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Dysregulation of autophagy in diabetic kidney disease (DKD) has been reported, but the underlying mechanism and its pathogenic role remain elusive. We show that autophagy was inhibited in DKD models and in human diabetic kidneys. Ablation of autophagy-related gene 7 (Atg7) from kidney proximal tubules led to autophagy deficiency and worse renal hypertrophy, tubular damage, inflammation, fibrosis, and albuminuria in diabetic mice, indicating a protective role of autophagy in DKD. Autophagy impairment in DKD was associated with the downregulation of unc-51-like autophagy-activating kinase 1 (ULK1), which was mediated by the upregulation of microRNA-214 (miR-214) in diabetic kidney cells and tissues. Ablation of miR-214 from kidney proximal tubules prevented a decrease in ULK1 expression and autophagy impairment in diabetic kidneys, resulting in less renal hypertrophy and albuminuria. Furthermore, blockade of p53 attenuated miR-214 induction in DKD, leading to higher levels of ULK1 and autophagy, accompanied by an amelioration of DKD. Compared with nondiabetic samples, renal biopsies from patients with diabetes showed induction of p53 and miR-214, associated with downregulation of ULK1 and autophagy. We found a positive correlation between p53/miR-214 and renal fibrosis, but a negative correlation between ULK1/LC3 and renal fibrosis in patients with diabetes. Together, these results identify the p53/miR-214/ULK1 axis in autophagy impairment in diabetic kidneys, pinpointing possible therapeutic targets for DKD.

BACKGROUNDProteinuria is considered an unfavorable clinical condition that accelerates renal and cardiovascular disease. However, it is not clear whether all forms of proteinuria are damaging. Mutations in CUBN cause Imerslund-Gräsbeck syndrome (IGS), which is characterized by intestinal malabsorption of vitamin B12 and in some cases proteinuria. CUBN encodes for cubilin, an intestinal and proximal tubular uptake receptor containing 27 CUB domains for ligand binding.METHODSWe used next-generation sequencing for renal disease genes to genotype cohorts of patients with suspected hereditary renal disease and chronic proteinuria. CUBN variants were analyzed using bioinformatics, structural modeling, and epidemiological methods.RESULTSWe identified 39 patients, in whom biallelic pathogenic variants in the CUBN gene were associated with chronic isolated proteinuria and early childhood onset. Since the proteinuria in these patients had a high proportion of albuminuria, glomerular diseases such as steroid-resistant nephrotic syndrome or Alport syndrome were often the primary clinical diagnosis, motivating renal biopsies and the use of proteinuria-lowering treatments. However, renal function was normal in all cases. By contrast, we did not found any biallelic CUBN variants in proteinuric patients with reduced renal function or focal segmental glomerulosclerosis. Unlike the more N-terminal IGS mutations, 37 of the 41 proteinuria-associated CUBN variants led to modifications or truncations after the vitamin B12-binding domain. Finally, we show that 4 C-terminal CUBN variants are associated with albuminuria and slightly increased GFR in meta-analyses of large population-based cohorts.CONCLUSIONCollectively, our data suggest an important role for the C-terminal half of cubilin in renal albumin reabsorption. Albuminuria due to reduced cubilin function could be an unexpectedly common benign condition in humans that may not require any proteinuria-lowering treatment or renal biopsy.FUNDINGATIP-Avenir program, Fondation Bettencourt-Schueller (Liliane Bettencourt Chair of Developmental Biology), Agence Nationale de la Recherche (ANR) Investissements d'avenir program (ANR-10-IAHU-01) and NEPHROFLY (ANR-14-ACHN-0013, to MS), Steno Collaborative Grant 2018 (NNF18OC0052457, to TSA and MS), Heisenberg Professorship of the German Research Foundation (KO 3598/5-1, to AK), Deutsche Forschungsgemeinschaft (DFG) Collaborative Research Centre (SFB) KIDGEM 1140 (project 246781735, to CB), and Federal Ministry of Education and Research (BMB) (01GM1515C, to CB).

