The progression of proteinuric kidney diseases is associated with podocyte loss, but the mechanisms underlying this process remain unclear. Podocytes reenter the cell cycle to repair double-stranded DNA breaks. However, unsuccessful repair can result in podocytes crossing the G1/S checkpoint and undergoing abortive cytokinesis. In this study, we identified Pfn1 as indispensable in maintaining glomerular integrity - its tissue-specific loss in mouse podocytes resulted in severe proteinuria and kidney failure. Our results suggest that this phenotype is due to podocyte mitotic catastrophe (MC), characterized histologically and ultrastructurally by abundant multinucleated cells, irregular nuclei, and mitotic spindles. Podocyte cell cycle reentry was identified using FUCCI2aR mice, and we observed altered expression of cell-cycle associated proteins, such as p21, p53, cyclin B1, and cyclin D1. Podocyte-specific translating ribosome affinity purification and RNA-Seq revealed the downregulation of ribosomal RNA-processing 8 (Rrp8). Overexpression of Rrp8 in Pfn1-KO podocytes partially rescued the phenotype in vitro. Clinical and ultrastructural tomographic analysis of patients with diverse proteinuric kidney diseases further validated the presence of MC podocytes and reduction in podocyte PFN1 expression within kidney tissues. These results suggest that profilin1 is essential in regulating the podocyte cell cycle and its disruption leads to MC and subsequent podocyte loss.

Significance Statement The loss of integrity of the glomerular filtration barrier results in proteinuria that is often attributed to podocyte loss. Yet how damaged podocytes are lost remains unknown. Germline loss of murine podocyte-associated Hdac1 and Hdac2 (Hdac1/2) results in proteinuria and collapsing glomerulopathy due to sustained double-stranded DNA damage. Hdac1/2 deletion induces loss of podocyte quiescence, cell cycle entry, arrest in G1, and podocyte senescence, observed both in vivo and in vitro. Through the senescence secretory associated phenotype, podocytes secrete proteins that contribute to their detachment. These results solidify the role of HDACs in cell cycle regulation and senescence, providing important clues in our understanding of how podocytes are lost following injury. Background Intact expression of podocyte histone deacetylases (HDAC) during development is essential for maintaining a normal glomerular filtration barrier because of its role in modulating DNA damage and preventing premature senescence. Methods Germline podocyte-specific Hdac1 and 2 (Hdac1/2) double-knockout mice were generated to examine the importance of these enzymes during development. Results Podocyte-specific loss of Hdac1/2 in mice resulted in severe proteinuria, kidney failure, and collapsing glomerulopathy. Hdac1/2-deprived podocytes exhibited classic characteristics of senescence, such as senescence-associated β-galactosidase activity and lipofuscin aggregates. In addition, DNA damage, likely caused by epigenetic alterations such as open chromatin conformation, not only resulted in podocyte cell-cycle entry as shown in vivo by Ki67 expression and by FUCCI-2aR mice, but also in p21-mediated cell-cycle arrest. Through the senescence secretory associated phenotype, the damaged podocytes secreted proinflammatory cytokines, growth factors, and matrix metalloproteinases, resulting in subsequent podocyte detachment and loss, evidenced by senescent podocytes in urine. Conclusions Hdac1/2 plays an essential role during development. Loss of these genes in double knockout mice leads to sustained DNA damage and podocyte senescence and loss.

Primary proteinuric kidney diseases (PPKD) as a group are an important cause of end-stage kidney disease (ESKD). Many receptor tyrosine kinases, including platelet-derived growth factor receptor (PDGFR), contribute to the progression of PPKDs to ESKD. ANG-3070 is a novel and proprietary inhibitor of multiple tyrosine kinases including PDGFR. This study evaluated the effects of ANG-3070 in a passive Heymann nephritis (PHN) rat model of membranous glomerulopathy.

