Unspliced XBP1 Counteracts ß-Catenin to Inhibit Vascular Calcification.

BACKGROUND: Vascular calcification is a prevalent complication in chronic kidney disease and contributes to increased cardiovascular morbidity and mortality. XBP1 (X-box binding protein 1), existing as the XBP1u (unspliced XBP1) and XBP1s (spliced XBP1) forms, is a key component of the endoplasmic reticulum stress involved in vascular diseases. However, whether XBP1u participates in the development of vascular calcification remains unclear. METHODS: We aim to investigate the role of XBP1u in vascular calcification. XBP1u protein levels were reduced in high phosphate-induced calcified vascular smooth muscle cells, calcified aortas from mice with adenine diet-induced chronic renal failure, and calcified radial arteries from patients with chronic renal failure. RESULTS: Inhibition of XBP1u rather than XBP1s upregulated in the expression of the osteogenic markers Runx2 (runt-related transcription factor 2) and Msx2 (msh homeobox 2), and exacerbated high phosphate-induced vascular smooth muscle cell calcification, as verified by calcium deposition and Alizarin red S staining. In contrast, XBP1u overexpression in high phosphate-induced vascular smooth muscle cells significantly inhibited osteogenic differentiation and calcification. Consistently, smooth muscle cell-specific XBP1 deficiency in mice markedly aggravated the adenine diet- and 5/6 nephrectomy-induced vascular calcification compared with that in the control littermates. Further interactome analysis revealed that XBP1u is bound directly to ß-catenin, a key regulator of vascular calcification, via amino acid (aa) 205-230 in its C-terminal degradation domain. XBP1u interacted with ß-catenin to promote its ubiquitin-proteasomal degradation and thus inhibited ß-catenin/TCF (T-cell factor)-mediated Runx2 and Msx2 transcription. Knockdown of ß-catenin abolished the effect of XBP1u deficiency on vascular smooth muscle cell calcification, suggesting a ß-catenin-mediated mechanism. Moreover, the degradation of ß-catenin promoted by XBP1u was independent of GSK-3ß (glycogen synthase kinase 3ß)-involved destruction complex. CONCLUSIONS: Our study identified XBP1u as a novel endogenous inhibitor of vascular calcification by counteracting ß-catenin and promoting its ubiquitin-proteasomal degradation, which represents a new regulatory pathway of ß-catenin and a promising target for vascular calcification treatment.

SDMA attenuates renal tubulointerstitial fibrosis through inhibition of STAT4.

BACKGROUND: Renal tubulointerstitial fibrosis is the hallmark of various chronic kidney diseases. Symmetric dimethylarginine (SDMA) is an independent cardiovascular risk factor in patients with chronic kidney diseases, which is mostly excreted through renal tubules. However, the effect of SDMA on kidneys in a pathological condition is currently unknown. In this study, we investigated the role of SDMA in renal tubulointerstitial fibrosis and explored its underlying mechanisms. METHODS: Mouse unilateral ureteral obstruction (UUO) and unilateral ischemia-reperfusion injury (UIRI) models were established to study renal tubulointerstitial fibrosis. SDMA was injected into kidneys through ureter retrogradely. TGF-ß stimulated human renal epithelial (HK2) cells were used as an in vitro model and treated with SDMA. Signal transducer and activator of transcription-4 (STAT4) was inhibited by berbamine dihydrochloride or siRNA or overexpressed by plasmids in vitro. Masson staining and Western blotting were performed to evaluate renal fibrosis. Quantitative PCR was performed to validate findings derived from RNA sequencing analysis. RESULTS: We observed that SDMA (from 0.01 to 10 µM) dose-dependently inhibited the expression of pro-fibrotic markers in TGF-ß stimulated HK2 cells. Intrarenal administration of SDMA (2.5 µmol/kg or 25 µmol/kg) dose-dependently attenuated renal fibrosis in UUO kidneys. A significant increase in SDMA concentration (from 19.5 to 117.7 nmol/g, p < 0.001) in mouse kidneys was observed after renal injection which was assessed by LC-MS/MS. We further showed that intrarenal administration of SDMA attenuated renal fibrosis in UIRI induced mouse fibrotic kidneys. Through RNA sequencing analysis, we found that the expression of STAT4 was reduced by SDMA in UUO kidneys, which was further confirmed by quantitative PCR and Western blotting analysis in mouse fibrotic kidneys and renal cells. Inhibition of STAT4 by berbamine dihydrochloride (0.3 mg/ml or 3.3 mg/ml) or siRNA reduced the expression of pro-fibrotic markers in TGF-ß stimulated HK2 cells. Furthermore, blockage of STAT4 attenuated the anti-fibrotic effect of SDMA in TGF-ß stimulated HK2 cells. Conversely, overexpression of STAT4 reversed the anti-fibrotic effect of SDMA in TGF-ß stimulated HK2 cells. CONCLUSION: Taken together, our study indicates that renal SDMA ameliorates renal tubulointerstitial fibrosis through inhibition of STAT4.

