Kidneys Of Diabetic Mice Research Articles

ODP200 Fyn is involved in the development of diabetic kidney disease by modulating autophagy

Abstract Diabetic kidney disease (DKD) is one of the major diabetic complications and the leading cause of the end stage renal disease. Recently autophagy was shown to regulate DKD. Previously we reported that Fyn regulates muscle mass by suppressing autophagy through Fyn-STAT3 signaling pathway. More recently, we demonstrated that Fyn also inhibits autophagy in HK2 cells, in vitro cell model of renal proximal tubular epithelial cells (RPTC). Moreover, we found Y369 of Tgm2, a known inhibitor of autophagy, is directory phosphorylated by Fyn both in vitro and in cells and Fyn-dependent phosphorylation of Tgm2 regulates autophagy in RPTC. It has been reported that Tgm2 forms complexes with p53 and p62 (a known autophagy regulator) to mediate the degradation of p53 at autophagosomes in cancer cells. To note is that p53 could function as a DKD inducer. We found that p53 expression was decreased in Tgm2 knock-downed HK2 cells, suggesting that Tgm2-p62-p53 complex also modulates autophagy in RPTC. Since these were only in vitro studies, we proceeded with those in vivo. In HFD fed mice, diet-induced rodent models of metabolic disorders, we found that protein expression of p53 was increased due to decreased levels of autophagy implicated by decreased p62 punctuations in RPTC. To confirm the role of Fyn in autophagy and p53 expression we took advantage of a diabetic mouse model of FynKO mice induced by Streptozocin(STZ). While p53 was increased in the kidney of diabetic WT mice induced by STZ, it was not the case in the kidney of diabetic FynKO, indicating that Fyn could regulate autophagic activity and p53 expression. Taken together, these data suggest that the metabolic status may regulate Fyn to phosphorylate Tgm2 and modulate Tgm2-p62-p53 complex leading to the development of DKD. This regulatory mechanism is not mediated by blood glucose and may lead to the new therapeutic targets for diabetic kidney disease regardless of control of diabetes. Presentation: No date and time listed

Enhanced SHP-1 Expression in Podocyturia Is Associated with Kidney Dysfunction in Patients with Diabetes

Diabetic kidney disease (DKD) remains the leading cause of end stage kidney disease worldwide. Despite significant advances in kidney care, there is a need to improve noninvasive techniques to predict the progression of kidney disease better for patients with diabetes. After injury, podocytes are shed in urine and may be used as a biologic tool. We previously reported that SHP-1 is upregulated in the kidney of diabetic mice, leading to podocyte dysfunction and loss. Our objective was to evaluate the expression levels of SHP-1 in urinary podocytes and kidney tissues of patients with diabetes. In this prospective study, patients with and without diabetes were recruited for the quantification of SHP-1 in kidney tissues, urinary podocytes, and peripheral blood monocytes. Immunochemistry and mass spectrometry techniques were applied for kidney tissues. Urinary podocytes were counted, and expression of SHP-1 and podocyte markers were measured by quantitative PCR. A total of 66 participants (diabetic n=48, nondiabetic n=18) were included in the analyses. Diabetes was associated with increased SHP-1 expression in kidney tissues (P=0.03). Nephrin and podocin mRNA was not significantly increased in urinary podocytes from patients with diabetes compared with those without diabetes, whereas levels of SHP-1 mRNA expression significantly correlated with HbA1c and estimated glomerular filtration rate (eGFR). Additionally, follow-up (up to 2 years post recruitment) evaluation indicated that SHP-1 mRNA expression continued to increase with eGFR decline. Levels of SHP-1 in urinary podocytes may serve as an additional marker of glomerular disease progression in this population.

Toward the renal vesicle: Ultrastructural investigation of the cap mesenchyme splitting process in the developing kidney.

