Dach1 is essential for maintaining normal mature podocytes.

Dach1 is highly expressed in normal podocytes, but this expression rapidly disappears after podocyte injury. To investigate the role of Dach1 in podocytes in vivo, we analyzed global, podocyte-specific, and inducible Dach1 knockout mice. Global Dach1 knockout (Dach1-/-) mice were assessed immediately after birth because they die within a day. The kidneys of Dach1-/- mice were slightly smaller than those of control mice but maintained a normal structure and normal podocyte phenotypes, including ultrastructure. To study the role of Dach1 in mature podocytes, we generated Dach1 knockout mice by mating Dach1fl/fl mice with Nphs1-Cre or ROSA-CreERT2 mice. Due to inefficient Cre recombination, only a small number of podocytes lacked Dach1 staining in these mice. However, all eleven Nphs1-Cre/Dach1fl/fl mice displayed abnormal albuminuria, and seven (63%) of them developed focal segmental glomerulosclerosis. Among 13 ROSA-CreERT2/Dach1fl/fl mice, eight (61%) exhibited abnormal albuminuria after treatment with tamoxifen, and five (38%) developed early sclerotic lesions. These results indicate that while Dach1 does not determine the fate of differentiation into podocytes, it is indispensable for maintaining the normal integrity of mature podocytes.

Dexamethasone promotes renal fibrosis by upregulating ILT4 expression in myeloid-derived suppressor cells.

Studies have shown that adoptive transfer of myeloid-derived suppressor cells (MDSCs) can alleviate various inflammatory diseases, including glomerulonephritis, but the long-term effects of the transferred MDSCs are still unclear. In addition, although glucocorticoids exert immunosuppressive effects on inflammatory diseases by inducing the expansion of MDSCs, the impact of glucocorticoids on the immunosuppressive function of MDSCs and their molecular mechanisms are unclear. In this study, we found that adoptive transfer of MDSCs to doxorubicin-induced focal segmental glomerulosclerosis (FSGS) mice for eight consecutive weeks led to an increase in serum creatinine and proteinuria and aggravation of renal interstitial fibrosis. Similarly, 8 weeks of high-dose dexamethasone administration exacerbated renal interstitial injury and interstitial fibrosis in doxorubicin-induced mice, manifested as an increase in serum creatinine and proteinuria, collagen deposition and α-SMA expression. On this basis, we found that dexamethasone could enhance MDSC expression and secretion of the fibrosis-related cytokines TGF-ß and IL-10. Mechanistically, we revealed that dexamethasone promotes the expression of immunoglobulin-like transcription factor 4 (ILT4), which enhances the T-cell inhibitory function of MDSCs and promotes the activation of STAT6, thereby strengthening the expression and secretion of TGF-ß and IL-10. Knocking down ILT4 alleviated renal fibrosis caused by adoptive transfer of MDSCs. Therefore, our findings demonstrate that the role and mechanism of dexamethasone mediate the expression and secretion of TGF-ß and IL-10 in MDSCs by promoting the expression of ILT4, thereby leading to renal fibrosis.

Analysis of glomerular deposition of IgM and C3 in patients with podocytopathies.

Minimal Change Disease (MCD) and Focal Segmental Glomerulosclerosis (FSGS) are the main causes of nephrotic syndrome in the world. The complement system appears to play an important role in the pathogenesis of these diseases. To evaluate the deposition of immunoglobulins and particles of the complement system in renal biopsies of patients with FSGS and MCD and relate to laboratory data, we selected 59 renal biopsies from patients with podocytopathies, 31 from patients with FSGS and 28 with MCD. Epidemiological, clinical, laboratory information and the prognosis of these patients were evaluated. Analysis of the deposition of IgM, IgG, C3, C1q and C4d in renal biopsies was performed. We related IgM and C3 deposition with laboratory parameters. Statistical analysis was performed using GraphPad Prism version 7.0. Glomerular deposition of IgM was significantly higher in the FSGS group, as was codeposition of IgM and C3. The clinical course of patients and laboratory data were also worse in cases of FSGS, with a higher percentage progressing to chronic kidney disease and death. Patients with C3 deposition had significantly higher mean serum creatinine and significantly lower eGFR, regardless of disease. Patients with FSGS had more IgM and C3 deposition in renal biopsies, worse laboratory data and prognosis than patients with MCD. C3 deposition, both in FSGS and MCD, appears to be related to worsening renal function.

