Unraveling the role of natriuretic peptide clearance receptor (NPR3) in glomerular diseases.

Natriuretic peptides (NPs) are cardio-derived hormones that have a crucial role in maintaining cardiovascular homeostasis. Physiological effects of NPs are mediated by binding to natriuretic peptide receptors 1 and 2 (NPR1/2), whereas natriuretic peptide receptor 3 (NPR3) acts as a clearance receptor that removes NPs from the circulation. Mouse studies have shown that local NP-signaling in the kidney glomerulus is important for the maintenance of renal homeostasis. In this study we examined the expression of NPR3 in kidney tissue and explored its involvement in renal physiology and disease by generating podocyte-specific knockout mice (NPR3podKO) as well as by using an NPR3 inhibitor (NPR3i) in rodent models of kidney disease. NPR3 was highly expressed by podocytes. NPR3podKO animals showed no renal abnormalities under healthy conditions and responded similarly to nephrotoxic serum (NTS) induced glomerular injury. However, NPR3i showed reno-protective effects in the NTS-induced model evidenced by decreased glomerulosclerosis and reduced podocyte loss. In a ZSF1 rat model of diabetic kidney injury, therapy alone with NPR3i did not have beneficial effects on renal function/histology, but when combined with losartan (angiotensin receptor blocker), NPR3i potentiated its ameliorative effects on albuminuria. In conclusion, these results suggest that NPR3 may contribute to kidney disease progression.

A Cross-Sectional Study of Glomerular Hyperfiltration in Polycystic Ovary Syndrome.

Glomerular hyperfiltration (GH) has been reported to be higher in women with polycystic ovary syndrome (PCOS) and is an independent risk factor for renal function deterioration, metabolic, and cardiovascular disease. The aim of this study was to determine GH in type A PCOS subjects and to identify whether inflammatory markers, markers of CKD, renal tubule injury markers, and complement system proteins were associated. In addition, a secondary cohort study was performed to determine if the eGFR had altered over time. In this comparative cross-sectional analysis, demographic, metabolic, and proteomic data from Caucasian women aged 18-40 years from a PCOS Biobank (137 with PCOS, 97 controls) was analyzed. Slow Off-rate Modified Aptamer (SOMA)-scan plasma protein measurement was undertaken for inflammatory proteins, serum markers of chronic kidney disease (CKD), tubular renal injury markers, and complement system proteins. A total of 44.5% of the PCOS cohort had GH (eGFR ≥ 126 mL/min/1.73 m2 (n = 55)), and 12% (n = 17) eGFR ≥ 142 mL/min/1.73 m2 (super-GH(SGH)). PCOS-GH women were younger and had lower creatinine and urea versus PCOS-nonGH. C-reactive protein (CRP), white cell count (WCC), and systolic blood pressure (SBP) were higher in PCOS versus controls, but CRP correlated only with PCOS-SGH alone. Complement protein changes were seen between controls and PCOS-nonGH, and decay-accelerator factor (DAF) was decreased between PCOS-nonGH and PCOS-GSGH (p < 0.05). CRP correlated with eGFR in the PCOS-SGH group, but not with other inflammatory or complement parameters. Cystatin-c (a marker of CKD) was reduced between PCOS-nonGH and PCOS-GSGH (p < 0.05). No differences in tubular renal injury markers were found. A secondary cohort notes review of the biobank subjects 8.2-9.6 years later showed a reduction in eGFR: controls -6.4 ± 12.6 mL/min/1.73 m2 (-5.3 ± 11.5%; decrease 0.65%/year); PCOS-nonGH -11.3 ± 13.7 mL/min/1.73 m2 (-9.7 ± 12.2%; p < 0.05, decrease 1%/year); PCOS-GH (eGFR 126-140 mL/min/17.3 m2) -27.1 ± 12.8 mL/min/1.73 m2 (-19.1 ± 8.7%; p < 0.0001, decrease 2%/year); PCOS-SGH (eGFR ≥ 142 mL/min/17.3 m2) -33.7 ± 8.9 mL/min/17.3 m2 (-22.8 ± 6.0%; p < 0.0001, decrease 3.5%/year); PCOS-nonGH eGFR versus PCOS-GH and PCOS-SGH, p < 0.001; no difference PCOS-GH versus PCOS-SGH. GH was associated with PCOS and did not appear mediated through tubular renal injury; however, cystatin-c and DAF were decreased, and CRP correlated positively with PCOS-SGH, suggesting inflammation may be involved at higher GH. There were progressive eGFR decrements for PCOS-nonGH, PCOS-GH, and PCOS-SGH in the follow-up period which, in the presence of additional factors affecting renal function, may be clinically important in the development of CKD in PCOS.

