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.

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.

In vivo characterization of a podocyte-expressed short podocin isoform.

The most common genetic causes of steroid-resistant nephrotic syndrome (SRNS) are mutations in the NPHS2 gene, which encodes the cholesterol-binding, lipid-raft associated protein podocin. Mass spectrometry and cDNA sequencing revealed the existence of a second shorter isoform in the human kidney in addition to the well-studied canonical full-length protein. Distinct subcellular localization of the shorter isoform that lacks part of the conserved PHB domain suggested a physiological role. Here, we analyzed whether this protein can substitute for the canonical full-length protein. The short isoform of podocin is not found in other organisms except humans. We therefore analysed a mouse line expressing the equivalent podocin isoform (podocinΔexon5) by CRISPR/Cas-mediated genome editing. We characterized the phenotype of these mice expressing podocinΔexon5 and used targeted mass spectrometry and qPCR to compare protein and mRNA levels of podocinwildtype and podocinΔexon5. After immunolabeling slit diaphragm components, STED microscopy was applied to visualize alterations of the podocytes' foot process morphology.Mice homozygous for podocinΔexon5 were born heavily albuminuric and did not survive past the first 24 h after birth. Targeted mass spectrometry revealed massively decreased protein levels of podocinΔexon5, whereas mRNA abundance was not different from the canonical form of podocin. STED microscopy revealed the complete absence of podocin at the podocytes' slit diaphragm and severe morphological alterations of podocyte foot processes. Mice heterozygous for podocinΔexon5 were phenotypically and morphologically unaffected despite decreased podocin and nephrin protein levels.The murine equivalent to the human short isoform of podocin cannot stabilize the lipid-protein complex at the podocyte slit diaphragm. Reduction of podocin levels at the site of the slit diaphragm complex has a detrimental effect on podocyte function and morphology. It is associated with decreased protein abundance of nephrin, the central component of the filtration-slit forming slit diaphragm protein complex.

A case report and literature study on Alport syndrome featuring nephrotic syndrome as its primary manifestation.

BACKGROUND: Historically, due to the lack of distinct clinical symptoms, Alport syndrome, a hereditary kidney disease prevalent in children and a leading cause of kidney failure, has often been misdiagnosed as other kidney conditions. CASE DESCRIPTION: This article presents a comprehensive review and analysis of clinical data concerning a child diagnosed with Alport syndrome, where nephrotic syndrome served as the primary manifestation. The male child in this case exhibited symptoms starting at the age of 6, initially diagnosed as nephrotic syndrome. Consequently, oral steroid medication was administered, proving ineffective. Due to persistent proteinuria and microscopic hematuria, a renal biopsy was performed. Immunofluorescence staining revealed no abnormal expression of the α3, α4, and α5 chains of type IV collagen. Notably, electron microscopy revealed the basement membrane to be partially torn and arachnoid. Genetic testing indicated a hemizygous COL4A5 acceptor-splice-site mutation c.4707-1(IVS50)G > A, inherited from his mother. CONCLUSION: This specific mutated locus, being the first of its kind reported, adds valuable information to the existing gene mutation spectrum of Alport syndrome. Consequently, it emphasizes the importance for clinicians to deepen their understanding of rare kidney diseases, contributing to enhanced diagnostic accuracy and improved patient care.

Engineering antioxidant ceria-zirconia nanomedicines for alleviating podocyte injury in rats with adriamycin-induced nephrotic syndrome.

BACKGROUND: Primary nephrotic syndrome (PNS) is characterized by edema, heavy proteinuria, hypoalbuminemia and hyperlipidemia. Moreover, podocyte injury is the key pathological change of PNS. Even though the pathophysiological etiology of PNS has not been fully understood, the production of excessive reactive oxygen species (ROS) plays an important role in the development and progression of the disease. Glucocorticoids are the first-line medications for patients with PNS, but their clinical use is hampered by dose-dependent side effects. Herein, we accelerated the rate of conversion from Ce4+ to Ce3+ by doping Zr4+ in ceria-zirconia nanomedicines to treat the PNS rat model by removal of ROS. RESULTS: The engineered Ce0.7Zr0.3O2 (7CZ) nanomedicines significantly improved the ROS scavenging ability of podocytes at a very low dose, enabling effective inhibition of podocyte apoptosis and actin cytoskeleton depolymerization induced by adriamycin (ADR). Accordingly, podocyte injury was effectively alleviated in rat models of ADR-induced nephrotic syndrome, as confirmed by serum tests and renal tissue staining. Moreover, the mRNA sequencing assay revealed the protective molecular signaling pathways of 7CZ nanomedicines in podocytes. CONCLUSION: 7CZ nanomedicines were highly effective in protecting against ADR-induced podocyte injury in vitro and in vivo at a very low concentration.