Background:A complex sequence of morphogenetic events leads to the development of the adult mouse kidney. In the present study, we investigated the morphological events that characterize the early stages of the mesenchymal-to-epithelial transition of cap mesenchymal cells, analyzing in depth the relationship between cap mesenchymal induction and ureteric bud (UB) branching.Design and methods:Normal kidneys of newborn non-obese diabetic (NOD) mice were excised and prepared for light and electron microscopic examination.Results:Nephrogenesis was evident in the outer portion of the renal cortex of all examined samples. This process was mainly due to the interaction of two primordial derivatives, the ureteric bud and the metanephric mesenchyme. Early renal developmental stages were initially characterized by the formation of a continuous layer of condensed mesenchymal cells around the tips of the ureteric buds. These caps of mesenchymal cells affected the epithelial cells of the underlying ureteric bud, possibly inducing their growth and branching.Conclusions:The present study provides morphological evidence of the reciprocal induction between the ureteric bud and the metanephric mesenchyme showing that the ureteric buds convert mesenchyme to epithelium that in turn stimulates the growth and the branching of the ureteric bud.

Blocking REDD1/TXNIP Complex Ameliorates HG-Induced Renal Tubular Epithelial Cell Apoptosis and EMT through Repressing Oxidative Stress.

Diabetic nephropathy (DN) has become the most common secondary kidney disease causing end-stage renal disease (ESRD). Nevertheless, the underlying mechanisms responsible for DN remain largely unknown. Regulated in development and DNA damage response 1 (REDD1) is a prooxidative molecule known to contribute to diabetes mellitus and its complications. However, it has not been previously examined whether and how REDD1 can further drive renal tubular epithelial cell (RTEC) apoptosis and epithelial-to-mesenchymal transition in DN. The expression of REDD1 was elevated in the kidneys of DN patients and diabetic mice in this study. By generating the DN model in REDD1 knockout mice, we demonstrated that REDD1 deficiency significantly improved apoptosis and EMT in diabetic mice. In vitro experiments showed that REDD1 generation was induced by high glucose (HG) in HK-2 cells. Similarly, the transfection of REDD1 siRNA plasmid also suppressed HG-induced apoptosis and EMT. Furthermore, we discovered that the inhibition of REDD1 suppressed the expression of Nox4-induced HG and reactive oxygen species (ROS) synthesis in HK-2 cells. In addition, HG could induce endogenous REDD1 and TXNIP to form a powerful complex. In summary, our findings demonstrate that blocking the REDD1/TXNIP complex can prevent HG-induced apoptosis and EMT by inhibiting ROS production, highlighting REDD1 as a valuable therapeutic priority site for DN.

Effect of BBT-877, a novel inhibitor of ATX, on a mouse model of type 1 diabetic nephropathy.

Diabetic nephropathy (DN) is one of the common microvascular complications of diabetes. Autotaxin (ATX) is an enzyme with lysophospholipase D activity, producing lysophosphatidic acid (LPA). LPA signaling has been implicated in renal fibrosis, thereby inducing renal dysfunction. BBT-877 is an orally administered small molecule inhibitor of ATX. However, its effect on DN has not been studied so far. In this study, we investigated the effect of BBT-877, a novel inhibitor of ATX, on the pathogenesis of DN in a mouse model of streptozotocin (STZ)-induced diabetes.BBT-877 treatment significantly reduced albuminuria, albumin-to-creatinine ratio (ACR), neutrophil gelatinase-associated lipocalin (NGAL), and glomerular volume compared to the STZ-vehicle group. Interestingly, BBT-877 treatment attenuated hyperglycemia and dyslipidemia in STZ-induced diabetes mice. In the liver, the expression levels of β-oxidation-related genes such as PPAR α and CPT1 were significantly decreased in STZ-induced diabetic mice. However, this effect was reversed by BBT-877 treatment. BBT-877 treatment also suppressed mRNA levels of pro-inflammatory cytokines IL-6, MCP-1, and TNF-α and protein levels of fibrotic factors (TGF-β, fibronectin, CTGF, and collagen type Ι alpha Ι (COL1A1)) in the kidneys of STZ-induced diabetic mice.In conclusion, our results indicate that BBT-877 is effective in preventing the pathogenesis of DN by reducing systemic blood glucose levels and inhibiting inflammation and fibrosis in the renal tissue of diabetes mice. These novel findings suggest that inhibition of ATX may be a potential therapeutic target for DN.

REDD1 Ablation Attenuates the Development of Renal Complications in Diabetic Mice.