Chronological Changes in the Histology of Infection-Related Glomerulonephritis in Renal Allograft: A Case Report.

We report the histological changes over time for a patient with infection-related glomerulonephritis (IRGN) that developed in a transplanted kidney. A 47-year-old man had undergone renal transplantation 3 years ago for end-stage kidney disease (ESKD). After several episodes of acute rejection, the patient was in a stable CKD condition. The abrupt development of severe microscopic hematuria and renal dysfunction was observed approximately 2 weeks after the onset of a phlegmon in his right leg. An allograft biopsy showed prominent glomerular endocapillary proliferation on light microscopy, granular C3 deposition on immunofluorescent microscopy, and subepithelial electron-dense deposits on electron microscopy, suggesting IRGN accompanied by moderate interstitial fibrosis and tubular atrophy (IFTA). Positive glomerular staining for nephritis-associated plasmin receptor (NAPlr) and plasmin activity, which are biomarkers of bacterial IRGN, supported the diagnosis. Although the infection was completely cured with antibiotic therapy, renal dysfunction persisted. A re-biopsy of the allograft 2 months later revealed resolution of the glomerular endocapillary proliferation and negative staining for NAPlr/plasmin activity, with worsening IFTA. We showed, for the first time, the chronological changes in infiltrating cells and histological markers of IRGN in transplanted kidneys. Glomerular changes, including NAPlr/plasmin activity staining, almost disappeared after the cessation of infection, while interstitial changes continuously progressed, contributing to ESKD progression.

Xanthine oxidoreductase inhibition ameliorates high glucose-induced glomerular endothelial injury by activating AMPK through the purine salvage pathway.

Xanthine oxidoreductase (XOR) contributes to reactive oxygen species production. We investigated the cytoprotective mechanisms of XOR inhibition against high glucose (HG)-induced glomerular endothelial injury, which involves activation of the AMP-activated protein kinase (AMPK). Human glomerular endothelial cells (GECs) exposed to HG were subjected to febuxostat treatment for 48 h and the expressions of AMPK and its associated signaling pathways were evaluated. HG-treated GECs were increased xanthine oxidase/xanthine dehydrogenase levels and decreased intracellular AMP/ATP ratio, and these effects were reversed by febuxostat treatment. Febuxostat enhanced the phosphorylation of AMPK, the activation of peroxisome proliferator-activated receptor (PPAR)-gamma coactivator (PGC)-1α and PPAR-α and suppressed the phosphorylation of forkhead box O (FoxO)3a in HG-treated GECs. Febuxostat also decreased nicotinamide adenine dinucleotide phosphate oxidase (Nox)1, Nox2, and Nox4 expressions; enhanced superoxide dismutase activity; and decreased malondialdehyde levels in HG-treated GECs. The knockdown of AMPK inhibited PGC-1α-FoxO3a signaling and negated the antioxidant effects of febuxostat in HG-treated GECs. Despite febuxostat administration, the knockdown of hypoxanthine phosphoribosyl transferase 1 (HPRT1) also inhibited AMPK-PGC-1α-FoxO3a in HG-treated GECs. XOR inhibition alleviates oxidative stress by activating AMPK-PGC-1α-FoxO3a signaling through the HPRT1-dependent purine salvage pathway in GECs exposed to HG conditions.

Preventive effects of vasohibin-2-targeting peptide vaccine for diabetic nephropathy.