Evaluation of C4d expression and staining patterns by immunohistochemistry in renal biopsy samples with focal segmental glomerulosclerosis and minimal change disease.

INTRODUCTION: C4d is an activation product of lectin pathway of complement. Glomerular deposition of C4d is associated with poor prognosis in different types of immune-related glomerulonephritis. The present study was conducted to investigate expression level of C4d and its staining pattern in renal biopsy of patients with focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) by immunohistochemistry method. MATERIALS AND METHODS: In this retrospective cross-sectional study, renal biopsy specimens from 46 samples of MCD, 47 samples of FSGS, and 15 samples without glomerular disease as the controls, were subjected to immunohistochemistry staining with C4d. Demographic characteristics and information obtained from light and electron microscopy (EM) of patients were also extracted from their files. RESULTS: C4d positive staining was observed in 97.9 % of FSGS and 43.5 % of MCD samples, which showed a statistically significant difference (P < 0.001). The sensitivity and specificity of C4d expression for diagnosing FSGS were 97.9 % and 56.5 %, respectively. There was no significant correlation between C4d expression and any of the light and electron microscopy findings, including presence of foam cells, mesangial matrix expansion, interstitial fibrosis and tubular atrophy, and basement membrane changes in MCD patients. Also, no significant correlation was observed between C4d expression and clinical symptoms of proteinuria or prolonged high level of creatinine in patients with MCD. DISCUSSION AND CONCLUSION: The expression of C4d marker had a good sensitivity and negative predictive value in the diagnosis of FSGS.

Drug-induced glomerular diseases.

Drug-induced kidney diseases represent a wide range of diseases that are responsible for a significant proportion of all acute kidney injuries and chronic kidney diseases. In the present review, we focused on drug-induced glomerular diseases, more precisely podocytopathies - minimal change diseases (MCD), focal segmental glomerulosclerosis (FSGS) - and membranous nephropathies (MN), from a physiological and a pharmacological point of view. The glomerular filtration barrier is composed of podocytes that form foot processes tightly connected and directly in contact with the basal membrane and surrounding capillaries. The common clinical feature of these diseases is represented by the loss of the ability of the filtration barrier to retain large proteins, leading to massive proteinuria and nephrotic syndrome. Drugs such as non-steroidal anti-inflammatory drugs (NSAIDs), D-penicillamine, tiopronin, trace elements, bisphosphonate, and interferons have been historically associated with the occurrence of MCD, FSGS, and MN. In the last ten years, the development of new anti-cancer agents, including tyrosine kinase inhibitors and immune checkpoint inhibitors, and research into their renal adverse effects highlighted these issues and have improved our comprehension of these diseases.

Podocyte injury at young age causes premature senescence and worsens glomerular aging.

