Diabetic Glomeruli Research Articles

RIPK3 causes mitochondrial dysfunction and albuminuria in diabetic podocytopathy through PGAM5-Drp1 signaling

BackgroundReceptor-interacting protein kinase (RIPK)3 is an essential molecule for necroptosis and its role in kidney fibrosis has been investigated using various kidney injury models. However, the relevance and the underlying mechanisms of RIPK3 to podocyte injury in albuminuric diabetic kidney disease (DKD) remain unclear. Here, we investigated the role of RIPK3 in glomerular injury of DKD. MethodsWe analyzed RIPK3 expression levels in the kidneys of patients with biopsy-proven DKD and animal models of DKD. Additionally, to confirm the clinical significance of circulating RIPK3, RIPK3 was measured by ELISA in plasma obtained from a prospective observational cohort of patients with type 2 diabetes, and estimated glomerular filtration rate (eGFR) and urine albumin-to-creatinine ratio (UACR), which are indicators of renal function, were followed up during the observation period. To investigate the role of RIPK3 in glomerular damage in DKD, we induced a DKD model using a high-fat diet in Ripk3 knockout and wild-type mice. To assess whether mitochondrial dysfunction and albuminuria in DKD take a Ripk3-dependent pathway, we used single-cell RNA sequencing of kidney cortex and immortalized podocytes treated with high glucose or overexpressing RIPK3. ResultsRIPK3 expression was increased in podocytes of diabetic glomeruli with increased albuminuria and decreased podocyte numbers. Plasma RIPK3 levels were significantly elevated in albuminuric diabetic patients than in non-diabetic controls (p = 0.002) and non-albuminuric diabetic patients (p = 0.046). The participants in the highest tertile of plasma RIPK3 had a higher incidence of renal progression (hazard ratio [HR] 2.29 [1.05–4.98]) and incident chronic kidney disease (HR 4.08 [1.10–15.13]). Ripk3 knockout improved albuminuria, podocyte loss, and renal ultrastructure in DKD mice. Increased mitochondrial fragmentation, upregulated mitochondrial fission-related proteins such as phosphoglycerate mutase family member 5 (PGAM5) and dynamin-related protein 1 (Drp1), and mitochondrial ROS were decreased in podocytes of Ripk3 knockout DKD mice. In cultured podocytes, RIPK3 inhibition attenuated mitochondrial fission and mitochondrial dysfunction by decreasing p-mixed lineage kinase domain-like protein (MLKL), PGAM5, and p-Drp1 S616 and mitochondrial translocation of Drp1. ConclusionsThe study demonstrates that RIPK3 reflects deterioration of renal function of DKD. In addition, RIPK3 induces diabetic podocytopathy by regulating mitochondrial fission via PGAM5-Drp1 signaling through MLKL. Inhibition of RIPK3 might be a promising therapeutic option for treating DKD.

Pharmacological Blockade of the Adenosine A2B Receptor Is Protective of Proteinuria in Diabetic Rats, through Affecting Focal Adhesion Kinase Activation and the Adhesion Dynamics of Podocytes.

Induction of the adenosine receptor A2B (A2BAR) expression in diabetic glomeruli correlates with an increased abundance of its endogenous ligand adenosine and the progression of kidney dysfunction. Remarkably, A2BAR antagonism protects from proteinuria in experimental diabetic nephropathy. We found that A2BAR antagonism preserves the arrangement of podocytes on the glomerular filtration barrier, reduces diabetes-induced focal adhesion kinase (FAK) activation, and attenuates podocyte foot processes effacement. In spreading assays using human podocytes in vitro, adenosine enhanced the rate of cell body expansion on laminin-coated glass and promoted peripheral pY397-FAK subcellular distribution, while selective A2BAR antagonism impeded these effects and attenuated the migratory capability of podocytes. Increased phosphorylation of the Myosin2A light chain accompanied the effects of adenosine. Furthermore, when the A2BAR was stimulated, the cells expanded more broadly and more staining of pS19 myosin was detected which co-localized with actin cables, suggesting increased contractility potential in cells planted onto a matrix with a stiffness similar to of the glomerular basement membrane. We conclude that A2BAR is involved in adhesion dynamics and contractile actin bundle formation, leading to podocyte foot processes effacement. The antagonism of this receptor may be an alternative to the intervention of glomerular barrier deterioration and proteinuria in the diabetic kidney disease.