Diabetic nephropathy remains the leading cause of end-stage kidney disease in many countries, and additional therapeutic targets are needed to prevent its development and progression. Some angiogenic factors are involved in the pathogenesis of diabetic nephropathy. Vasohibin-2 (VASH2) is a novel proangiogenic factor, and our previous study showed that glomerular damage is inhibited in diabetic Vash2 homozygous knockout mice. Therefore, we established a VASH2-targeting peptide vaccine as a tool for anti-VASH2 therapy in diabetic nephropathy. In this study, the preventive effects of the VASH2-targeting peptide vaccine against glomerular injury were examined in a streptozotocin (STZ)-induced diabetic mouse model. The mice were subcutaneously injected with the vaccine at two doses 2 wk apart and then intraperitoneally injected with 50 mg/kg STZ for 5 consecutive days. Glomerular injury was evaluated 20 wk after the first vaccination. Treatment with the VASH2-targeting peptide vaccine successfully induced circulating anti-VASH2 antibody without inflammation in major organs. Although the vaccination did not affect blood glucose levels, it significantly prevented hyperglycemia-induced increases in urinary albumin excretion and glomerular volume. The vaccination did not affect increased VASH2 expression but significantly inhibited renal angiopoietin-2 (Angpt2) expression in the diabetic mice. Furthermore, it significantly prevented glomerular macrophage infiltration. The preventive effects of vaccination on glomerular injury were also confirmed in db/db mice. Taken together, the results of this study suggest that the VASH2-targeting peptide vaccine may prevent diabetic glomerular injury in mice by inhibiting Angpt2-mediated microinflammation.NEW & NOTEWORTHY This study demonstrated preventive effects of VASH2-targeting peptide vaccine therapy on albuminuria and glomerular microinflammation in STZ-induced diabetic mouse model by inhibiting renal Angpt2 expression. The vaccination was also effective in db/db mice. The results highlight the importance of VASH2 in the pathogenesis of early-stage diabetic nephropathy and the practicability of anti-VASH2 strategy as a vaccine therapy.

A zebrafish model of diabetic nephropathy shows hyperglycemia, proteinuria and activation of the PI3K/Akt pathway.

Diabetic nephropathy (DN), as a complication of diabetes, is a substantial healthcare challenge owing to the high risk of morbidity and mortality involved. Although significant progress has been made in understanding the pathogenesis of DN, more efficient models are required to develop new therapeutics. Here, we created a DN model in zebrafish by crossing diabetic Tg(acta1:dnIGF1R-EGFP) and proteinuria-tracing Tg(l-fabp::VDBP-GFP) lines, named zMIR/VDBP. Overfed adult zMIR/VDBP fish developed severe hyperglycemia and proteinuria, which were not observed in wild-type zebrafish. Renal histopathology revealed human DN-like characteristics, such as glomerular basement membrane thickening, foot process effacement and glomerular sclerosis. Glomerular dysfunction was restored upon calorie restriction. RNA sequencing analysis demonstrated that DN zebrafish kidneys exhibited transcriptional patterns similar to those seen in human DN pathogenesis. Notably, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was activated, a phenomenon observed in the early phase of human DN. In addition, metformin improved hyperglycemia and proteinuria in DN zebrafish by modulating Akt phosphorylation. Our results indicate that zMIR/VDBP fish are suitable for elucidating the mechanisms underlying human DN and could be a powerful tool for therapeutic discovery.

Characterizing Glomerular Barrier Dysfunction with Patient-Derived Serum in Glomerulus-on-a-Chip Models: Unveiling New Insights into Glomerulonephritis.

Glomerulonephritis (GN) is characterized by podocyte injury or glomerular filtration dysfunction, which results in proteinuria and eventual loss of kidney function. Progress in studying the mechanism of GN, and developing an effective therapy, has been limited by the absence of suitable in vitro models that can closely recapitulate human physiological responses. We developed a microfluidic glomerulus-on-a-chip device that can recapitulate the physiological environment to construct a functional filtration barrier, with which we investigated biological changes in podocytes and dynamic alterations in the permeability of the glomerular filtration barrier (GFB) on a chip. We also evaluated the potential of GN-mimicking devices as a model for predicting responses to human GN. Glomerular endothelial cells and podocytes successfully formed intact monolayers on opposite sides of the membrane in our chip device. Permselectivity analysis confirmed that the chip was constituted by a functional GFB that could accurately perform differential clearance of albumin and dextran. Reduction in cell viability resulting from damage was observed in all serum-induced GN models. The expression of podocyte-specific marker WT1 was also decreased. Albumin permeability was increased in most models of serum-induced IgA nephropathy (IgAN) and membranous nephropathy (MN). However, sera from patients with minimal change disease (MCD) or lupus nephritis (LN) did not induce a loss of permeability. This glomerulus-on-a-chip system may provide a platform of glomerular cell culture for in vitro GFB in formation of a functional three-dimensional glomerular structure. Establishing a disease model of GN on a chip could accelerate our understanding of pathophysiological mechanisms of glomerulopathy.

Sirt7/HIC1 complex participates in hyperglycaemia-mediated EndMT via modulation of SDC1 expression in diabetic kidney disease and metabolic memory.