As life expectancy continues to rise, age-related diseases are becoming more prevalent. For example, proteinuric glomerular diseases typified by podocyte injury have worse outcomes in the elderly compared with young patients. However, the reasons are not well understood. We hypothesized that injury to nonaged podocytes induces senescence, which in turn augments their aging processes. In primary cultured human podocytes, injury induced by a cytopathic antipodocyte antibody, adriamycin, or puromycin aminonucleoside increased the senescence-related genes CDKN2A (p16INK4a/p14ARF), CDKN2D (p19INK4d), and CDKN1A (p21). Podocyte injury in human kidney organoids was accompanied by increased expression of CDKN2A, CDKN2D, and CDKN1A. In young mice, experimental focal segmental glomerulosclerosis (FSGS) induced by adriamycin and antipodocyte antibody increased the glomerular expression of p16, p21, and senescence-associated ß-galactosidase (SA-ß-gal). To assess the long-term effects of early podocyte injury-induced senescence, we temporally followed young mice with experimental FSGS through adulthood (12 m of age) and middle age (18 m of age). p16 and Sudan black staining were higher at middle age in mice with earlier FSGS compared with age-matched mice that did not get FSGS when young. This was accompanied by lower podocyte density, reduced canonical podocyte protein expression, and increased glomerular scarring. These results are consistent with injury-induced senescence in young podocytes, leading to increased senescence of podocytes by middle age accompanied by lower podocyte lifespan and health span.NEW & NOTEWORTHY Glomerular function is decreased by aging. However, little is known about the molecular mechanisms involved in age-related glomerular changes and which factors could contribute to a worse glomerular aging process. Here, we reported that podocyte injury in young mice and culture podocytes induced senescence, a marker of aging, and accelerates glomerular aging when compared with healthy aging mice.

Scaffold protein SH3BP2 signalosome is pivotal for immune activation in nephrotic syndrome.

Despite clinical use of immunosuppressive agents, the immunopathogenesis of minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) remains unclear. Src homology 3-binding protein 2 (SH3BP2), a scaffold protein, forms an immune signaling complex (signalosome) with 17 other proteins, including phospholipase Cγ2 (PLCγ2) and Rho-guanine nucleotide exchange factor VAV2 (VAV2). Bioinformatic analysis of human glomerular transcriptome (Nephrotic Syndrome Study Network cohort) revealed upregulated SH3BP2 in MCD and FSGS. The SH3BP2 signalosome score and downstream MyD88, TRIF, and NFATc1 were significantly upregulated in MCD and FSGS. Immune pathway activation scores for Toll-like receptors, cytokine-cytokine receptor, and NOD-like receptors were increased in FSGS. Lower SH3BP2 signalosome score was associated with MCD, higher estimated glomerular filtration rate, and remission. Further work using Sh3bp2KI/KI transgenic mice with a gain-in-function mutation showed ~6-fold and ~25-fold increases in albuminuria at 4 and 12 weeks, respectively. Decreased serum albumin and unchanged serum creatinine were observed at 12 weeks. Sh3bp2KI/KI kidney morphology appeared normal except for increased mesangial cellularity and patchy foot process fusion without electron-dense deposits. SH3BP2 co-immunoprecipitated with PLCγ2 and VAV2 in human podocytes, underscoring the importance of SH3BP2 in immune activation. SH3BP2 and its binding partners may determine the immune activation pathways resulting in podocyte injury leading to loss of the glomerular filtration barrier.

Progression of albuminuria and podocyte injury in focal segmental glomerulosclerosis inhibited by enhanced glycosphingolipid GM3 via valproic acid.

Focal segmental glomerulosclerosis, characterized by decreased numbers of podocytes in glomeruli, is a common cause of refractory nephrotic syndrome. Recently, we showed that enhanced glycosphingolipid GM3 expression after administration of valproic acid, an upregulator of ST3GAL5/St3gal5, was effective in preventing albuminuria and podocyte injury. We also revealed the molecular mechanism for this preventive effect, which involves GM3 directly binding nephrin that then act together in glycolipid-enriched membrane (GEM) fractions under normal conditions and in non-GEM fractions under nephrin injury conditions. Kidney disease is frequently referred to as a "silent killer" because it is often difficult to detect subjective symptoms. Thus, primary treatment for these diseases is initiated after the onset of disease progression. Consequently, the efficacy of enhanced levels of GM3 induced by valproic acid needs to be evaluated after the onset of the disease with severe albuminuria such as focal segmental glomerulosclerosis. Here, we report the therapeutic effect of enhanced GM3 expression induced via administration of valproic acid on albuminuria and podocyte injury after the onset focal segmental glomerulosclerosis in anti-nephrin antibody treated mice. Our findings suggest elevated levels of GM3 following treatment with valproic acid has therapeutic utility for kidney disease associated with severe albuminuria and podocyte injury.