Chronic hyperglycemia contributes to development of diabetic kidney disease by promoting glomerular injury. In this study, we evaluated the hypothesis that hyperglycemic conditions promote expression of the stress response protein regulated in development and DNA damage response 1 (REDD1) in the kidney in a manner that contributes to the development of oxidative stress and renal injury. After 16 weeks of streptozotocin-induced diabetes, albuminuria and renal hypertrophy were observed in wild-type (WT) mice coincident with increased renal REDD1 expression. In contrast, diabetic REDD1 knockout (KO) mice did not exhibit impaired renal physiology. Histopathologic examination revealed that glomerular damage including mesangial expansion, matrix deposition, and podocytopenia in the kidneys of diabetic WT mice was reduced or absent in diabetic REDD1 KO mice. In cultured human podocytes, exposure to hyperglycemic conditions enhanced REDD1 expression, increased reactive oxygen species (ROS) levels, and promoted cell death. In both the kidney of diabetic mice and in podocyte cultures exposed to hyperglycemic conditions, REDD1 deletion reduced ROS and prevented podocyte loss. Benefits of REDD1 deletion were recapitulated by pharmacological GSK3β suppression, supporting a role for REDD1-dependent GSK3β activation in diabetes-induced oxidative stress and renal defects. The results support a role for REDD1 in diabetes-induced renal complications.

Identification of novel key genes and potential candidate small molecule drugs in diabetic kidney disease using comprehensive bioinformatics analysis.

Objective: The currently established diagnostic and prognostic tools for diabetic kidney disease (DKD) have limitations, which demands the necessity to find new genes and pathways associated with diagnosis and treatment. Our study aims to reveal the gene expression alteration and discover critical genes involved in the development of DKD, thus providing novel diagnostic molecular markers and therapeutic targets. Materials and methods: The differences of infiltrating immune cells within kidney were compared between healthy living donors and DKD patients. Besides, differentially expressed genes (DEGs) within kidney from healthy living donor, early stage DKD and advanced stage DKD samples were detected. Furthermore, the weighted co-expressed network (WGCNA) and protein-protein interaction (PPI) network were constructed, followed by recognition of core hub genes and module analysis. Receiver operating characteristic (ROC) curve analysis was implemented to determine the diagnostic value of hub genes, correlation analysis was employed to explore the association between hub genes and infiltrating immune cells, and certain hub genes was validated by quantitative real-time PCR and immunohistochemistry staining in cultured tubule cells and diabetic mice kidney. Finally, the candidate small molecules as potential drugs to treat DKD were anticipated through utilizing virtual screening and molecular docking investigation. Results: Our study revealed significantly higher proportion of infiltrating immune cells within kidney from DKD patients via probing the immune landscape by single-cell transcriptomics. Besides, 126 commonly shared DEGs identified among three group samples were enriched in immune biological process. In addition, the ROC curve analysis demonstrated the strong diagnostic accuracy of recognized hub genes (NFKB1, DYRK2, ATAD2, YAP1, and CHD3) from PPI network. Correlation analysis further confirmed the positive association between these hub genes with infiltrating natural killer cells. More importantly, the mRNA transcripts and protein abundance of YAP1 were significantly higher in high glucose-treated renal tubule cells and diabetic mice kidney, and the small molecules exhibiting the best binding affinities with YAP1 were predicted and acquired. Conclusion: Our findings for the first time indicate that NFKB1, DYRK2, ATAD2, YAP1, and CHD3 might be potential novel biomarkers and therapeutic targets for DKD, providing insights into the molecular mechanisms underlying the pathogenesis of DKD.

Integrated analysis of miRNA and mRNA expression profiles in diabetic mouse kidney treated to Korean Red Ginseng

Integrated analysis of miRNA and mRNA expression profiles in diabetic mouse kidney treated to Korean Red Ginseng

Susceptibility of renal fibrosis in diabetes: Role of hypoxia inducible factor-1.

