Autoantibodies Targeting Nephrin in Podocytopathies.

BACKGROUND: Minimal change disease and primary focal segmental glomerulosclerosis in adults, along with idiopathic nephrotic syndrome in children, are immune-mediated podocytopathies that lead to nephrotic syndrome. Autoantibodies targeting nephrin have been found in patients with minimal change disease, but their clinical and pathophysiological roles are unclear. METHODS: We conducted a multicenter study to analyze antinephrin autoantibodies in adults with glomerular diseases, including minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, IgA nephropathy, antineutrophil cytoplasmic antibody-associated glomerulonephritis, and lupus nephritis, as well as in children with idiopathic nephrotic syndrome and in controls. We also created an experimental mouse model through active immunization with recombinant murine nephrin. RESULTS: The study included 539 patients (357 adults and 182 children) and 117 controls. Among the adults, antinephrin autoantibodies were found in 46 of the 105 patients (44%) with minimal change disease, 7 of 74 (9%) with primary focal segmental glomerulosclerosis, and only in rare cases among the patients with other conditions. Of the 182 children with idiopathic nephrotic syndrome, 94 (52%) had detectable antinephrin autoantibodies. In the subgroup of patients with active minimal change disease or idiopathic nephrotic syndrome who were not receiving immunosuppressive treatment, the prevalence of antinephrin autoantibodies was as high as 69% and 90%, respectively. At study inclusion and during follow-up, antinephrin autoantibody levels were correlated with disease activity. Experimental immunization induced a nephrotic syndrome, a minimal change disease-like phenotype, IgG localization to the podocyte slit diaphragm, nephrin phosphorylation, and severe cytoskeletal changes in mice. CONCLUSIONS: In this study, circulating antinephrin autoantibodies were common in patients with minimal change disease or idiopathic nephrotic syndrome and appeared to be markers of disease activity. Their binding at the slit diaphragm induced podocyte dysfunction and nephrotic syndrome, which highlights their pathophysiological significance. (Funded by Deutsche Forschungsgemeinschaft and others.).

The Dual-specificity Phosphatase 3 (DUSP3): A Potential Target Against Renal Ischemia/Reperfusion Injury.

Renal ischemia/reperfusion (I/R) injury is a common clinical challenge faced by clinicians in kidney transplantation. I/R is the leading cause of acute kidney injury, and it occurs when blood flow to the kidney is interrupted and subsequently restored. I/R impairs renal function in both short and long terms. Renal ischemic preconditioning refers to all maneuvers intended to prevent or attenuate ischemic damage. In this context, the present review focuses on the dual-specificity phosphatase 3 (DUSP3), also known as vaccinia H1-related phosphatase, an uncommon regulator of mitogen-activated protein kinase (MAPK) phosphorylation. DUSP3 has different biological functions: (1) it acts as a tumor modulator and (2) it is involved in the regulation of immune response, thrombosis, hemostasis, angiogenesis, and genomic stability. These functions occur either through MAPK-dependent or MAPK-independent mechanisms. DUSP3 genetic deletion dampens kidney damage and inflammation caused by I/R in mice, suggesting DUSP3 as a potential target for preventing renal I/R injury. Here, we discuss the putative role of DUSP3 in ischemic preconditioning and the potential mechanisms of such an attenuated inflammatory response via improved kidney perfusion and adequate innate immune response.

An integrated organoid omics map extends modeling potential of kidney disease.

Kidney organoids are a promising model to study kidney disease, but their use is constrained by limited knowledge of their functional protein expression profile. Here, we define the organoid proteome and transcriptome trajectories over culture duration and upon exposure to TNFα, a cytokine stressor. Older organoids increase deposition of extracellular matrix but decrease expression of glomerular proteins. Single cell transcriptome integration reveals that most proteome changes localize to podocytes, tubular and stromal cells. TNFα treatment of organoids results in 322 differentially expressed proteins, including cytokines and complement components. Transcript expression of these 322 proteins is significantly higher in individuals with poorer clinical outcomes in proteinuric kidney disease. Key TNFα-associated protein (C3 and VCAM1) expression is increased in both human tubular and organoid kidney cell populations, highlighting the potential for organoids to advance biomarker development. By integrating kidney organoid omic layers, incorporating a disease-relevant cytokine stressor and comparing with human data, we provide crucial evidence for the functional relevance of the kidney organoid model to human kidney disease.