Objective: To provide evidence on the efficacy and safety of Chinese herbal medicine (CHM) as interventions for systemic lupus erythematosus (SLE). Methods: Seven electronic databases, including the Cochrane Library, Chinese National Knowledge Infrastructure (CNKI), Chinese Scientific Journal Database (VIP), Chinese Biomedical Literature Service System (SinoMed), Wanfang, Embase, and PubMed, were comprehensively searched, from their inception to August 16, 2020, for all randomized controlled trials (RCTs) that focused on CHM used alone or in combination with conventional medicine for SLE. Outcomes were SLE activity index (SLEDAI), traditional Chinese medicine symptom/syndrome score (TCMSS), dosage of glucocorticoids, main serological testing, and incidence of adverse events. Data were extracted and pooled using Review Manager 5.3 software. Results: A total of 13 RCTs enrolling 856 participants met our inclusion criteria. Meta-analyses showed that, compared to placebo, CHM had statistically significant effect on reducing SLEDAI score (MD=-1.74, 95% CI: -2.29 to -1.18), diminishing TCMSS (SMD=-0.89, 95% CI: -1.16 to -0.62), decreasing dosage of glucocorticoids (MD=-2.41 mg/d, 95% CI: -3.34 to -1.48), lowering erythrocyte sedimentation rate (MD=-4.78 mm/h, 95% CI: -8.86 to -0.71), and increasing serum complement C4 level (MD=0.03 mg/dL, 95% CI: 0.00 to 0.06). No significant difference was found between CHM and placebo on adverse events. Conclusions: CHM provided signifificant beneficial effect on controlling disease activity and reducing dose of glucocorticoids used among SLE patients. Future advanced designed RCTs for CHM treating moderate to severe SLE with multicenter and longer follow-up are urgently needed.

Impaired cellular cholesterol efflux is a key factor in the progression of renal, cardiovascular, and autoimmune diseases. Here we describe a class of 5-arylnicotinamide compounds, identified through phenotypic drug discovery, that upregulate ABCA1-dependent cholesterol efflux by targeting Oxysterol Binding Protein Like 7 (OSBPL7). OSBPL7 was identified as the molecular target of these compounds through a chemical biology approach, employing a photoactivatable 5-arylnicotinamide derivative in a cellular cross-linking/immunoprecipitation assay. Further evaluation of two compounds (Cpd A and Cpd G) showed that they induced ABCA1 and cholesterol efflux from podocytes in vitro and normalized proteinuria and prevented renal function decline in mouse models of proteinuric kidney disease: Adriamycin-induced nephropathy and Alport Syndrome. In conclusion, we show that small molecule drugs targeting OSBPL7 reveal an alternative mechanism to upregulate ABCA1, and may represent a promising new therapeutic strategy for the treatment of renal diseases and other disorders of cellular cholesterol homeostasis.

Evidence for reduced expression of cyclin G associated kinase (GAK) in glomeruli of patients with chronic kidney disease was observed in the Nephroseq human database, and GAK was found to be associated with the decline in kidney function. To examine the role of GAK, a protein that functions to uncoat clathrin during endocytosis, we generated podocyte-specific Gak-knockout mice (Gak-KO), which developed progressive proteinuria and kidney failure with global glomerulosclerosis. We isolated glomeruli from the mice carrying the mutation to perform messenger RNA profiling and unearthed evidence for dysregulated podocyte calpain protease activity as an important contributor to progressive podocyte damage. Treatment with calpain inhibitor III specifically inhibited calpain-1/-2 activities, mitigated the degree of proteinuria and glomerulosclerosis, and led to a striking increase in survival in the Gak-KO mice. Podocyte-specific deletion of Capns1, essential for calpain-1 and calpain-2 activities, also improved proteinuria and glomerulosclerosis in Gak-KO mice. Increased podocyte calpain activity–mediated proteolysis of IκBα resulted in increased NF-κB p65–induced expression of growth arrest and DNA-damage-inducible 45 beta in the Gak-KO mice. Our results suggest that loss of podocyte-associated Gak induces glomerular injury secondary to calcium dysregulation and aberrant calpain activation, which when inhibited, can provide a protective role.

Background:Clathrin-mediated endocytosis (CME) plays a fundamental role in podocyte health. Genetic ablation of genes implicated in CME has been shown to cause severe proteinuria and foot process effacement in mice. However, little is known about the cargo of clathrin-coated vesicles (CCVs) in podocytes. The goal of this study was to isolate CCVs from podocytes and identify their cargo by proteomic analysis.Methods:Glomeruli isolated from Podocin-Cre Rosa-DTRflox mouse kidneys were seeded and treated with diphtheria toxin to obtain pure primary podocyte cultures. CCVs were isolated by differential gradient ultracentrifugation, and enrichment of CCVs was assessed by immunoblotting and electron microscopy (EM). Liquid chromatography-mass spectrometry (LC-MS) was performed for proteomic analysis. Proteins with higher abundance than transferrin receptor protein 1 were evaluated for CCV cargo potential against previously published literature. Immunofluorescence staining of identified cargo proteins and CCVs was performed in podocytes for further verification.Results:Immunoblotting for multiple protein markers of CME revealed enrichment in the CCV fraction. Enrichment of CCVs among other small vesicles was observed via EM. Proteomics yielded a total of >1200 significant proteins. Multiple-step data analysis revealed 36 CCV-associated proteins, of which 10 represent novel, highly abundant cargo proteins in podocytes. Colocalization of cargo proteins and CCVs on immunostaining was observed.Conclusions:Our identification of podocyte CCV cargo proteins helps to elucidate the importance of endocytic trafficking for podocyte health and maintenance of the glomerular environment.