Characteristics of sodium and water retention in rats with nephrotic syndrome induced by puromycin aminonucleoside.

INTRODUCTION: Nephrotic syndrome (NS) is characterized by renal sodium and water retention. The mechanisms are not fully elucidated. METHODS: The NS rat model was established by single intraperitoneal injection of 100 mg/kg puromycin aminonucleoside (PAN). The plasma electrolyte level and urinary sodium excretion were monitored dynamically. The changes of some sodium transporters, including epithelial Na+ channel (ENaC), Na+/H+ exchanger 3 (NHE3), Na+-K+-2Cl- cotransporter 2 (NKCC2) and Na+-Cl- cotransporter (NCC) in renal cortex at different time points and the level of peripheral circulation factors were detected. RESULTS: The urinary sodium excretion of the model group increased significantly on the first day, then decreased compared with the control group, and there was no significant difference between the model group and the control group on the 12th day. The changes of peripheral circulation factors were not obvious. Some sodium transporters in renal cortex increased in varying degrees, while NKCC2 decreased significantly compared with the control group. CONCLUSIONS: The occurrence of NS edema may not be related to the angiotensin system. The decrease of urinary sodium excretion is independent of the development of albuminuria. During the 18 days of observation, it can be divided into three stages: sodium retention, sodium compensation, and simple water retention. The mechanism is related to the increased expression of α-ENaC, γ-ENaC, NHE3 and NCC in a certain period of time, the compensatory decrease of NKCC2 expression and the continuous increase of aquaporin 2 (AQP2) expression.

"Idiopathic" minimal change nephrotic syndrome: a podocyte mystery nears the end.

The discovery of zinc fingers and homeoboxes (ZHX) transcriptional factors and the upregulation of hyposialylated angiopoietin-like 4 (ANGPTL4) in podocytes have been crucial in explaining the cardinal manifestations of human minimal change nephrotic syndrome (MCNS). Recently, uncovered genomic defects upstream of ZHX2 induce a ZHX2 hypomorph state that makes podocytes inherently susceptible to mild cytokine storms resulting from a common cold. In ZHX2 hypomorph podocytes, ZHX proteins are redistributed away from normal transmembrane partners like aminopeptidase A (APA) toward alternative binding partners like IL-4Rα. During disease relapse, high plasma soluble IL-4Rα (sIL-4Rα) associated with chronic atopy complements the cytokine milieu of a common cold to displace ZHX1 from podocyte transmembrane IL-4Rα toward the podocyte nucleus. Nuclear ZHX1 induces severe upregulation of ANGPTL4, resulting in incomplete sialylation of part of the ANGPTL4 protein, secretion of hyposialylated ANGPTL4, and hyposialylation-related injury in the glomerulus. This pattern of injury induces many of the classic manifestations of human minimal change disease (MCD), including massive and selective proteinuria, podocyte foot process effacement, and loss of glomerular basement membrane charge. Administration of glucocorticoids reduces ANGPTL4 upregulation, which reduces hyposialylation injury to improve the clinical phenotype. Improving sialylation of podocyte-secreted ANGPTL4 also reduces proteinuria and improves experimental MCD. Neutralizing circulating TNF-α, IL-6, or sIL-4Rα after the induction of the cytokine storm in Zhx2 hypomorph mice reduces albuminuria, suggesting potential new therapeutic targets for clinical trials to prevent MCD relapse. These studies collectively lay to rest prior suggestions of a role of single cytokines or soluble proteins in triggering MCD relapse.

Minnelide Markedly Reduces Proteinuria in Mice with Adriamycin Nephropathy by Protecting Against Podocyte Injury.