Expansion-enhanced super-resolution radial fluctuations enable nanoscale molecular profiling of pathology specimens.

Expansion microscopy physically enlarges biological specimens to achieve nanoscale resolution using diffraction-limited microscopy systems1. However, optimal performance is usually reached using laser-based systems (for example, confocal microscopy), restricting its broad applicability in clinical pathology, as most centres have access only to light-emitting diode (LED)-based widefield systems. As a possible alternative, a computational method for image resolution enhancement, namely, super-resolution radial fluctuations (SRRF)2,3, has recently been developed. However, this method has not been explored in pathology specimens to date, because on its own, it does not achieve sufficient resolution for routine clinical use. Here, we report expansion-enhanced super-resolution radial fluctuations (ExSRRF), a simple, robust, scalable and accessible workflow that provides a resolution of up to 25 nm using LED-based widefield microscopy. ExSRRF enables molecular profiling of subcellular structures from archival formalin-fixed paraffin-embedded tissues in complex clinical and experimental specimens, including ischaemic, degenerative, neoplastic, genetic and immune-mediated disorders. Furthermore, as examples of its potential application to experimental and clinical pathology, we show that ExSRRF can be used to identify and quantify classical features of endoplasmic reticulum stress in the murine ischaemic kidney and diagnostic ultrastructural features in human kidney biopsies.

Accumulation of α-synuclein mediates podocyte injury in Fabry nephropathy.

Current therapies for Fabry disease are based on reversing intracellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosomal dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease, remains unclear. In this study, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/Cas9-mediated α-galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified α-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.

Single-cell transcriptomics reveals a mechanosensitive injury signaling pathway in early diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is the leading cause of end-stage renal disease, and histopathologic glomerular lesions are among the earliest structural alterations of DN. However, the signaling pathways that initiate these glomerular alterations are incompletely understood. METHODS: To delineate the cellular and molecular basis for DN initiation, we performed single-cell and bulk RNA sequencing of renal cells from type 2 diabetes mice (BTBR ob/ob) at the early stage of DN. RESULTS: Analysis of differentially expressed genes revealed glucose-independent responses in glomerular cell types. The gene regulatory network upstream of glomerular cell programs suggested the activation of mechanosensitive transcriptional pathway MRTF-SRF predominantly taking place in mesangial cells. Importantly, activation of MRTF-SRF transcriptional pathway was also identified in DN glomeruli in independent patient cohort datasets. Furthermore, ex vivo kidney perfusion suggested that the regulation of MRTF-SRF is a common mechanism in response to glomerular hyperfiltration. CONCLUSIONS: Overall, our study presents a comprehensive single-cell transcriptomic landscape of early DN, highlighting mechanosensitive signaling pathways as novel targets of diabetic glomerulopathy.

VPS34-dependent control of apical membrane function of proximal tubule cells and nutrient recovery by the kidney.

