Renal Injury In Diabetic Nephropathy Research Articles

Sodium butyrate improves renal injury in diabetic nephropathy through AMPK/SIRT1/PGC-1α signaling pathway

Diabetic nephropathy (DN) is a prototypical chronic energy metabolism imbalance disease. The AMPK/Sirt1/PGC-1α signaling pathway plays a pivotal role in regulating energy metabolism throughout the body. Gut microbiota ferment indigestible carbohydrates to produce a variety of metabolites, particularly short-chain fatty acids (SCFAs), which exert positive effects on energy metabolism. However, the potential for SCFAs to ameliorate DN-associated renal injury via the AMPK/Sirt1/PGC-1α pathway remains a matter of debate. In this study, we investigated the effects of sodium butyrate (NaB), a SCFA, on energy metabolism in mice with spontaneous DN at two different doses. Body weight, blood glucose and lipid levels, urinary protein excretion, liver and kidney function, interleukin-6 (IL-6) levels, and the expressions of AMPK, phosphorylated AMPK (p-AMPK), mitofusin 2 (MFN2), optic atrophy 1 (OPA1), and glucagon-like peptide-1 receptor (GLP-1R) were monitored in mice. Additionally, butyrate levels, gut microbiota composition, and diversity in colonic stool were also assessed. Our findings demonstrate that exogenous NaB supplementation can improve hyperglycemia and albuminuria, reduce renal tissue inflammation, inhibit extracellular matrix accumulation and glomerular hypertrophy, and could alter the gut microbiota composition in DN. Furthermore, NaB was found to upregulate the expressions of MFN2, OPA1, p-AMPK, and GLP-1R in DN renal tissue. These results suggest that NaB could improve the composition of gut microbiota in DN, activate the AMPK/Sirt1/PGC-1α signaling pathway, and enhance mitochondrial function to regulate energy metabolism throughout the body. Collectively, our findings indicate that NaB may be a novel therapeutic agent for the treatment of DN.

HNRNP I promotes IRAK1 degradation to reduce podocyte apoptosis and inflammatory response alleviating renal injury in diabetic nephropathy

Podocytes maintain renal filtration integrity when the glomerular filtration barrier (GFB) is integrated. Impairment or attrition of podocytes, leading to compromised GFB permeability, constitutes the primary etiology of proteinuria and is a hallmark pathological feature of diabetic nephropathy (DN). This study centers on Heterogeneous Nuclear Ribonucleoprotein I (HNRNP I), an RNA-binding protein, delineating its role in facilitating DN-induced renal damage by modulating podocyte health. Comparative analyses in renal biopsy specimens from DN patients and high-glucose-challenged podocyte models in vitro revealed a marked downregulation of HNRNP I expression relative to normal renal tissues and podocytes. In vitro assays demonstrated that high-glucose conditions precipitated a significant reduction in podocyte viability and an escalation in markers indicative of apoptosis. Conversely, HNRNP I overexpression was found to restore podocyte viability and attenuate apoptotic indices. IRAK1, a gene encoding a protein integral to inflammatory signaling, was shown to interact with HNRNP I, which promotes IRAK1 degradation. This interaction culminates in suppressing the PI3K/AKT/mTOR signaling pathway, thereby diminishing podocyte apoptosis and mitigating renal damage in DN. This investigation unveils the mechanistic role of HNRNP I in DN for the first time, potentially informing novel therapeutic strategies for DN renal impairment.

Acetyl-CoA synthetase 2 promotes diabetic renal tubular injury in mice by rewiring fatty acid metabolism through SIRT1/ChREBP pathway.