Minimal change disease (MCD) is the most common cause of idiopathic nephrotic syndrome in children. The current major therapy is hormones for most steroid-sensitive patients. However, many patients have recurrent relapses of the disease and require long-term immunosuppression, leading to significant morbidity due to the side effects of the drugs. Therefore, better drugs need to be urgently explored to treat nephrotic syndrome while avoiding the side effects of drugs. Minnelide, a water-soluble prodrug of triptolide, has been proved to be effective in treating cancers in many clinical trials. This study aimed to investigate the therapeutic effect of minnelide in mice with adriamycin (ADR) nephropathy, its underlying protection mechanisms, and its reproductive toxicity. Minnelide was administered intraperitoneally to 6-8-week female mice with adriamycin nephropathy for 2 weeks, and the urine, blood, and kidney tissues were taken to analyze the therapeutic effect. In addition, we evaluated reproductive toxicity by measuring the levels of gonadal hormones and observing the histological changes in ovaries and testes. Primary mouse podocytes were exposed to puromycin (PAN) to damage the cytoskeleton and induce apoptosis, and then, triptolide was used to evaluate the therapeutic effect and underlying protection mechanisms in vitro. It was observed that minnelide dramatically alleviated proteinuria and apoptosis in mice with adriamycin nephropathy. In vitro, triptolide ameliorated puromycin-induced cytoskeletal rearrangement and apoptosis via reactive oxygen species-mediated mitochondrial pathway. In addition, minnelide caused no reproductive toxicity to male and female mice. The results suggested that minnelide might be a promising drug for nephrotic syndrome.

Loss of surface transport is a main cellular pathomechanism of CRB2 variants causing podocytopathies.

Crumbs2 (CRB2) is a central component of the renal filtration barrier and part of the slit diaphragm, a unique cell contact formed by glomerular podocytes. Some CRB2 variants cause recessive inherited forms of steroid-resistant nephrotic syndrome. However, the disease-causing potential of numerous CRB2 variants remains unknown. Here, we report the establishment of a live-cell imaging-based assay, allowing a quantitative evaluation of the pathogenic potential of so far non-categorized CRB2 variants. Based on in silico data analysis and protein prediction software, putative disease-associated CRB2 missense variants were selected, expressed as CRB2-GFP fusion proteins, and analyzed in reporter cell lines with BFP-labeled plasma membrane. We found that in comparison with PM-localized WT, disease-associated CRB2 variants remained predominantly at the ER. Accumulation at the ER was also present for several non-characterized CRB2 variants and variants in which putative disulfide bridge-forming cysteines were replaced. Strikingly, WT CRB2 retained inside the ER in cells lacking protein disulfide isomerase A3, indicating that posttranslational modification, especially the formation of disulfide bridges, is a crucial step for the CRB2 PM transport.

ANGPTL4 promotes nephrotic syndrome by downregulating podocyte expression of ACTN4 and podocin.

BACKGROUND: lipopolysaccharide (LPS) can induce nephrotic syndrome-like features such as massive proteinuria, hyperlipidemia, and fusion of glomerular podocytes with foot processes (FPs) in mice. Angiopoietin-like protein 4 (ANGPTL4) neutralized the negative charge of glomerular basement membrane charge and aggravated renal injury. The mechanism of ANGPTL4 aggravating podocyte injury has not been well clarified. In this study, we aimed to investigate the potential role of ANGPTL4 on podocyte FPs fusion and podocyte signal molecules. METHODS: We built angptl4 gene knocked out in C57BL6 mice using CRISPR/Cas9 technique. Nephrotic model was built by LPS in wild type and angptl4-/- mice. Expression of ACTN4, podocin and TRPC6 in the glomerulus were determined by immunohistochemistry. RESULTS: In physical condition, the wild type and angptl4-/- mice showed no significant differences in biochemical indicators and kidney pathology. But in nephrotic condition, compared with wild type mice hyperlipidemia and proteinuria with the angptl4-/- mice was significantly relieved. Moreover, the degree of FPs fusion was notably improved in the nephrotic mice knocked out angptl4 gene. Expression of ACTN4 and podocin decreased drastically in the glomerulus of wild-type nephrotic mice. Different from wild-type, the ACTN4 and podocin expression showed slight weakening in angptl4-/- nephrotic mice. As transient receptor potential cation channel subfamily member, TRPC6 expression had no visible change in glomerulus of each group. CONCLUSIONS: ANGPTL4 induces hyperlipidemia and podocyte injury in nephrotic mice, thereby promoting the formation of proteinuria. Its molecular mechanism may be related to ANGPTL4 down-regulating actin cytoskeletal regulatory signals ACTN4 and podocin.

Kidney Organoids Generated Using an Allelic Series of NPHS2 Point Variants Reveal Distinct Intracellular Podocin Mistrafficking.

