Nephrin Protein Research Articles

Renal Implications of Psoriasis: Urinary Podocyte Markers and Disease Progression

Introduction: Psoriasis may lead to glomerular inflammatory damage and disruption of the podocyte barrier, allowing podocyte degradation products to leak into the urine. Objectives: We aimed to analyze the concentrations of podocyte surface glycoprotein podocalyxin (PDX) and podocyte slit protein nephrin in spot urine samples of patients diagnosed with mild, moderate, or severe psoriasis vulgaris. Methods: A total of 78 participants, including 58 patients diagnosed with mild, moderate, or severe psoriasis vulgaris and 20 controls, were included in the study. The diagnosis of psoriasis was made considering the typical morphological appearance of the lesions. Morning urine samples were used to evaluate urine PDX and nephrin levels. Albuminuria was evaluated by calculating the albumin-creatinine ratio (uACR). Results: Urinary PDX and nephrin levels of psoriasis groups were significantly higher than the control group. Urinary PDX and nephrin levels of mild and moderate psoriasis groups were similar but higher than the control group. Urinary PDX and nephrin levels of the severe psoriasis group were significantly higher than the mild and moderate psoriasis groups. Microalbuminuria rates were similar in the psoriasis and control groups. A positive significant correlation was detected between urine PDX, nephrin, uACR, and SBP. After adjustment for age, BMI and gender, urine PDX and nephrin were found to be independent risk factors for microalbuminuria. Conclusion: This study showed that podocyte damage in psoriasis patients begins in the early stage of the disease and increases significantly in the severe stage of the disease.

Potential and pitfalls of measuring circulating anti-nephrin autoantibodies in glomerular diseases.

Recent studies have identified autoantibodies targeting the podocyte protein nephrin in patients with primary podocytopathies such as minimal change disease, primary focal segmental glomerulosclerosis (FSGS), post-transplant recurrent FSGS and childhood idiopathic nephrotic syndrome. These antibodies bind nephrin and directly influence nephrin downstream signaling, with immense effect on the podocytes' cellular structure and function, substantially changing our understanding of antibody-mediated podocytopathies and disease classification. Their presence correlates with disease activity and holds great potential as a novel biomarker of anti-nephrin-associated podocytopathy. However, the detection of these potentially low-titre autoantibodies has proven challenging. In this review, we highlight and explain distinct detection methodologies with their advantages and disadvantages and discuss the potential of anti-nephrin autoantibodies as a novel biomarker in nephrotic syndrome for diagnosis, prognostication and therapeutic guidance in patients with nephrotic syndrome.

Podocyte-Specific Protein Expression in Urine Exosome Acts as a Marker for Renal Injury in Post-COVID State.

Introduction: Severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) has been associated with the development of COVID-19. COVID-19 may cause endothelial cell dysfunction (ECD), which can lead to cardiometabolic diseases and podocytopathy. In this study, we explored whether presence of hyperglycemia predisposes to SARS-CoV-2 infection, in vitro, and whether COVID-19 can put an individual at a higher risk of persistent renal damage in the long-term following acute COVID infection. To estimate renal damage, we evaluated albuminuria and podocytopathy. Podocytopathy was estimated by measuring podocyte-specific protein levels in urine-derived exosomes from patients who were admitted with acute COVID-19 at 10 days, 6 months, and 12 months post-acute SARS-CoV-2 infection. Methods: Blood and urine samples from patients with SARS-CoV-2 post-infection were procured from the George Washington University COVID repository. Peripheral blood mononuclear cells and urine exosomes were isolated. Podocyte-specific proteins Podocalyxin (PODXL) and Nephrin (NEPH) were identified from urine exosomes. Results: Urine exosomal podocalyxin levels were significantly high at 10 week (n = 18; P = 0.001), 6 month (n = 25; P = 0.003) and 12 month (n = 14; P = 0.0001) time points. Nephrin levels were also noted to be high at 10 week (n = 18; P = 0.001) and 12 month (n = 14; P = 0.007) time points, compared with urine samples obtained from type 2 diabetes subjects who never had COVID-19. Though urinary podocyte-specific proteins were high, compared to control, there were no significant differences noted on urine albumin:creatinine ratios (UACR) between the groups. Conclusion: Persistent high levels of podocyte-specific proteins noted in urinary exosomes even at 12 months post-Covid may lead to the development of chronic kidney disease.