Btg2 Promotes Focal Segmental Glomerulosclerosis via Smad3-Dependent Podocyte-Mesenchymal Transition.

Podocyte injury plays a critical role in the progression of focal segmental glomerulosclerosis (FSGS). Here, it is reported that B-cell translocation gene 2 (Btg2) promotes Adriamycin (ADR)-induced FSGS via Smad3-dependent podocyte-mesenchymal transition. It is found that in FSGS patients and animal models, Btg2 is markedly upregulated by podocytes and correlated with progressive renal injury. Podocyte-specific deletion of Btg2 protected against the onset of proteinuria and glomerulosclerosis in ADR-treated mice along with inhibition of EMT markers such as α-SMA and vimentin while restoring epithelial marker E-cadherin. In cultured MPC5 podocytes, overexpression of Btg2 largely promoted ADR and TGF-ß1-induced EMT and fibrosis, which is further enhanced by overexpressing Btg2 but blocked by disrupting Btg2. Mechanistically, Btg2 is rapidly induced by TGF-ß1 and then bound Smad3 but not Smad2 to promote Smad3 signaling and podocyte EMT, which is again exacerbated by overexpressing Btg2 but blocked by deleting Btg2 in MPC5 podocytes. Interestingly, blockade of Smad3 signaling with a Smad3 inhibitor SIS3 is also capable of inhibiting Btg2 expression and Btg2-mediated podocyte EMT, revealing a TGF-ß/Smad3-Btg2 circuit mechanism in Btg2-mediated podocyte injury in FSGS. In conclusion, Btg2 is pathogenic in FSGS and promotes podocyte injury via a Smad3-dependent EMT pathway.

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. In vivo, 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. In vitro, 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.

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

Introduction: 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. Aims: To assess the expression of CD44 in MCNS and FSGS and to evaluate its association with the known clinical and histopathological prognostic factors. Materials and Methods: 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. Results: 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. Conclusions: 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.

Enhanced Levels of Glycosphingolipid GM3 Delay the Progression of Diabetic Nephropathy.

We recently found that albuminuria levels in patients with minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) inversely correlate with glycosphingolipid GM3 expression levels in glomerular podocytes. Moreover, we showed enhanced expression of GM3 via activation of the GM3 synthase gene upon administration of valproic acid (VPA) is effective in suppressing albuminuria and podocyte injury in mice with anti-nephrin antibody-induced podocytopathy. However, the therapeutic effect of GM3 on diabetic nephropathy, which is the most common underlying disease in patients undergoing dialysis and with podocyte injury, remains unclear. Here, we investigated the therapeutic effect of enhanced GM3 expression via VPA on podocyte injury using streptozotocin-induced diabetic nephropathy model mice. Administration of VPA clearly decreased levels of albuminuria and glomerular lesions and inhibited the loss of podocytes and expansion in the mesangial area. Furthermore, we found that albuminuria levels in patients with diabetic nephropathy inversely correlate with the expression of GM3 in podocytes. These results indicate that maintaining GM3 expression in podocytes by administration of VPA may be effective in treating not only podocyte injury, such as MCD and FSGS, but also the late stage of diabetic nephropathy.

Uncovering pharmacological mechanisms of Phellinus linteus on focal segmental glomeruloscleosis rats through tandem mass tag-based quantitative proteomic analysis, network pharmacology analysis and experimental validation.