SENP1 protects cisplatin-induced AKI by attenuating apoptosis through regulation of HIF-1α.

BACKGROUND: Acute kidney injury is a clinical syndrome with both high morbidity and mortality. However, the underlying molecular mechanism of AKI is still largely unknown. The role of SENP1 in AKI is unclear, while one of its substrates, HIF-1α possesses nephroprotective effect in AKI. Herein, this study aimed to reveal the role of SENP1/HIF-1α axis in AKI by using both cell and animal models. METHODS: We investigated the effects of AKI on SENP1 expression using clinical samples, and cisplatin-induced AKI model based on mice or HK-2 cells. The influence of SENP1 knockdown or over-expression on cisplatin-induced AKI was studied in vitro and in vivo. Following the exploration of the change in HIF-1α expression brought by AKI, the synergistic effects of SENP1 knockdown and HIF-1α over-expression on AKI were examined. RESULTS: The results showed the up-regulation of SENP1 in clinical specimens, as well as cell and animal models. The knockdown or over-expression of SENP1 in HK-2 cells could promote or inhibit AKI through regulating cell apoptosis, respectively. Moreover, SENP1+/- mice suffered from much more serious AKI compared with mice in wild type group. Furthermore, we found that HIF-1α over-expression could attenuate the promoted cell apoptosis as well as AKI induced by SENP1 knockdown. CONCLUSIONS: we showed that SENP1 provided protection for kidney in AKI via regulating cell apoptosis and through the regulation of HIF-1α. This study could benefit for the understanding of the pathogenesis of AKI and provide potential therapeutic target for AKI treatment.

Salvianolic acid B attenuates tubulointerstitial fibrosis by inhibiting EZH2 to regulate the PTEN/Akt pathway.

CONTEXT: Salvianolic acid B (SAB) can alleviate renal fibrosis and improve the renal function. OBJECTIVE: To investigate the effect of SAB on renal tubulointerstitial fibrosis and explore its underlying mechanisms. MATERIALS AND METHODS: Male C57 mice were subjected to unilateral ureteric obstruction (UUO) and aristolochic acid nephropathy (AAN) for renal fibrosis indication. Vehicle or SAB (10 mg/kg/d, i.p.) were given consecutively for 2 weeks in UUO mice while 4 weeks in AAN mice. The serum creatinine (Scr) and blood urine nitrogen (BUN) were measured. Masson's trichrome staining and the fibrotic markers (FN and α-SMA) were used to evaluate renal fibrosis. NRK-49F cells exposed to 2.5 ng/mL TGF-ß were treated with SAB in the presence or absence of 20 µM 3-DZNep, an inhibitor of EZH2. The protein expression of EZH2, H3k27me3 and PTEN/Akt signaling pathway in renal tissue and NRK-49F cells were measured by Western blots. RESULTS: SAB significantly improved the levels of Scr by 24.3% and BUN by 35.7% in AAN mice. SAB reduced renal interstitial collagen deposition by 34.7% in UUO mice and 72.8% in AAN mice. Both in vivo and in vitro studies demonstrated that SAB suppressed the expression of FN and α-SMA, increased PTEN and decreased the phosphorylation of Akt, which were correlated with the down-regulation of EZH2 and H3k27me3. The inhibition of EZH2 attenuated the anti-fibrotic effects of SAB in NRK-49Fs. CONCLUSION: SAB might have therapeutic potential on renal fibrosis of CKD through inhibiting EZH2, which encourages further clinical trials.