Is There a Diagnostic and Prognostic Role for Anti-Nephrin Autoantibodies in Diabetic Nephropathy?

Diabetic nephropathy (DN) is one of the key causes of end-stage kidney disease worldwide, especially in developed countries. The classic pathogenic development of DN is characterized by microalbuminuria which would progress to nephrotic-range proteinuria and loss of kidney function. The degree of albuminuria is considered an independent risk factor for all-cause mortality in patients with DN. It is now well established that albuminuria stems from disruptions in podocyte structure and function. Podocytes play a major role in the glomerular filtration barrier. The nephrin protein has been identified as a core component of the slit diaphragm in podocytes, and as such, the downregulation of nephrin expression has been described well in various proteinuric glomerulopathies, including DN. Previous studies have shown that the presence of urinary nephrin potentially signifies an early marker of podocyte injury in DN. More recently, there have been increasing bodies of evidence which suggest that circulating autoantibodies targeting nephrin contributes to the pathogenesis of podocytopathies. However, the functional significance of these circulating autoantibodies in patients with DN is not well understood. In this review, we aim to evaluate the significance of nephrin dysregulation in the pathogenesis of DN based on the current available literature and provide an overview on the application of circulating anti-nephrin autoantibodies in relation to its diagnostic as well as prognostic role in podocytopathies, including DN.

Podocyte specific knockout of the natriuretic peptide clearance receptor is podocyte protective in focal segmental glomerulosclerosis.

Natriuretic peptides (NPs) bind to glomerular podocytes and attenuate glomerular injury. The beneficial effects of NPs are negatively regulated by the NP clearance receptor (NPRC), which is highly expressed in podocytes. To determine if inhibiting NPRC is podocyte protective, we examined the effects of deleting NPRC in both cultured podocytes and in vivo. We found that: 1.Both atrial NP and C-type NP inhibit podocyte apoptosis in cultured podocytes, but these podocyte protective effects are significantly attenuated in cells expressing NPRC, and 2. Atrial NP was significantly more effective than CNP at inhibiting the apoptotic response. Consistent with the protective actions of NPs, podocyte specific knockout of NPRC reduced albuminuria, glomerular sclerosis and tubulointerstitial inflammation in a mouse model of focal segmental glomerulosclerosis. These beneficial actions were associated with: 1. Decreased expression of the myofibroblast marker alpha-smooth muscle actin, 2. Reduced expression of the extracellular matrix proteins collagen 4-alpha-1 and fibronectin, and 3. Preserved expression of the podocyte proteins nephrin and podocin. Inhibiting NP clearance may be a useful therapeutic approach to treat glomerular diseases.

Changes in the immunohistochemical expression of nephrin protein in renal corpuscle of rats in response to sleepdisturbance.

Sleep is an essential health requirement; human body needs sufficient amount and quality of sleep to ensure its health. Sleep disturbance led to deterioration in renal functions. This study aimed to asses effect of sleep disturbance on nephrin protein in renal corpuscle. A sample of thirty adult male albino rats, subjected to sleep disturbance by light, divided into three groups; control group with normal sleep rhythm of 12-12 h dark- light phases, group A: subjected to interruption of sleep by light at three intervals, group B: rats were exposed to a reduction in sleep time by continuous light stimulation for 7 h. Animals were sacrificed by euthanasia, their kidneys were dissected and prepared for paraffin, sections stained for Nephrin protein, and the immunohistochemical intensity was quantified by Aperio Image Scope analysis software. This study showed variations in the effect of sleep disturbance patterns by light exposure on nephrin protein expression in renal corpuscles; in the control group a strong patchy distribution of Nephrine in the peripheral region of the glomerulus, group A showed a significant reduction compared to the control group, and group B a weak expression of nephrin protein in the glomerulus, with significant changes between group B and group A, but no significant changes between group B and control. These changes reflect that sleep disturbance affects the structural integrity of the slit diaphragm and nephrin protein expression, which is considered a novel protein for the slit diaphragm structural integrity, and a sign of podocyte injury.

Anti-nephrin autoantibodies in steroid-resistant nephrotic syndrome may inform treatment strategy.