Diabetic kidney disease (DKD), a primary microvascular complication arising from diabetes, may result in end-stage renal disease. Epigenetic regulation of endothelial mesenchymal transition (EndMT) has been recently reported to exert function in metabolic memory and DKD. Here, we investigated the mechanism which Sirt7 modulated EndMT in human glomerular endothelial cells (HGECs) in the occurrence of metabolic memory in DKD. Lower levels of SDC1 and Sirt7 were noted in the glomeruli of both DKD patients and diabetes-induced renal injury rats, as well as in human glomerular endothelial cells (HGECs) with high blood sugar. Endothelial-to-mesenchymal transition (EndMT) was sustained despite the normalization of glycaemic control. We also found that Sirt7 overexpression associated with glucose normalization promoted the SDC1 expression and reversed EndMT in HGECs. Furthermore, the sh-Sirt7-mediated EndMT could be reversed by SDC1 overexpression. The ChIP assay revealed enrichment of Sirt7 and H3K18ac in the SDC1 promoter region. Furthermore, hypermethylated in cancer 1 (HIC1) was found to be associated with Sirt7. Overexpression of HIC1 with normoglycaemia reversed high glucose-mediated EndMT in HGECs. The knockdown of HIC1-mediated EndMT was reversed by SDC1 upregulation. In addition, the enrichment of HIC1 and Sirt7 was observed in the same promoter region of SDC1. The overexpressed Sirt7 reversed EndMT and improved renal function in insulin-treated diabetic models. This study demonstrated that the hyperglycaemia-mediated interaction between Sirt7 and HIC1 exerts a role in the metabolic memory in DKD by inactivating SDC1 transcription and mediating EndMT despite glucose normalization in HGECs.

Esm-1 mediates transcriptional polarization associated with diabetic kidney disease.

Esm-1, endothelial cell-specific molecule-1, is a susceptibility gene for diabetic kidney disease (DKD) and is a secreted proteoglycan, with notable expression in kidney, which attenuates inflammation and albuminuria. However, little is known about Esm1 expression in mature tissues in the presence or absence of diabetes. We utilized publicly available single-cell RNA sequencing data to characterize Esm1 expression in 27,786 renal endothelial cells (RECs) obtained from three mouse and four human databases. We validated our findings using bulk transcriptome data from 20 healthy subjects and 41 patients with DKD and using RNAscope. In both mice and humans, Esm1 is expressed in a subset of all REC types and represents a minority of glomerular RECs. In patients, Esm1(+) cells exhibit conserved enrichment for blood vessel development genes. With diabetes, these cells are fewer in number and shift expression toward chemotaxis pathways. Esm1 correlates with a majority of genes within these pathways, delineating a glomerular transcriptional polarization reflected by the magnitude of Esm1 deficiency. Diabetes correlates with lower Esm1 expression and with changes in the functional characterization of Esm1(+) cells. Thus, Esm1 appears to be a marker for glomerular transcriptional polarization in DKD.NEW & NOTEWORTHY Esm-1 is primarily expressed in glomerular endothelium in humans. Cells expressing Esm1 exhibit a high degree of conservation in the enrichment of genes related to blood vessel development. In the context of diabetes, these cells are reduced in number and show a significant transcriptional shift toward the chemotaxis pathway. In diabetes, there is a transcriptional polarization in the glomerulus that is reflected by the degree of Esm1 deficiency.

Podocyte-specific silencing of acid sphingomyelinase gene to abrogate hyperhomocysteinemia-induced NLRP3 inflammasome activation and glomerular inflammation.