Diabetic nephropathy (DN) is characterized by chronic low-grade renal inflammatory responses, which greatly contribute to disease progression. Abnormal glucose metabolism disrupts renal lipid metabolism, leading to lipid accumulation, nephrotoxicity, and subsequent aseptic renal interstitial inflammation. In this study, we investigated the mechanisms underlying the renal inflammation in diabetes, driven by glucose-lipid metabolic rearrangement with a focus on the role of acetyl-CoA synthetase 2 (ACSS2) in lipid accumulation and renal tubular injury. Diabetic models were established in mice by the injection of streptozotocin and in human renal tubular epithelial HK-2 cells cultured under a high glucose (HG, 30 mmol/L) condition. We showed that the expression levels of ACSS2 were significantly increased in renal tubular epithelial cells (RTECs) from the diabetic mice and human diabetic kidney biopsy samples, and ACSS2 was co-localized with the pro-inflammatory cytokine IL-1β in RTECs. Diabetic ACSS2-deficient mice exhibited reduced renal tubular injury and inflammatory responses. Similarly, ACSS2 knockdown or inhibition of ACSS2 by ACSS2i (10 µmol/L) in HK-2 cells significantly ameliorated HG-induced inflammation, mitochondrial stress, and fatty acid synthesis. Molecular docking revealed that ACSS2 interacted with Sirtuin 1 (SIRT1). In HG-treated HK-2 cells, we demonstrated that ACSS2 suppressed SIRT1 expression and activated fatty acid synthesis by modulating SIRT1-carbohydrate responsive element binding protein (ChREBP) activity, leading to mitochondrial oxidative stress and inflammation. We conclude that ACSS2 promotes mitochondrial oxidative stress andrenal tubular inflammation in DN by regulating the SIRT1-ChREBP pathway. This highlights the potential therapeutic value of pharmacological inhibition of ACSS2 for alleviating renal inflammation and dysregulation of fatty acid metabolic homeostasis in DN. Metabolic inflammation in the renal region, driven by lipid metabolism disorder, is a key factor in renal injury in diabetic nephropathy(DN). Acetyl-CoA synthetase 2 (ACSS2) is abundantly expressed in renal tubular epithelial cells (RTECs) and highly upregulated in diabetic kidneys. Deleting ACSS2 reduces renal fatty acid accumulation and markers of renal tubular injury in diabetic mice. We demonstrate that ACSS2 deletion inhibits ChREBP-mediated fatty acid lipogenesis, mitochondrial oxidative stress, and inflammatory response in RTECs, which play a major role in the progression of diabetic renal tubular injury in the kidney. These findings support the potential use of ACSS2 inhibitors in treating patients with DN.

Sex and strain differences in renal hemodynamics in mice.

The present study was to examine sex and strain differences in glomerular filtration rate (GFR) and renal blood flow (RBF) in C57BL6, 129/Sv, and C57BLKS/J mice, three commonly used mouse strains in renal research. GFR was measured by transdermal measurement of FITC-sinitrin clearance in conscious mice. RBF was measured by a flow probe placed in the renal artery under an anesthetic state. In C57BL6 mice, there were no sex differences in both GFR and RBF. In 129/Sv mice, females had significantly greater GFR than males at age of 24 weeks, but not at 8 weeks. However, males had higher RBF and lower renal vascular resistance (RVR). Similar to 129/Sv, female C57BLKS/J had significantly greater GFR at both 8 and 24 weeks, lower RBF, and higher RVR than males. Across strains, male 129/Sv had lower GFR and higher RBF than male C57BL6, but no significant difference in GFR and greater RBF than male C57BLKS/J. No significant difference in GFR or RBF was observed between C57BL6 and C57BLKS/J mice. Deletion of eNOS in C57BLKS/J mice reduced GFR in both sexes, but decreased RBF in males. Furthermore, there were no sex differences in the severity of renal injury in eNOS-/- dbdb mice. Taken together, our study suggests that sex differences in renal hemodynamics in mice are strain and age dependent. eNOS was not involved in the sex differences in GFR, but in RBF. Furthermore, the sexual dimorphism did not impact the severity of renal injury in diabetic nephropathy.

Thrombomodulin activation driven by LXR agonist attenuates renal injury in diabetic nephropathy.

Inflammation and thrombosis are recognized as interrelated biological processes. Both thrombomodulin (TM) and factor XIII-A (FXIII-A) are involved in inflammation and coagulation process. However, their role in the pathogenesis of diabetic nephropathy (DN) remains unclear. In vitro study, the liver X receptor (LXR) agonist T0901317 can up-regulate the expression of TM in glomerular endothelial cells. Now we evaluated the interaction between TM activation and FXIII-A and their effects against renal injury. We first evaluated the serum levels of FXIII-A and TM and the expression of TM, LXR-α and FXIII-A in renal tissues of patients with biopsy-proven DN. We then analyzed the expression of TM, LXR-α and FXIII-A in renal tissues of db/db DN mice after upregulating TM expression via T0901317 or downregulating its expression via transfection of TM shRNA-loaded adenovirus. We also investigated the serum levels of Tumor necrosis factor (TNF)-α, Interleukin (IL)-6, creatinine, and urinary microalbumin level in db/db mice. Our study showed that elevations in serum levels of FXIII-A positively correlated to the serum levels of TM and were also associated with end-stage kidney disease in patients with DN. The number of TM+ cells in the renal tissues of patients with DN negatively correlated with the number of FXIII-A+ cells and positively correlated with the number of LXR-α+ cells and estimated glomerular filtration rate (eGFR), whereas the number of FXIII-A+ cells negatively correlated with the eGFR. Thrombomodulin activation with T0901317 downregulated FXIII-A expression in the kidney tissue and alleviated renal injury in db/db mice.