Emodin ameliorates tubulointerstitial fibrosis in obstructed kidneys by inhibiting EZH2.

Emodin, a major component of Chinese herbal rhubarb, delays the progression of chronic renal failure. However, the effect and working mechanisms of Emodin on renal tubulointerstitial fibrosis remains elusive. We hypothesized that emodin inhibits renal tubulointerstitial fibrosis through EZH2, a histone methyltransferase. Our in vivo and in vitro studies demonstrate that emodin reduced extracellular collagen deposition and inhibited Smad3 and CTGF pro-fibrotic signaling pathways, which were correlated with the down-regulation of EZH2 and reduced trimethylation of histone H3 on lysine 27 (H3k27me3) in NRK-49F fibrotic cells and UUO kidneys. Inhibition of EZH2 by 3-DZNeP blocked or attenuated the anti-fibrotic effect of emodin in UUO kidneys and NRK-49F cells. These data indicate that emodin inhibits renal tubulointerstitial fibrosis in obstructed kidneys and this effect is mediated through EZH2.

Reduced asymmetric dimethylarginine accumulation through inhibition of the type I protein arginine methyltransferases promotes renal fibrosis in obstructed kidneys.

The role of asymmetric dimethylarginine (ADMA) in chronic kidney disease (CKD) is unclear. Through inhibition of type I protein arginine methyltransferases (PRMTs), a novel strategy, we aimed to determine the effect of ADMA on renal fibrosis and explore its underlying working mechanisms. After sham or unilateral ureter ligation (UUO) operation, 20-25 g male c57 mice were treated with vehicle or PT1001B, an inhibitor of type I PRMTs, for 13 d. Moreover, human kidney 2 (HK2) and normal rat kidney 49F (NRK-49F) cells were treated with various concentrations of PT1001B or ADMA in the presence of 2.5 ng/ml TGF-ß. We found that treatment with PT1001B increased the deposition of extracellular matrix proteins, the expression of α smooth muscle actin, and connective tissue growth factor in UUO-induced fibrotic kidneys, which is correlated with reduced expression of PRMT1, reduced the production of ADMA, and increased expression of uromodulin. In TGF-ß-stimulated HK2 and NRK-49F cells, PT1001B dose-dependently inhibited ADMA production, increased NO concentrations, and enhanced the expression of profibrotic proteins. Exogenous addition of ADMA inhibited the expression of profibrotic proteins dose-dependently and attenuated the profibrotic effect of PT1001B. Moreover, ADMA reduced the NO concentration in PT1001B-treated HK2 cells. Finally, we conclude that ADMA has an antifibrotic effect in obstructed kidneys, and future application of type I PRMT inhibitor should be done cautiously for patients with CKD.-Wu, M., Lin, P., Li, L., Chen, D., Yang, X., Xu, L., Zhou, B., Wang, C., Zhang, Y., Luo, C., Ye, C. Reduced asymmetric dimethylarginine accumulation through inhibition of the type I protein arginine methyltransferases promotes renal fibrosis in obstructed kidneys.

Magnesium lithospermate B improves renal hemodynamics and reduces renal oxygen consumption in 5/6th renal ablation/infarction rats.