Acid sphingomyelinase (ASM) has been reported to increase tissue ceramide and thereby mediate hyperhomocysteinemia (hHcy)-induced glomerular nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation, inflammation, and sclerosis. In the present study, we tested whether somatic podocyte-specific silencing of Smpd1 gene (mouse ASM gene code) attenuates hHcy-induced NLRP3 inflammasome activation and associated extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. In vivo, somatic podocyte-specific Smpd1 gene silencing almost blocked hHcy-induced glomerular NLRP3 inflammasome activation in Podocre (podocyte-specific expression of cre recombinase) mice compared with control littermates. By nanoparticle tracking analysis (NTA), floxed Smpd1 shRNA transfection was found to abrogate hHcy-induced elevation of urinary EV excretion in Podocre mice. In addition, Smpd1 gene silencing in podocytes prevented hHcy-induced immune cell infiltration into glomeruli, proteinuria, and glomerular sclerosis in Podocre mice. Such protective effects of podocyte-specific Smpd1 gene silencing were mimicked by global knockout of Smpd1 gene in Smpd1-/- mice. On the contrary, podocyte-specific Smpd1 gene overexpression exaggerated hHcy-induced glomerular pathological changes in Smpd1trg/Podocre (podocyte-specific Smpd1 gene overexpression) mice, which were significantly attenuated by transfection of floxed Smpd1 shRNA. In cell studies, we also confirmed that Smpd1 gene knockout or silencing prevented homocysteine (Hcy)-induced elevation of EV release in the primary cultures of podocyte isolated from Smpd1-/- mice or podocytes of Podocre mice transfected with floxed Smpd1 shRNA compared with WT/WT podocytes. Smpd1 gene overexpression amplified Hcy-induced EV secretion from podocytes of Smpd1trg/Podocre mice, which was remarkably attenuated by transfection of floxed Smpd1 shRNA. Mechanistically, Hcy-induced elevation of EV release from podocytes was blocked by ASM inhibitor (amitriptyline, AMI), but not by NLRP3 inflammasome inhibitors (MCC950 and glycyrrhizin, GLY). Super-resolution microscopy also showed that ASM inhibitor, but not NLRP3 inflammasome inhibitors, prevented the inhibition of lysosome-multivesicular body interaction by Hcy in podocytes. Moreover, we found that podocyte-derived inflammatory EVs (released from podocytes treated with Hcy) induced podocyte injury, which was exaggerated by T cell coculture. Interstitial infusion of inflammatory EVs into renal cortex induced glomerular injury and immune cell infiltration. In conclusion, our findings suggest that ASM in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy and that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effect.NEW & NOTEWORTHY In the present study, we tested whether podocyte-specific silencing of Smpd1 gene attenuates hyperhomocysteinemia (hHcy)-induced nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and associated inflammatory extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. Our findings suggest that acid sphingomyelinase (ASM) in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy. Based on our findings, it is anticipated that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effects.

Mice with a Pax2 missense variant display impaired glomerular repair.

PAX2 regulates kidney development, and its expression persists in parietal epithelial cells (PECs), potentially serving as a podocyte reserve. We hypothesized that mice with a Pax2 pathogenic missense variant (Pax2A220G/+) have impaired PEC-mediated podocyte regeneration. Embryonic wild-type mouse kidneys showed overlapping expression of PAX2/Wilms' tumor-1 (WT-1) until PEC and podocyte differentiation, reflecting a close lineage relationship. Embryonic and adult Pax2A220G/+ mice have reduced nephron number but demonstrated no glomerular disease under baseline conditions. Pax2A220G/+ mice compared with wild-type mice were more susceptible to glomerular disease after adriamycin (ADR)-induced podocyte injury, as demonstrated by worsened glomerular scarring, increased podocyte foot process effacement, and podocyte loss. There was a decrease in PAX2-expressing PECs in wild-type mice after adriamycin injury accompanied by the occurrence of PAX2/WT-1-coexpressing glomerular tuft cells. In contrast, Pax2A220G/+ mice showed no changes in the numbers of PAX2-expressing PECs after adriamycin injury, associated with fewer PAX2/WT-1-coexpressing glomerular tuft cells compared with injured wild-type mice. A subset of PAX2-expressing glomerular tuft cells after adriamycin injury was increased in Pax2A220G/+ mice, suggesting a pathological process given the worse outcomes observed in this group. Finally, Pax2A220G/+ mice have increased numbers of glomerular tuft cells expressing Ki-67 and cleaved caspase-3 compared with wild-type mice after adriamycin injury, consistent with maladaptive responses to podocyte loss. Collectively, our results suggest that decreased glomerular numbers in Pax2A220G/+ mice are likely compounded with the inability of their mutated PECs to regenerate podocyte loss, and together these two mechanisms drive the worsened focal segmental glomerular sclerosis phenotype in these mice.NEW & NOTEWORTHY Congenital anomalies of the kidney and urinary tract comprise some of the leading causes of kidney failure in children, but our previous study showed that one of its genetic causes, PAX2, is also associated with adult-onset focal segmental glomerular sclerosis. Using a clinically relevant model, our present study demonstrated that after podocyte injury, parietal epithelial cells expressing PAX2 are deployed into the glomerular tuft to assist in repair in wild-type mice, but this mechanism is impaired in Pax2A220G/+ mice.

Sparsentan ameliorates glomerular hypercellularity and inflammatory-gene networks induced by IgA1-IgG immune complexes in a mouse model of IgA nephropathy.