English

BACKGROUND Increased levels of urinary biomarkers can be detected in type 2 diabetic patients before the onset of significant albuminuria and may be used as an early marker of renal injury in diabetic nephropathy (DN) which would play a significant role for the effective management and treatment approaches in diabetic care. We wanted to evaluate cystatin C and microalbumin as effective early biomarkers in assessing nephropathy in patients with type 2 diabetes mellitus in this study. METHODS A cross-sectional study was conducted among 180 subjects grouped into healthy controls, clinically proven T2DM without nephropathy and type 2 DM with nephropathy comprising 60 participants in each group. Fasting and postprandial blood samples and urine samples were collected and analysed by standard methods. eGFR was calculated using CKD-EPI 2012 equation. IBM - SPSS version 20 was used for statistical analysis. RESULTS Diabetic nephropathy patients had significantly elevated serum cystatin C and microalbumin (2.43 ± 0.59, 700.5 ± 591.8 mg / L, respectively), compared to T2DM (0.98 ± 0.26, 63.7 ± 102.9 mg / L, respectively), and the control study subjects (0.81 ± 0.16, 11.15 ± 8.9 mg / L, respectively). Serum cystatin C showed AUC of 0.994 (95 % CI, 0.986 - 1.00) whereas microalbumin showed 0.944 (95 % CI, 0.907 - 0.981). Serum cystatin C showed a sensitivity of 96.7 % and a specificity of 91.7 % at a cutoff point of 1.34 mg / L whereas at a cut-off point of 138.5 mg / L for microalbumin, the sensitivity and specificity were 90 % and 83.3 % respectively. CONCLUSIONS Serum cystatin C and microalbumin both could be considered as markers for early detection of nephropathy in T2DM patients. The more prominent rise in serum cystatin C values provide an earlier diagnosis of diabetic nephropathy among T2DM patients. KEY WORDS Biomarker, Type 2 Diabetes Mellitus, Cystatin C, Diabetic Nephropathy, Microalbumin

Protective effect of dexmedetomidine against renal injury in diabetic nephropathy rats through inhibiting NF-κB pathway.

The aim of this study was to investigate the protective effect of dexmedetomidine (Dex) against renal injury in diabetic nephropathy (DN) rats by inhibiting the nuclear factor-κB (NF-κB) pathway. A total of 36 Sprague-Dawley rats were randomly divided into three groups, including: normal group (n=12), model group (n=12) and Dex group (n=12). The rats underwent no treatment in normal group. In model group, the diabetes model was successfully established, and normal saline was intraperitoneally injected after operation. In Dex group, the diabetes model was established as well, and Dex was intraperitoneally injected after operation. After intervention for 2 weeks, the samples were taken for use. Blood urea nitrogen (BUN) and serum creatinine (Cr) were detected using a full-automatic biochemical analyzer. The expression of Caspase-3 was detected via immunohistochemistry. Western blotting was conducted to detect the protein expression of NF-κB. The apoptosis was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. In addition, the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined via enzyme-linked immunosorbent assay (ELISA). The levels of BUN and Cr were significantly higher in model group and Dex group than those in normal group (p<0.05). However, they were significantly lower in Dex group than those in the model group (p<0.05). Immunohistochemistry results showed that the mean optical density of Caspase-3 positive expression increase remarkably in model group and Dex group when compared with normal group (p<0.05). However, it significantly declined in Dex group when compared with the model group (p<0.05). The results of Western blotting revealed that model group and Dex group exhibited evidently higher relative protein expression of NF-κB than normal group (p<0.05). However, Dex group displayed notably lower relative protein expression of NF-κB than model group (p<0.05). TUNEL assay demonstrated that the apoptosis rate increased significantly in the model group and Dex group when compared with normal group (p<0.05). However, it remarkably declined in Dex group in comparison with the model group (p<0.05). Finally, ELISA assay indicated that model group and Dex group had markedly higher levels of IL-6 and TNF-α than normal group (p<0.05). However, the levels of IL-6 and TNF-α were significantly lower in Dex group than model group (p<0.05). Dex inhibits inflammation and apoptosis by suppressing the NF-κB signaling pathway, thereby exerting a protective effect against renal injury in DN rats.