Diabetic nephropathy is the most common cause of end-stage renal disease. Shu Wakino and colleagues now show that high-glucose conditions in the renal proximal tubules result in downregulation of Sirt1 expression there and in the glomeruli, resulting in epigentic upregulation of Claudin-1 in the glomeruli and thus proteinuria. They also show that genetic or chemical targeting of Sirt1 in the kidney is sufficient to improve kidney function in a mouse model of diabetic nephropathy. Sirtuin 1 (Sirt1), a NAD + -regulated deacetylase with numerous known positive effects on cellular and whole-body metabolism, is expressed in the renal cortex and medulla. It is known to have protective effects against age-related disease, including diabetes. Here we investigated the protective role of Sirt1 in diabetic renal damage. We found that Sirt1 in proximal tubules (PTs) was downregulated before albuminuria occurred in streptozotocin-induced or obese ( db / db ) diabetic mice. PT-specific SIRT1 transgenic and Sirt1 knockout mice showed prevention and aggravation of the glomerular changes that occur in diabetes, respectively, and nondiabetic knockout mice exhibited albuminuria, suggesting that Sirt1 in PTs affects glomerular function. Downregulation of Sirt1 and upregulation of the tight junction protein Claudin-1 by SIRT1-mediated epigenetic regulation in podocytes contributed to albuminuria. We did not observe these phenomena in 5/6 nephrectomized mice. We also demonstrated retrograde interplay from PTs to glomeruli using nicotinamide mononucleotide (NMN) from conditioned medium, measurement of the autofluorescence of photoactivatable NMN and injection of fluorescence-labeled NMN. In human subjects with diabetes, the levels of SIRT1 and Claudin-1 were correlated with proteinuria levels. These results suggest that Sirt1 in PTs protects against albuminuria in diabetes by maintaining NMN concentrations around glomeruli, thus influencing podocyte function.

Monocyte/macrophage recruitment correlates strongly with the progression of diabetic nephropathy. Tumor necrosis factor-α (TNF-α) is produced by monocytes/macrophages but the direct role of TNF-α and/or macrophage-derived TNF-α in the progression of diabetic nephropathy remains unclear. Here we tested whether inhibition of TNF-α confers kidney protection in diabetic nephropathy via a macrophage-derived TNF-α-dependent pathway. Compared to vehicle-treated mice, blockade of TNF-α with a murine anti-TNF-α antibody conferred kidney protection in Ins2Akita mice as indicated by reductions in albuminuria, plasma creatinine, histopathologic changes, kidney macrophage recruitment, and plasma inflammatory cytokine levels at 18 weeks of age. To assess the direct role of macrophage-derived TNF-α in diabetic nephropathy, we generated macrophage-specific TNF-α-deficient mice (CD11bCre/TNF-αFlox/Flox). Conditional ablation of TNF-α in macrophages significantly reduced albuminuria, the increase in plasma creatinine and blood urea nitrogen, histopathologic changes, and kidney macrophage recruitment compared to diabetic TNF-αFlox/Flox control mice after 12 weeks of streptozotocin-induced diabetes. Thus, production of TNF-α by macrophages plays a major role in diabetic renal injury. Hence, blocking TNF-α could be a novel therapeutic approach for treatment of diabetic nephropathy.

Abstract Background Obesity and type 2 diabetes (T2D) are correlated risk factors for chronic kidney disease (CKD). Methods Using summary data from GIANT (Genetic Investigation of Anthropometric Traits), DIAGRAM (DIAbetes Genetics Replication And Meta-analysis), and CKDGen (CKD Genetics), we examined causality and directionality of the association between obesity and kidney function. Bidirectional 2-sample Mendelian randomization (MR) estimated the total causal effects of body mass index (BMI) and waist-to-hip ratio (WHR) on kidney function, and vice versa. Effects of adverse obesity and T2D were examined by stratifying BMI variants by their association with WHR and T2D. Multivariable MR estimated the direct causal effects of BMI and WHR on kidney function. The inverse variance weighted random-effects MR for Europeans was the main analysis, accompanied by several sensitivity MR analyses. Results One standard deviation (SD ≈ 4.8 kg/m2) genetically higher BMI was associated with decreased estimated glomerular filtration rate (eGFR) [β=−0.032 (95% confidence intervals: −0.036, −0.027) log[eGFR], P = 1 × 10−43], increased blood urea nitrogen (BUN) [β = 0.010 (0.005, 0.015) log[BUN], P = 3 × 10−6], increased urinary albumin-to-creatinine ratio [β = 0.199 (0.067, 0.332) log[urinary albumin-to-creatinine ratio (UACR)], P = 0.003] in individuals with diabetes, and increased risk of microalbuminuria [odds ratios (OR) = 1.15 [1.04–1.28], P = 0.009] and CKD [1.13 (1.07–1.19), P = 3 × 10−6]. Corresponding estimates for WHR and for trans-ethnic populations were overall similar. The associations were driven by adverse obesity, and for microalbuminuria additionally by T2D. While genetically high BMI, unlike WHR, was directly associated with eGFR, BUN, and CKD, the pathway to albuminuria was likely through T2D. Genetically predicted kidney function was not associated with BMI or WHR. Conclusions Genetically high BMI is associated with impaired kidney function, driven by adverse obesity, and for albuminuria additionally by T2D.

