patient (Figure 1G and Figure S4). Further studies are clearly needed to confirm our preliminary results; however, our current findings indicate that circulating nephrin autoantibodies detected in plasma from a recurrent patient induce nephrin tyrosine phosphorylation that leads to increased ShcA expression, altered nephrin distribution, and podocyte FPE very early after kidney transplantation. Furthermore, circulating nephrin autoantibodies could be a possible candidate for CFs in the pathogenesis of recurrent FSGS. We thank the affected individuals, their families, and their physicians who contributed to this study. In this research work, we used instruments in the Medical Research Institute, Tokyo Women’s Medical University. This work was supported in part by Grants-in-Aid Scientific Research (C) (JP18K07857 [MH], JP21K07829 [KM], and JP21K07857 [NK]) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Blocking ribosomal protein S6 phosphorylation inhibits podocyte hypertrophy and focal segmental glomerulosclerosis

Ribosomal protein S6 (rpS6) phosphorylation mediates the hypertrophic growth of kidney proximal tubule cells. However, the role of rpS6 phosphorylation in podocyte hypertrophy and podocyte loss during the pathogenesis of focal segmental glomerulosclerosis (FSGS) remains undefined. Here, we examined rpS6 phosphorylation levels in kidney biopsy specimens from patients with FSGS and in podocytes from mouse kidneys with Adriamycin-induced FSGS. Using genetic and pharmacologic approaches in the mouse model of FSGS, we investigated the role of rpS6 phosphorylation in podocyte hypertrophy and loss during development and progression of FSGS. Phosphorylated rpS6 was found to be markedly increased in the podocytes of patients with FSGS and Adriamycin-induced FSGS mice. Genetic deletion of the Tuberous sclerosis 1 gene in kidney glomerular podocytes activated mammalian target of rapamycin complex 1 signaling to rpS6 phosphorylation, resulting in podocyte hypertrophy and pathologic features similar to those of patients with FSGS including podocyte loss, leading to segmental glomerulosclerosis. Since protein phosphatase 1 is known to negatively regulate rpS6 phosphorylation, treatment with an inhibitor increased phospho-rpS6 levels, promoted podocyte hypertrophy and exacerbated formation of FSGS lesions. Importantly, blocking rpS6 phosphorylation (either by generating congenic rpS6 knock-in mice expressing non-phosphorylatable rpS6 or by inhibiting ribosomal protein S6 kinase 1-mediated rpS6 phosphorylation with an inhibitor) significantly blunted podocyte hypertrophy, inhibited podocyte loss, and attenuated formation of FSGS lesions. Thus, our study provides genetic and pharmacologic evidence indicating that specifically targeting rpS6 phosphorylation can attenuate the development of FSGS lesions by inhibiting podocyte hypertrophy and associated podocyte depletion.

The role of cationic channels of the potential TRPC receptor in the pathogenesis of idiopathic nephrotic syndrome in children

Idiopathic nephrotic syndrome is the most common glomerulopathy in children, with a prevalence of approximately 16 per 100,000 of child population worldwide. Any chronic glomerular disease has the same type of development mechanism. Regardless of the damaging factor, after the death of a significant part of the nephrons, there occurs a steady decrease in the glomerular filtration rate, while morphologically we most often determine focal segmental glomerulosclerosis. Studying the causes of focal segmental glomerulosclerosis is an urgent problem in pediatric nephrology. Recently, there has been discussed the role of the cation channels of the potential receptor TRPC of podocytes in the development of proteinuria and focal segmental glomerulosclerosis. The article provides data on the role of TRPC receptors in the pathogenesis of focal segmental glomerulosclerosis. The authors present their our own data demonstrating gene expression of the cationic channels family of the potential receptor TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 in children with idiopathic nephrotic syndrome, depending on the morphological picture of the disease and sensitivity to steroid therapy.