DsbA-L mediated renal tubulointerstitial fibrosis in UUO mice

Recent studies have reported that upregulation of disulfide-bond A oxidoreductase-like protein (DsbA-L) prevented lipid-induced renal injury in diabetic nephropathy (DN). However, the role and regulation of proximal tubular DsbA-L for renal tubulointerstitial fibrosis (TIF) remains unclear. In current study, we found that a proximal tubules-specific DsbA-L knockout mouse (PT-DsbA-L-KO) attenuated UUO-induced TIF, renal cell apoptosis and inflammation. Mechanistically, the DsbA-L interacted with Hsp90 in mitochondria of BUMPT cells which activated the signaling of Smad3 and p53 to produce connective tissue growth factor (CTGF) and then resulted in accumulation of ECM of BUMPT cells and mouse kidney fibroblasts. In addition, the progression of TIF caused by UUO, ischemic/reperfusion (I/R), aristolochic acid, and repeated acute low-dose cisplatin was also alleviated in PT-DsbA-L-KO mice via the activation of Hsp90 /Smad3 and p53/CTGF axis. Finally, the above molecular changes were verified in the kidney biopsies from patients with obstructive nephropathy (Ob). Together, these results suggest that DsbA-L in proximal tubular cells promotes TIF via activation of the Hsp90 /Smad3 and p53/CTGF axis.

Liraglutide suppresses production of extracellular matrix proteins and ameliorates renal injury of diabetic nephropathy by enhancing Wnt/β-catenin signaling.

The Wnt/β-catenin signaling pathway is involved in production of the extracellular matrix (ECM) by mesangial cells (MCs). Recent studies by us and others have demonstrated that glucagon-like peptide-1 receptor agonists (GLP-1RAs) have protective effects against diabetic nephropathy. The purpose of the present study was to investigate whether the Wnt/β-catenin signaling in MCs contributes to GLP-1RA-induced inhibition of ECM accumulation and mitigation of glomerular injury in diabetic nephropathy. In cultured human mesangial cells, liraglutide (a GLP-1RA) treatment significantly reduced high glucose (HG)-stimulated production of fibronectin, collagen type IV, and α-smooth muscle actin, and the liraglutide effects were significantly attenuated by XAV-939, a selective inhibitor of Wnt/β-catenin signaling. Furthermore, HG treatment significantly decreased protein abundance of Wnt4, Wnt5a, phospho-glycogen synthase kinase-3β, and β-catenin. These HG effects on Wnt/β-catenin signaling proteins were significantly blunted by liraglutide treatment. For in vivo experiments, we administered liraglutide (200 μg·kg-1·12 h-1) by subcutaneous injection to streptozocin-induced type 1 diabetic rats for 8 wk. Administration of liraglutide significantly improved elevated blood urine nitrogen, serum creatinine, and urinary albumin excretion rate and alleviated renal hypertrophy, mesangial expansion, and glomerular fibrosis in type 1 diabetic rats, whereas blood glucose level and body weight did not have significant changes. Consistent with the in vitro experiments, liraglutide treatment significantly reduced the diabetes-induced increases in glomerular fibronectin, collagen type IV, and α-smooth muscle actin and decreases in glomerular Wnt/β-catenin signaling proteins. These results suggest that liraglutide alleviated glomerular ECM accumulation and renal injury in diabetic nephropathy by enhancing Wnt/β-catenin signaling.

Effect of Alpina oxyphylla extract on streptozotocin-induced kidney injure via regulating TGF-\u03b21 and MyD88