BACKGROUND:Epsins, a family of evolutionarily conserved membrane proteins, play an essential role in endocytosis and signaling in podocytes.METHODS:Podocyte-specific Epn1, Epn2, Epn3 triple-knockout mice were generated to examine downstream regulation of serum response factor (SRF) by cell division control protein 42 homolog (Cdc42).RESULTS:Podocyte-specific loss of epsins resulted in increased albuminuria and foot process effacement. Primary podocytes isolated from these knockout mice exhibited abnormalities in cell adhesion and spreading, which may be attributed to reduced activation of cell division control protein Cdc42 and SRF, resulting in diminished β1 integrin expression. In addition, podocyte-specific loss of Srf resulted in severe albuminuria and foot process effacement, and defects in cell adhesion and spreading, along with decreased β1 integrin expression.CONCLUSIONS:Epsins play an indispensable role in maintaining properly functioning podocytes through the regulation of Cdc42 and SRF-dependent β1 integrin expression.

Introduction: As the standard of care for diabetic kidney disease (DKD), angiotensin receptor blockers (ARBs) does not prevent the development of end-stage kidney disease (ESKD) in DKD patients. Tripterygium glucosides (TG) have been widely used for the treatment of various kidney diseases. The additional administration of TG to ARB may provide extra benefit for DKD patients than ARB alone. The aim of this study was to assess the evidence and evaluate the additional benefit and potential harms of TG combined with ARB for the treatment of DKD. Methods: We comprehensively searched six electronic databases for randomized controlled trials (RCTs) with treatment of DKD with TG and ARB. All studies meeting our selection criteria were included and evaluated. Meta-analyses were conducted by using RevMan software. Results: 18 RCTs involving 1160 participants were included. Meta-analyses demonstrated that compared with ARB alone, TG combined with ARB provided a further beneficial therapeutic effect on clinically relevant end points including reduced urinary protein (-0.95 g/24 h, 95 %CI: -1.17 to -0.74), and improved serum albumin (3.53 g/L, 95 %CI: 2.44 to 4.62), but with higher incidence of adverse events (RR: 2.22, 95 %CI:1.32 to 3.73). Conclusion: The combination treatment of TG and ARB showed promising results regarding significant proteinuria reduction and serum albumin improvement for DKD, but with a higher risk of adverse events. Further higher-quality studies are necessary to provide solid evidence to determine a rational treatment strategy including TG, while maximizing antiproteinuric effects and minimizing adverse events for DKD patients.

Defective cholesterol metabolism primarily linked to reduced ATP-binding cassette transporter A1 (ABCA1) expression is closely associated with the pathogenesis and progression of kidney diseases, including diabetic kidney disease and Alport Syndrome. However, whether the accumulation of free or esterified cholesterol contributes to progression in kidney disease remains unclear. Here, we demonstrate that inhibition of sterol-O-acyltransferase-1 (SOAT1), the enzyme at the endoplasmic reticulum that converts free cholesterol to cholesterol esters, which are then stored in lipid droplets, effectively reduced cholesterol ester and lipid droplet formation in human podocytes. Furthermore, we found that inhibition of SOAT1 in podocytes reduced lipotoxicity-mediated podocyte injury in diabetic kidney disease and Alport Syndrome in association with increased ABCA1 expression and ABCA1-mediated cholesterol efflux. In vivo, Soat1 deficient mice did not develop albuminuria or mesangial expansion at 10-12 months of age. However, Soat1 deficiency/inhibition in experimental models of diabetic kidney disease and Alport Syndrome reduced cholesterol ester content in kidney cortices and protected from disease progression. Thus, targeting SOAT1-mediated cholesterol metabolism may represent a new therapeutic strategy to treat kidney disease in patients with diabetic kidney disease and Alport Syndrome, like that suggested for Alzheimer's disease and cancer treatments.