GDF-15 alleviates diabetic nephropathy via inhibiting NEDD4L-mediated IKK/NF-κB signalling pathways

Podocyte inflammatory injury has been indicated to play a pivotal role in the occurrence and development of diabetic nephropathy (DN). However, the pathogenesis of inflammation remains unclear. Recent researches have shown that GDF-15, a member of the transforming growth factor-β superfamily, were elevated under pathological conditions, such as myocardial ischemia, cancer, as well as inflammation. Here, we demonstrated that GDF-15 could alleviate podocyte inflammatory injury by modulating the NF-κB pathway. GDF-15 and other pro-inflammatory factors, such as TNF-α, IL-1β, and IL-6 were upregulated in the serum of HFD/STZ rat models. GDF-15 was also elevated in diabetic glomeruli and hyperglycemic stimuli treated-podocytes. The silence of GDF-15 in HG-stimulated podocytes further augmented inflammation and podocyte injury, while overexpression of GDF-15 significantly reduced the inflammatory response in podocytes. Mechanistically, we demonstrated that GDF-15 could inhibit the nuclear translocation of NF-κB through IKK and IκBα by interaction with ubiquitin ligase NEDD4L. Taken together, our data suggested a protective mechanism of elevated GDF-15 in DN through obstruction of ubiquitin degradation of IKK by inhibiting NEDD4L expression, thus decreasing the activation of NF-κB and relieving the inflammation. GDF-15 could serve as a potential therapeutic target for DN.

Identification and Validation of Glomeruli Cellular Senescence-Related Genes in Diabetic Nephropathy by Multiomics.

Accumulating evidence indicates that cellular premature senescence of the glomerulus, including endothelial cells, mesangial cells, and podocytes leads to diabetic nephropathy (DN), and DN is regarded as a clinical model of premature senescence. However, the role of cellular senescence-associated genes in the glomerulus in DN progression remains unclear. Therefore, this work aims to identify and validate potential cellular aging-related genes in the glomerulus in DN to provide novel clues for DN treatment based on anti-aging. The microarray GSE96804 dataset, including 41 diabetic glomeruli and 20 control glomeruli, is retrieved from the Gene Expression Omnibus (GEO) database and cellular senescence-related genes (CSRGs) are obtained from the GeneCards database and literature reports. Subsequently, PPI, GO, and KEGG enrichment are analyzed by screening the intersection between differentially expressed genes (DEGs) and CSRGs. scRNA-seq dataset GSE127235 is used to verify core genes expression in glomerulocytes of mice. Finally, db/db mice are utilized to validate the hub gene expression in the glomeruli, and high glucose-induced mesangial cells are used to confirm key gene expression. This study reveals that FOS and ZFP36 may play an anti-aging role in DN to ameliorate cell intracellular premature aging in mesangial cells of glomeruli.

Deletion of protein tyrosine phosphatase SHP-1 restores SUMOylation of podocin and reverses the progression of diabetic kidney disease

Both clinical and experimental data suggest that podocyte injury is involved in the onset and progression of diabetic kidney disease (DKD). Although the mechanisms underlying the development of podocyte loss are not completely understood, critical structural proteins such as podocin play a major role in podocyte survival and function. We have reported that the protein tyrosine phosphatase SHP-1 expression increased in podocytes of diabetic mice and glomeruli of patients with diabetes. However, the invivo contribution of SHP-1 in podocytes is unknown. Conditional podocyte-specific SHP-1-deficient mice (Podo-SHP-1-/-) were generated to evaluate the impact of SHP-1 deletion at four weeks of age (early) prior to the onset of diabetes and after 20 weeks (late) of diabetes (DM; Ins2+/C96Y) on kidney function (albuminuria and glomerular filtration rate) and kidney pathology. Ablation of the SHP-1 gene specifically in podocytes prevented and even reversed the elevated albumin/creatinine ratio, glomerular filtration rate progression, mesangial cell expansion, glomerular hypertrophy, glomerular basement membrane thickening and podocyte foot process effacement induced by diabetes. Moreover, podocyte-specific deletion of SHP-1 at an early and late stage prevented diabetes-induced expression of collagen IV, fibronectin, transforming growth factor-β, transforming protein RhoA, and serine/threonine kinase ROCK1, whereas it restored nephrin, podocin and cation channel TRPC6 expression. Mass spectrometry analysis revealed that SHP-1 reduced SUMO2 post-translational modification of podocin while podocyte-specific deletion of SHP-1 preserved slit diaphragm protein complexes in the diabetic context. Thus, our data uncovered a new role of SHP-1 in the regulation of cytoskeleton dynamics and slit diaphragm protein expression/stability, and its inhibition preserved podocyte function preventing DKD progression.