IgA nephropathy (IgAN) is characterized by glomerular deposition of immune complexes (ICs) consisting of IgA1 with O-glycans deficient in galactose (Gd-IgA1) and Gd-IgA1-specific IgG autoantibodies. These ICs induce kidney injury, and in the absence of disease-specific therapy, up to 40% of patients with IgAN progress to kidney failure. IgA1 with its clustered O-glycans is unique to humans, which hampered development of small-animal models of IgAN. Here, we used a model wherein engineered ICs (EICs) formed from human Gd-IgA1 and recombinant human IgG autoantibody are injected into nude mice to induce glomerular injury mimicking human IgAN. In this model, we assessed the protective effects of sparsentan, a single-molecule dual endothelin angiotensin receptor antagonist (DEARA) versus vehicle on EIC-induced glomerular proliferation and dysregulation of gene expression in the kidney. Oral administration of sparsentan (60 or 120 mg/kg daily) to mice intravenously injected with EIC attenuated the EIC-induced glomerular hypercellularity. Furthermore, analysis of changes in the whole kidney transcriptome revealed that key inflammatory and proliferative biological genes and pathways that are upregulated in this EIC model of IgAN were markedly reduced by sparsentan, including complement genes, integrin components, members of the mitogen-activated protein kinase family, and Fc receptor elements. Partial overlap between mouse and human differentially expressed genes in IgAN further supported the translational aspect of the immune and inflammatory components from our transcriptional findings. In conclusion, our data indicate that in the mouse model of IgAN, sparsentan targets immune and inflammatory processes leading to protection from mesangial hypercellularity.NEW & NOTEWORTHY The mechanisms by which deposited IgA1 immune complexes cause kidney injury during early phases of IgA nephropathy are poorly understood. We used an animal model we recently developed that involves IgA1-IgG immune complex injections and determined pathways related to the induced mesangioproliferative changes. Treatment with sparsentan, a dual inhibitor of endothelin type A and angiotensin II type 1 receptors, ameliorated the induced mesangioproliferative changes and the associated alterations in the expression of inflammatory genes and networks.

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.

Podocyte Ercc1 is indispensable for glomerular integrity.

As life expectancy continues to increase, age-related kidney diseases are becoming more prevalent. Chronic kidney disease (CKD) is not only a consequence of aging but also a potential accelerator of aging process. Here we report the pivotal role of podocyte ERCC1, a DNA repair factor, in maintaining glomerular integrity and a potential effect on multiple organs. Podocyte-specific ERCC1-knockout mice developed severe proteinuria, glomerulosclerosis, and renal failure, accompanied by a significant increase in glomerular DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). ERCC1 gene transfer experiment in the knockout mice attenuated proteinuria and glomerulosclerosis with reduced DNA damage. Notably, CD44+CD8+ memory T cells, indicative of T-cell senescence, were already elevated in the peripheral blood of knockout mice at 10 weeks old. Additionally, levels of senescence-associated secretory phenotype (SASP) factors were significantly increased in both the circulation and multiple organs of the knockout mice. In older mice and human patients, we observed an accumulation of DSBs and an even greater buildup of SSBs in glomeruli, despite no significant reduction in ERCC1 expression with age in mice. Collectively, our findings highlight the crucial role of ERCC1 in repairing podocyte DNA damage, with potential implications for inflammation in various organs.

Adiponectin Reduces Glomerular Endothelial Glycocalyx Disruption and Restores Glomerular Barrier Function in a Mouse Model of Type 2 Diabetes.

Adiponectin has vascular anti-inflammatory and protective effects. Although adiponectin protects against the development of albuminuria, historically, the focus has been on podocyte protection within the glomerular filtration barrier (GFB). The first barrier to albumin in the GFB is the endothelial glycocalyx (eGlx), a surface gel-like barrier covering glomerular endothelial cells (GEnCs). In diabetes, eGlx dysfunction occurs before podocyte damage; hence, we hypothesized that adiponectin could protect from eGlx damage to prevent early vascular damage in diabetic kidney disease (DKD). Globular adiponectin (gAd) activated AMPK signaling in human GEnCs through AdipoR1. It significantly reduced eGlx shedding and the tumor necrosis factor-α (TNF-α)-mediated increase in syndecan-4 (SDC4) and MMP2 mRNA expression in GEnCs in vitro. It protected against increased TNF-α mRNA expression in glomeruli isolated from db/db mice and against expression of genes associated with glycocalyx shedding (namely, SDC4, MMP2, and MMP9). In addition, gAd protected against increased glomerular albumin permeability (Ps'alb) in glomeruli isolated from db/db mice when administered intraperitoneally and when applied directly to glomeruli (ex vivo). Ps'alb was inversely correlated with eGlx depth in vivo. In summary, adiponectin restored eGlx depth, which was correlated with improved glomerular barrier function, in diabetes.