BACKGROUND:Inhibition of the renin-angiotensin system remains a cornerstone in reducing proteinuria and progression of kidney failure, effects believed to be the result of reduction in BP and glomerular hyperfiltration. However, studies have yielded conflicting results on whether podocyte-specific angiotensin II (AngII) signaling directly induces podocyte injury. Previous research has found that after AngII stimulation, β-arrestin-bound angiotensin II receptor type 1 (AT1R) is internalized in a clathrin- and dynamin-dependent manner, and that Dynamin1 and Dynamin2 double-knockout mice exhibit impaired clathrin-mediated endocytosis. METHODS:We used podocyte-specific Dyn double-knockout mice to examine AngII-stimulated AT1R internalization and signaling in primary podocytes and controls. We also examined the in vivo effect of AngII in these double-knockout mice through renin-angiotensin system blockers and through deletion of Agtr1a (which encodes the predominant AT1R isoform expressed in kidney, AT1aR). We tested calcium influx, Rac1 activation, and lamellipodial extension in control and primary podocytes of Dnm double-knockout mice treated with AngII. RESULTS:We confirmed augmented AngII-stimulated AT1R signaling in primary Dnm double-knockout podocytes resulting from arrest of clathrin-coated pit turnover. Genetic ablation of podocyte Agtr1a in Dnm double-knockout mice demonstrated improved albuminuria and kidney function compared with the double-knockout mice. Isolation of podocytes from Dnm double-knockout mice revealed abnormal membrane dynamics, with increased Rac1 activation and lamellipodial extension, which was attenuated in Dnm double-knockout podocytes lacking AT1aR. CONCLUSIONS:Our results indicate that inhibiting aberrant podocyte-associated AT1aR signaling pathways has a protective effect in maintaining the integrity of the glomerular filtration barrier.

Sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) is a lipid raft enzyme that regulates plasma membrane (PM) fluidity. Here we report that SMPDL3b excess, as observed in podocytes in diabetic kidney disease (DKD), impairs insulin receptor isoform B-dependent pro-survival insulin signaling by interfering with insulin receptor isoforms binding to caveolin-1 in the PM. SMPDL3b excess affects the production of active sphingolipids resulting in decreased ceramide-1-phosphate (C1P) content as observed in human podocytes in vitro and in kidney cortexes of diabetic db/db mice in vivo. Podocyte-specific Smpdl3b deficiency in db/db mice is sufficient to restore kidney cortex C1P content and to protect from DKD. Exogenous administration of C1P restores IR signaling in vitro and prevents established DKD progression in vivo. Taken together, we identify SMPDL3b as a modulator of insulin signaling and demonstrate that supplementation with exogenous C1P may represent a lipid therapeutic strategy to treat diabetic complications such as DKD.

Fibroblasts from patients with Tangier disease carrying ATP-binding cassette A1 (ABCA1) loss-of-function mutations are characterized by cardiolipin accumulation, a mitochondrial-specific phospholipid. Suppression of ABCA1 expression occurs in glomeruli from patients with diabetic kidney disease (DKD) and in human podocytes exposed to DKD sera collected prior to the development of DKD. We demonstrated that siRNA ABCA1 knockdown in podocytes led to reduced oxygen consumption capabilities associated with alterations in the oxidative phosphorylation (OXPHOS) complexes and with cardiolipin accumulation. Podocyte-specific deletion of Abca1 (Abca1(fl/fl)) rendered mice susceptible to DKD, and pharmacological induction of ABCA1 improved established DKD. This was not mediated by free cholesterol, as genetic deletion of sterol-o-acyltransferase-1 (SOAT1) in Abca1(fl/fl) mice was sufficient to cause free cholesterol accumulation but did not cause glomerular injury. Instead, cardiolipin mediates ABCA1-dependent susceptibility to podocyte injury, as inhibition of cardiolipin peroxidation with elamipretide improved DKD in vivo and prevented ABCA1-dependent podocyte injury in vitro and in vivo. Collectively, we describe a pathway definitively linking ABCA1 deficiency to cardiolipin-driven mitochondrial dysfunction. We demonstrated that this pathway is relevant to DKD and that ABCA1 inducers or inhibitors of cardiolipin peroxidation may each represent therapeutic strategies for the treatment of established DKD.