Rho-kinase inhibitor restores glomerular fatty acid metabolism in diabetic kidney disease

The small GTPase Rho and its effector Rho-kinase (ROCK) are activated in the diabetic kidney, and recent studies decade have demonstrated that ROCK signaling is an integral pathway in the progression of diabetic kidney disease. We previously identified the distinct role of ROCK1, an isoform of ROCK, in fatty acid metabolism in diabetic glomeruli. However, the effect of pharmacological intervention for ROCK1 is not clear. In the present study, we show that the inhibition of ROCK1 by Y-27632 and fasudil restores fatty acid oxidation in the glomeruli. Mechanistically, these compounds optimize fatty acid utilization and redox balance in mesangial cells via AMPK phosphorylation and the subsequent induction of PGC-1α. A further in vivo study showed that the inhibition of ROCK1 suppressed the downregulation of the fatty acid oxidation-related gene expression in glomeruli and mitochondrial fragmentation in the mesangial cells of db/db mice. These observations indicate that ROCK1 could be a promising therapeutic target for diabetic kidney disease through a mechanism that improves glomerular fatty acid metabolism.

Highly water-soluble diacetyl chrysin ameliorates diabetes-associated renal fibrosis and retinal microvascular abnormality in db/db mice.

Chronic or intermittent hyperglycemia is associated with the development of diabetic complications. Oxidative stress and inflammation can be altered by hyperglycemia in diverse tissues, including kidneys and eyes, and play a pivotal role in diabetic complications. Our previous studies showed that the water-insoluble 5,7-dihydroxyflvone chrysin effectively combats diabetic damages incurred in diabetic kidneys and retinas. The current study employed the newly-synthesized 5.7-di-O-acetylchrysin, having higher solubility than chrysin, to compare the effects on diabetes-associated renal fibrosis and abnormal retinal neovascularization. In the in vivo study, db/db mice as animal models of type 2 diabetes were orally administrated 10 mg/kg BW diacetylchrysin, daily for 10 weeks. Unlike chrysin, oral administration of 10 mg/kg diacetylchrysin did not lower the blood glucose level and 24 h urine volume in db/db mice. Nevertheless, the urinary albumin excretion was markedly reduced. The administration of diacetylchrysin also diminished the deposition of collagen fibers in diabetic glomeruli and tubules by suppressing the induction of connective tissue growth factor and collagen IV in diabetic kidneys. Supplying diacetylchrysin enhanced the membrane type-1 matrix metalloproteinase (MMP) expression reduced in diabetic kidneys, while the tissue inhibitor of MMP-2 induction was attenuated in diacetylchrysin-challenged diabetic kidneys. In addition, supplementing diacetylchrysin to diabetic mice ameliorated renal injury due to glomerulosclerosis and tubular interstitial fibrosis. Furthermore, the reduced retinal inductions of Zonula occludens-1 and vascular endothelial cadherin in db/db mice were elevated in the retinal tissues of diacetylchrysin-treated animals. Oral administration of diacetylchrysin curtailed the induction of vascular endothelial growth factor (VEGF) and VEGF receptor 2 in db/db mice, ultimately retarding diabetes-associated retinal neovascularization. Additionally, the retinal formation of acellular capillaries with leaky vessels was reduced in diacetylchrysin-treated db/db mice. Diacetylchrysin may act as a potent pro-health agent for treating renal fibrosis-associated diabetic nephropathy and retinal neovascularization-associated diabetic retinopathy.