BACKGROUND: NPHS2 variants are the most common cause of steroid-resistant nephrotic syndrome in children >1 month old. Missense NPHS2 variants were reported to cause mistrafficking of the encoded protein, PODOCIN, but this conclusion was on the basis of overexpression in some nonpodocyte cell lines. METHODS: We generated a series of human induced pluripotent stem cell (iPSC) lines bearing pathogenic missense variants of NPHS2 , encoding the protein changes p.G92C, p.P118L, p.R138Q, p.R168H, and p.R291W, and control lines. iPSC lines were also generated from a patient with steroid-resistant nephrotic syndrome (p.R168H homozygote) and a healthy heterozygous parent. All lines were differentiated into kidney organoids. Immunofluorescence assessed PODOCIN expression and subcellular localization. Podocytes were transcriptionally profiled and PODOCIN-NEPHRIN interaction interrogated. RESULTS: All variant lines revealed reduced levels of PODOCIN protein in the absence of reduced transcription. Although wild-type PODOCIN localized to the membrane, distinct variant proteins displayed unique patterns of subcellular protein trafficking, some unreported. P118L and R138Q were preferentially retained in the endoplasmic reticulum (ER); R168H and R291W accumulated in the Golgi. Podocyte profiling demonstrated minimal disease-associated transcriptional change. All variants displayed podocyte-specific apoptosis, which was not linked to ER stress. NEPHRIN-PODOCIN colocalization elucidated the variant-specific effect on NEPHRIN association and hence NEPHRIN trafficking. CONCLUSIONS: Specific variants of endogenous NPHS2 result in distinct subcellular PODOCIN localization within organoid podocytes. Understanding the effect of each variant on protein levels and localization and the effect on NEPHRIN provides additional insight into the pathobiology of NPHS2 variants. PODCAST: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_01_05_JASN2022060707.mp3.

Danshen injection induces autophagy in podocytes to alleviate nephrotic syndrome via the PI3K/AKT/mTOR pathway.

BACKGROUND: Danshen injection (DSI) is an agent extracted from the Salvia miltiorrhiza Bunge, a natural drug commonly used to alleviate kidney diseases. However, the material basis and therapeutic effects of DSI on nephrotic syndrome (NS) remain unclear. PURPOSE: To investigate the material basis of DSI and the therapeutic effects and underlying mechanisms of NS. METHODS: NS models were established using adriamycin-induced BALB/c mice and lipopolysaccharide-induced mouse podocytes (MPC-5). Following DSI and prednisone administration, kidney coefficients, 24 h urine protein, blood urea nitrogen, and serum creatinine levels were tested. Histomorphology was observed by periodic acid-Schiff staining and hematoxylin and eosin staining of the kidney sections. The glomerular basement membrane and autophagosomes of the kidneys were observed using transmission electron microscopy. Nephrin and desmin levels in the glomeruli were tested using immunohistochemistry. The viability of MPC-5 cells was tested using cell counting kit-8 after chloroquine and rapamycin administration in combination with DSI. The in vivo and in vitro protein levels of phosphatidylinositol 3-kinase (PI3K), AKT, phosphorylated AKT (Ser473), mammalian target of rapamycin (mTOR), microtubule-associated protein light chain 3 (LC3), beclin1, cleaved caspase-3, and caspase-3 were detected using western blotting. RESULTS: Our results showed that DSI contained nine main components: caffeic acid, danshensu, lithospermic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B, salvianolic acid C, salvianolic acid D, and 3, 4-Dihydroxybenzaldehyde. In in vivo studies, the NS mice showed renal function and pathological impairment. Podocytes were damaged, with decreased levels of autophagy and apoptosis, accompanied by inhibition of the PI3K/AKT/mTOR signaling. DSI administration resulted in improved renal function and pathology in NS mice, with the activation of autophagy and PI3K/AKT/mTOR signaling in the kidneys. Additionally, podocytes were less damaged and intracellular autophagosomes were markedly increased. In vitro studies have shown that DSI activated MPC-5 autophagy and reduced apoptosis via the PI3K/AKT/mTOR pathway. CONCLUSION: Collectively, this study demonstrated that DSI activated podocyte autophagy and reduced apoptosis via the PI3K/AKT/mTOR signaling, ultimately attenuating NS. Our study clarified the main components of DSI and elucidated its therapeutic effects and potential mechanisms for NS, providing new targets and agents for the clinical treatment of NS.

