Astragaloside IV attenuates high glucose-induced NF-κB-mediated inflammation through activation of PI3K/AKT-ERK-dependent Nrf2/ARE signaling pathway in glomerular mesangial cells.

Inflammation is a key contributor to diabetic kidney disease pathogenesis, including reactive oxidation stress (ROS)-mediated nuclear factor-κB (NF-κB) signaling pathway. In this study, we examined the effect of Astragaloside IV (AS-IV) on anti-inflammatory and anti-oxidative properties under high glucose (HG) condition and the potential mechanism in glomerular mesangial cells (GMCs). We showed that AS-IV concentration-dependently reduced GMCs proliferation, restrained ROS release and hydrogen peroxide content, and suppressed pro-inflammatory cytokines as well as pro-fibrotic factors expression, which were associated with the inhibition of NF-κB and nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling activation. Accordingly, both NF-κB overexpression by using RNA plasmid and Nrf2 gene silencing by using RNA interference weakened the ability of AS-IV to ameliorate HG-induced oxidative stress, inflammation, and cell proliferation. Furthermore, phosphatidylinositide 3-kinases (PI3K)/serine/threonine protein kinase (Akt) and extracellular regulated protein kinases (ERK) signaling pathway regulated the process of AS-IV-induced Nrf2 activation and antioxidant capacity, which evidenced by using PI3K inhibitor LY294002 or ERK inhibitor PD98059 that largely abolished the AS-IV efficacy. Taken together, these results indicated that AS-IV protected against HG-induced GMCs damage by inhibiting ROS/NF-kB-induced increases of inflammatory cytokines, fibrosis biomarkers, and cell proliferation via up-regulation of Nrf2-dependent antioxidant enzyme expression, which were mediated by PI3K/Akt and ERK signaling pathway activation.

Serum chromogranin A correlated with albuminuria in diabetic patients and is associated with early diabetic nephropathy.

BACKGROUND: The kidney is the main site for the removal of chromogranin A (CgA). Previous studies have found that patients with renal impairment displayed elevated concentrations of CgA in plasma and that CgA concentrations reflect a deterioration of renal function. In this study, we aimed to estimate serum CgA levels and to evaluate the role of serum CgA in the early diagnosis of diabetic nephropathy (DN). METHODS: A total of 219 patients with type 2 diabetes mellitus (T2DM) were included in this cross-sectional study. These patients were classified into normoalbuminuria (n = 121), microalbuminuria (n = 73), or macroalbuminuria (n = 25) groups based on their urine albumin to creatinine ratios (UACRs). The degree of DN is reflected by UACR. A control group consisted of 45 healthy subjects. The serum CgA levels were measured by ELISA, and other key parameters were assayed. RESULTS: Serum CgA levels were higher in patients with T2DM than in control subjects, and a statistically significant difference among the studied subgroups regarding CgA was found (P < 0.05). The levels of serum CgA increased gradually with the degree of DN (P < 0.001). Serum CgA levels showed a moderate-intensity positive correlation with UACRs (P < 0.001). A cutoff level of 3.46 ng/ml CgA showed 69.86% sensitivity and 66.12% specificity to detect DN in the early stage. CONCLUSION: The levels of serum CgA increased gradually with the degree of DN and can be used as a biomarker in the early detection of DN.

Klotho ameliorates diabetic nephropathy by activating Nrf2 signaling pathway in podocytes.

Oxidative stress plays a key role in the pathogenesis of diabetic nephropathy (DN). The anti-aging protein Klotho has been demonstrated to have antioxidant capacity. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a central transcription factor regulating antioxidant responses. The present study aimed to explore the effects of Klotho on DN and the underlying mechanisms related to Nrf2. Low glucose (LG) or high glucose (HG) medium-cultured podocytes and diabetic db/db mice were overexpressed with Klotho via adenoviral transfer to evaluate the effects of Klotho on Nrf2 signaling, oxidative stress, podocyte apoptosis, and renal function and histopathology. Klotho overexpression significantly induced the expression and activation of Nrf2 as well as its downstream targets SOD2 and NQO1 in podocytes. Moreover, Klotho overexpression inhibited HG-induced oxidative stress and apoptosis in podocytes. Co-treatment with Nrf2 inhibitor trigonelline prevented Klotho-induced expression of SOD2 and NQO1, and abolished Klotho-conferred antioxidant and anti-apoptotic effects. In db/db mice, Klotho overexpression also activated Nrf2 signaling, and suppressed diabetes-induced oxidative stress and podocyte apoptosis, which were accompanied by improved renal function and decreased glomerulosclerosis. Our data highlight a novel Nrf2-mediated antioxidant mechanism underlying the protective effects of Klotho in podocytes and indicate the therapeutic potential of targeting Klotho to activate Nrf2 in DN.