PS-B09-9: ENOS-NO PATHWAY REGULATES INFLAMMASOME ACTIVATION IN DIABETIC MICE VIA MODULATION OF CEBP TRANSLOCATION

Background: Diabetic kidney disease (DKD) is one of the most common vascular complications of diabetes mellitus (DM). Endothelial dysfunction is also a common pathophysiologic mechanism of diabetic vascular complications. Nitric oxide (NO) produced in the endothelial cells by endothelial nitric oxide synthase (eNOS) is an important mediator for the maintenance of vascular homeostasis. In persons with diabetes, eNOS produces superoxide anion rather than nitric oxide, referred to as eNOS uncoupling which may contribute to endothelial dysfunction, albuminuria, and diabetic nephropathy. eNOS uncoupling can then lead to dysfunction of the eNOS-NO-pathway in diabetic glomeruli. To determine the mechanism of regulation of inflammasome activation by eNOS-NO pathway in DKD, we used eNOS deficient mice and eNOS-ASC double knockout mice. Methods: We used wild-type (WT) and eNOS-deficient mice (eNOSKO) to determine the role of the eNOS-NO pathway. Diabetes was induced by a single intraperitoneal injection of streptozocin. We then divided the mice into four groups: WT, WT/STZ, eNOSKO, and eNOS/STZ. Four weeks after the induction of DM, the mice were sacrificed, and their kidney tissues were harvested. Urinary albumin excretion was checked before the sacrifice, and glomerular damage was assessed by periodic acid-Schiff (PAS) staining. To determine the role of inflammasome on the development of glomerulopathy, we generated eNOS-ASC double knockout mice (eNOS-ASC-DKO). Also, to investigate inflammasome activation in glomeruli, the glomeruli were isolated from the kidney tissue by using Dynabeads. Results: Urinary albumin excretion was increased in WT/STZ compared with WT. These urinary albumin excretions were increased much more in eNOS/STZ than in WT/STZ, while the glomeruli were more damaged in eNOSKO/STZ than in WT/STZ. Urinary albumin excretion was lower in eNOS-ASC-DKO/STZ compared with eNOSKO/STZ, and the glomerular damage also was ameliorated in eNOS-ASC-DKO after treatment with STZ. S-Nitroso glutathione (GSNO) attenuated NLRP3 inflammasome activation after treatment with LPS-ATP. These results indicate that NO directly inhibits NLRP3-inflammasome activation. In addition, GSNO decreased the expression of inflammasome-related genes. To investigate the detailed the mechanism (epigenetic regulation), we performed Assay for Transposase-Accessible Chromatin-Sequencing (ATAC-seq) analysis. The binding region of the transcription factor CCAAT/ enhancer binding protein (C/EBP) β; was closed in the LPS+GSNO group significantly, compared with the LPS group. Conclusion: In eNOS-deficient mice, inflammasomes were activated, and glomerular injuries were exacerbated. However in eNOS-ASC-STZ mice, the glomerular damage were attenuated. Our data suggested that eNOS/NO signaling attenuates inflammasome activation via regulation of CEBP β;, leading to the suppressed development of DKD.

Glucosamine inhibits extracellular matrix accumulation in experimental diabetic nephropathy.

Glucosamine, the intermediate metabolite of the hexosamine biosynthesis pathway (HBP), is widely used as a supplementary drug in patients with osteoarthritis. However, its consequences in such patients concomitantly suffering from diabetic nephropathy is unknown. The aim of the study was to investigate the effect of exogenous administration of glucosamine in the diabetic kidney. A mouse model of streptozotocin-induced diabetic nephropathy in vivo and cultured endothelial cells in vitro were used in the study. The mice were treated with glucosamine for 6 months. Renal function was evaluated by metabolic cage, and histology of the kidney was estimated by periodic acid-schiff (PAS) staining. The expression of related genes was assessed by real-time PCR, immunofluorescence staining, immunoblotting and ELISA. There was no significant difference in urinary albumin secretion, relative kidney weight, or creatinine clearance between the groups treated with glucosamine and controls. Assessment of the kidney demonstrated reduction in mesangial expansion and fibronectin expression in the diabetic glomeruli treated with glucosamine. Glucosamine treatment significantly decreased α-smooth muscle actin (α-SMA) protein expression in both diabetic and control kidneys, whereas the expression of other fibrosis-related genes and inflammatory factors was unaltered. Moreover, α-SMA colocalized with the endothelial marker CD31 in the diabetic and control kidneys, and glucosamine reduced α-SMA+ ECs in the diabetic glomeruli. In addition, glucosamine suppressed α-SMA expression in endothelial cells treated with or without high glucose. In summary, this is the first report to show that glucosamine reduces mesangial expansion and inhibits endothelial-mesenchymal transition in diabetic nephropathy. The underlying mechanisms need to be further investigated.

Integrative transcriptome analysis reveals TEKT2 and PIAS2 involvement in diabetic nephropathy.