OBJECTIVE: To explore the underlying molecular mechanism of (). METHODS: We used a tandem mass tag-based quantitative proteomic method to determine the differentially expressed proteins. Network pharmacology analysis was used to analysis the main components of and construct the compound-target network. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were used to validate the analyses results. RESULTS: The expression levels of thrombospondin-1 (TSP-1) and transforming growth factor (TGF)-ß1/Smad3 signaling pathway proteins were significantly upregulated in focal segmental glomeruloscleosis (FSGS) rats. The reduced the expression levels of TSP-1 and TGF-ß1 signaling pathway proteins. Network pharmacology analysis revealed that protocatechualdehyde was the main active component. Subsequent and experiments validated the results of proteomic and network pharmacology analyses. CONCLUSIONS: Our results suggested that may inhibit renal sclerosis by inhibiting TSP-1-activated TGF-ß1 signaling and may have potential applications in the treatment of FSGS.

Inhibition of Importin- α -Mediated Nuclear Localization of Dendrin Attenuates Podocyte Loss and Glomerulosclerosis.

SIGNIFICANCE STATEMENT: Nuclear translocation of dendrin is observed in injured podocytes, but the mechanism and its consequence are unknown. In nephropathy mouse models, dendrin ablation attenuates proteinuria, podocyte loss, and glomerulosclerosis. The nuclear translocation of dendrin promotes c-Jun N -terminal kinase phosphorylation in podocytes, altering focal adhesion and enhancing cell detachment-induced apoptosis. We identified mediation of dendrin nuclear translocation by nuclear localization signal 1 (NLS1) sequence and adaptor protein importin- α . Inhibition of importin- α prevents nuclear translocation of dendrin, decreases podocyte loss, and attenuates glomerulosclerosis in nephropathy models. Thus, inhibiting importin- α -mediated nuclear translocation of dendrin is a potential strategy to halt podocyte loss and glomerulosclerosis. BACKGROUND: Nuclear translocation of dendrin is observed in the glomeruli in numerous human renal diseases, but the mechanism remains unknown. This study investigated that mechanism and its consequence in podocytes. METHODS: The effect of dendrin deficiency was studied in adriamycin (ADR) nephropathy model and membrane-associated guanylate kinase inverted 2 ( MAGI2 ) podocyte-specific knockout ( MAGI2 podKO) mice. The mechanism and the effect of nuclear translocation of dendrin were studied in podocytes overexpressing full-length dendrin and nuclear localization signal 1-deleted dendrin. Ivermectin was used to inhibit importin- α . RESULTS: Dendrin ablation reduced albuminuria, podocyte loss, and glomerulosclerosis in ADR-induced nephropathy and MAGI2 podKO mice. Dendrin deficiency also prolonged the lifespan of MAGI2 podKO mice. Nuclear dendrin promoted c-Jun N -terminal kinase phosphorylation that subsequently altered focal adhesion, reducing cell attachment and enhancing apoptosis in cultured podocytes. Classical bipartite nuclear localization signal sequence and importin- α mediate nuclear translocation of dendrin. The inhibition of importin- α / ß reduced dendrin nuclear translocation and apoptosis in vitro as well as albuminuria, podocyte loss, and glomerulosclerosis in ADR-induced nephropathy and MAGI2 podKO mice. Importin- α 3 colocalized with nuclear dendrin in the glomeruli of FSGS and IgA nephropathy patients. CONCLUSIONS: Nuclear translocation of dendrin promotes cell detachment-induced apoptosis in podocytes. Therefore, inhibiting importin- α -mediated dendrin nuclear translocation is a potential strategy to prevent podocyte loss and glomerulosclerosis.

CD44 Expression in Renal Tissue Is Associated with an Increase in Urinary Levels of Complement Components in Chronic Glomerulopathies.