Assessment of Brain-Derived Neurotrophic Factor (BDNF) Concentration in Children with Idiopathic Nephrotic Syndrome.

Idiopathic nephrotic syndrome (INS) is a chronic disease affecting children in early childhood. It is characterized by proteinuria, hypoalbuminemia, edema and hyperlipidemia. To date, the diagnosis is usually established at an advanced stage of proteinuria. Therefore, new methods of early INS detection are desired. This study was designed to assess brain-derived neurotrophic factor (BDNF) as a potential marker in the early diagnosis of INS. The study group included patients with a diagnosis of idiopathic nephrotic syndrome (n = 30) hospitalized in Clinical Hospital No. 1 in Zabrze, from December 2019 to December 2021. Our study shows that serum BDNF concentration decreased and urine BDNF concentration increased in a group of patients with INS, compared with healthy controls. Such outcomes might be related to loss of the BDNF contribution in podocyte structure maintenance. Moreover, we anticipate the role of BDNF in urine protein concentration increase, which could be used as a direct predictor of urine protein fluctuations in clinical practice. Moreover, the ROC curve has also shown that serum BDNF and urine BDNF levels might be useful as an INS marker.

Nephrotic Syndrome Gene TBC1D8B Is Required for Endosomal Maturation and Nephrin Endocytosis in Drosophila.

BACKGROUND: Variants in TBC1D8B cause nephrotic syndrome. TBC1D8B is a GTPase-activating protein for Rab11 (RAB11-GAP) that interacts with nephrin, but how it controls nephrin trafficking or other podocyte functions remains unclear. METHODS: We generated a stable deletion in Tbc1d8b and used microhomology-mediated end-joining for genome editing. Ex vivo functional assays utilized slit diaphragms in podocyte-like Drosophila nephrocytes. Manipulation of endocytic regulators and transgenesis of murine Tbc1d8b provided a comprehensive functional analysis of Tbc1d8b. RESULTS: A null allele of Drosophila TBC1D8B exhibited a nephrocyte-restricted phenotype of nephrin mislocalization, similar to patients with isolated nephrotic syndrome who have variants in the gene. The protein was required for rapid nephrin turnover in nephrocytes and for endocytosis of nephrin induced by excessive Rab5 activity. The protein expressed from the Tbc1d8b locus bearing the edited tag predominantly localized to mature early and late endosomes. Tbc1d8b was required for endocytic cargo processing and degradation. Silencing Hrs, a regulator of endosomal maturation, phenocopied loss of Tbc1d8b. Low-level expression of murine TBC1D8B rescued loss of the Drosophila gene, indicating evolutionary conservation. Excessive murine TBC1D8B selectively disturbed nephrin dynamics. Finally, we discovered four novel TBC1D8B variants within a cohort of 363 patients with FSGS and validated a functional effect of two variants in Drosophila, suggesting a personalized platform for TBC1D8B-associated FSGS. CONCLUSIONS: Variants in TBC1D8B are not infrequent among patients with FSGS. TBC1D8B, functioning in endosomal maturation and degradation, is essential for nephrin trafficking.

A Novel Mouse Model of Idiopathic Nephrotic Syndrome Induced by Immunization with the Podocyte Protein Crb2.

BACKGROUND: The cause of podocyte injury in idiopathic nephrotic syndrome (INS) remains unknown. Although recent evidence points to the role of B cells and autoimmunity, the lack of animal models mediated by autoimmunity limits further research. We aimed to establish a mouse model mimicking human INS by immunizing mice with Crb2, a transmembrane protein expressed at the podocyte foot process. METHODS: C3H/HeN mice were immunized with the recombinant extracellular domain of mouse Crb2. Serum anti-Crb2 antibody, urine protein-to-creatinine ratio, and kidney histology were studied. For signaling studies, a Crb2-expressing mouse podocyte line was incubated with anti-Crb2 antibody. RESULTS: Serum anti-Crb2 autoantibodies and significant proteinuria were detected 4 weeks after the first immunization. The proteinuria reached nephrotic range at 9-13 weeks and persisted up to 29 weeks. Initial kidney histology resembled minimal change disease in humans, and immunofluorescence staining showed delicate punctate IgG staining in the glomerulus, which colocalized with Crb2 at the podocyte foot process. A subset of mice developed features resembling FSGS after 18 weeks. In glomeruli of immunized mice and in Crb2-expressing podocytes incubated with anti-Crb2 antibody, phosphorylation of ezrin, which connects Crb2 to the cytoskeleton, increased, accompanied by altered Crb2 localization and actin distribution. CONCLUSION: The results highlight the causative role of anti-Crb2 autoantibody in podocyte injury in mice. Crb2 immunization could be a useful model to study the immunologic pathogenesis of human INS, and may support the role of autoimmunity against podocyte proteins in INS.