MicroRNAs (miRNAs) are short non-coding RNA species which are important post-transcriptional regulators of gene expression and play an important role in the pathogenesis of diabetic nephropathy. miRNAs are present in urine in a remarkably stable form packaged in extracellular vesicles, predominantly exosomes. In the present study, urinary exosomal miRNA profiling was conducted in urinary exosomes obtained from 8 healthy controls (C), 8 patients with type II diabetes (T2D) and 8 patients with type II diabetic nephropathy (DN) using Agilent´s miRNA microarrays. In total, the expression of 16 miRNA species was deregulated (>2-fold) in DN patients compared to healthy donors and T2D patients: the expression of 14 miRNAs (miR-320c, miR-6068, miR-1234-5p, miR-6133, miR-4270, miR-4739, miR-371b-5p, miR-638, miR-572, miR-1227-5p, miR-6126, miR-1915-5p, miR-4778-5p and miR-2861) was up-regulated whereas the expression of 2 miRNAs (miR-30d-5p and miR-30e-5p) was down-regulated. Most of the deregulated miRNAs are involved in progression of renal diseases. Deregulation of urinary exosomal miRNAs occurred in micro-albuminuric DN patients but not in normo-albuminuric DN patients. We used qRT-PCR based analysis of the most strongly up-regulated miRNAs in urinary exosomes from DN patients, miRNAs miR-320c and miR-6068. The correlation of miRNA expression and micro-albuminuria levels could be replicated in a confirmation cohort. In conclusion, urinary exosomal miRNA content is altered in type II diabetic patients with DN. Deregulated miR-320c, which might have an impact on the TGF-β-signaling pathway via targeting thrombospondin 1 (TSP-1) shows promise as a novel candidate marker for disease progression in type II DN that should be evaluated in future studies.

Risk variants of APOL1 associated with human chronic kidney disease have been identified, but causality has been unclear. Transgenic expression in mice now shows that such alleles can indeed cause renal disease. African Americans have a heightened risk of developing chronic and end-stage kidney disease, an association that is largely attributed to two common genetic variants, termed G1 and G2, in the APOL1 gene. Direct evidence demonstrating that these APOL1 risk alleles are pathogenic is still lacking because the APOL1 gene is present in only some primates and humans; thus it has been challenging to demonstrate experimental proof of causality of these risk alleles for renal disease. Here we generated mice with podocyte-specific inducible expression of the APOL1 reference allele (termed G0) or each of the risk-conferring alleles (G1 or G2). We show that mice with podocyte-specific expression of either APOL1 risk allele, but not of the G0 allele, develop functional (albuminuria and azotemia), structural (foot-process effacement and glomerulosclerosis) and molecular (gene-expression) changes that closely resemble human kidney disease. Disease development was cell-type specific and likely reversible, and the severity correlated with the level of expression of the risk allele. We further found that expression of the risk-variant APOL1 alleles interferes with endosomal trafficking and blocks autophagic flux, which ultimately leads to inflammatory-mediated podocyte death and glomerular scarring. In summary, this is the first demonstration that the expression of APOL1 risk alleles is causal for altered podocyte function and glomerular disease in vivo .