It is suggested that activated CD44+ cells play a profibrogenic role in the pathogenesis of active glomerulopathies. Complement activation is also involved in renal fibrogenesis. The aim of the study was to evaluate the role of the activation of CD44+ cells in the kidney tissue and complement components' filtration to the urine as factors of renal tissue fibrosis in patients with glomerulopathies. In total, 60 patients with active glomerulopathies were included in our study: 29 patients with focal segmental glomerulosclerosis (FSGS), 10 patients with minimal change disease (MCD), 10 patients with membranous nephropathy (MN), and 11 patients with IgA nephropathy. The immunohistochemical peroxidase method was used to study the expression of CD44+ in kidney biopsies. Components of complement were analyzed in urine by the multiple reaction monitoring (MRM) approach using liquid chromatography. Strong CD44 expression was noted predominantly in PEC and mesangial cells (MC) in patients with FSGS, and to a lesser extent, in patients with MN and IgA nephropathy, and it was absent in patients with MCD. Expression of profibrogenic CD44+ in glomeruli correlated with the levels of proteinuria and complement C2, C3, and C9 components, and CFB and CFI in urine. The CD44+ expression scores in the renal interstitium correlated with the level of C3 and C9 components of complement in the urine and the area of tubulo-interstitial fibrosis. The strongest expression of CD44+ was found in the glomeruli (MC, PEC, and podocytes) of patients with FSGS compared with other glomerulopathies. The CD44 expression score in the glomeruli and interstitium is associated with high levels of complement components in the urine and renal fibrosis.

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.

The Angiogenesis Inhibitor Isthmin-1 (ISM1) Is Overexpressed in Experimental Models of Glomerulopathy and Impairs the Viability of Podocytes.

Focal segmental glomerulosclerosis (FSGS) is a major cause of end-stage renal disease and remains without specific treatment. To identify new events during FSGS progression, we used an experimental model of FSGS associated with nephroangiosclerosis in rats injected with L-NAME (Nω-nitro-L-arginine methyl ester). After transcriptomic analysis we focused our study on the role of Isthmin-1 (ISM1, an anti-angiogenic protein involved in endothelial cell apoptosis. We studied the renal expression of ISM1 in L-NAME rats and other models of proteinuria, particularly at the glomerular level. In the L-NAME model, withdrawal of the stimulus partially restored basal ISM1 levels, along with an improvement in renal function. In other four animal models of proteinuria, ISM1 was overexpressed and localized in podocytes while the renal function was degraded. Together these facts suggest that the glomerular expression of ISM1 correlates directly with the progression-recovery of the disease. Further in vitro experiments demonstrated that ISM1 co-localized with its receptors GRP78 and integrin αvß5 on podocytes. Treatment of human podocytes with low doses of recombinant ISM1 decreased cell viability and induced caspase activation. Stronger ISM1 stimuli in podocytes dropped mitochondrial membrane potential and induced nuclear translocation of apoptosis-inducing factor (AIF). Our results suggest that ISM1 participates in the progression of glomerular diseases and promotes podocyte apoptosis in two different complementary ways: one caspase-dependent and one caspase-independent associated with mitochondrial destabilization.

Emerging Perspectives on the Rare Tubulopathy Dent Disease: Is Glomerular Damage a Direct Consequence of ClC-5 Dysfunction?

Dent disease (DD1) is a rare tubulopathy caused by mutations in the CLCN5 gene. Glomerulosclerosis was recently reported in DD1 patients and ClC-5 protein was shown to be expressed in human podocytes. Nephrin and actin cytoskeleton play a key role for podocyte functions and podocyte endocytosis seems to be crucial for slit diaphragm regulation. The aim of this study was to analyze whether ClC-5 loss in podocytes might be a direct consequence of the glomerular damage in DD1 patients. Three DD1 kidney biopsies presenting focal global glomerulosclerosis and four control biopsies were analyzed by immunofluorescence (IF) for nephrin and podocalyxin, and by immunohistochemistry (IHC) for ClC-5. ClC-5 resulted as down-regulated in DD1 vs. control (CTRL) biopsies in both tubular and glomerular compartments (p < 0.01). A significant down-regulation of nephrin (p < 0.01) in DD1 vs. CTRL was demonstrated. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Caspase9) gene editing of CLCN5 in conditionally immortalized human podocytes was used to obtain clones with the stop codon mutation p.(R34Efs*14). We showed that ClC-5 and nephrin expression, analyzed by quantitative Reverse Transcription/Polymerase Chain Reaction (qRT/PCR) and In-Cell Western (ICW), was significantly downregulated in mutant clones compared to the wild type ones. In addition, F-actin staining with fluorescent phalloidin revealed actin derangements. Our results indicate that ClC-5 loss might alter podocyte function either through cytoskeleton disorganization or through impairment of nephrin recycling.