Diabetes may prevent kidney repair and sensitize the kidney to fibrosis or scar formation. To test this possibility, we examined renal fibrosis induced by unilateral ureteral obstruction (UUO) in diabetic mouse models. Indeed, UUO induced significantly more renal fibrosis in both Akita and STZ-induced diabetic mice than in nondiabetic mice. The diabetic mice also had more apoptosis and interstitial macrophage infiltration during UUO. In vitro, hypoxia induced higher expression of the fibrosis marker protein fibronectin in high glucose-conditioned renal tubular cells than in normal glucose cells. Mechanistically, hypoxia induced significantly more hypoxia-inducible factor-1 α (HIF-1 α) in high glucose cells than in normal glucose cells. Inhibition of HIF-1 attenuated the expression of fibronectin induced by hypoxia in high-glucose cells. Consistently, UUO induced significantly higher HIF-1α expression along with fibrosis in diabetic mice kidneys than in nondiabetic kidneys. The increased expression of fibrosis induced by UUO in diabetic mice was diminished in proximal tubule-HIF-1α-knockout mice. Together, these results indicate that diabetes sensitizes kidney tissues and cells to fibrogenesis probably by enhancing HIF-1 activation.

Protective and health-promoting impact of Washingtonia filifera oil on the kidney of STZ-induced diabetic mice

Diabetes kidney damage (DKD) is a chronic inflammatory disease of the kidney induced with continuous hyperglycemia as the most prevalent consequence of diabetes. Washingtonia filifera seed oil (WFO) was used as a traditional medicine to cure various diseases in ancient Saudi. This work was carried out to investigate the potential protective impact of WFO against DKD on streptozotocin (STZ)-induced type 2 diabetic mice (C57BL/6 mice). The mice were randomly split into groups: C, C + WFO (200 mg/Kg B.W.), T2D, and T2D + WFO (200 mg/Kg B.W.). Diabetes was created in mice groups except for the control group after 6 weeks of high-fat diet (HFD) feeding. Treatments with STZ (60 mg/kg body weight) were administered three times for 6 weeks, and after that, mice were sacrificed. Kidney tissues and serum were obtained to analyze levels of insulin, metabolism of lipids [triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and free fatty acids (FFA)], antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)], creatine, and blood urea nitrogen (BUN). In addition, H&E staining had been used to investigate the histological changes of the kidneys. In T2D mice, WFO corrected aberrant serum lipids (TG, TC, HDL, LDL, and FFA), elevated antioxidative enzyme levels (CAT, SOD, and GPx), and inhibited GST to various degrees. In addition, WFO improves kidney pathological traits such as fibrosis of the kidney, hypertrophy of glomeruli, and basement membrane thickness of glomeruli. Through hypoglycemic, hypolipidemic, antioxidative, and anti-inflammatory actions, WFO might ameliorate diabetic alterations in T2D mice. WFO could significantly reduce AGE buildup in the T2D mice kidneys, therefore alleviating kidney oxidative stress and inflammatory kidney damage.

Intestinal Bacterial Translocation Contributes to Diabetic Kidney Disease.

In recent years, many studies have focused on the intestinal environment to elucidate pathogenesis of various diseases, including kidney diseases. Impairment of the intestinal barrier function, the "leaky gut," reportedly contributes to pathologic processes in some disorders. Mitochondrial antiviral signaling protein (MAVS), a component of innate immunity, maintains intestinal integrity. The effects of disrupted intestinal homeostasis associated with MAVS signaling in diabetic kidney disease remains unclear. To evaluate the contribution of intestinal barrier impairment to kidney injury under diabetic conditions, we induced diabetic kidney disease in wild-type and MAVS knockout mice through unilateral nephrectomy and streptozotocin treatment. We then assessed effects on the kidney, intestinal injuries, and bacterial translocation. MAVS knockout diabetic mice showed more severe glomerular and tubular injuries compared with wild-type diabetic mice. Owing to impaired intestinal integrity, the presence of intestine-derived Klebsiella oxytoca and elevated IL-17 were detected in the circulation and kidneys of diabetic mice, especially in diabetic MAVS knockout mice. Stimulation of tubular epithelial cells with K. oxytoca activated MAVS pathways and the phosphorylation of Stat3 and ERK1/2, leading to the production of kidney injury molecule-1 (KIM-1). Nevertheless, MAVS inhibition induced inflammation in the intestinal epithelial cells and KIM-1 production in tubular epithelial cells under K. oxytoca supernatant or IL-17 stimulation. Treatment with neutralizing anti-IL-17 antibody treatment had renoprotective effects. In contrast, LPS administration accelerated kidney injury in the murine diabetic kidney disease model. Impaired MAVS signaling both in the kidney and intestine contributes to the disrupted homeostasis, leading to diabetic kidney disease progression. Controlling intestinal homeostasis may offer a novel therapeutic approach for this condition.