The lipid kinase VPS34 orchestrates autophagy, endocytosis, and metabolism and is implicated in cancer and metabolic disease. The proximal tubule in the kidney is a key metabolic organ that controls reabsorption of nutrients such as fatty acids, amino acids, sugars, and proteins. Here, by combining metabolomics, proteomics, and phosphoproteomics analyses with functional and superresolution imaging assays of mice with an inducible deficiency in proximal tubular cells, we revealed that VPS34 controlled the metabolome of the proximal tubule. In addition to inhibiting pinocytosis and autophagy, VPS34 depletion induced membrane exocytosis and reduced the abundance of the retromer complex necessary for proper membrane recycling and lipid retention, leading to a loss of fuel and biomass. Integration of omics data into a kidney cell metabolomic model demonstrated that VPS34 deficiency increased ß-oxidation, reduced gluconeogenesis, and enhanced the use of glutamine for energy consumption. Furthermore, the omics datasets revealed that VPS34 depletion triggered an antiviral response that included a decrease in the abundance of apically localized virus receptors such as ACE2. VPS34 inhibition abrogated SARS-CoV-2 infection in human kidney organoids and cultured proximal tubule cells in a glutamine-dependent manner. Thus, our results demonstrate that VPS34 adjusts endocytosis, nutrient transport, autophagy, and antiviral responses in proximal tubule cells in the kidney.

A high-throughput drug discovery pipeline to optimize kidney normothermic machine perfusion.

Kidney transplantation is the only definitive therapy for end-stage kidney disease. The shortage of organs for transplantation is the main limitation of this life-saving treatment. Normothermic machine perfusion (NMP) is a novel preservation technique with the potential to increase the number of transplantable kidneys through reducing delayed graft function and organ evaluation under physiological conditions. To date, the cellular effects and possible pharmacological interventions during machine perfusion are incompletely understood. A major limitation is the technically complex, time-consuming, and small-scale replication of NMP in rodent models. To overcome this, we developed a 3D-printed, high throughput ex-vivo mouse kidney slice incubator (KSI) mimicking mouse kidney NMP by working under closely resembling conditions. KSI significantly reduced the time per experiment and increased the sample throughput (theoretical: 54 incubations with n = 500/day). The model recapitulated the cellular responses during NMP, namely increased endoplasmic reticulum stress (ER stress). Using KSI, five pharmacological interventions against ER stress taken from the literature were tested. While four were ineffective and excluded, one, ß-Nicotinamide-adenine-dinucleotide (NADH), ameliorated ER stress significantly during KSI. The test of NADH in mouse kidney NMP replicated the positive effects against ER stress. This suggests that testing the addition of NADH during clinical kidney NMP might be warranted.

A slit-diaphragm-associated protein network for dynamic control of renal filtration.

The filtration of blood in the kidney which is crucial for mammalian life is determined by the slit-diaphragm, a cell-cell junction between the foot processes of renal podocytes. The slit-diaphragm is thought to operate as final barrier or as molecular sensor of renal filtration. Using high-resolution proteomic analysis of slit-diaphragms affinity-isolated from rodent kidney, we show that the native slit-diaphragm is built from the junction-forming components Nephrin, Neph1 and Podocin and a co-assembled high-molecular weight network of proteins. The network constituents cover distinct classes of proteins including signaling-receptors, kinases/phosphatases, transporters and scaffolds. Knockout or knock-down of either the core components or the selected network constituents tyrosine kinase MER (MERTK), atrial natriuretic peptide-receptor C (ANPRC), integral membrane protein 2B (ITM2B), membrane-associated guanylate-kinase, WW and PDZ-domain-containing protein1 (MAGI1) and amyloid protein A4 resulted in target-specific impairment or disruption of the filtration process. Our results identify the slit-diaphragm as a multi-component system that is endowed with context-dependent dynamics via a co-assembled protein network.

Rodent models to study sodium retention in experimental nephrotic syndrome.