Autophagy and podocytopathy.

Autophagy is a complex process of lysosomal-dependent degradation of unwanted cellular material. In response to endogenous or exogenous stimuli, autophagy is induced and regulated by two kinases: the AMP activated kinase and the mammalian target of rapamycin (mTOR). Cells activated by Unc-51-like kinase 1 form a double membrane complex that sequesters the cargo (phagophore) and elongates producing spherical vesicles (autophagosomes). These reach and fuse with lysosomes, which degrade the cargo (autolysosomes). The resulting macromolecules are released back and recycled in the cytosol for reuse. In the podocyte, autophagy is a homeostatic mechanism that contributes to the formation and preservation of the morphological and functional integrity of actin cytoskeleton. Podocytes, fenestrated endothelial cells and glomerular basement membrane compose the glomerular filtration barrier. Podocyte damage may cause dysfunction of the glomerular barrier, proteinuria and glomerulosclerosis in different glomerular diseases and particularly in so-called podocytopathies, namely minimal change disease and focal segmental glomerulosclerosis. Several drugs and molecules may activate autophagic function in murine models. Among them, aldosterone inhibitors, mineralocorticoid inhibitors and vitamin D3 were proven to protect podocyte from injury and reduce proteinuria in clinical studies. However, no clinical trial with autophagy regulators in podocytopathies has been conducted. Caution is needed with other autophagy activators, such as mTOR inhibitors and metformin, because of potential adverse events.

TYRO3 agonist as therapy for glomerular disease.

Podocyte injury and loss are key drivers of primary and secondary glomerular diseases, such as focal segmental glomerulosclerosis (FSGS) and diabetic kidney disease (DKD). We previously demonstrated the renoprotective role of protein S (PS) and its cognate tyrosine-protein kinase receptor, TYRO3, in models of FSGS and DKD and that their signaling exerts antiapoptotic and antiinflammatory effects to confer protection against podocyte loss. Among the 3 TAM receptors (TYRO3, AXL, and MER), only TYRO3 expression is largely restricted to podocytes, and glomerular TYRO3 mRNA expression negatively correlates with human glomerular disease progression. Therefore, we posited that the agonistic PS/TYRO3 signaling could serve as a potential therapeutic approach to attenuate glomerular disease progression. As PS function is not limited to TYRO3-mediated signal transduction but includes its anticoagulant activity, we focused on the development of TYRO3 agonists as an optimal therapeutic approach to glomerular disease. Among the small-molecule TYRO3 agonistic compounds screened, compound 10 (C-10) showed a selective activation of TYRO3 without any effects on AXL or MER. We also confirmed that C-10 directly binds to TYRO3, but not the other receptors. In vivo, C-10 attenuated proteinuria, glomerular injury, and podocyte loss in mouse models of Adriamycin-induced nephropathy and a db/db model of type 2 diabetes. Moreover, these renoprotective effects of C-10 were lost in Tyro3-knockout mice, indicating that C-10 is a selective agonist of TYRO3 activity that mitigates podocyte injury and glomerular disease. Therefore, C-10, a TYRO3 agonist, could be potentially developed as a new therapy for glomerular disease.