Astragaloside IV inhibits palmitic acid-induced apoptosis through regulation of calcium homeostasis in mice podocytes.

Loss of podocytes is a hallmark of diabetic nephropathy, and a growing body of evidence indicates that podocytes are susceptible to palmitic acid (PA). We have previously shown that AS-IV inhibited PA-induced podocyte apoptosis by activating sarcoendoplasmic reticulum Ca2+ ATPase (SERCA), which indicate calcium regulation may involve in the process. Immunofluorescence staining, Western blot and flow cytometry were used to measure the protective efficacy of AS-IV to ameliorate PA-induced ER stress and podocyte apoptosis. Meanwhile, AS-IV inhibited cytochrome c release, decreased mitochondrial membrane potential, accompany with the depletion of endoplasmic reticulum Ca2+ and elevation of cytosolic and mitochondrial Ca2+. Sequestration of cytosolic calcium with BAPTA-AM limited the response of podocyte apoptosis, while during the process the effect of AS-IV was also restrained. In contrast, elevation of cytosolic calcium with calcium ionophore ionomycin was depressed by AS-IV addition. Furthermore, inhibiting TRPC6 expression with SKF96365 or TRPC6 siRNA counteracted the beneficial effect of AS-IV. Our study provides further evidence to conclude the inhibitory effect of AS-IV to podocyte apoptosis is Ca2+-dependent. And the efficacy correlates with inhibiting TRPC6-mediated Ca2+ influx, and then cellular Ca2+ disturbance was coordinated.

ApoL1 induces kidney inflammation through RIG-I/NF-κB activation.

The genetic variations of the apolipoprotein L1 (APOL1) gene are associated with non-diabetic kidney diseases. However, very little is known about the role of ApoL1 in glomerular damage. Here, we aimed to identify the function and mechanism of ApoL1 in glomerular damage. The mice were randomly divided into two groups: one group was intraperitoneally injected with phosphate buffer saline (PBS), while the other group was intraperitoneally injected with recombinant ApoL1 every other day for 3 months. Hematoxylin and eosin (HE) and periodic acid Schiff (PAS) staining were used to demonstrate the effects of ApoL1 on kidney inflammation and injury. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses revealed that ApoL1-treated mice exhibited enhanced expression of various inflammation markers in the kidney and serum compared to the PBS-treated mice. Immunofluorescence staining revealed that ApoL1 accumulated in kidney podocytes. Treatment with ApoL1 dose-dependently increased the expression of inflammation markers and apoptotic markers. The abnormal gene expression associated with ApoL1-mediated podocyte inflammation was evaluated using microarray analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the upregulated genes were enriched in the inflammation-related processes, such as the RIG-I/NF-κB signaling pathway. Consistently, the knockdown of RIG-I significantly mitigated the ApoL1-induced upregulation of inflammatory and apoptotic markers in the human podocytes. Additionally, the ApoL1-induced glomerular damage was attenuated in AAV-shRIG-I mice. Therefore, the effects of ApoL1 on glomerular damage may be, at least partially, through inducing abnormal expression of inflammatory molecules, which may have important implications for treatment of kidney diseases.

Calcium Uptake via Mitochondrial Uniporter Contributes to Palmitic Acid-Induced Apoptosis in Mouse Podocytes.