Sodium retention and edema are hallmarks of nephrotic syndrome (NS). Different experimental rodent models have been established for simulating NS, however, not all of them feature sodium retention which requires proteinuria to exceed a certain threshold. In rats, puromycin aminonucleoside nephrosis (PAN) is a classic NS model introduced in 1955 that was adopted as doxorubicin-induced nephropathy (DIN) in 129S1/SvImJ mice. In recent years, mice with inducible podocin deletion (Nphs2Δipod ) or podocyte apoptosis (POD-ATTAC) have been developed. In these models, sodium retention is thought to be caused by activation of the epithelial sodium channel (ENaC) in the distal nephron through aberrantly filtered serine proteases or proteasuria. Strikingly, rodent NS models follow an identical chronological time course after the development of proteinuria featuring sodium retention within days and spontaneous reversal thereafter. In DIN and Nphs2Δipod mice, inhibition of ENaC by amiloride or urinary serine protease activity by aprotinin prevents sodium retention, opening up new and promising therapeutic approaches that could be translated into the treatment of nephrotic patients. However, the essential serine protease(s) responsible for ENaC activation is (are) still unknown. With the use of nephrotic rodent models, there is the possibility that this (these) will be identified in the future. This review summarizes the various rodent models used to study experimental nephrotic syndrome and the insights gained from these models with regard to the pathophysiology of sodium retention.

Loss of the collagen IV modifier prolyl 3-hydroxylase 2 causes thin basement membrane nephropathy.

The glomerular filtration barrier (GFB) produces primary urine and is composed of a fenestrated endothelium, a glomerular basement membrane (GBM), podocytes, and a slit diaphragm. Impairment of the GFB leads to albuminuria and microhematuria. The GBM is generated via secreted proteins from both endothelial cells and podocytes and is supposed to majorly contribute to filtration selectivity. While genetic mutations or variations of GBM components have been recently proposed to be a common cause of glomerular diseases, pathways modifying and stabilizing the GBM remain incompletely understood. Here, we identified prolyl 3-hydroxylase 2 (P3H2) as a regulator of the GBM in an a cohort of patients with albuminuria. P3H2 hydroxylates the 3' of prolines in collagen IV subchains in the endoplasmic reticulum. Characterization of a P3h2ΔPod mouse line revealed that the absence of P3H2 protein in podocytes induced a thin basement membrane nephropathy (TBMN) phenotype with a thinner GBM than that in WT mice and the development of microhematuria and microalbuminuria over time. Mechanistically, differential quantitative proteomics of the GBM identified a significant decrease in the abundance of collagen IV subchains and their interaction partners in P3h2ΔPod mice. To our knowledge, P3H2 protein is the first identified GBM modifier, and loss or mutation of P3H2 causes TBMN and focal segmental glomerulosclerosis in mice and humans.

Collateral Effects and Mortality of Kidney Transplant Recipients during the COVID-19 Pandemic.

Background: Collateral effects and consequences of the coronavirus disease 19 (COVID-19) pandemic on kidney transplant recipients remain widely unknown. Methods: This retrospective cohort study examined changes in admission rates, incidences of diseases leading to hospitalization, in-patient procedures, and maintenance medication in long-term kidney transplant recipients with functioning graft during the early COVID-19 pandemic in Germany. Data were derived from a nationwide health insurance database. Analysis was performed from March 15 to September 30 and compared the years 2019 and 2020. Effects on mortality and adverse allograft events were compared with COVID-19-attributed effects. Results: A total of 7725 patients were included in the final analysis. Admissions declined in 2020 by 17%, with the main dip during a 3-month lockdown (-31%) but without a subsequent rebound. Incidences for hospitalization did not increase for any investigated disease entities, whereas decreasing trends were noted for non-COVID-19 pulmonary and urogenital infections (incidence rate ratio 0.8, 95% CI, 0.62 to 1.03, and 0.82, 95% CI, 0.65 to 1.04, respectively). Non-COVID-19 hospital stays were 0.6 days shorter (P=0.03) and not complicated by increased dialysis, ventilation, or intensive care treatment rates. In-hospital and 90-day mortality remained stable. Incidences of severe COVID-19 requiring hospitalization was 0.09 per 1000 patient-days, and in-hospital mortality was 9%. A third (31%) of patients with calcineurin-inhibitor medication and without being hospitalized for COVID-19 reduced doses by at least 25%, which was associated with an increased allograft rejection risk (adjusted hazard ratio 1.29, 95% CI, 1.02 to 1.63). COVID-19 caused 17% of all deaths but had no significant association with allograft rejections. All-cause mortality remained stable (incidence rate ratio 1.15, 95% CI, 0.91 to 1.46), also when restricting analysis to patients with no or outpatient-treated COVID-19 (0.97, 95% CI, 0.76 to 1.25). Conclusion: Despite significant collateral effects, mortality remained unchanged during the early COVID-19 pandemic. Considerable temporary reductions in admissions are safe, whereas reducing immunosuppression results in increased allograft rejection risk.