BACKGROUND: Magnesium lithospermate B (MLB) can promote renal microcirculation. The aim of the current project was to study whether MLB improves renal hemodynamics, oxygen consumption and subsequently attenuates hypoxia in rats induced by 5/6th renal Ablation/Infarction(A/I). METHODS: Chronic renal failure (CRF) was induced in male SD rats by the 5/6 (A/I) surgery. 30 rats were randomly divided into three groups: sham group, 5/6 (A/I) + vehicle group (CRF group) and 5/6 (A/I) + MLB (CRF + MLB) group. 28 days after the surgery, rats were given with saline or 100 mg/kg MLB by i.p. injection for 8 weeks. The 24-h urinary protein (24hUp), serum creatinine (Scr), blood urine nitrogen (BUN), systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured. The protein expression of Fibronectin (FN), Collagen-I (Col-I), Connective Tissue Growth Factor(CTGF) and Interleukin-6 (IL-6) were measured by Western blot. Renal blood flow (RBF) and renal O2 consumption (QO2) indicated as sodium reabsorption (QO2/TNa) were detected before sacrifice. Renal hypoxia was assessed by measuring the protein expression of nNOS, HIF-1α and VEGF. RESULTS: MLB significantly reduced 24hUp, Scr, BUN, SBP and DBP levels in rats with CRF. The expression of FN, Col-I, CTGF and IL-6 were down-regulated by MLB treatment in rats with CRF. In comparison to sham operated rats, 5/6 (A/I) rats had significantly lower RBF, and MLB significantly increased RBF in rats with CRF. Moreover, QO2/TNa was higher in the CRF group as compared to that in the sham group, and it was significantly attenuated in the CRF + MLB group. MLB reversed the expression of nNOS (neuronal nitric oxide synthase), HIF-1α (hypoxia inducible factor-1) and VEGF in rats with CRF. CONCLUSIONS: MLB improves renal function, fibrosis and inflammation in CRF rats induced by 5/6 (A/I), which is probably related to the increase in RBF, reduction of oxygen consumption and attenuation of renal hypoxia in the remnant kidney with CRF.

Yue-bi-tang attenuates adriamycin-induced nephropathy edema through decreasing renal microvascular permeability via inhibition of the Cav-1/ eNOS pathway.

Edema is one of the most typical symptoms of nephrotic syndrome. Increased vascular permeability makes a significant contribution to the progression of edema. Yue-bi-tang (YBT) is a traditional formula with excellent clinical efficacy in the treatment of edema. This study investigated the effect of YBT on renal microvascular hyperpermeability-induced edema in nephrotic syndrome and its mechanism. In our study, the content of target chemical components of YBT was identified using UHPLC-Q-Orbitrap HRMS analysis. A nephrotic syndrome model was replicated based on male Sprague-Dawley rats with Adriamycin (6.5 mg/kg) by tail vein injection. The rats were randomly divided into control, model, prednisone, and YBT (22.2 g/kg, 11.1 g/kg, and 6.6 g/kg) groups. After 14 d of treatment, the severity of renal microvascular permeability, edema, the degree of renal injury, and changes in the Cav-1/eNOS pathway were assessed. We found that YBT could regulate renal microvascular permeability, alleviate edema, and reduce renal function impairment. In the model group, the protein expression of Cav-1 was upregulated, whereas VE-cadherin was downregulated, accompanied by the suppression of p-eNOS expression and activation of the PI3K pathway. Meanwhile, an increased NO level in both serum and kidney tissues was observed, and the above situations were improved with YBT intervention. It thus indicates YBT exerts therapeutic effects on the edema of nephrotic syndrome, as it improves the hyperpermeability of renal microvasculature, and that YBT is engaged in the regulation of Cav-1/eNOS pathway-mediated endothelial function.

Protective effects and regulatory mechanisms of Shen-shuai-yi recipe on renal fibrosis in unilateral ureteral obstruction-induced mice.