TangShenWeiNing Formula Prevents Diabetic Nephropathy by Protecting Podocytes Through the SIRT1/HIF-1α Pathway.

BackgroundDiabetic nephropathy (DN) represents a major complication of diabetes, and podocyte injury has a critical function in DN development. TangShenWeiNing formula (TSWN) has been demonstrated to efficiently decrease proteinuria and protect podocytes in DN. This work aimed to explore the mechanism by which TSWN alleviates DN and protects podocytes.MethodsThe major bioactive components of TSWN were detected by mass spectrometry (MS) and pharmacological databases. Eight-week-old male C57BLKS/J db/m and db/db mice were provided pure water, valsartan, low dose TSWN, middle dose TSWN and high dose TSWN by gavage for 12 weeks, respectively.ResultsMS and network pharmacology analyses suggested that TSWN might prevent DN through the sirtuin (SIRT)1/hypoxia-inducible factor (HIF)-1α pathway. Diabetic mice showed elevated urinary albumin in comparison with non-diabetic mice, and TSWN decreased urinary albumin in diabetic mice. Histological injury increased in the kidney in diabetic mice, which could be improved by TSWN. Fibrosis and collagen I expression were induced in the diabetic mouse kidney in comparison with the non-diabetic mouse kidney; TSWN alleviated these effects. Apoptosis and cleaved caspase-3 were induced in the diabetic mouse kidney in comparison with the non-diabetic mouse kidney, and TSWN blunted these effects. Podocytes were damaged in the diabetic mouse kidney, which was improved by TSWN. Podocin and nephrin amounts were decreased in the diabetic mouse kidney in comparison with the non-diabetic mouse kidney, and podocalyxin was increased in urine of diabetic animals in comparison with non-diabetic counterparts. After TSWN treatment, podocin and nephrin were raised in the diabetic mouse kidney, and urinary podocalyxin was depressed in diabetic animals. Diabetic mice had lower SIRT1 and higher HIF-1α amounts in kidney specimens in comparison with non-diabetic mice, and TSWN promoted SIRT1 and inhibited HIF-1α in the diabetic mouse kidney. Moreover, co-staining of SIRT1 and podocin revealed that SIRT1 decreased in podocytes from diabetic mice in comparison with those from non-diabetic mice, and TSWN elevated SIRT1 in podocytes.ConclusionsThis study indicated that TSWN alleviates DN by improving podocyte injury through the SIRT1/HIF-1α pathway in diabetic mouse kidneys.

IGFBP5 promotes diabetic kidney disease progression by enhancing PFKFB3-mediated endothelial glycolysis

Renal inflammation is a critical pathophysiological characteristic of diabetic kidney disease (DKD). The mechanism of the inflammatory response is complicated, and there are few effective treatments for renal inflammation that can be used clinically. Insulin-like growth factor-binding protein 5 (IGFBP5) is an important secretory protein that is related to inflammation and fibrosis in several tissues. Studies have shown that the IGFBP5 level is significantly upregulated in DKD. However, the function of IGFBP5 and its mechanism in DKD remain unclear. Here, we showed that IGFBP5 levels were significantly increased in the kidneys of diabetic mice. Ablation of IGFBP5 alleviated kidney inflammation in DKD mice. Mechanistically, IGFBP5 increased glycolysis, which was characterized by increases in lactic acid and the extracellular acidification rate, by activating the transcription factor early growth response 1 (EGR1) and enhancing the expression of PFKFB3 in endothelial cells. Furthermore, a mutation in PFKFB3 attenuated renal inflammation in DKD mice. Taken together, we provided evidence that IGFBP5 enhanced kidney inflammation through metabolic reprogramming of glomerular endothelial cells. Our results provide new mechanistic insights into the effect of IGFBP5 on kidney and highlight potential therapeutic opportunities for IGFBP5 and the metabolic regulators involved in DKD.