BackgroundAbnormal renal metabolism is closely related to the development of chronic kidney disease. It is well known that renal inflammation plays an important role in the occurrence and development of tubulointerstitial damage in the renal tubules. The purpose of the experiment was to observe the bioactivity of Alpina oxyphylla extract (AOE) on renal injury in diabetic nephropathy (DN) rats induced by streptozotocin (STZ).MethodsThirty male Wistar rats were randomly divided into five group (n = 6): (1) intact control (non-diabetic, ND); (2) intact diabetic (STZ), (3) diabetic rats treated with gliclazide 5 mg/kg (STZ-gli), (4) diabetic rats treated with AOE 400 mg/kg (AOE 400), (5) diabetic rats treated with AOE 800 mg/kg (AOE 800). The diabetic nephropathy rat model was established by single intraperitoneal injected 50 mg/kg STZ. Fasting blood glucose (FBG) and body weight was observed at 1、3、6 weeks. After 6 weeks, the renal function parameters of five groups and 24 h urinary protein were detected. Expression of transforming growth factor-beta1 (TGF-β1) and myeloid differentiation factor 88 (MyD88) were assessed by Western Blot.ResultsThe STZ group showed hyperglycemia, proteinuria, renal function damage, and the levels of 24 h urinary protein, fasting blood glucose (FBG), blood urea nitrogen (BUN), serum creatinine (Scr), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and interleukin-6 (IL-6) in the STZ group increased significantly compared with the ND group. The expression of TGF-β1 in STZ group was increase (p < 0.01), and the expression of MyD88 was significantly lower than in ND group (p < 0.05). The treatment of DN rats with AOE attenuated DN-associated in the serum biochemical index and the expression of TGF-β1.ConclusionsAOE can effectively protect kidney tissues of diabetic nephropathy, and probably through regulating level of TGF-β1/MyD88.

Astragalus mongholicus Bunge and Panax notoginseng (Burkill) F.H. Chen Formula for Renal Injury in Diabetic Nephropathy-In Vivo and In Vitro Evidence for Autophagy Regulation.

BackgroundDiabetic nephropathy (DN) is a serious complication of diabetes mellitus (DM) with limited treatment options. DN leads to progressive renal failure and accelerates rapidly into end-stage renal disease. Astragalus mongholicus Bunge and Panax notoginseng (Burkill) F.H. Chen formula (APF) is a traditional Chinese medicine (TCM) formula widely used to treat chronic kidney diseases (CKD) in the clinic in the southwest of China. The aim of this study is to explore how APF and its related TCM theory work on DN and whether mTOR/PINK1/Parkin signaling plays a part in this process.MethodsHPLC was used for preliminary chemical analysis and quantitative analysis of the five components of APF. An in vivo autophagy deficiency model was established in C57BL/6 mice by streptozocin (STZ) combined with a high-fat and high-sugar diet, while the in vitro autophagy deficiency model was induced with high glucose (HG) in renal mesangial cells (RMCs). Renal histopathology staining was performed to investigate the extents of inflammation and injury. Real time-PCR and Western blotting techniques were utilized to assess autophagy-related proteins.ResultsAPF significantly ameliorated renal injury in DN mice, specifically restoring blood urea nitrogen, serum creatinine, and 24-hour albuminuria. APF also reduced the mRNA and protein expressions of TNFα, IL-1β, and IL-6 in STZ-induced DN mice. Furthermore, APF improved the autophagy deficiency induced by STZ in vivo or HG in vitro, as revealed by changes in the expressions of mTOR, PINK1, Parkin, Beclin 1, p62, and LC3B. Notably, inhibition of autophagy with 3-methyladenine in APF-treated RMCs aggravated cellular damage and altered mTOR/PINK1/Parkin signaling, indicating that APF rescued HG damage through promoting autophagy.ConclusionAPF may protect the kidneys from inflammation injuries in DN by upregulating autophagy via suppressing mTOR and activating PINK1/Parkin signaling. This experimental evidence strongly supports APF as a potential option for the prevention and treatment of DN.

Exploring the role of the metabolite-sensing receptor GPR109a in diabetic nephropathy

Alterations in gut homeostasis may contribute to the progression of diabetic nephropathy. There has been recent attention on the renoprotective effects of metabolite-sensing receptors in chronic renal injury, including the G-protein-coupled-receptor (GPR)109a, which ligates the short chain fatty acid butyrate. However, the role of GPR109a in the development of diabetic nephropathy, a milieu of diminished microbiome-derived metabolites, has not yet been determined. This study aimed to assess the effects of insufficient GPR109a signalling via genetic deletion of GPR109a on the development of renal injury in diabetic nephropathy. Gpr109a-/- mice or their wildtype littermates (Gpr109a+/+) were rendered diabetic with streptozotocin (STZ). Mice received a control diet or an isocaloric high fiber diet (12.5% resistant starch) for 24 weeks and gastrointestinal permeability and renal injury were determined. Diabetes was associated with increased albuminuria, glomerulosclerosis and inflammation. In comparison, Gpr109a-/- mice with diabetes did not show an altered renal phenotype. Resistant starch supplementation did not afford protection from renal injury in diabetic nephropathy. Whilst diabetes was associated with alterations in intestinal morphology, intestinal permeability assessed in vivo using the FITC-dextran test was unaltered. GPR109a deletion did not worsen gastrointestinal permeability. Further, 12.5% resistant starch supplementation, at physiological concentrations, had no effect on intestinal permeability or morphology. These studies indicate that GPR109a does not play a critical role in intestinal homeostasis in a model of type 1 diabetes or in the development of diabetic nephropathy.