Podocytes are component cells of the glomerular filtration barrier, and their loss by apoptosis is the main cause of proteinuria that leads to diabetic nephropathy (DN). Therefore, insights into podocyte apoptosis mechanism would allow a better understanding of DN pathogenesis and thus help develop adequate therapeutic strategies. Here, we investigated the molecular mechanism of palmitic acid-inhibited cell death in mouse podocytes, and found that palmitic acid increased cell death in a dose- and time-dependent manner. Palmitic acid induces apoptosis in podocytes through upregulation of cytosolic and mitochondrial Ca2+ , mitochondrial membrane potential (MMP), cytochrome c release, and depletion of endoplasmic reticulum (ER) Ca2+ . The intracellular calcium chelator, 1,2-bis (2-aminophenoxy) ethane-N,N,N, N'-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM), partially prevented this upregulation whereas 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-triphosphate receptor (IP3R) inhibitor; dantrolene, a ryanodine receptor (RyR) inhibitor; and 4,4'-diisothiocyanatostibene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor, had no effect. Interestingly, ruthenium red and Ru360, both inhibitors of the mitochondrial Ca2+ uniporter (MCU), blocked palmitic acid-induced mitochondrial Ca2+ elevation, cytochrome c release from mitochondria to cytosol, and apoptosis. siRNA to MCU markedly reduced palmitic acid-induced apoptosis. These data indicate that Ca2+ uptake via mitochondrial uniporter contributes to palmitic acid-induced apoptosis in mouse podocytes. J. Cell. Biochem. 118: 2809-2818, 2017. © 2017 Wiley Periodicals, Inc.

Ursodeoxycholic acid and 4-phenylbutyrate prevent endoplasmic reticulum stress-induced podocyte apoptosis in diabetic nephropathy.

Endoplasmic reticulum (ER) stress, resulting from the accumulation of misfolded and/or unfolded proteins in ER membranes, is involved in the pathogenesis of diabetic nephropathy (DN). The aim of this study was to investigate the role of ER stress inhibitors ursodeoxycholic acid (UDCA) and 4-phenylbutyrate (4-PBA) in the treatment of DN in db/db mice. Findings have revealed that diabetic db/db mice were more hyperglycemic than their non-diabetic controls, and exhibited a marked increase in body weight, water intake, urine volume, fasting plasma glucose, systolic blood pressure, glucose and insulin tolerance. UDCA (40 mg/kg/day) or 4-PBA (100 mg/kg/day) treatment for 12 weeks resulted in an improvement in these biochemical and physical parameters. Moreover, UDCA or 4-PBA intervention markedly decreased urinary albuminuria and attenuated mesangial expansion in diabetic db/db mice, compared with db/db mice treated with vehicle. These beneficial effects of UDCA or 4-PBA on DN were associated with the inhibition of ER stress, as evidenced by the decreased expression of BiP, phospho-IRE1α, phospho-eIF2α, CHOP, ATF-6 and spliced X-box binding protein-1 in vitro and in vivo. UDCA or 4-PBA prevented hyperglycemia-induced or high glucose (HG)-induced apoptosis in podocytes in vivo and in vitro via the inhibition of caspase-3 and caspase-12 activation. Autophagy deficiency was also seen in glomeruli in diabetic mice and HG-incubated podocytes, exhibiting decreased expression of LC3B and Beclin-1, which could be restored by UDCA or 4-PBA treatment. Taken together, our results have revealed an important role of ER stress in the development of DN, and UDCA or 4-PBA treatment may be a potential novel therapeutic approach for the treatment of DN.

HuangQi Decoction Ameliorates Renal Fibrosis via TGF-ß/Smad Signaling Pathway In Vivo and In Vitro.

OBJECTIVE: Traditional Chinese Medicine compound HuangQi decoction is widely used in clinical treatment of chronic kidney disease, but its role on renal interstitial fibrosis and the underlying mechanism remains unclear. The aim of this study is to investigate the effect of HuangQi decoction on renal interstitial fibrosis and its association with the TGF-ß/Smad signaling pathway Methods: A total of 120 C57/BL mice were randomly divided into six groups: sham group, sham plus high-dose HuangQi decoction (1.08g/kg) group, unilateral ureteral obstruction (UUO) model group, and UUO model plus low to high doses of HuangQi decoction (0.12g/kg, 0.36g/kg and 1.08g/kg respectively) groups. Animals were sacrificed 14 days after the administration and ipsilateral kidney tissue was sampled for pathologic examinations. Immunohistochemistry, PCR and western blot were used to detect the expressions of related molecules in the TGF-ß/Smad signaling pathway. TGF-ß1 was used in in vitro experiments to induce human kidney proximal tubule epithelial cells (HK2). RESULTS: HuangQi decoction improved ipsilateral kidney fibrosis in UUO mice and downregulated the expressions of TGF-ß1, TßRI, TßRII, Smad4, Smad2/3, P-Smad2/3, α-SMA, collagen type I, III and IV in a dose-dependent manner while upregulated the expression of Smad7 in the same fashion. Similar results were found in in vitro studies. CONCLUSION: The protective effect of HuangQi decoction for unilateral ureteral obstruction kidney damage in mice was mediated by downregulating the TGF-ß/Smad signaling pathway.