We identified 2 genes, histone deacetylase 1 (HDAC1) and HDAC2, contributing to the pathogenesis of proteinuric kidney diseases, the leading cause of end-stage kidney disease. mRNA expression profiling from proteinuric mouse glomeruli was linked to Connectivity Map databases, identifying HDAC1 and HDAC2 with the differentially expressed gene set reversible by HDAC inhibitors. In numerous progressive glomerular disease models, treatment with valproic acid (a class I HDAC inhibitor) or SAHA (a pan-HDAC inhibitor) mitigated the degree of proteinuria and glomerulosclerosis, leading to a striking increase in survival. Podocyte HDAC1 and HDAC2 activities were increased in mice podocytopathy models, and podocyte-associated Hdac1 and Hdac2 genetic ablation improved proteinuria and glomerulosclerosis. Podocyte early growth response 1 (EGR1) was increased in proteinuric patients and mice in an HDAC1- and HDAC2-dependent manner. Loss of EGR1 in mice reduced proteinuria and glomerulosclerosis. Longitudinal analysis of the multicenter Veterans Aging Cohort Study demonstrated a 30% reduction in mean annual loss of estimated glomerular filtration rate, and this effect was more pronounced in proteinuric patients receiving valproic acid. These results strongly suggest that inhibition of HDAC1 and HDAC2 activities may suppress the progression of human proteinuric kidney diseases through the regulation of EGR1.

Cell-matrix interactions and podocyte intercellular junctions are key for maintaining the glomerular filtration barrier. Vinculin, a cytoplasmic protein, couples actin filaments to integrin-mediated cell-matrix adhesions and to cadherin-based intercellular junctions. Here, we examined the role of vinculin in podocytes by the generation of a podocyte-specific knockout mouse. Mice lacking podocyte vinculin had increased albuminuria and foot process effacement following injury in vivo. Analysis of primary podocytes isolated from the mutant mice revealed defects in cell protrusions, altered focal adhesion size and signaling, as well as impaired cell migration. Furthermore, we found a marked mislocalization of the intercellular junction protein zonula occludens-1. In kidney sections from patients with focal segmental glomerulosclerosis, minimal change disease and membranous nephropathy, we observed dramatic differences in the expression levels and localization of vinculin. Thus, our results suggest that vinculin is necessary to maintain the integrity of the glomerular filtration barrier by modulating podocyte foot processes and stabilizing intercellular junctions.

Esterified cholesterol content is often lower than free cholesterol content in biological systems and thus the determination of the esterified cholesterol content of cells is often challenging. Traditional methods use enzymatic assays in which an indirect measurement of the esterified cholesterol content is obtained by subtracting the measurements of the free from the total cholesterol content. However, this approach fails in the case where the total cholesterol content of cells is unchanged while the ratio of free to esterified cholesterol changes such that total and free cholesterol content are very similar and thus the difference may fall within the background noise of the enzymatic assay. To overcome this challenge, we here describe a method that utilizes a TLC-based technique to isolate esterified cholesterol. Isolated esterified cholesterol can then be measured using traditional enzymatic methods. Therefore, this method provides a practical and more sensitive assay to measure esterified cholesterol content in cellular extracts.

High levels of circulating TNF and its receptors, TNFR1 and TNFR2, predict the progression of diabetic kidney disease (DKD), but their contribution to organ damage in DKD remains largely unknown. Here, we investigated the function of local and systemic TNF in podocyte injury. We cultured human podocytes with sera collected from DKD patients, who displayed elevated TNF levels, and focal segmental glomerulosclerosis (FSGS) patients, whose TNF levels resembled those of healthy patients. Exogenous TNF administration or local TNF expression was equally sufficient to cause free cholesterol-dependent apoptosis in podocytes by acting through a dual mechanism that required a reduction in ATP-binding cassette transporter A1-mediated (ABCA1-mediated) cholesterol efflux and reduced cholesterol esterification by sterol-O-acyltransferase 1 (SOAT1). TNF-induced albuminuria was aggravated in mice with podocyte-specific ABCA1 deficiency and was partially prevented by cholesterol depletion with cyclodextrin. TNF-stimulated free cholesterol-dependent apoptosis in podocytes was mediated by nuclear factor of activated T cells 1 (NFATc1). ABCA1 overexpression or cholesterol depletion was sufficient to reduce albuminuria in mice with podocyte-specific NFATc1 activation. Our data implicate an NFATc1/ABCA1-dependent mechanism in which local TNF is sufficient to cause free cholesterol-dependent podocyte injury irrespective of TNF, TNFR1, or TNFR2 serum levels.