Cell heterogeneity has impeded the accurate interpretation of the bulk transcriptome data from patients with diabetic nephropathy (DN). We performed an analysis by integrating bulk and single-cell transcriptome datasets to uncover novel mechanisms leading to DN, especially in the podocytes. Microdissected glomeruli and tubules transcriptome datasets were selected from Gene Expression Omnibus (GEO). Then the consistency between datasets was evaluated. The analysis of the bulk dataset and single-nucleus RNA dataset was integrated to reveal the cell type-specific responses to DN. The candidate genes were validated in kidney tissues from DN patients and diabetic mice. We compared 4 glomerular and 4 tubular datasets and found considerable discrepancies among datasets regarding the deferentially expressed genes (DEGs), involved signaling pathways, and the hallmark enrichment profiles. Deconvolution of the bulk data revealed that the variations in cell-type proportion contributed greatly to this discrepancy. The integrative analysis uncovered that the dysregulation of spermatogenesis-related genes, including TEKT2 and PIAS2, was involved in the development of DN. Importantly, the mRNA level of TEKT2 was negatively correlated with the mRNA levels of NPHS1 (r= -.66, p <.0001) and NPHS2 (r= -.85, p <.0001) in human diabetic glomeruli. Immunostaining confirmed that the expression of TEKT2 and PIAS2 were up-regulated in podocytes of DN patients and diabetic mice. Knocking down TEKT2 resisted high glucose-induced cytoskeletal remodeling and down-regulation of NPHS1 protein in the cultured podocyte. In conclusion, the integrative strategy can help us efficiently use the publicly available transcriptomics resources. Using this approach and combining it with classical research methods, we identified TEKT2 and PIAS2, two spermatogenesis-related genes involved in the pathogenesis of DN. Furthermore, TEKT2 is involved in this pathogenesis by regulating the podocyte cytoskeleton.

POS-135 GROWTH HORMONE INDUCES TNF-ALPHA IN PODOCYTES AND CONTRIBUTES TO MONOCYTE-TO-MACROPHAGE DIFFERENTIATION: IMPLICATIONS IN DIABETIC KIDNEY DISEASE

Diabetes shortens the life expectancy by more than a decade, and the excess mortality in diabetes is correlated with the incidence of kidney disease. Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease. In human biopsies and experimental models of DKD, macrophage accumulation predicts the severity of kidney damage. The mechanism of macrophage accumulation in diabetic glomeruli, however, is unknown. Increased growth hormone (GH) levels in type 1 diabetes and acromegalic patients had deleterious effects on glomerular biology.

LncRNA H19 deficiency protects against the structural damage of glomerular endothelium in diabetic nephropathy via Akt/eNOS pathway

Objective: This study aimed to investigate the functions of lncRNA H19 on glomerular endothelial structural damage of diabetic nephropathy (DN). Materials and Methods: Rats were fed a high sugar and fat high feed die, and intraperitoneally administrated with streptozotocin (30 mg/kg) to induce DN model. Meanwile, rat glomerular endothelial cells (rGEnCs) were treated with high a level of glucose （HG, 30 mM glucose）to induce structural damage. Results: Our results showed that H19 level was drastically increased in diabetic glomeruli and high-glucose (HG)-stimulated rat glomerular endothelial cells (rGEnCs). Deficiency of H19 ameliorated microalbumin, creatinine, BUN, and histopathological alterations in diabetic rats. In addition, H19 deficiency significantly attenuated the damage of endothelial structure by upregulating the expression of junction proteins ZO-1 and Occludin, glycolcalyx protein Syndecan-1, and endothelial activation marker sVCAM-1 and sICAM-1 in diabetic rats. The in vitro results also showed that H19-siRNA alleviated glycocalyx shedding, tight junctions damage, and endothelial activation in HG-stimulated rGEnCs. Moreover, H19 deficiency significantly enhanced the expression of p-Akt and p-eNOS and NO concentration in vitro and in vivo. Pre-treatment with Akt inhibitor LY294002 abrogated these favourable effects mediated by H19 deficiency. Discussion and Conclusion: These results indicate that H19 deficiency could mitigate the structural damage of glomerular endothelium in DN via activating Akt/eNOS pathway.