Dose-response regulation system for improving renal injury in diabetic nephropathy by buckwheat hull flavonoids

Abstract Diabetic nephropathy (DN) is the most devastating for patients’ quality of life and survival. In this study, biochemical indicators and differences in protein expression by proteomics analysis and post-translational modifications (PTMs) of kidney of db/db mice were determined at low (LBHF, 10 mg/kg), middle (MBHF, 30 mg/kg) and high (HBHF, 60 mg/kg) Buckwheat hull flavonoids (BHF) doses intervention for 9 weeks. The results showed that BHF can significantly reduce SCr, SUA, IL-6 and IL-1β levels. Proteomics analysis revealed that the down-regulated differentially expressed proteins (DE protein) in the MBHF group were moreover 2 times than that in LBHF and HBHF group. BHF significantly down-regulated the pathway associated with the Immune system, Transport and catabolism, Environmental information processing. Decreasing lysine succinylation, acetylation and crotonylation of kidney protein were detected significantly in the MBHF group. In conclusion, BHF may be a potential natural product candidate for the effective treatment of DN.

Calpain-10 drives podocyte apoptosis and renal injury in diabetic nephropathy.

BackgroundDiabetic nephropathy (DN) is a progressive microvascular complication of diabetes mellitus (DM), driven largely by podocyte apoptosis. The cysteine protease Calpain 10 is known to augment apoptosis and necrosis, and is a potential therapeutic target in DN.MethodsType 2 diabetes was induced in SD rats by high-fat diet (HFD) feeding and streptozotocin (STZ) injections, and simulated in vitro by culturing conditionally immortalized mouse podocytes in hyperlipidemic (PA, 100 μM) conditions. The rate of apoptosis in the renal tissues and cultured podocytes was determined by TUNEL assay. The expression of Calpain 10 and its biological effects were assayed by real-time PCR, Western blotting, immunofluorescence and electron microscopy.ResultsCalpain 10 was up-regulated in the kidneys of DN rats, as well as immortalized mouse podocytes. High levels of Calpain 10 was associated with renal dysfunction and tissue destruction, and podocyte injury and apoptosis. Knockdown of Calpain 10 protected podocytes by decreasing apoptosis rate, and upregulated nephrin.ConclusionCalpain 10 is a pro-apoptotic factor in DN, and can be targeted for treating glomerular diseases.

Overexpression of Sirt6 promotes M2macrophage transformation, alleviating renal injury in diabetic nephropathy.

In this study, we aimed to investigate the associations between Sirt6, macrophages and diabetic nephropathy (DN). Immunohistochemical, western blot and RT-qPCR analyses were performed to detect the expression levels of Sirt6, the markers of podocytes and monocytes and related inflammatory factors in the tissues of rats with streptozocin-induced DN. A series of cell experiments in isolated culture or the co-culture of macrophages and podocytes were conducted to examine the effects of the overexpression of Sirt6 on macrophage transformation, podocyte apoptosis and associated genes, and analyses were performed using RT-qPCR, flow cytometry and western blot analysis, where appropriate. In the rat model of DN, injured podocytes were represented by the decreased protein expression levels of Nephrin and Sirt6, and by an increased Desmin expression. Additionally, the M1 phenotype transformation of macrophages was evidenced by the increased expression levels of CD86, tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS), and by the decreased expression levels of CD206, Sirt6, interleukin (IL)-4 and IL-10. In vitro assays of macrophages and podocytes demonstrated that glucose promoted macrophage M1 transformation and podocyte apoptosis in a dose-dependent manner and attenuated Sirt6 expression. Macrophages transformed into the M2 phenotype following the overexpression of Sirt6 by the successful transfection of macrophages with a Sirt6 overexpression plasmid. Sirt6 was also overexpressed in podocytes. In a Transwell co-culture system, the overexpression of Sirt6 in macrophages (but not the overexpression of Sirt6 in podocytes) protected the podocytes from high-glucose-induced injury. However, the apoptosis of the podocytes overexpressing Sirt6 (induced by transfection with a Sirt6 overexpression plasmid) still increased when these podocytes were co-cultured with macrophages in high-glucose medium. These protective effects were evidenced by the inhibition of apoptosis, the upregulation of the expression levels of Bcl-2 and CD206, as well as by the decreased expression levels of Bax and CD86. On the whole, the findings of this study suggest that Sirt6 protects podocytes against injury in a mimicked diabetic kidney microenvironment by activating M2 macrophages, indicating that Sirt6 can act as an immune response regulatory factor in DN-associated renal inflammatory injury.