Oral bacteria induce IgA autoantibodies against a mesangial protein in IgA nephropathy model mice.

IgA nephropathy (IgAN) is caused by deposition of IgA in the glomerular mesangium. The mechanism of selective deposition and production of IgA is unclear; however, we recently identified the involvement of IgA autoantibodies. Here, we show that CBX3 is another self-antigen for IgA in gddY mice, a spontaneous IgAN model, and in IgAN patients. A recombinant antibody derived from gddY mice bound to CBX3 expressed on the mesangial cell surface in vitro and to glomeruli in vivo. An elemental diet and antibiotic treatment decreased the levels of autoantibodies and IgAN symptoms in gddY mice. Serum IgA and the recombinant antibody from gddY mice also bound to oral bacteria of the mice and binding was competed with CBX3. One species of oral bacteria was markedly decreased in elemental diet-fed gddY mice and induced anti-CBX3 antibody in normal mice upon immunization. These data suggest that particular oral bacteria generate immune responses to produce IgA that cross-reacts with mesangial cells to initiate IgAN.

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.

Low dose cadmium inhibits syndecan-4 expression in glycocalyx of glomerular endothelial cells.

Cadmium (Cd) is one of the most polluting heavy metal in the environment. Cd exposure has been elucidated to cause dysfunction of the glomerular filtration barrier (GFB). However, the underlying mechanism remains unclear. C57BL/6J male mice were administered with 2.28 mg/kg cadmium chloride (CdCl2) dissolved in distilled water by oral gavage for 14 days. The expression of SDC4 in the kidney tissues was detected. Human renal glomerular endothelial cells (HRGECs) were exposed to varying concentrations of CdCl2 for 24 h. The mRNA levels of SDC4, along with matrix metalloproteinase (MMP)-2 and 9, were analyzed by quantitative PCR. Additionally, the protein expression levels of SDC4, MMP-2/9, and both total and phosphorylated forms of Smad2/3 (P-Smad2/3) were detected by western blot. The extravasation rate of fluorescein isothiocyanate-dextran through the Transwell was used to evaluate the permeability of HRGECs. SB431542 was used as an inhibitor of transforming growth factor (TGF)-ß signaling pathway to further investigate the role of TGF-ß. Cd reduced SDC4 expression in both mouse kidney tissues and HRGECs. In addition, Cd exposure increased permeability and upregulated P-Smad2/3 levels in HRGECs. SB431542 treatment inhibited the phosphorylation of Smad2/3, Cd-induced SDC4 downregulation, and hyperpermeability. MMP-2/9 levels increased by Cd exposure was also blocked by SB431542, demonstrating the involvement of TGF-ß/Smad pathway in low-dose Cd-induced SDC4 reduction in HRGECs. Given that SDC4 is an essential component of glycocalyx, protection or repair of endothelial glycocalyx is a potential strategy for preventing or treating kidney diseases associated with environmental Cd exposure.

ANGPT2/CAV1 regulates albumin transcytosis of glomerular endothelial cells under high glucose exposure and is impaired by losartan / ANGPT2/CAV1 regula la transcitosis de la albúmina de las células endoteliales glomerulares bajo exposición a hiperglucemia, alterándose con la intervención de losartan

Background Microalbuminuria is a common clinical symptom that manifests in the early stages of diabetic kidney disease (DKD) and is also the main feature of glomerular endothelial cells (GECs) injury. There is increasing evidence that the transcytosis of albumin across GECs is closely related to the formation of albuminuria. Our previous studies have shown that angiopoietin 2 (ANGPT2) can inhibit albumin transcytosis across renal tubular epithelial cells by activating caveolin 1 (CAV1) phosphorylation during high glucose (HG) exposure. The role of ANGPT2 in albumin transcytosis across GECs remains unclear. Losartan significantly reduces albuminuria, but the mechanism has not been clarified. Methods We established an in vitro albumin transcytosis model to investigate the change in albumin transcytosis across human renal glomerular endothelial cells (hrGECs) under normal glucose (NG), high glucose (HG) and losartan intervention. We knocked down ANGPT2 and CAV1 to evaluate their roles in albumin transcytosis across hrGECs and verified the relationship between them. In vivo, DKD mouse models were established and treated with different doses of losartan. Immunohistochemistry and Western blot were used to detect the expression of ANGPT2 and CAV1. Results In vitro, the transcytosis of albumin across hrGECs was significantly increased under high glucose stimulation, and losartan inhibited this process. The expression of ANGPT2 and CAV1 were both increased in hrGECs under HG conditions and losartan intervention reduced the expression of them. Moreover, ANGPT2 downregulation reduced albumin transcytosis in hrGECs by regulating CAV1 expression. In vivo, the expression of ANGPT2 and CAV1 in the glomerulus was both increased significantly in DKD mice. Compared with DKD mice, losartan treatment reduced albuminuria and decreased the expression of ANGPT2 and CAV1 in a dose-dependent manner (AU)