24-OR: Podocyte Targeted Pyruvate Kinase M2 (PKM2) Activation Normalized Glomerular VEGF Expression, Mitochondrial Function, Fuel Metabolism, and Diabetic Nephropathy

Diabetic nephropathy (DN) is the result of abnormal systemic and local changes in metabolism and hemodynamics. We have reported that many glycolytic enzymes, such as pyruvate kinase M2 (PKM2) , were elevated in the renal glomeruli of type 1 and type 2 diabetic patients who were protected from DN. TEPP46, a small-molecule which activates PKM2 by inducing oligomerization, reversed glomerular pathology in diabetic mice. Here, mice with PKM2-specific overexpression in podocytes (PPKM2Tg) were generated to uncover its renal protective function as potential therapeutic target, which prevented elevated albumin-creatinine ratio (ACR) , mesangial expansion, basement membrane thickness and podocyte foot process effacement after 7-months of STZ-induced diabetes. Further, diabetes-induced impairment of glycolytic rate and mitochondrial function were normalized in diabetic PPKM2Tg glomeruli, in concordance with elevated Ppargc1a and Vegf expressions. Restored VEGF expression improved glomerular maximal mitochondrial function in diabetic PPKM2Tg and WT mice. Elevated VEGF levels were observed in the glomeruli of DN-protected patients with chronic type 1 diabetes, and clinically correlated with estimated GFR, but not glycemic control. Mechanistically, the preservations of mitochondrial function and VEGF expression were dependent on tetrameric structure and enzymatic activities of PKM2 in podocyte. Thus, these findings show that PKM2 activation in the podocyte can regulate mitochondrial metabolism of the entire glomeruli and prevent mitochondrial dysfunction induced by chronic diabetes and even reverse metabolic memory. PKM2 structures and enzymatic activities are necessary to mediate glomerular metabolism against toxicity of hyperglycemia via paracrine factors such as VEGF. Thus, PKM2 activation in podocyte is a potential therapeutic target to protect against DN. Disclosure J.Fu: None. T.Shinjo: None. Q.Li: None. K.Park: None. M.Yu: None. H.Yokomizo: None. Q.Huang: None. I.Wu: None. G.L.King: Advisory Panel; Medtronic, Research Support; Janssen Pharmaceuticals, Inc.

Rho-associated, coiled-coil–containing protein kinase 1 regulates development of diabetic kidney disease via modulation of fatty acid metabolism

Dysregulation of fatty acid utilization is increasingly recognized as a significant component of diabetic kidney disease. Rho-associated, coiled-coil-containing protein kinase (ROCK) is activated in the diabetic kidney, and studies over the past decade have illuminated ROCK signaling as an essential pathway in diabetic kidney disease. Here, we confirmed the distinct role of ROCK1, an isoform of ROCK, in fatty acid metabolism using glomerular mesangial cells and ROCK1 knockout mice. Mesangial cells with ROCK1 deletion were protected from mitochondrial dysfunction and redox imbalance driven by transforming growth factor β, a cytokine upregulated in diabetic glomeruli. We found that high-fat diet-induced obese ROCK1 knockout mice exhibited reduced albuminuria and histological abnormalities along with the recovery of impaired fatty acid utilization and mitochondrial fragmentation. Mechanistically, we found that ROCK1 regulates the induction of critical mediators in fatty acid metabolism, including peroxisome proliferator-activated receptor gamma coactivator 1α, carnitine palmitoyltransferase 1, and widespread program-associated cellular metabolism. Thus, our findings highlight ROCK1 as an important regulator of energy homeostasis in mesangial cells in the overall pathogenesis of diabetic kidney disease.

Regeneration of glomerular metabolism and function by podocyte pyruvate kinase M2 in diabetic nephropathy.

Diabetic nephropathy (DN) arises from systemic and local changes in glucose metabolism and hemodynamics. We have reported that many glycolytic and mitochondrial enzymes, such as pyruvate kinase M2 (PKM2), were elevated in renal glomeruli of DN-protected patients with type 1 and type 2 diabetes. Here, mice with PKM2 overexpression specifically in podocytes (PPKM2Tg) were generated to uncover the renal protective function of PPKM2Tg as a potential therapeutic target that prevented elevated albumin/creatinine ratio (ACR), mesangial expansion, basement membrane thickness, and podocyte foot process effacement after 7 months of streptozotocin-induced (STZ-induced) diabetes. Furthermore, diabetes-induced impairments of glycolytic rate and mitochondrial function were normalized in diabetic PPKM2Tg glomeruli, in concordance with elevated Ppargc1a and Vegf expressions. Restored VEGF expression improved glomerular maximal mitochondrial function in diabetic PPKM2Tg and WT mice. Elevated VEGF levels were observed in the glomeruli of DN-protected patients with chronic type 1 diabetes and clinically correlated with estimated glomerular filtration (GFR) — but not glycemic control. Mechanistically, the preservations of mitochondrial function and VEGF expression were dependent on tetrameric structure and enzymatic activities of PKM2 in podocytes. These findings demonstrate that PKM2 structure and enzymatic activation in podocytes can preserve the entire glomerular mitochondrial function against toxicity of hyperglycemia via paracrine factors such as VEGF and prevent DN progression.