HuangQi Decoction Improves Renal Tubulointerstitial Fibrosis in Mice by Inhibiting the Up-Regulation of Wnt/ß-Catenin Signaling Pathway.

OBJECT: To explore the effects of HuangQi decoction on tubulointerstitial fibrosis in mice and the Wnt/ß-catenin signaling pathway. METHODS: Unilateral ureteral obstruction (UUO) model was used. A total of 120 C57/BL mice were randomly divided into 6 groups, sham group, sham+HuangQi decoction group (1.08 g/kg), UUO group, UUO+HuangQi decoction group (0.12, 0.36, 1.08 g/kg). Immunohistochemical analysis, RT-PCR and Western blot were employed to examine the proteins and genes related to the Wnt/ß-catenin signaling pathway. RESULTS: In UUO mice models, expression levels of Wnt3,4, Frizzled4, LRP5,6, ß-catenin, LEF-1, TCF-1, Snail, MMP2,7 genes were positively correlated with the degree of renal tubulointerstitial fibrosis, while expression levels of GSK-3ß, Axin, APC, CK1 were negatively correlated. HuangQi decoction could down-regulate expression levels of Wnt3,4, Frizzled4, LRP5,6, ß-catenin, LEF-1, TCF-1, Snail, Twist, MMP2,7 and up-regulate expression levels of GSK-3ß, Axin, APC, CK1 and E-cadherin. CONCLUSION: HuangQi decoction could effectively inhibit the up-regulation of Wnt/ß-catenin signaling pathway induced by UUO, implying a possible role in improving renal interstitial fibrosis.

Advanced IgA nephropathy with impaired renal function benefits from losartan treatment in rats.

BACKGROUND: Treatment with angiotensin receptor blockers (ARBs) is successful in mitigating IgA nephropathy (IgAN), independent of blood pressure changes, but the therapeutic role of ARB in advanced IgAN with impaired renal function is to be ascertained. The present study was performed to investigate the effect of losartan on advanced IgAN induced by staphylococcal enterotoxin B (SEB) combined with 5/6 nephrectomy in rats. METHODS: Fifty-four male SD rats were randomly divided into three group: Rats in the model group were treated with SEB plus 5/6 nephrectomy, and those in the losartan group were gavaged with losartan (33.3 mg kg(-1 )d(-1)) besides the treatment with SEB plus 5/6 nephrectomy. The urine and blood biochemical changes of rats were tested. IgA, IgG, IgM and C3 depositions were studied dynamically with immunofluorescence. The renal tissue structures were observed under light microscopy. The expressions of TGF-ß1, FN, alpha-SMA and FGF-1 in rat renal tissues were determined with immunohistochemical methods and real-time PCR. RESULTS: At 12 weeks, rats with SEB treatment plus 5/6 nephrectomy showed gradually increased urinary red blood cell (URBC) with a gradual elevation of the 24 h urinary protein, serum BUN and Scr, but losartan treatment lowered the levels of 24 h urinary protein, serum BUN and Scr. A large number of IgA depositions in the mesangial area, glomerulosclerosis and tubulointerstitial fibrosis were found in the model group, and the losartan group showed relieved injury. The expressions of TGF-ß1, FN, alpha-SMA and FGF-1 were significantly elevated in the model. Losartan lessened their expressions. CONCLUSION: Losartan treatment can delay the progression of advanced IgA nephropathy with impaired renal function.

Astragaloside IV attenuates podocyte apoptosis through ameliorating mitochondrial dysfunction by up-regulated Nrf2-ARE/TFAM signaling in diabetic kidney disease.