Autoantibodies against the podocyte protein nephrin were recently identified in a pediatric cohort primarily comprising steroid-sensitive (SSNS) and steroid-dependent (SDNS) nephrotic syndrome (NS). However, their prevalence across all NS subtypes, particularly in steroid-resistant nephrotic syndrome (SRNS), and their relation to therapy response need to be determined to advance pathophysiological understanding and refine treatment strategies. A multicenter cohort study measuring anti-nephrin autoantibodies in samples from children with SSNS, SDNS, nongenetic and genetic SRNS was conducted. Sixty-nine of 101 (68%) patients with SSNS, 19 of 67 (28%) patients with SDNS, 14 of 103 patients (14%) with non-genetic SRNS, and 1 of 62 patients (2%) with genetic SRNS were positive for anti-nephrin autoantibodies. The prevalence of anti-nephrin autoantibodies increased with presence of active disease in cases of SSNS and SDNS. Within the group of non-genetic SRNS patients with active disease, anti-nephrin positivity was found in 13 of 74 (18%) patients responding to intensified immunosuppression compared to none of 17 patients with multidrug-resistant SRNS. The prevalence of anti-nephrin antibodies is substantially higher in children with steroid responsive NS than in those with SRNS, suggesting that anti-nephrin antibodies primarily drive SSNS/SDNS. In contrast, NS due to podocyte gene mutations is primarily genotype-caused. Anti-nephrin autoantibodies may serve as a positive prognostic marker in pediatric NS, indicating a favorable response to immunosuppressive therapy.

Yiqi Yangyin Huazhuo Tongluo Formula alleviates diabetic podocyte injury by regulating miR-21a-5p/FoxO1/PINK1-mediated mitochondrial autophagy.

To investigate the protective effect of Yiqi Yangyin Huazhuo Tongluo Formula (YYHT) against high glucose-induced injury in mouse renal podocytes (MPC5 cells) and the possible mechanism. Adult Wistar rats were treated with 19, 38, and 76 g/kg YYHT or saline via gavage for 7 days to prepare YYHT-medicated or blank sera for treatment of MPC5 cells cultured in high glucose (30 mmol/L) prior to transfection with a miR-21a-5p inhibitor or a miR-21a-5p mimic. The changes in miR-21a-5p expressions and the mRNA levels of FoxO1, PINK1, and Parkin in the treated cells were detected with qRT-PCR, and the protein levels of nephrin, podocin, FoxO1, PINK1, and Parkin were detected with Western blotting. Autophagic activity in the cells were evaluated with MDC staining. The effect of miR-21a-5p mimic on FoxO1 transcription and the binding of miR-21a-5p to FoxO1 were examined with luciferase reporter gene assay and radioimmunoprecipitation assay. MPC5 cells exposed to high glucose showed significantly increased miR-21a-5p expression, lowered expressions of FoxO1, PINK1, and Parkin1 mRNAs, and reduced levels of FoxO1, PINK1, parkin, nephrin, and podocin proteins and autophagic activity. Treatment of the exposed cells with YYHT-medicated sera and miR-21a-5p inhibitor both significantly enhanced the protein expressions of nephrin and podocin, inhibited the expression of miR-21a-5p, increased the mRNA and protein expressions of FoxO1, PINK1 and Parkin, and upregulated autophagic activity of the cells. Transfection with miR-21a-5p mimic effectively inhibited the transcription of FoxO1 and promoted the binding of miR-21a-5p to FoxO1 in MPC5 cells, and these effects were obviously attenuated by treatment with YYHT-medicated sera. YYHT-medicated sera alleviate high glucose-induced injury in MPC5 cells by regulating miR-21a-5p/FoxO1/PINK1-mediated mitochondrial autophagy.