Asparaginyl endopeptidase protects against podocyte injury in diabetic nephropathy through cleaving cofilin-1

Podocyte injury and loss are critical events in diabetic nephropathy (DN); however, the underlying molecular mechanisms remain unclear. Here, we demonstrate that asparaginyl endopeptidase (AEP) protects against podocyte injury through modulating the dynamics of the cytoskeleton. AEP was highly upregulated in diabetic glomeruli and hyperglycemic stimuli treated-podocytes; however, AEP gene knockout and its compound inhibitor treatment accelerated DN in streptozotocin-induced diabetic mice, whereas specific induction of AEP in glomerular cells attenuated podocyte injury and renal function deterioration. In vitro, elevated AEP was involved in actin cytoskeleton maintenance and anti-apoptosis effects. Mechanistically, we found that AEP directly cleaved the actin-binding protein cofilin-1 after the asparagine 138 (N138) site. The protein levels of endogenous cofilin-1 1-138 fragments were upregulated in diabetic podocytes, consistent with the changes in AEP levels. Importantly, we found that cofilin-1 1-138 fragments were remarkably unphosphorylated than full-length cofilin-1, indicating the enhanced cytoskeleton maintenance activity of cofilin-1 1-138. Then we validated cofilin-1 1-138 could rescue podocytes from cytoskeleton disarrangement and injury in diabetic conditions. Taken together, our data suggest a protective role of elevated AEP in podocyte injury during DN progression through cleaving cofilin-1 to maintain podocyte cytoskeleton dynamics and defend damage.

POS-337 ASPARAGINYL ENDOPEPTIDASE PROTECTS AGAINST PODOCYTE INJURY IN DIABETIC NEPHROPATHY THROUGH CLEAVING COFILIN-1

Asparaginyl endopeptidase (AEP) is a lysosomal cysteine protease that specifically cleaves its substrates after asparagine residues. Physiologically, AEP is most abundant in kidney proximal tubule and is required for normal kidney physiology and homeostasis. Recent studies reported that AEP exerts anti-fibrotic effect through degrading of matrix fibronection during unilateral ureteral obstruction (UUO) animal model. However, its role and mechanism in podocyte injury during diabetic nephropathy (DN) remains unclear. Diabetic mice model was established by intraperitoneal injection of streptozotocin (STZ) and oral administration of AEP inhibitor was adopted to inhibit AEP activity, and also, AEP knockout mice were employed. In vitro study, human podocyte cell line was employed, and subjected to different treatments after differentiation. RNA interfere and adenovirus expression vector were utilized to knockdown AEP or overexpression of AEP and cofilin 1 fragments respectively. Moreover, AEP recombinase was used to detect its substrate---cofilin 1 cleavage activity in vitro. In the present study, AEP was highly upregulated in diabetic glomeruli and hyperglycemic stimuli treated-podocytes; however, AEP gene knockout and its compound inhibitor treatment accelerated DN in streptozotocin-induced diabetic mice, whereas specific induction of AEP in glomerular cells attenuated podocyte injury and renal function deterioration. In vitro, elevated AEP was involved in actin cytoskeleton maintenance and anti-apoptosis effects. Mechanistically, we found that AEP directly cleaved the actin-binding protein cofilin-1 after the asparagine 138 (N138) site. The protein levels of endogenous cofilin-1 1-138 fragments were upregulated in diabetic podocytes, consistent with the changes in AEP levels. Importantly, we found that cofilin-1 1-138 fragments were remarkably unphosphorylated than full-length cofilin-1, indicating the enhanced cytoskeleton maintenance activity of cofilin-1 1-138. Then we validated cofilin-1 1-138 could rescue podocytes from cytoskeleton disarrangement and injury in diabetic conditions. Taken together, our data suggests a protective role of elevated AEP in podocyte injury during DN progression through cleaving cofilin-1 to maintain podocyte cytoskeleton dynamics and defend damage.