Adenosine/adenosine type 1 receptor signaling pathway did not play dominant roles on the influence of sodium-glucose cotransporter 2 inhibitor in the kidney of bovine serum albumin-overloaded streptozotocin-induced diabetic mice.

Aims/IntroductionSodium–glucose cotransporter 2 inhibitors (SGLT2i) have been shown to display excellent renoprotective effects in diabetic kidney disease with macroalbuminuria/proteinuria. Regarding the renoprotective mechanism of SGLT2i, a sophisticated hypothesis was made by explaining the suppression of glomerular hypertension/hyperfiltration through the adenosine/adenosine type 1 receptor (A1R) signaling‐mediated restoration of the tubuloglomerular feedback mechanism; however, how such A1R signaling is relevant for renoprotection by SGLT2i in diabetic kidney disease with proteinuria has not been elucidated.Materials and MethodsStreptozotocin‐induced diabetic CD‐1 mice were injected with bovine serum albumin (BSA) and treated with SGLT2i in the presence/absence of A1R inhibitor administration.ResultsWe found that the influences of SGLT2i are essentially independent of the activation of A1R signaling in the kidney of BSA‐overloaded streptozotocin‐induced diabetic mice. BSA‐overloaded diabetic mice showed the trend of kidney damage with higher glomerular filtration rate (GFR) and the significant induction of fibrogenic genes, such as transforming growth factor‐β2 and collagen type III. SGLT2i TA‐1887 suppressed diabetes‐induced GFR in BSA‐overloaded diabetic mice was associated with the significant suppression of transforming growth factor‐β2 and collagen type III; A1R‐specific inhibitor 8‐cyclopentyl‐1,3‐dipropylxanthine did not cancel the effects of TA‐1887 on either GFR or associated gene levels. Both TA‐1887 and 8‐cyclopentyl‐1,3‐dipropylxanthine‐treated BSA‐overloaded diabetic mice showed suppressed glycated hemoglobin levels associated with the increased food intake. When analyzing the association among histological evaluation, GFR and potential fibrogenic gene levels, each group of mice showed distinct correlation patterns.ConclusionsA1R signaling activation was not the dominant mechanism on the influence of SGLT2i in the kidney of BSA‐overloaded diabetic mice.

Peroxisome-generated succinate induces lipid accumulation and oxidative stress in the kidneys of diabetic mice

Diabetes normally causes lipid accumulation and oxidative stress in the kidneys, which plays a critical role in the onset of diabetic nephropathy; however, the mechanism by which dysregulated fatty acid metabolism increases lipid and reactive oxygen species (ROS) formation in the diabetic kidney is not clear. As succinate is remarkably increased in the diabetic kidney, and accumulation of succinate suppresses mitochondrial fatty acid oxidation and increases ROS formation, we hypothesized that succinate might play a role in inducing lipid and ROS accumulation in the diabetic kidney. Here we demonstrate a novel mechanism by which diabetes induces lipid and ROS accumulation in the kidney of diabetic animals. We show that enhanced oxidation of dicarboxylic acids by peroxisomes leads to lipid and ROS accumulation in the kidney of diabetic mice via the metabolite succinate. Furthermore, specific suppression of peroxisomal β-oxidation improved diabetes-induced nephropathy by reducing succinate generation and attenuating lipid and ROS accumulation in the kidneys of the diabetic mice. We suggest that peroxisome-generated succinate acts as a pathological molecule inducing lipid and ROS accumulation in kidney, and that specifically targeting peroxisomal β-oxidation might be an effective strategy in treating diabetic nephropathy and related metabolic disorders.

Midazolam Ameliorates Hyperglycemia-Induced Glomerular Endothelial Dysfunction by Inhibiting Transglutaminase 2 in Diabetes.