DsbA-L ameliorates high glucose induced tubular damage through maintaining MAM integrity.

BackgroundThe mitochondrial associated endoplasmic reticulum (ER) membrane (MAM) provides a platform for communication between the mitochondria and ER, and it plays a vital role in many biological functions. Disulphide-bond A oxidoreductase-like protein (DsbA-L), expressed in the MAM, serves as an antioxidant and reduces ER stress. However, the role of DsbA-L and MAM in kidney pathobiology remains unclear.MethodsMolecular biology techniques, transmission electron microscopy (TEM), in situ proximity ligation assays (PLAs), confocal microscopy, TUNEL staining and flow cytometry were utilized to analyse apoptosis and status of MAM in DsbA-L mutant mice.FindingsWe showed that MAM was significantly reduced in the kidneys of streptozotocin-induced diabetic mice, which correlated with the extent of renal injury. We also observed a correlation between the loss of MAM integrity and increased apoptosis and renal injury in diabetic nephropathy (DN). These alterations were further exacerbated in diabetic DsbA-L gene-deficient mice (DsbA-L−/−). In vitro, overexpression of DsbA-L in HK-2 cells restored MAM integrity and reduced apoptosis induced by high-glucose ambience. These beneficial effects were partially blocked by overexpression of FATE-1, a MAM uncoupling protein. Finally, the expression of DsbA-L was positively correlated with MAM integrity in the kidneys of DN patients but negatively correlated with apoptosis and renal injury.InterpretationOur results indicate that DsbA-L exerts an antiapoptotic effect by maintaining MAM integrity, which is apparently disrupted in DN.FundThis work was supported by the National Natural Science Foundation of China (81730018), the National Key R&D Program of China (2016YFC1305501) and NIH (DK60635).

STC-1 ameliorates renal injury in diabetic nephropathy by inhibiting the expression of BNIP3 through the AMPK/SIRT3 pathway

Diabetic kidney disease (DKD) is the leading cause of morbidity and mortality in individuals with diabetes, and it is the leading cause of end-stage renal disease (ESRD) worldwide. Stanniocalcin-1 (STC-1) is present in various tissues, and it has antioxidant and anti-apoptotic activities, which play a role in kidney protection, including diabetic nephropathy (DN). However, the mechanism that underlies these effects remains unknown. This study suggests that STC-1 ameliorates oxidative stress and cell apoptosis in the kidneys of db/db mice and high glucose (HG)-treated BUMPT cells by inhibiting Bnip3 expression through AMPK/Sirt3 pathway activation. In the clinic, DKD patients with high levels of STC-1 have a better prognosis than patients with low STC-1 levels. Thus, we concluded that STC-1 ameliorates renal injury in DN by inhibiting the expression of Bnip3 through the AMPK/SIRT3 pathway and that serum STC-1 is independently associated with DKD progression in patients with type 2 diabetes. As high STC-1 levels indicate a better prognosis, synthetic STC-1 may become a potential drug for the treatment of DKD patients.

Influence of the Klotho/FGF23/Egr1 signaling pathway on calcium-phosphorus metabolism in diabetic nephropathy and the intervention of Shenyuan granules.