Aims/hypothesis Changes in podocyte morphology and function are associated with albuminuria and progression of diabetic nephropathy. NADPH oxidase 4 (NOX4) is the main source of reactive oxygen species (ROS) in the kidney and Nox4 is upregulated in podocytes in response to high glucose. We assessed the role of NOX4-derived ROS in podocytes in vivo in a model of diabetic nephropathy using a podocyte-specific NOX4-deficient mouse, with a major focus on the development of albuminuria and ultra-glomerular structural damage. Methods Streptozotocin-induced diabetes-associated changes in renal structure and function were studied in male floxed Nox4 and podocyte-specific, NOX4 knockout (pod Nox4 KO) mice. We assessed albuminuria, glomerular extracellular matrix accumulation and glomerulosclerosis, and markers of ROS and inflammation, as well as glomerular basement membrane thickness, effacement of podocytes and expression of the podocyte-specific protein nephrin. Results Podocyte-specific Nox4 deletion in streptozotocin-induced diabetic mice attenuated albuminuria in association with reduced vascular endothelial growth factor (VEGF) expression and prevention of the diabetes-induced reduction in nephrin expression. In addition, podocyte-specific Nox4 deletion reduced glomerular accumulation of collagen IV and fibronectin, glomerulosclerosis and mesangial expansion, as well as glomerular basement membrane thickness. Furthermore, diabetes-induced increases in renal ROS, glomerular monocyte chemoattractant protein-1 (MCP-1) and protein kinase C alpha (PKC-α) were attenuated in podocyte-specific NOX4-deficient mice. Conclusions/interpretation Collectively, this study shows the deleterious effect of Nox4 expression in podocytes by promoting podocytopathy in association with albuminuria and extracellular matrix accumulation in experimental diabetes, emphasising the role of NOX4 as a target for new renoprotective agents.

Albuminuria is a hallmark of glomerular disease of various etiologies. It is not only a symptom of glomerular disease but also a cause leading to glomerulosclerosis, interstitial fibrosis, and eventually, a decline in kidney function. The molecular mechanism underlying albuminuria-induced kidney injury remains poorly defined. In our genetic model of nephrotic syndrome (NS), we have identified CHOP (C/EBP homologous protein)-TXNIP (thioredoxin-interacting protein) as critical molecular linkers between albuminuria-induced ER dysfunction and mitochondria dyshomeostasis. TXNIP is a ubiquitously expressed redox protein that binds to and inhibits antioxidant enzyme, cytosolic thioredoxin 1 (Trx1), and mitochondrial Trx2. However, very little is known about the regulation and function of TXNIP in NS. By utilizing Chop −/− and Txnip −/− mice as well as 68Ga-Galuminox, our molecular imaging probe for detection of mitochondrial reactive oxygen species (ROS) in vivo, we demonstrate that CHOP up-regulation induced by albuminuria drives TXNIP shuttling from nucleus to mitochondria, where it is required for the induction of mitochondrial ROS. The increased ROS accumulation in mitochondria oxidizes Trx2, thus liberating TXNIP to associate with mitochondrial nod-like receptor protein 3 (NLRP3) to activate inflammasome, as well as releasing mitochondrial apoptosis signal-regulating kinase 1 (ASK1) to induce mitochondria-dependent apoptosis. Importantly, inhibition of TXNIP translocation and mitochondrial ROS overproduction by CHOP deletion suppresses NLRP3 inflammasome activation and p-ASK1–dependent mitochondria apoptosis in NS. Thus, targeting TXNIP represents a promising therapeutic strategy for the treatment of NS.

Emerging evidence has established primary nephrotic syndrome (NS), including focal segmental glomerulosclerosis (FSGS), as a primary podocytopathy. Despite the underlying importance of podocyte endoplasmic reticulum (ER) stress in the pathogenesis of NS, no treatment currently targets the podocyte ER. In our monogenic podocyte ER stress-induced NS/FSGS mouse model, the podocyte type 2 ryanodine receptor (RyR2)/calcium release channel on the ER was phosphorylated, resulting in ER calcium leak and cytosolic calcium elevation. The altered intracellular calcium homeostasis led to activation of calcium-dependent cytosolic protease calpain 2 and cleavage of its important downstream substrates, including the apoptotic molecule procaspase 12 and podocyte cytoskeletal protein talin 1. Importantly, a chemical compound, K201, can block RyR2-Ser2808 phosphorylation-mediated ER calcium depletion and podocyte injury in ER-stressed podocytes, as well as inhibit albuminuria in our NS model. In addition, we discovered that mesencephalic astrocyte-derived neurotrophic factor (MANF) can revert defective RyR2-induced ER calcium leak, a bioactivity for this ER stress-responsive protein. Thus, podocyte RyR2 remodeling contributes to ER stress-induced podocyte injury. K201 and MANF could be promising therapies for the treatment of podocyte ER stress-induced NS/FSGS.