Beneficial effects of procyanidin B2 on adriamycin-induced nephrotic syndrome mice: the multi-action mechanism for ameliorating glomerular permselectivity injury.

Despite considerable advances in prevention, diagnosis, and therapy, nephrotic syndrome (NS) remains a significant cause of high morbidity and mortality globally. As a result, there is an urgent need to identify novel effective preventative and therapeutic agents for NS. NS is implicated in glomerular permselectivity injury, which can be attributed to oxidative distress, inflammation, lipid nephrotoxicity, podocyte apoptosis, autophagy dysfunction, and slit diaphragm (SLD) dysfunction. In addition to its well-documented antioxidant potency, procyanidin B2 (PB2) may exhibit pleiotropic effects by targeting various canonical signaling events, such as NF-κB, PPARs, PI3K/Akt, mTOR, and the caspase family. As a result, PB2 may be a promising therapeutic target against NS. To test this hypothesis, we established an Adriamycin (ADR)-induced NS mouse model to evaluate the pleiotropic renoprotective effects of PB2 on NS. Here, we demonstrated that PB2 improves podocyte injury via inhibition of NOX4/ROS and Hsp90/NF-κB to exhibit antioxidant and anti-inflammatory potency, respectively. We also show that PB2 indirectly activates the PI3K/Akt axis by regulating SLD protein levels, resulting in normalized podocyte apoptosis and autophagy function. Further, loss of albumin (ALB) induces lipid nephrotoxicity, which we found to be alleviated by PB2 via activation of PPARα/ß-mediated lipid homeostasis and the cholesterol efflux axis. Interestingly, our results also suggested that PB2 reduces electrolyte abnormalities and edema. In addition, PB2 may contribute protective effects against trace element dys-homeostasis, which, through alleviating serum ALB loss, leads to a protective effect on glomerular permselectivity injury. Taken together, our results reveal that the identified mechanisms of PB2 on NS are multifactorial and involve inhibition of oxidative distress and inflammatory responses, as well as improvements in podocyte apoptosis and autophagy dysfunction, amelioration of lipid nephrotoxicity, and modulation of electrolyte abnormalities and edema. Thus, we provide a theoretical basis for the clinical application of PB2 against NS.

Protective effects of rituximab on puromycin-induced apoptosis, loss of adhesion and cytoskeletal alterations in human podocytes.

Podocytes are highly specialized cells playing a key role in the filtration function of the kidney. A damaged podocyte ultrastructure is associated with a reorganization of the actin cytoskeleton and accompanied with a loss of adhesion to the glomerular basement membrane leading to proteinuria in many forms of glomerular diseases, e.g. nephrotic syndrome. If the first-line therapy with glucocorticoids fails, alternative immunosuppressive agents are used, which are known to have the potential to stabilize the actin cytoskeleton. A new option for preventing relapses in steroid dependent nephrotic syndrome is the monoclonal antibody rituximab, which, in addition to its B-cell depleting effect, is assumed to have direct effects on podocytes. We here provide data on the non-immunological off-target effects of the immunosuppressant rituximab on podocyte structure and dynamics in an in vitro puromycin aminonucleoside model of podocyte injury. A conditionally immortalized human podocyte cell line was used. Differentiated podocytes were treated with puromycin aminonucleoside and rituximab. Our studies focussed on analyzing the structure of the actin cytoskeleton, cellular adhesion and apoptosis using immunofluorescence staining and protein biochemistry methods. Treatment with rituximab resulted in a stabilization of podocyte actin stress fibers in the puromycin aminonucleoside model, leading to an improvement in cell adhesion. A lower apoptosis rate was observed after parallel treatment with puromycin aminonucleoside and rituximab visualized by reduced nuclear fragmentation. Consistent with this data, Western-blot analyses demonstrated that rituximab directly affects the caspase pathways by inhibiting the activation of Caspases-8, -9 and -3, suggesting that rituximab may inhibit apoptosis. In conclusion, our results indicate an important role of the immunosuppressant rituximab in terms of stability and morphogenesis of podocytes, involving apoptosis pathways. This could help to improve therapeutical concepts for patients with proteinuria mediated by diseased podocytes.