Defective antioxidant system as well as mitochondrial dysfunction contributes to the pathogenesis and progression of diabetic kidney disease (DKD). Nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated signaling is the central defensive mechanism against oxidative stress and therefore pharmacological activation of Nrf2 is a promising therapeutic strategy. In this study, using molecular docking we found that Astragaloside IV (AS-IV), an active ingredient from traditional formula of Huangqi decoction (HQD), exerted a higher potential to promote Nrf2 escape from Keap1-Nrf2 interaction via competitively bind to amino acid sites in Keap1. When podocyte exposed to high glucose (HG) stimulation, mitochondrial morphological alterations and podocyte apoptosis were presented and accompanied by Nrf2 and mitochondrial transcription factor A (TFAM) downregulation. Mechanistically, HG promoted a decrease in mitochondria-specific electron transport chain (ETC) complexes, ATP synthesis and mtDNA content as well as increased ROS production. Conversely, all these mitochondrial defects were dramatically alleviated by AS-IV, but suppression of Nrf2 with inhibitor or siRNA and TFAM siRNA simultaneously alleviated the AS-IV efficacy. Moreover, experimental diabetic mice exhibited significant renal injury as well as mitochondrial disorder, corresponding with the decreased expression of Nrf2 and TFAM. On the contrary, AS-IV reversed the abnormality and the Nrf2 and TFAM expression were also restored. Taken together, the present findings demonstrate the improvement of AS-IV on mitochondrial function, thereby resistance to oxidative stress-induced diabetic kidney injury and podocyte apoptosis, and the process is closely associated with activation of Nrf2-ARE/TFAM signaling.

Losartan alleviates renal fibrosis by down-regulating HIF-1α and up-regulating MMP-9/TIMP-1 in rats with 5/6 nephrectomy.

BACKGROUND: This study investigated the effects of losartan intervention on the expressions of hypoxia-inducible factor-1α (HIF-1α), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in renal fibrosis in rats with 5/6 nephrectomy. METHODS: Sprague Dawley rats were randomly divided into three groups. Rats in the losartan group were gavaged with losartan (33.3 mg/kg/day) from 1 week after 5/6 nephrectomy, and those in the sham group and the model group only received an equal volume of saline solution by gavage. Rats were sacrificed at the ends of the 4, 8 and 12 weeks, respectively. Urinary N-acetyl-glucosaminidase (NAG), 24-h urinary protein, serum cystatin C, blood urea nitrogen (BUN), and serum creatinine (Scr) levels were assessed. Kidney tissues were observed under light and electron microscope. The expressions of HIF-1α, transforming growth factor-ß1 (TGF-ß1), MMP-9, and TIMP-1 were determined by immunohistochemistry and Western blotting. RESULTS: Twenty-four hour urinary protein, urinary NAG, serum cystatin C, BUN, and Scr levels in the model group were significantly higher than those in the sham group (p < 0.05), but losartan treatment improved these changes. The apparent glomerular sclerosis and tubulointerstitial fibrosis were also found in the model group, which were ameliorated by losartan. The expressions of HIF-1α, TGF-ß1, MMP-9, and TIMP-1 were elevated and MMp-9/TIMP-1 ratio was lowered in the model group (p < 0.05), but losartan increased the expression of MMP-9 and MMp-9/TIMP-1 ratio (p < 0.05) and lessened the expressions of HIF-1α, TGF-ß1, and TIMP-1 (p < 0.05). CONCLUSION: Losartan may ameliorate renal fibrosis partly by down-regulating HIF-1α and up-regulating MMP-9/TIMP-1 in rats with 5/6 nephrectomy.

Renal microvascular injury in chronic aristolochic acid nephropathy and protective effects of Cozaar.

BACKGROUND: To investigate the renal microvascular injury in chronic aristolochic acid nephropathy (AAN) and the protective effects of Cozaar. METHODS: Male Sprague-Dawley rats were randomized into three groups. Rats in the model group received Caulis Aristolochiae manshuriensis (CAM) decoction by gavage (10 mL/kg/day); those in the Cozaar group were gavaged with CAM and Cozaar (33.3 mg/kg/day); and those in the control group only received an equal daily volume of saline solution by gavage. Kidney tissues were observed under a light and electron microscope. CD34, caspase-3, and bone morphogenetic protein-7 (BMP-7) were determined by immunohistochemistry, and expressions of angiopoietin (Ang) 1 and 2, Tie-2, BMP-7, and vascular endothelial growth factor (VEGF) mRNA were monitored via real-time polymerase chain reaction (PCR). RESULTS: (1) The kidney tissue injury in the chronic AAN model group was apparent, compared to the normal structure in the normal control group, and the Cozaar group showed relieved injury. (2) The expression of caspase-3 in the model group was elevated, while expressions of BMP-7 and CD34 were decreased (p < 0.05). Cozaar lessened caspase-3 expression (p < 0.05) and promoted BMP-7 and CD34 expressions (p < 0.05). (3) Real-time PCR demonstrated a downregulation of Ang-1, Tie-2, BMP-7, and VEGF mRNA (p < 0.05) and an upregulation of Ang-2 mRNA (p < 0.01) in the renocortex, while Cozaar upregulated the expression of Ang-1, Tie-2, BMP-7, and VEGF mRNA (p < 0.05). CONCLUSION: Renal microvascular injury was observed in chronic AAN, which was hypothetically correlated with a lack in the expressions of Ang-1, BMP-7, Tie-2, and VEGF and an excess in caspase-3 and Ang-2. Cozaar can significantly ameliorate the renal microvascular injury and protect renal function.