Role of fibroblast specific protein 1 expression in the progression of adriamycin-induced glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a commonly occurring cause of steroid-resistant nephrotic syndrome and frequently progresses to renal failure. Podocyte epithelial-mesenchymal transition (EMT) is thought to induce podocyte detachment in glomerular diseases, and severe degeneration and shedding of glomerular podocytes plays a major role in the progression of FSGS. We showed that fibroblast specific protein 1 (FSP1), an EMT marker, is strongly expressed in podocytes of FSGS patients, but the significance of podocyte expression of FSP1 to the pathophysiology of FSGS remained unclear. Here, we investigated FSP1 expression in podocytes from mice with adriamycin (ADR)-induced nephropathy, a murine model of FSGS. The number of FSP1-positive (FSP1+) podocytes was increased in ADR-treated mice and positively correlated with the degree of proteinuria and glomerulosclerosis in ADR-treated mice. ADR-induced FSGS and the attendant proteinuria were significantly ameliorated in FSP1 knockout mice as compared to wild type mice. These findings indicate that podocyte expression of FSP1 plays a crucial role in the pathogenesis of FSGS, which makes FSP1 a potential target for treatment of FSGS.

Hsa_circ_0001461 as a novel biomarker in patients with primary focal segmental glomerulosclerosis

IntroductionCircular RNAs (circRNAs), a novel class of non-coding RNAs, have been implicated in various diseases and are considered to play an important role in physiological and pathological processes. We aimed to profile renal circRNA in children with primary focal segmental glomerulosclerosis (FSGS) and predict the potential mechanism underlying the pathogenesis of FSGS.Material and methodsA global circRNA expression analysis was performed in renal tissues from patients with FSGS (n=3) and controls (n=3). Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to validate the expression levels of three circRNAs in a cohort of 44 patients with FSGS, 44 age-matched healthy controls, and 15 patients with minimal change disease (MCD).ResultsA total of 152 circRNAs were differentially expressed in patients with FSGS compared with controls. Hsa_circ_0001461 and hsa_circ_0000605 were significantly increased in patients with FSGS compared with controls, as determined by qRT-PCR (p &lt; 0.001). The receiver operating characteristic (ROC) curve areas of hsa_circ_0001461 and hsa_circ_0000605 were 0.948 and 0.934, respectively. Hsa_circ_0001461 was significantly elevated in patients with FSGS compared with those with MCD and was correlated with 24 h urinary protein in patients with FSGS (r = 0.732, p &lt; 0.001). Bioinformatics analysis predicted mir-30a interaction with hsa_circ_0001461. Mir-30a was negatively correlated with the renal hsa_circ_0001461 level (r = -0.90, p &lt; 0.001).ConclusionsHsa_circ_0001461 is identified as a novel biomarker in children with FSGS and might provide insights into the mechanism of FSGS pathogenesis.

Nuclear exclusion of YAP exacerbates podocyte apoptosis and disease progression in Adriamycin-induced focal segmental glomerulosclerosis

AbstractFocal segmental glomerulosclerosis (FSGS) is a chronic glomerular disease with poor clinical outcomes. Podocyte loss via apoptosis is one important mechanism underlying the pathogenesis of FSGS. Recently, Yes-associated-protein (YAP), a key downstream protein in the Hippo pathway, was identified as an activator for multiple gene transcriptional factors in the nucleus to control cell proliferation and apoptosis. To investigate the potential role of YAP in the progression of FSGS, we examined kidney samples from patients with minimal change disease or FSGS and found that increases in podocyte apoptosis is positively correlated with the cytoplasmic distribution of YAP in human FSGS. Utilizing an established mT/mG transgenic mouse model and primary cultured podocytes, we found that YAP was distributed uniformly in nucleus and cytoplasm in the podocytes of control animals. Adriamycin treatment induced gradual nuclear exclusion of YAP with enhanced phospho-YAP/YAP ratio, accompanied by the induction of podocyte apoptosis both in vivo and in vitro. Moreover, we used verteporfin to treat an Adriamycin-induced FSGS mouse model, and found YAP inhibition by verteporfin induced nuclear exclusion of YAP, thus increasing podocyte apoptosis and accelerating disease progression. Therefore, our findings suggest that YAP nuclear distribution and activation in podocytes is an important endogenous anti-apoptotic mechanism during the progression of FSGS.An increase in podocyte apoptosis is positively correlated with the cytoplasmic distribution of YAP in focal segmental glomerulosclerosis (FSGS). YAP inhibition by verteporfin induces nuclear exclusion of YAP, thus increasing the podocyte apoptosis and accelerating disease progression. Therefore, this study suggests that YAP activation and nuclear distribution in podocytes is an endogenous anti-apoptotic mechanism during the progression of FSGS.