Investigating the inflammatory mechanism of notoginsenoside R1 in Diabetic nephropathy via ITGB8 based on network pharmacology and experimental validation

BackgroundDiabetes often causes diabetic nephropathy (DN), a serious long-term complication. It is characterized by chronic proteinuria, hypertension, and kidney function decline, can progress to end-stage renal disease, lowering patients’ quality of life and lifespan. Inflammation and apoptosis are key to DN development. Network pharmacology, clinical correlation, and basic experimental validation to find out how NGR1 might work to reduce inflammation in DN treatment. The study aims to improve DN treatment with new findings.MethodsTo determine how NGR1 treats DN, this study used network pharmacology, clinical correlation, and basic experimental validation. Three methods were used to predict NGR1 drug targets: ChEMBL, SuperPred, and Swiss Target Prediction. Drug targets are linked to diseases by molecular docking. A clinical correlation analysis using the Nephroseq Classic (V4) database looked at the strong link between medication targets and the development, progression, and renal function of DN. Additional research showed that NGR1 reduces high blood sugar-induced podocyte inflammation.ResultsThe integrin subunit beta 8 (ITGB8) protein is a potential NGR1 therapeutic target for DN. It may be linked to inflammatory proteins like caspase 3 and IL-18. Validation of the molecular docking showed that SER-407, ALA-22, Ala-343, and TYR-406 form hydrogen bonds with NGR1 and ITGB8. These interactions represent pharmacodynamic targets. Clinical correlation showed that DN patients had significantly lower ITGB8 expression levels than healthy individuals. Between 50 and 80 years old, DN patients’ ITGB8 expression levels decreased. ITGB8 expression was lowest in renal function conditions, with eGFR values of 15–29 ml/min/1.73 m2. In the db/db mouse model, downregulation of ITGB8 expression in renal tissue was associated with renal inflammatory damage. The hyperglycemic group had significantly lower levels of nephrin and caspase-3 protein, but higher levels of cleaved caspase-1 protein. Giving NGR1 in different amounts (1, 3, 10, and 30 µM) greatly decreased the expression of caspase3, stopped the expression of cleaved caspase1, and lowered the damage caused by NLRP3 in podocytes.ConclusionWe identified several NGR1 pharmacological targets and found that the ITGB8 protein is a key drug target linked to inflammation and DN. ITGB8 is critical for DN development and can help to reduce high blood sugar-induced podocyte inflammation.

Renal protective effect of Isaria felina mycelium powder on diet and STZ-induced diabetes mice and the identification of major chemical constituents

Renal protective effect of Isaria felina mycelium powder on diet and STZ-induced diabetes mice and the identification of major chemical constituents

Glomerular Injury Is Associated with Severe Courses of Orthohantavirus Infection.

Hemorrhagic fever with renal syndrome (HFRS) induced by Eurasian pathogenic orthohantaviruses is characterized by acute kidney injury (AKI) with often massive proteinuria. The mechanisms of the organ-specific manifestation are not completely understood. To analyze the role of glomerular and tubular damage in kidney injury induced by HFRS, we measured specific markers in urine samples of patients with acute Puumala virus (PUUV) infection and determined their correlation with disease severity. Levels of α1-microglobulin (α1-MG) and kidney injury molecule 1 (KIM-1), which is expressed by injured tubular epithelial cells, were measured to detect tubular dysfunction and injury. Immunoglobulin G (IgG) and the podocyte specific protein nephrin served as markers for glomerular injury. All four markers were elevated on admission. Markers of glomerular injury, IgG and nephrin, correlated with markers of disease severity such as length of hospitalization, serum creatinine, and proteinuria. In contrast, tubular injury did not correlate with these severity markers. Our results demonstrate that hantavirus infection induces both glomerular and tubular injury early in the clinical course. However, the glomerular dysfunction and podocyte injury seem to contribute directly to disease severity and to play a more central role in HFRS pathogenicity than direct damage to tubular epithelial cells.

Therapeutic Potential of Blocking Nephrin Phosphorylation to Improve Pancreatic β-cell Function.

The phosphorylation of the transmembrane protein nephrin has been shown to play an important role in signaling in kidney podocytes, and it has now been shown to also play a key role in regulating pancreatic β-cell function. Williamson et al have recently shown that the loss of nephrin tyrosine phosphorylation on its 3 cytoplasmic YDxV motifs can enhance insulin release in aged female mice. These studies suggest that blocking nephrin phosphorylation may be an effective treatment option for improving β-cell function.