This study aimed to explore the effects of Shenyuan granules on the Klotho/FGFR23/Egr1 signaling pathway and calcium-phosphorus metabolism in diabetic mice models with impairment of renal function. Streptozotocin-induced diabetic nephropathy (DN) mice models were randomly divided into three groups: Shenyuan granules group (n=10), model control group (n=10), and blank control group (n=10). Corresponding drugs were given by gavage for 8 weeks. Blood glucose and serum creatinine (SCr), urea nitrogen (BUN), calcium (Ca), phosphorus (P) and mLAB were detected before and after administration. Moreover, RT-qPCR was performed to detect the expression of CYP24 and CYP27 mRNA in kidney tissue. Blood FGF23 was detected by ELISA. Western-blot and immunohistochemistry were performed to detect the expressions of Klotho, FGFR1, Egr1, CYP24, CYP27, ERK1/2 and p-ERK1/2. Compared with the blank control group, in the model control group serum FGF23,P, SCr and 24-hour proteinuria levels increased (P<0.05), serum Ca significantly decreased (P<0.05), expressionss of Egr1, CYP24, CYP27 and p-ERK1/2 were up-regulated (P<0.05), and the expressions of Klotho and FGFR1 were down-regulated (P<0.05). After treatment, compared with the model control group, in the Shenyuan granule group serum FGF23, P, SCr levels decreased (P<0.05), serum Ca increased (P<0.05), expressions of Egr-1, CYP24, CYP27 and p-ERK1/2 were down-regulated (P<0.05), and the expressions of Klotho and FGFR1 were up-regulated (P<0.05). Shenyuan granules may partly intervene in the expressions of CYP24 and CYP27 through the Klotho/FGF23/Egr1 signaling pathway, thereby improving calcium and phosphorus metabolism and alleviating renal injury in diabetic nephropathy.

Glucose fluctuation increased mesangial cell apoptosis related to AKT signal pathway.

IntroductionBlood glucose fluctuation is an important factor for the development of diabetic complications. Glucose fluctuation aggravated the renal injury in diabetic nephropathy. In the present study, our aim was to investigate the effects of blood glucose fluctuation on the glomerular mesangal cells and its related mechanism.Material and methodsMesangial cells were divided into four groups: the normal glucose group (NG) cells were incubated in normal glucose conditions (5.6 mmol/l); the high glucose group (HG) cells were treated with 25 mmol/l; the glucose fluctuation (FG) group received 5.6 mmol/l and 25 mmol/l glucose repeated 3 times; the mannitol group (MG) received 5.6 mmol/l glucose plus 24.4 mmol/l mannitol as a control. Cell viability and apoptosis were detected, reactive oxygen species (ROS) level, superoxide dismutase (SOD) activity and malonaldehyde (MDA) levels were measured. Phosphorylated ser/thr protein kinase (P-AKT, phosphor-Ser473), phosphorylated glycogen synthase kinase-3β (P-GSK-3β, phosphor-Ser9) and cleaved cysteinyl aspartate-specific proteinase-3 (cleaved caspase-3) levels were assessed using western blot.ResultsData suggested that mesangial cells in the FG group show higher cell viability in 12 h, and lower cell viability from 48 h. The FG group showed cell apoptosis accompanied by a significant MDA level increase and SOD activity decrease in 48 h. More importantly, glucose fluctuation could aggravate oxidative stress in glomerular mesangial cells. Furthermore, the P-AKT level was lower, and increased P-GSK-3β and cleaved caspase-3 levels were higher in the FG group than in the HG group.ConclusionsGlucose fluctuation aggravates mesangial cell apoptosis, which may be partly induced by activating oxidative stress and inhibiting the AKT signaling pathway.

Fangchinoline Protects Against Renal Injury in Diabetic Nephropathy by Modulating the MAPK Signaling Pathway.

In this study, we evaluated the nephroprotective effects of fangchinoline in rats with diabetic nephropathy (DN). DN was induced by feeding a high-fat diet for 4 weeks and administering a single dose of streptozotocin (STZ) (30 mg/kg) intraperitoneally. The rats were split into groups; one group received oral fangchinoline (3 mg/kg) per day for 8 weeks. After completion of the 8-week study period, biomedical and inflammatory markers were evaluated in serum and urine, and oxidative stress was estimated in kidney tissues. In addition, Western blot assays, reverse transcription-polymerase chain reaction, and immunohistochemical analyses were performed in the kidney tissues of DN rats. The results suggest that treatment with fangchinoline attenuated the biochemical marker changes induced by DN in blood and urine. Moreover, a significant (p<0.01) reduction in inflammatory markers in serum was found in the fangchinoline group compared to the controls. Immunohistochemical analyses also revealed that treatment with fangchinoline significantly reduced the expression of collagen IV and CD31 in the kidneys compared to the control group. The expression of p38 MAPK, TNF-α, COX-2, and MMP-9 was also attenuated by fangchinoline treatment in the kidney tissues of DN rats. Together, the results of this study suggest that fangchinoline protects against nephron damage by attenuating alterations in the p38 MAPK pathway, thereby reducing oxidative stress and inflammation in DN rats.