Knowledge-Based Discovery of the Role and Mechanism of Resveratrol in Improving Glomerular Tether Cell Proliferation and Apoptosis in Diabetic Nephropathy.

To investigate the effects and mechanisms of resveratrol on glucolipid metabolism in diabetic humans. In this paper, we introduced the knowledge discovery theory into the data processing of the factors related to the pathogenesis of type 2 diabetes for the first time, and identified valid, potentially useful, and understandable pathogenesis patterns from a large amount of measured data. A data mining C4.5 algorithm was used to classify 17072 validated cross-sectional health survey data from the whole population according to the characteristics of type ρ diabetes data. A human model of diabetes mellitus was prepared by high sugar and high fat diet plus low dose streptozotocin (STZ, 35 mg/kg) and randomly grouped into four groups: the normal control group, the model group, the resveratrol group, and the pioglitazone group. 8 animals in each group were treated with the corresponding drugs for 8 weeks. Hepatic steatosis and damage were significantly reduced compared with the model group as observed by HE staining. Resveratrol has obvious effects on regulating glucolipid metabolism, and its mechanism of action is associated with its ability to increase the antioxidant activity of the body, activate the Akt signaling pathway, and improve liver pathological damage.

Candidate metabolite markers of peripheral neuropathy in Chinese patients with type 2 diabetes.

OBJECTIVES: To analyze the serum and urine metabolites present in type 2 diabetes mellitus (T2DM) patients and T2DM patients with diabetic peripheral neuropathy (DPN) and to select differentially expressed biomarkers for early diagnosis of DPN. METHODS: Serum and urine metabolites from 74 T2DM patients with peripheral neuropathy and 41 without peripheral neuropathy were analyzed using gas chromatograph system with time-of-flight mass spectrometer metabolomics to detect biomarkers of peripheral neuropathy in T2DM. RESULTS: There were increased serum triglycerides, alanine aminotransferase, and decreased C-peptide, and total cholesterol levels in T2DM patients with DPN compared to those without peripheral neuropathy. Metabolomic analysis revealed visible differences in metabolic characteristics between two groups, and overall 53 serum differential metabolites and 56 urine differential metabolites were identified with variable influence on projection (VIP) >1 and P<0.05. To further analyze the correlation between the identified metabolites and DPN, four serum metabolites and six urine metabolites were selected with VIP>2, and fold change (FC) >1, including serum ß-alanine, caproic acid, ß-alanine/L-aspartic acid, and L-arabinose/L-arabitol, and urine gluconic acid, erythritol, galactonic acid, guanidoacetic acid, cytidine, and aminoadipic acid. Furthermore, five serum biomarkers and six urine biomarkers were found to show significant changes (P<0.05, VIP>1, and FC>1) respectively in patients with mild, moderate, and severe DPN. In addition, we found that glyoxylate and dicarboxylate metabolism was a differential metabolic pathway not only between T2DM and DPN, but also among different degrees of DPN. The differential metabolites such as ß-alanine and caproic acid are expected to be biomarkers for DPN patients, and the significant changes in glyoxylate and dicarboxylate metabolism may be related to the pathogenesis of DPN. CONCLUSION: There were serum and urine spectrum metabolomic differences in patients with DPN, which could serve as biomarkers for T2DM and DPN patients.

Astragaloside IV prevents endothelial dysfunction by improving oxidative stress in streptozotocin-induced diabetic mouse aortas.