Application of Lycium Barbarum Polysaccharide Liposome Nanoparticles to Improve the Slow Healing of Refractory Wounds in Diabetic Foot

Refractory wounds in diabetic foot heal slowly. Lycium barbarum polysaccharides has been found to have the effect of lowering blood sugar. At the same time, the role of CXCL12/CXCR4 signaling in the healing process of diabetic foot has attracted much more attention. This study aimed to explore the mechanism by which Lycium barbarum polysaccharide liposome nanoparticles improve slow healing of refractory wounds in diabetic feet through CXCL12/CXCR4 signaling axis. A rat model of diabetic foot trauma was constructed and lipid nanoparticles-Lycium barbarum polysaccharides (LNP-LBP) nanocomposite was prepared and administrated into the rats. During the administration process, wound healing conditions were observed and recorded. HE staining was performed on each group, and inflammatory factors, CXCR4, and podocyte marker protein Nephrin were observed. Compared with control group, the blood sugar levels and inflammatory factor IL-6 levels of mice in the Lycium barbarum polysaccharide liposome nanoparticles group were reduced, and the wound healing speed was significantly accelerated (P < 0.05). LNP-LBP significantly reduced the levels of CXCL12 and CXCR4 in mouse wound tissues (P < 0.05). Moreover, when LNP-LBP and CXCL12/CXCR4 signaling axis inhibitors were used in combination, the wound healing speed was further accelerated and IL-6 levels were significantly increased. LNP-LBP can reduce the blood sugar level of diabetic foot rats, reduce the inflammatory response of diabetic foot wounds and swelling of wound podocytes, promote cell autophagy to speed up metabolism, thereby promoting refractory wounds healing in diabetic foot. The effect is related to inhibiting the expression of CXCL12/CXCR4 signaling.

#993 The molecular effects of SGLT2i (Empagliflozin) on α-Klotho and Nephrin/Podocin proteins in type II diabetic mice model

Abstract Background and Aims Type 2 diabetes mellitus (T2DM), especially hyperglycemia, is associated with increased glucose cell toxicity and oxidative stress that can lead to irreversible damage in the kidney such as diabetic nephropathy (DN). DN is one of the most common microvascular complications of diabetes and is the leading cause of end-stage renal disease (ESRD) and replacement therapy. Several works in the setting of early experimental diabetic nephropathy using the recent anti-diabetic drugs such as sodium-glucose transporter type 2 inhibitors in the prevention, development or amelioration of renal injury. The exact mechanisms, by which these drugs ameliorate glomerular injury from hyperglycemia, had not been clarified well. Klotho and Podocin-Nephrin proteins are an important proteins involved in the tubule-glomerular injury. The Klotho is a circulating anti-ageing hormone with anti-oxidative and anti-inflammatory properties with vascular protective effects in animal studies. Podocin & Nephrin are important component of the glomerular foot-process membrane, the slit-diaphragm. Recently, several experimental reports showed decreased α-Klotho, Nephrin and Podocin expression in different models of diabetic nephropathy. Method We used BTBR mouse strain with the ob/ob leptin-deficiency mutation, which develops T2DM with hyperglycemia and DN; C57/BL mice were the control (CON). EMPA was administrated to T2DM mice (T2DM+EMPA) via drinking water for 12 weeks. Blood parameters were measured repeatedly, and at sacrifice, mice kidneys were harvested for histological and biochemical analyses. We evaluated renal glomerular structure of Nephrin, Podocin and α-Klotho proteins expression. Results EMPA treatment reduced T2DM blood glucose vs C57 group (P < 0:01), and increase urine glucose in EMPA+DM group (P < 0:001) EMPA vs. C57BL/6 and DM). Histological analyses in kidney sections, revealed decreased α-Klotho expression in glomeruli of DM mice vs Control mice (15.95 ± 0.64 vs 36 ± 2.42, (P < 0:01), and restored back in DM+ EMPA mice vs control mice (27.64 ± 0.94 vs 15.95 ± 0.64, P < 0:01). Podocin expression in glomeruli: control mice 31.3 ± 3.7 VS DM mice 21.26 ± 1.22 and DM + EMPA mice 28.22 ± 0.50 VS DM mice 21.26 ± 1.22 Conclusion Hyperglycemia (DM type II) reduces Podocin-Nephrin and α-Klotho proteins expression in the glomeruli kidney and upregulated by EMPA treatment. SGLT2i (EMPA) treatment in DM patients can significantly reduce progression of DN and onset of ESRD probably through its effect on α-Klotho/Podocin-Nephrin proteins.