Proteinuric chronic kidney disease is associated with altered red blood cell lifespan, deformability and metabolism.

Anemia is a common complication of chronic kidney disease, affecting the quality of life of patients. Among various factors, such as iron and erythropoietin deficiency, reduced red blood cell (RBC) lifespan has been implicated in the pathogenesis of anemia. However, mechanistic data on in vivo RBC dysfunction in kidney disease are lacking. Herein, we describe the development of chronic kidney disease-associated anemia in mice with proteinuric kidney disease resulting from either administration of doxorubicin or an inducible podocin deficiency. In both experimental models, anemia manifested at day 10 and progressed at day 30 despite increased circulating erythropoietin levels and erythropoiesis in the bone marrow and spleen. Circulating RBCs in both mouse models displayed altered morphology and diminished osmotic-sensitive deformability together with increased phosphatidylserine externalization on the outer plasma membrane, a hallmark of RBC death. Fluorescence-labelling of RBCs at day 20 of mice with doxorubicin-induced kidney disease revealed premature clearance from the circulation. Metabolomic analyses of RBCs from both mouse models demonstrated temporal changes in redox recycling pathways and Lands' cycle, a membrane lipid remodeling process. Anemic patients with proteinuric kidney disease had an increased proportion of circulating phosphatidylserine-positive RBCs. Thus, our observations suggest that reduced RBC lifespan, mediated by altered RBC metabolism, reduced RBC deformability, and enhanced cell death contribute to the development of anemia in proteinuric kidney disease.

Role of mTOR Signaling for Tubular Function and Disease.

The mechanistic target of rapamycin (mTOR) forms two distinct intracellular multiprotein complexes that control a multitude of intracellular processes linked to metabolism, proliferation, actin cytoskeleton, and survival. Recent studies have identified the importance of these complexes for transport regulation of ions and nutrients along the entire nephron. First reports could link altered activity of these complexes to certain disease entities, i.e. diabetic nephropathy, acute kidney injury or hyperkalemia.

Essential role of DNA-PKcs and plasminogen for the development of doxorubicin-induced glomerular injury in mice.

Susceptibility to doxorubicin-induced nephropathy (DIN), a toxic model for the induction of proteinuria in mice, is related to the single-nucleotide polymorphism (SNP) C6418T of the Prkdc gene encoding for the DNA-repair enzyme DNA-PKcs. In addition, plasminogen (Plg) has been reported to play a role in glomerular damage. Here, we investigated the interdependence of both factors for the development of DIN. Genotyping confirmed the SNP of the Prkdc gene in C57BL/6 (PrkdcC6418/C6418) and 129S1/SvImJ (PrkdcT6418/T6418) mice. Intercross of heterozygous 129SB6F1 mice led to 129SB6F2 hybrids with Mendelian inheritance of the SNP. After doxorubicin injection, only homozygous F2 mice with PrkdcT6418/T6418 developed proteinuria. Genetic deficiency of Plg (Plg-/-) in otherwise susceptible 129S1/SvImJ mice led to resistance to DIN. Immunohistochemistry revealed glomerular binding of Plg in Plg+/+ mice after doxorubicin injection involving histone H2B as Plg receptor. In doxorubicin-resistant C57BL/6 mice, Plg binding was absent. In conclusion, susceptibility to DIN in 129S1/SvImJ mice is determined by a hierarchical two-hit process requiring the C6418T SNP in the Prkdc gene and subsequent glomerular binding of Plg. This article has an associated First Person interview with the first author of the paper.