A critical role of the podocyte cytoskeleton in the pathogenesis of glomerular proteinuria and autoimmune podocytopathies.

Selective glomerular filtration relies on the membrane separating the glomerular arterioles from the Bowman space. As a major component of the glomerular filtration barrier, podocytes form foot processes by the actin cytoskeleton, which dynamically adjusts in response to environmental changes to maintain filtration barrier integrity. The slit diaphragms bridge the filtration slits between neighboring foot processes and act as signaling hubs interacting with the actin cytoskeleton. Focal adhesions relay signals to regulate actin dynamics while allowing podocyte adherence to the basement membrane. Mutations in actin regulatory and signaling proteins may disrupt the actin cytoskeleton, resulting in foot process retraction, effacement, and proteinuria. Large-scale gene expression profiling platforms, transgenic animal models, and other in vivo gene delivery methods now enhance our understanding of the interactions among podocyte focal adhesions, slit diaphragms, and actin dynamics. In addition, our team found that at least 66% of idiopathic nephrotic syndrome (INS) children have podocyte autoantibodies, which was defined as a new disease subgroup-, autoimmune podocytopathies. This review outlines the pathophysiological mechanisms of podocyte cytoskeleton protein interactions in proteinuria and glomerular podocytopathy.

Nephrinuria and podocytopathies.

The discovery of nephrin in 1998 has launched a new era in glomerular diseases research, emphasizing its crucial role in the structure and function of the glomerular filtration barrier. In the past 20 years, substantial advances have been made in understanding podocyte structure and function as well as the discovery of several podocyte-related proteins including nephrin. The glomerular filtration barrier is comprised of podocytes, the glomerular basement membrane and endothelial cells. Podocytes, with their specialized slit diaphragm, form the essential backbone of the glomerular filtration barrier. Nephrin is a crucial structural and functional feature of the slit diaphragm that prevents plasma protein, blood cell and macromolecule leakage into the urine. Podocyte damage results in nephrin release. Podocytopathies are kidney diseases in which podocyte damage drives proteinuria, i.e., nephrotic syndrome. Many kidney diseases involve podocytopathy including congenital nephrotic syndrome of Finnish type, diffuse mesangial sclerosis, minimal change disease, focal segmental glomerulosclerosis, collapsing glomerulonephropathy, diabetic nephropathy, lupus nephropathy, hypertensive nephropathy and preeclampsia. Recently, urinary nephrin measurement has become important in the early detection of podocytopathies. In this chapter, we elaborate the main structural and functional features of nephrin as a podocyte-specific protein, pathomechanisms of podocytopathies which result in nephrinuria, highlight the most commonly used methods for detecting urinary nephrin and investigate the diagnostic, prognostic and potential therapeutic relevance of urinary nephrin in primary and secondary proteinuric kidney diseases.

Isolated steroid-resistant nephrotic syndrome in a Chinese child carrying a de novo mutation in WT1 gene:a case report and literature review.

BACKGROUND: Isolated steroid-resistant nephrotic syndrome (ISRNS) is caused by mutations in the Wilms' tumor-1 (WT1) gene, which encodes glomerular podocytes and podocyte slit diaphragm.We report a novel 8-year-old female patient with ISRNS carrying a de novo missense mutation in WT1 gene and presenting a new type of pathology, have never been reported.We also systematically review previous reports of ISRNS in Chinese children. CASE PRESENTATION: A 8-year-old Chinese patient who had steroid-resistant nephrotic syndrome,responded poorly to immunosuppressant, and had no extrarenal manifestations. The patient had a female phenotype and karyotype of 46, XX. A new type of renal pathology, proliferative sclerosing glomerulonephritis (PSG),and a de novo missense mutation in WT1 gene, c.748C > T (p.R250W),which have not yet been reported, were identified. She was diagnosed with ISRNS.The patient progressed to end-stage renal disease at the age of 10 years,underwent dialysis and kidney transplant. Renal function and urine protein were normal during 4-year follow-up. CONCLUSIONS: WT1 gene testing should be performed to guide treatment for patients with steroid-resistant nephrotic syndrome, especially for isolated cases and female patients.