MAD2B-mediated cell cycle reentry of podocytes is involved in the pathogenesis of FSGS.

Rationale: Focal segmental glomerulosclerosis (FSGS) is characterized by the dysfunction of “post-mitotic” podocytes. The reentry of podocytes in the cell cycle will ultimately result in cell death. Mitotic arrest deficient 2-like protein 2 (MAD2B), an inhibitor of anaphase-promoting complex (APC)/cyclosome, precisely controls the metaphase to anaphase transition and ordered cell cycle progression. However, the role of MAD2B in FSGS podocyte injury remains unknown.Methods: To explore MAD2B function in podocyte cell cycle reentry, we used conditional mutant mice lacking MAD2B selectively in podocytes in ADR-induced FSGS murine model. Additionally, KU-55933, a specific inhibitor of ataxia-telangiectasia mutated (ATM) was utilized in vivo and in vitro to explore the role of ATM in regulating MAD2B.Results: The expression of MAD2B in podocytes was dramatically increased in patients with FSGS and ADR-treated mice along with podocyte cell cycle reentry. Podocyte-specific knockout of MAD2B effectively attenuated proteinuria, podocyte injury, and prevented the aberrant cell cycle reentry. By bioinformatics analysis we revealed that ATM kinase is a key upstream regulator of MAD2B. Furthermore, inhibition of ATM kinase abolished MAD2B-driven cell cycle reentry and alleviated podocyte impairment in FSGS murine model. In vitro studies by site-directed mutagenesis and immunoprecipitation we revealed ATM phosphorylated MAD2B and consequently hampered the ubiquitination of MAD2B in a phosphorylation-dependent manner.Conclusions: ATM kinase-MAD2B axis importantly contributes to the cell cycle reentry of podocytes, which is a novel pathogenic mechanism of FSGS, and may shed light on the development of its therapeutic approaches.

Circulating and cellular levels of miR-193 and miR-217 in patients with common glomerular diseases

Introduction: The definitive diagnosis of the common types of glomerular disease including FSGS (focal segmental glomerulosclerosis) and MN (membranous nephropathy) is still performed by biopsy studies, which has high risks and complications. MicroRNAs (miRNAs) can open a new horizon for the treatment and diagnosis of glomerular diseases. Objectives: In the present study, we focused on miR-217, miR-193-3p, and miR-124 expression in patients with FSGS and MN. Patients and Methods: Sixty cases (30 FSGS and 30 MN) were included based on strict criteria. A group of healthy controls were also included. The relative expression of the microRNAs was evaluated in the plasma and peripheral blood mononuclear cells (PBMCs) by quantitative real-time PCR. The association between the expression levels of microRNAs and clinicopathological parameters were also assessed. Results: There were significant differences in miR-193-3p levels between FSGS and MN group in plasma samples (P = 0.036). Furthermore, significantly decreased levels of miR-217 were observed in plasma samples of patients with NS (P = 0.026) and MN (P = 0.036) groups. Conclusion: The studied miRNAs are dysregulated in clinical samples of patients with nephrotic syndrome and they may be involved in the pathogenesis of FSGS and MN. More research is needed for understanding the relationship between these microRNAs and the pathogenesis of FSGS and MGN.