Midazolam is an anesthetic widely used for anxiolysis and sedation; however, to date, a possible role for midazolam in diabetic kidney disease remains unknown. Here, we investigated the effect of midazolam on hyperglycemia-induced glomerular endothelial dysfunction and elucidated its mechanism of action in kidneys of diabetic mice and human glomerular microvascular endothelial cells (HGECs). We found that, in diabetic mice, subcutaneous midazolam treatment for 6 weeks attenuated hyperglycemia-induced elevation in urine albumin/creatinine ratios. It also ameliorated hyperglycemia-induced adherens junction disruption and subsequent microvascular leakage in glomeruli of diabetic mice. In HGECs, midazolam suppressed high glucose-induced vascular endothelial-cadherin disruption and endothelial cell permeability via inhibition of intracellular Ca2+ elevation and subsequent generation of reactive oxygen species (ROS) and transglutaminase 2 (TGase2) activation. Notably, midazolam also suppressed hyperglycemia-induced ROS generation and TGase2 activation in glomeruli of diabetic mice and markedly improved pathological alterations in glomerular ultrastructure in these animals. Analysis of kidneys from diabetic Tgm2−/− mice further revealed that TGase2 played a critical role in microvascular leakage. Overall, our findings indicate that midazolam ameliorates hyperglycemia-induced glomerular endothelial dysfunction by inhibiting ROS-mediated activation of TGase2.

Etoposide-induced protein 2.4 ameliorates high glucose-induced epithelial-mesenchymal transition by activating adenosine monophosphate-activated protein kinase pathway in renal tubular cells.

Epithelial-mesenchymal transition (EMT), known as the transition of tubular epithelial cells into fibroblasts, is one of the potential mechanisms of renal fibrosis, which promotes the development of diabetic kidney disease (DKD). Etoposide-induced protein 2.4 (EI24) is known as an endoplasmic reticulum (ER)-localized Bcl-2-binding transmembrane protein with various functions that can affect autophagy, apoptosis and differentiation. However, whether EI24 is involved in EMT of renal tubular epithelial cells and the exact mechanism is still not known. In this study, we first reported that EI24 expression was significantly downregulated in the kidneys of diabetic mice and in high glucose-stimulated HK2 cells. Knockdown of EI24 led to EMT of HK2 cells, as indicated by decreased E-cadherin and increased α-smooth muscle actin (α-SMA). Meanwhile, overexpression of EI24 ameliorated high glucose-induced EMT of HK2 cells via activation of the adenosine monophosphate-activated protein kinase (AMPK) pathway. Then, DNA methyltransferase (DNMT) inhibitor 5-Aza-2'-deoxycytidine (5-Aza) treatment enhanced EI24 expression and alleviated EMT in high glucose-treated HK2 cells and the kidneys of diabetic mice. Furthermore, DNMT1 and DNMT3a upregulation were found to be involved in the decrease of EI24 in high glucose-stimulated HK2 cells. Silencing of DNMT1 and DNMT3a effectively reversed high glucose-induced downregulation of EI24 and aggravation of EMT. Our findings demonstrate that the DNA methyltransferase-regulated EI24 affects EMT of renal tubular cells via AMPK signaling pathway. It is suggested that EI24 may be a potential therapeutic target for diabetic renal injury.

Pterostilbene, a Resveratrol Derivative, Improves Ectopic Lipid Deposition in the Kidneys of Mice Induced by a High-Fat Diet

Background: Diabetic kidney disease is a major cause of global end-stage renal diseases. Ectopic lipid deposition in the renal tissues of diabetic kidney disease is one major factor leading to renal fibrosis and chronic kidney disease. Pterostilbene has been reported to display lipid-lowing activity and participate in many kidney diseases. However, the influence of pterostilbene on the ectopic lipid deposition is unclear. We intend to explore the influence of pterostilbene on the ectopic lipid deposition in the kidneys of mice induced by high fat. Methods: A high-fat diet-induced diabetic mouse model was established to detect the alleviative effect of pterostilbene on the ectopic lipid deposition in the kidneys of diabetic mice. A biochemical analysis was performed to examine the levels of urine albumin, urine creatinine, serum creatinine, and blood urea nitrogen in mice after pterostilbene treatment. Histological analysis was conducted to detect the degree of renal injury and fibrosis. Oil red O staining and immunohistochemical staining were carried out to evaluate lipid droplets and the expression of adipose differentiation-related protein in renal tissues of the mice treated by pterostilbene. The protein levels were assessed by Western blotting. Results: Pterostilbene inhibits the expression of the TGF-β1 and p-smad3 and suppresses the protein levels of SREBP-1 and FAS, and it ultimately reduces the ectopic lipid deposition, alleviates the renal tubular damage and renal fibrosis in the kidneys of diabetic mice induced by high fat, and improves kidney function. Conclusion: Pterostilbene alleviates renal fibrosis and ectopic lipid deposition in the kidneys of diabetic mice induced by high-fat diet by inhibiting the TGF-β1/smad3 signaling.