Antecedentes La microalbuminuria es un síntoma clínico común que se manifiesta en las fases tempranas de la enfermedad renal diabética (ERD), y también es característica del daño de las células endoteliales glomerulares (GEC). Existe evidencia creciente en cuanto a que la transcitosis de la albúmina a través de las GEC está estrechamente relacionada con la formación de albuminuria. Nuestros estudios previos reflejaron que angiopoyetina 2 (ANGPT2) puede inhibir la transcitosis de la albúmina a través de las células epiteliales tubulares renales activando la fosforilación de caveolina 1 (CAV1) durante la exposición a hiperglucemia (HG). El rol de ANGPT2 en la transcitosis de la albúmina a través de las GEC resulta incierto. Losartan reduce considerablemente la albuminuria, aunque no se ha esclarecido el mecanismo. Métodos Establecimos un modelo in vitro de transcitosis de la albúmina para investigar el cambio de dicho mecanismo a través de las células endoteliales glomerulares renales humanas (hrGEC) en condiciones de glucosa normal (GN), hiperglucemia (HG) e intervención de losartan. Realizamos breakdown de ANGPT2 y CAV1 para evaluar sus roles en la transcitosis de la albúmina a través de las hrGEC, y verificamos la relación entre ellas. Se establecieron modelos in vivo de ratones con ERD, tratados con diferentes dosis de losartan. Se utilizaron pruebas de inmunohistoquímica e inmunotransferencia para detectar la expresión de ANGPT2 y CAV1. Resultados In vitro, la transcitosis de la albúmina a través de hrGEC se incrementó considerablemente en condiciones de estimulación de la hiperglucemia, inhibiendo losartan este proceso. La expresión de ANGPT2 y CAV1 se incrementó en las hrGEC en condiciones de HG, reduciendo la intervención de losartan la expresión de ambas (AU)

Fasting mimicking diet in diabetic mice partially preserves glomerular endothelial glycocalyx coverage, without changing the diabetic metabolic environment.

Intermittent fasting has become of interest for its possible metabolic benefits and reduction of inflammation and oxidative damage, all of which play a role in the pathophysiology of diabetic nephropathy. We tested in a streptozotocin (60 mg/kg)-induced diabetic apolipoprotein E knockout mouse model whether repeated fasting mimicking diet (FMD) prevents glomerular damage. Diabetic mice received 5 FMD cycles in 10 wk, and during cycles 1 and 5 caloric measurements were performed. After 10 wk, glomerular endothelial morphology was determined together with albuminuria, urinary heparanase-1 activity, and spatial mass spectrometry imaging to identify specific glomerular metabolic dysregulation. During FMD cycles, blood glucose levels dropped while a temporal metabolic switch was observed to increase fatty acid oxidation. Overall body weight at the end of the study was reduced together with albuminuria, although urine production was dramatically increased without affecting urinary heparanase-1 activity. Weight loss was found to be due to lean mass and water, not fat mass. Although capillary loop morphology and endothelial glycocalyx heparan sulfate contents were preserved, hyaluronan surface expression was reduced together with the presence of UDP-glucuronic acid. Mass spectrometry imaging further revealed reduced protein catabolic breakdown products and increased oxidative stress, not different from diabetic mice. In conclusion, although FMD preserves partially glomerular endothelial glycocalyx, loss of lean mass and increased glomerular oxidative stress argue whether such diet regimes are safe in patients with diabetes.NEW & NOTEWORTHY Repeated fasting mimicking diet (FMD) partially prevents glomerular damage in a diabetic mouse model; however, although endothelial glycocalyx heparan sulfate contents were preserved, hyaluronan surface expression was reduced in the presence of UDP-glucuronic acid. The weight loss observed was of lean mass, not fat mass, and increased glomerular oxidative stress argue whether such a diet is safe in patients with diabetes.