#2407 Multiomics analysis of isolated zebrafish glomeruli to unravel the impact of alternative splicing in glomerular diseases

Abstract Background and Aims Alternative splicing (AS) is a known key factor in gene regulation. There is evidence that changes in the alternative mRNA splicing can lead to the onset of various diseases. However, the potential role of AS in the pathogenesis of glomerular diseases has only been investigated to a very limited extent. In the past, the zebrafish larva (Danio rerio) has been established as an animal model for the in vivo investigation of kidney diseases, since the glomeruli of zebrafish larvae are genetically, molecularly, and functionally similar to those of mammals. As part of the Sys_CARE (Systems Medicine Investigation of AS in Cardiac and Renal Diseases) project, we wanted to identify specific AS events that might be involved in the development of focal segmental glomerulosclerosis (FSGS). Method For all experiments, the double transgenic strain [Tg (nphs2: GAL4-VP16); (UAS:Eco.nfsB-mCherry)] was used. At 4 dpf (days post fertilization), the zebrafish larvae were divided into two groups. The control group was treated with 0.1% dimethyl sulfoxide (DMSO) in E3 medium. To induce FSGS, zebrafish larvae were treated with 50 nM nifurpirinol (NFP) in 0.1% DMSO in E3 medium for 24 hours. Glomeruli were manually isolated at 5 and 6 dpf and analyzed together with lysates of whole larvae by LC-MS/MS and RNA-Seq. To identify AS events, RNA-Seq data were analyzed by MAJIQ, Multivariate Analysis of Transcript Splicing (rMATS), leafcutter, Whippet as well as IsoformSwitchAnalyzeR. Gene set enrichment analysis (GSEA) identified proteins and genes which were classified according to their related biological processes. Results Proteomics and transcriptomics showed significant differences in the glomerular protein and transcript levels between NFP-treated larvae and the control group. The abundance of the podocyte-specific protein nephrin as well as the podocin promotor-driven expression of the fluorescent dye mCherry was significantly reduced after NFP treatment confirming the onset of the injury. Subsequent analyses identified more than 1000 transcripts that were significantly differentially expressed, as well as several splicing events in the larvae with a FSGS-like damage. There was also a marked shift in isoform patterns in larvae with FSGS. Gene set enrichment analysis indicated an enrichment in the up-regulated transcripts and proteins involved in metabolic processes, especially those related to rRNA and ncRNA processing. Conversely, transcripts and proteins that were down-regulated showed significant clusters in catabolic processes, extracellular matrix, and cytoskeleton organization as well as in nephron and glomerulus development. Conclusion Using our zebrafish model, in-depth transcriptomics and proteomics analysis provided novel insights into the pathogenesis of podocyte-associated glomerular diseases. Data suggest, that pathological changes are accompanied by alternative splice events which might therefore be a new therapeutic approach in glomerular nephropathy.

Biophysical characterization and insights into the oligomeric nature of CD2-associated protein.

Glomerular podocytes are specialized epithelial cells localized to the blood-urine interface of the kidney. Podocyte slit-diaphragm (SD), a size-and-charge-selective junction, is instrumental in blood ultrafiltration and the formation of protein-free urine. The SD consists of macromolecular complexes of several proteins, such as nephrin, podocin, and CD2-associated protein (CD2AP). CD2AP is an adapter protein and is considered to be crucial for the integrity of SD. Mutations in the SD proteins cause nephrotic syndrome (NS), characterized by proteinuria. SD proteins' structural features must be elucidated to understand the mechanism of proteinuria in NS. In this study, we expressed, purified, and biophysically characterized heterologously expressed human CD2AP. Codon-optimized human CD2AP was expressed in E. coli Rosetta cells. The recombinant protein was induced with 1 mM IPTG and purified by Ni-NTA affinity chromatography. Analytical size-exclusion chromatography, blue native-PAGE, circular dichroism, and fluorescence spectroscopy were performed to decipher the oligomeric nature, secondary structural content, and tertiary packing of CD2AP. Our analysis revealed that CD2AP adopts a predominantly disordered secondary structure despite exhibiting moderate tertiary packing, characterized by low helical and β-sheet content. CD2AP readily assembles into homo-oligomers, with octamers and tetramers constituting the primary population. Interestingly, the inherent flexibility of CD2AP's secondary structural elements appears resistant to thermal denaturation. Frameshift mutation (p.K579Efs*7) that leads to loss of the coiled-coil domain promotes aberrant oligomerization of CD2AP through SH3 domains. We successfully expressed full-length human CD2AP in a heterologous system, wherein the secondary structure of CD2AP is predominantly disordered. CD2AP can form higher-order oligomers, and the significance of these oligomers and the impact of mutations in the context of size-selective permeability of SD needs further investigation.