GPR43 activation-mediated lipotoxicity contributes to podocyte injury in diabetic nephropathy by modulating the ERK/EGR1 pathway.

Background: G-protein-coupled receptor 43 (GPR43) is a posttranscriptional regulator involved in cholesterol metabolism. This study aimed to investigate the possible roles of GPR43 activation in podocyte lipotoxicity in diabetic nephropathy (DN) and explore the potential mechanisms.Methods: The experiments were conducted by using diabetic GPR43-knockout mice and a podocyte cell culture model. Lipid deposition and free cholesterol levels in kidney tissues were measured by BODIPY staining and quantitative cholesterol assays, respectively. The protein expression of GPR43, LC3II, p62, beclin1, low-density lipoprotein receptor (LDLR) and early growth response protein 1 (EGR1) in kidney tissues and podocytes was measured by real-time PCR, immunofluorescent staining and Western blotting.Results: There were increased LDL cholesterol levels in plasma and cholesterol accumulation in the kidneys of diabetic mice. However, GPR43 gene knockout inhibited these changes. An in vitro study further demonstrated that acetate treatment induced cholesterol accumulation in high glucose-stimulated podocytes, which was correlated with increased cholesterol uptake mediated by LDLR and reduced cholesterol autophagic degradation, as characterized by the inhibition of LC3 maturation, p62 degradation and autophagosome formation. Gene knockdown or pharmacological inhibition of GPR43 prevented these effects on podocytes. Furthermore, GPR43 activation increased extracellular regulated protein kinases 1/2 (ERK1/2) activity and EGR1 expression in podocytes, which resulted in an increase in cholesterol influx and autophagy inhibition. In contrast, after GPR43 deletion, these changes in podocytes were improved, as shown by the in vivo and in vitro results.Conclusion: GPR43 activation-mediated lipotoxicity contributes to podocyte injury in DN by modulating the ERK/EGR1 pathway.

Marein Regulates Insulin Resistance in Diabetic Nephropathy Mice by Inducing Autophagy

Marein is the main active substance of Coreopsis tinctoria nutt. It not only has anti-oxidation and anti-tumor effects, but also can lower blood lipid, prevent high blood glucose, improve insulin resistance, inhibit gluconeogenesis and promote glycogen synthesis. However, the exact mechanism of its action is still unclear. Here, we explored the effect and mechanism of Marein on insulin resistance. The mice were divided into db/m, db/db, metformin+db/db, and marein+db/db groups. The body weight and kidney weight were recorded. Serum biochemical and renal function tests were measured after 8 weeks of continuous administration. Kidney tissues were subjected to HE staining, PAS staining, and Masson staining. The effect of marein on PI3K/Akt signal and autophagy pathway was detected by Western blot. After 8 weeks of Marein intervention, the body weight and kidney weight of mice did not change significantly, but the fasting blood glucose and blood lipid levels were significantly reduced than db/db group. Marein significantly improved the insulin resistance index, increased serum adiponectin and improved glucose and lipid metabolism disorders of db/db mice. Moreover, marein improved the basement membrane thickness of glomeruli and tubules, improved glomerular sclerosis and tubular fibrosis, as well as renal insufficiency, thereby protecting kidney function and delaying the pathological damage. Furthermore, marein increased the expression of PI3K and the phosphorylation of Akt/Akt (Ser473), and promoted the expression of LC3II/I, Beclin1 and ATG5. Additionally, it promoted the expression of FGFR1 in the kidney of db/db mice, and promoted the increase of serum FGF21 and FGF23. Marein has a protective effect on the kidneys of diabetic mice. It protects diabetic nephropathy by regulating the IRS1/PI3K/Akt signaling pathway to improve insulin resistance. Therefore, marein may be an insulin sensitizer.