Oxidative stress serves a role in endothelial dysfunction exhibited by patients with diabetes mellitus. Astragaloside IV (AS-IV) is a major active ingredient of Radix Astragali, which is considered to exhibit vasoprotective effects through unknown mechanisms. Thus, the current study was performed to investigate the protective effects of AS-IV in streptozotocin (STZ)-induced endothelial dysfunction and to explore whether antioxidant mechanisms were involved. The protective effects of AS-IV on the endothelium-dependent relaxation and contraction of aortic rings were determined by isometric tension recordings. NADPH subunits and endothelial nitric oxide synthase (eNOS) expression was identified via western blotting. Superoxide dismutase and malondialdehyde levels were assayed using ELISA. Furthermore, the generation of reactive oxygen species (ROS) and nitric oxide (NO) was detected via dihydroethidium and 4,5-diaminofluorescein diacetate staining, respectively. The results revealed that STZ-injected mice exhibited increased aortic endothelium-dependent vasoconstriction and decreased vasorelaxation to acetylcholine. However, AS-IV treatment reversed these effects. NG-nitro-L-arginine was subsequently used to completely inhibit impaired relaxation. Accordingly, impaired NO generation was restored following AS-IV treatment by increasing eNOS phosphorylation levels. Furthermore, ROS formation was also depressed following AS-IV treatment compared with that in STZ-injected mice. AS-IV also decreased the expression of various NADPH subunits, including human neutrophil cytochrome b light chain, neutrophil cytosolic factor 1, NADPH oxidase (NOX)2, NOX4 and Rac-1. The results of the current study may provide novel evidence that diabetes-induced vascular injury arises from either the inhibition of eNOS or the activation of NOX-derived ROS generation. In addition, the results warrant further investigation into the application of AS-IV treatment, leading to the improvement of oxidative stress, in patients with diabetes exhibiting endothelial dysfunction.

Astragaloside IV protects against podocyte apoptosis by inhibiting oxidative stress via activating PPARγ-Klotho-FoxO1 axis in diabetic nephropathy.

AIMS: Podocyte apoptosis plays an important role in the pathogenesis of diabetic nephropathy (DN). Astragaloside IV (AS-IV) has been shown to protect against podocyte apoptosis. Here we aim to investigate the mechanism responsible for the protective effects of AS-IV. MAIN METHODS: Diabetic db/db mice and high glucose (HG)-cultured podocytes were treated with AS-IV. Renal function and histopathological changes were measured to evaluate the therapeutic effects of AS-IV against DN. Adenovirus-mediated Klotho overexpression, Klotho siRNA, and PPARγ inhibitor were applied in vitro to investigate the potential mechanism. The expression levels of mRNA and proteins were analyzed by qRT-PCR, western blot or immunofluorescence. Intracellular ROS and mitochondrial superoxide were detected by DHE and MitoSOx Red, respectively. Cell apoptosis was evaluated by TUNEL staining and flow cytometry. KEY FINDINGS: AS-IV improved renal function and ameliorated podocyte injury in db/db mice accompanied with enhanced Klotho expression in glomerular podocytes. In vitro, AS-IV inhibited HG-induced podocyte apoptosis and restored HG-inhibited Klotho expression, whereas Klotho knockdown abrogated the anti-apoptosis action of AS-IV. Further study showed that adenovirus-mediated Klotho overexpression enhanced Forkhead transcription factor O1 (FoxO1)-dependent antioxidant activity and attenuated HG-evoked oxidative stress and apoptosis. AS-IV prevented HG-induced FoxO1 inhibition and oxidative stress, whereas Klotho knockdown reversed these effects. Cotreatment with PPARγ inhibitor T0070907 abolished AS-IV-induced Klotho expression and anti-apoptosis action. SIGNIFICANCE: These data suggested that AS-IV attenuated podocyte apoptosis presumably by inhibiting oxidative stress via activating PPARγ-Klotho-FoxO1 signaling pathway, thereby ameliorating DN. This study provided new insights into the molecular mechanisms of AS-IV against DN.

Serum and urine metabolomics reveal potential biomarkers of T2DM patients with nephropathy.