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.

The Role of Podocyte Cells in Diabetic Nephropathy: A Narrative Literature Review

The main cells affected in diabetic nephropathy are podocytes and proximal tubular cells. One of the main functional proteins in the podocyte slit diaphragm is podocin which podocytes need to express together with several specific proteins in the correct way for their differentiation, as well as to maintain their complex anatomy. Podocytes are highly specialized epithelial cells. They line the urinary surface of the glomerular capillary bundles. Podocytes are part of the filtration barrier along with capillary endothelial cells and GBM. Podocytes ensure selective permeability of the glomerular capillary walls. Podocin is a membrane protein with a molecular weight of 42kD which is located in the foot processes, and also forms part of the SD with the nephrin protein. Urinary podocin levels more specifically indicate ongoing glomerular damage because they were significantly increased in the normoalbuminuria group compared with the control group.

Huangkui capsule alleviates doxorubicin-induced proteinuria via protecting against podocyte damage and inhibiting JAK/STAT signaling.

Huangkui capsule alleviates doxorubicin-induced proteinuria via protecting against podocyte damage and inhibiting JAK/STAT signaling.

Therapeutic potential of a novel peripherally restricted CB1R inverse agonist on the progression of diabetic nephropathy.

This study assessed the efficacy of INV-202, a novel peripherally restricted cannabinoid type-1 receptor (CB1R) inverse agonist, in a streptozotocin-induced type-1 diabetes nephropathy mouse model. Diabetes was induced in 8-week-old C57BL6/J male mice via intraperitoneal injection of streptozotocin (45 mg/kg/day for 5 days); nondiabetic controls received citrate buffer. Diabetic mice were randomized to 3 groups based on blood glucose, polyuria, and albuminuria, and administered daily oral doses for 28-days of INV-202 at 0.3 or 3 mg/kg or vehicle. INV-202 did not affect body weight but decreased kidney weight compared with the vehicle group. While polyuria was unaffected by INV-202 treatment, urinary urea (control 30.77 ± 14.93; vehicle 189.81 ± 31.49; INV-202 (0.3 mg/kg) 127.76 ± 20; INV-202 (3 mg/kg) 93.70 ± 24.97 mg/24h) and albumin (control 3.06 ± 0.38; vehicle 850.08 ± 170.50; INV-202 (0.3 mg/kg) 290.65 ± 88.70; INV-202 (3 mg/kg) 111.29 ± 33.47 µg/24h) excretion both decreased compared with vehicle-treated diabetic mice. Compared with the vehicle group, there was a significant improvement in the urinary albumin to creatinine ratio across INV-202 groups. Regardless of the dose, INV-202 significantly reduced angiotensin II excretion in diabetic mice. The treatment also decreased Agtr1a renal expression in a dose-dependent manner. Compared with nondiabetic controls, the glomerular filtration rate was increased in the vehicle group and significantly decreased by INV-202 at 3 mg/kg. While the vehicle group showed a significant loss in the mean number of podocytes per glomerulus, INV-202 treatment limited podocyte loss in a dose-dependent manner. Moreover, in both INV-202 groups, expression of genes coding for podocyte structural proteins nephrin (Nphs1), podocin (Nphs2), and podocalyxin (Pdxl) were restored to levels similar to nondiabetic controls. INV-202 partially limited the proximal tubular epithelial cell (PTEC) hyperplasia and normalized genetic markers for PTEC lesions. INV-202 also reduced expression of genes contributing to oxidative stress (Nox2, Nox4, and P47phox) and inflammation (Tnf). In addition, diabetes-induced renal fibrosis was significantly reduced by INV-202. INV-202 reduced glomerular injury, preserved podocyte structure and function, reduced injury to PTECs, and ultimately reduced renal fibrosis in a streptozotocin-induced diabetic nephropathy mouse model. These results suggest that INV-202 may represent a new therapeutic option in the treatment of diabetic kidney disease.