POS-328 SUPPRESSION OF ER-ASSOCIATED DEGRADATION BY INTRAGLOMERULAR CROSS-COMMUNICATION BETWEEN MESANGIAL CELLS AND PODOCYTES CAUSES PODOCYTE INJURY IN DIABETIC KIDNEY DISEASE

Mesangial lesions and podocyte injury are essential manifestations of the progression of diabetic kidney disease (DKD). Although cross-communication between mesangial cells (MCs) and podocytes has recently been suggested by the results of single cell analyses, the molecular mechanisms and its role in disease progression still remain elusive. Furthermore, our cDNA microarray data of diabetic mouse glomeruli suggested the involvement of endoplasmic reticulum (ER) stress in DKD pathophysiology. Thus, we hypothesized that the cross-communication between MCs and podocytes may affect the ER stress responses of these cells and thereby participate in the progression of DKD, especially in podocyte injury.

MAD2B promotes podocyte injury through regulating Numb-dependent Notch 1 pathway in diabetic nephropathy.

Rationale: Recent studies have demonstrated that the loss of podocyte is a critical event in diabetic nephropathy (DN). Previously, our group have found that the mitotic arrest deficient protein MAD2B was involved in high glucose (HG)-induced podocyte injury by regulating APC/C activity. However, the exact mechanism of MAD2B implicated in podocyte injury is still lacking.Methods: The experiments were conducted by using kidney tissues from streptozotocin (STZ) induced diabetic mice with or without podocyte-specific deletion of MAD2B and the cultured podocytes exposed to different treatments. Glomerular pathological injury was evaluated by periodic acid-Schiff staining and transmission electron microscopy. The endogenous interaction between MAD2B and Numb was discovered by yeast two-hybrid analysis and co-immunoprecipitation assay. The expressions of MAD2B, Numb and related pathway were detected by western blot, immunochemistry and immunofluorescence.Results: The present study revealed that MAD2B was upregulated in diabetic glomeruli and cultured podocytes under hyperglycemic conditions. Podocyte-specific deletion of MAD2B alleviated podocyte injury and renal function deterioration in mice of diabetic nephropathy. Afterwards, MAD2B was found to interact with Numb, which was downregulated in diabetic glomeruli and HG-stimulated cultured podocytes. Interestingly, MAD2B genetic deletion could partly reverse the decline of Numb in podocytes exposed to HG and in diabetic mice, and the expressions of Numb downstream molecules such as NICD and Hes-1 were decreased accordingly. In addition, overexpression of Numb ameliorated HG-induced podocyte injury.Conclusions: The present findings suggest that upregulated MAD2B expression contributes to Numb depletion and activation of Notch 1 signaling pathway, which ultimately leads to podocyte injury during DN progression.

Glomerular Endothelial Cells Are the Coordinator in the Development of Diabetic Nephropathy.

The prevalence of diabetes is consistently rising worldwide. Diabetic nephropathy is a leading cause of chronic renal failure. The present study aimed to explore the crosstalk among the different cell types inside diabetic glomeruli, including glomerular endothelial cells, mesangial cells, podocytes, and immune cells, by analyzing an online single-cell RNA profile (GSE131882) of patients with diabetic nephropathy. Differentially expressed genes in the glomeruli were processed by gene enrichment and protein-protein interactions analysis. Glomerular endothelial cells, as well as podocytes, play a critical role in diabetic nephropathy. A subgroup of glomerular endothelial cells possesses characteristic angiogenesis genes, indicating that angiogenesis takes place in the progress of diabetic nephropathy. Immune cells such as macrophages, T lymphocytes, B lymphocytes, and plasma cells also contribute to the disease progression. By using iTALK, the present study reports complicated cellular crosstalk inside glomeruli. Dysfunction of glomerular endothelial cells and immature angiogenesis result from the activation of both paracrine and autocrine signals. The present study reinforces the importance of glomerular endothelial cells in the development of diabetic nephropathy. The exploration of the signaling pathways involved in aberrant angiogenesis reported in the present study shed light on potential therapeutic target(s) for diabetic nephropathy.