BACKGROUND: Diabetes is a metabolic disease and is often accompanied by severe microvascular and macrovascular complications. A comprehensive understanding of its complex mechanisms can help prevent type 2 diabetes mellitus (T2DM) complications, such as diabetic nephropathy (DN). METHODS: To reveal the systemic metabolic changes related to renal injury, clinical information of T2DM patients with or without nephropathy was collected, and it was found that serum urea levels of DN patients were significantly higher in T2DM patients without nephropathy. Further along the disease progression, the serum urea levels also gradually increased. We used gas chromatograph coupled with time-of-flight mass spectrometry (GC-TOFMS) metabolomics to analyze the serum and urine metabolites of T2DM patients with or without nephropathy to study the metabolic changes associated with the disease. RESULTS: Finally, we identified 61 serum metabolites and 46 urine metabolites as potential biomarkers related to DN (P<0.05, VIP >1). In order to determine which metabolic pathways were major altered in DN, we summarized pathway analysis based on P values from their impact values and enrichment. There were 9 serum metabolic pathways and 12 urine metabolic pathways with significant differences in serum and urine metabolism, respectively. CONCLUSIONS: This study emphasizes that GC-TOFMS-based metabolomics provides insight into the potential pathways in the pathogenesis and progression of DN.

Stimulatory Role of SPAK Signaling in the Regulation of Large Conductance Ca2+-Activated Potassium (BK) Channel Protein Expression in Kidney.

SPS1-related proline/alanine-rich kinase (SPAK) plays important roles in regulating the function of numerous ion channels and transporters. With-no-lysine (WNK) kinase phosphorylates SPAK kinase to active the SPAK signaling pathway. Our previous studies indicated that WNK kinases regulate the activity of the large-conductance Ca2+-activated K+ (BK) channel and its protein expression via the ERK1/2 signaling pathway. It remains largely unknown whether SPAK kinase directly modulates the BK protein expression in kidney. In this study, we investigated the effect of SPAK on renal BK protein expression in both HEK293 cells and mouse kidney. In HEK293 cells, siRNA-mediated knockdown of SPAK expression significantly reduced BK protein expression and increased ERK1/2 phosphorylation, whereas overexpression of SPAK significantly enhanced BK expression and decreased ERK1/2 phosphorylation in a dose-dependent manner. Knockdown of ERK1/2 prevented SPAK siRNA-mediated inhibition of BK expression. Similarly, pretreatment of HEK293 cells with either the lysosomal inhibitor bafilomycin A1 or the proteasomal inhibitor MG132 reversed the inhibitory effects of SPAK knockdown on BK expression. We also found that there is no BK channel activity in PCs of CCD in SPAK KO mice using the isolated split-open tubule single-cell patching. In addition, we found that BK protein abundance in the kidney of SPAK knockout mice was significantly decreased and ERK1/2 phosphorylation was significantly enhanced. A high-potassium diet significantly increased BK protein abundance and SPAK phosphorylation levels, while reducing ERK1/2 phosphorylation levels. These findings suggest that SPAK enhances BK protein expression by reducing ERK1/2 signaling-mediated lysosomal and proteasomal degradations of the BK channel.

Huangqi decoction inhibits hyperglycemia-induced podocyte apoptosis by down-regulated Nox4/p53/Bax signaling in vitro and in vivo.

BACKGROUND: Podocyte dysfunction is associated with the progression of diabetic nephropathy (DN). Huangqi decoction (HQD), a traditional Chinese medical formula, has been used to improve diabetes-related syndrome in China. The present study was to investigate the protective effect of HQD on podocyte apoptosis and the underlying molecular mechanism. METHODS: Podocyte was used to measure the efficacy of HQD on cell apoptosis, activities of NADPH oxidases, ROS generation and mitochondrial membrane potential (MMP), and the activation of Nox4/p53/Bax signaling pathway with HQD treatment were also investigated in vitro. Renal pathological morphology, renal function, podocyte apoptosis and Nox4/p53/Bax signaling pathway were investigated with STZ-induced diabetic mice in vivo. RESULTS: HQD increased the cell proliferation and MMP level, while the ROS production and activities of NADPH oxidases were decreased. Meanwhile, Nox4/p53/Bax signaling was down-regulated. Contrarily, overexpression of Nox4 or p53 significantly abolished those efficacies of HQD. Accordingly, in vivo study showed that the progressive albuminuria, glomerulosclerosis and loss of podocytes were significantly alleviated with HQD treatment in diabetic mice, which paralleled by the marked inhibition of Nox4/p53/Bax signaling. CONCLUSION: Collectively, we provide further evidence that HQD had a renoprotective effect in preventing podocyte apoptosis, which was mediated at least in part by down-regulation of Nox4/p53/Bax signaling.