Renal fibrosis (RF) is a common pathological feature of chronic kidney disease (CKD), which remains a major public health problem. As now, there is still lack of chemical or biological drugs to reverse RF. Shen-shuai-yi Recipe (SSYR) is a classical Chinese herbal formula for the treatment of CKD. However, the effects and mechanisms of SSYR in treating RF are still not clear. In this study, the active constituents SSYR for treating RF were explored by UHPLC-Q-Orbitrap HRMS. Bioinformatics analyses were employed to analyze the key pharmacological targets and the core active constituents of SSYR in the treatment of RF. In experimental validation, vehicle or SSYR at doses of 2.12 g/kg/d and 4.25 g/kg/d were given by orally to unilateral ureteric obstruction (UUO) mice. 13 days after treatment, we detected the severity of renal fibrosis, extracellular collagen deposition and pre-fibrotic signaling pathways. Bioinformatics analysis suggested that signal transducer and activator of transcription 3 (STAT3) was the core target and lenticin, luteolin-7-O-rutinoside, hesperidin, kaempferol-3-O-rutinoside, and 3,5,6,7,8,3',4'-heptamethoxyflavone were the key constituents in SSYR for treating RF. SSYR significantly reduced the expressions of fibronectin (FN), α-smooth muscle actin (α-SMA), collagen-I and alleviated renal interstitial collagen deposition in UUO kidneys. In mechanism, SSYR potently blocked the phosphorylation of STAT3 and Smad3 and suppressed the expression of connective tissue growth factor (CTGF). Collectively, SSYR can ameliorate RF via inhibiting the phosphorylation of STAT3 and its downstream and reducing the collagen deposition, suggesting that SSYR can be developed as a novel medicine for treating RF.

Compatibility Study of Danggui Buxue Tang on Chemical Ingredients, Angiogenesis and Endothelial Function.

Danggui Buxue Tang (DBT) is a classic Chinese herbal formula which consists of Astragali mongholici Radix and Angelica sinensis Radix (ASR). For chemical ingredients, HPLC were performed. Results showed compared with single herbs, DBT decoction could promote the dissolution of ingredients such as ferulic acid and calycosin. Furthermore, when ratio of AMR to ASR was 5 to 1, synthetic score was the best. For angiogenesis, normal and injured zebrafish model were applied. Results showed DBT and its ingredients had angiogenesis effects on Sub Intestinal vessels (SIVs) of normal zebrafish. Meanwhile, DBT and its single herbs could also recover Inter-Segmental Vessels (ISVs) injured by VRI. Angiogenesis effects of DBT on ISVs were better than single herbs. AMR extract, Total Saponins of AMR, Polysaccharide of ASR, ferulic acid, calycosin and calycosin-7-glucoside could be effective ingredients for angiogenisis. For endothelium functions, Lysoph-Osphatidyl choline was used to damage rat endothelial function of thoracic aorta. The results showed DBT and its single herbs could improve endothelial dysfunctions in dose-dependence. Both ferulic acid and calycosin-7-glucoside could also improve endothelium dysfunction in dose dependence. Therefore, compatibility of DBT was reasonable. Compared with single herbs, DBT could promote dissolution of effective ingredients, enhance angiogenesis and relieve endothelial dysfunction.

Effects of Shizhifang on NLRP3 Inflammasome Activation and Renal Tubular Injury in Hyperuricemic Rats.

OBJECTIVE: Uric acid (UA) activates the NLRP3-ASC-caspase-1 axis and triggers cascade inflammatory that leads to hyperuricemic nephropathy and hyperuricemia-induced renal tubular injury. The original study aims to verify the positive effects of the traditional Chinese medicinal formula Shizhifang (SZF) on ameliorating the hyperuricemia, tubular injury, and inflammasome infiltration in the kidneys of hyperuricemic lab rats. METHOD: Twenty-eight male Sprague-Dawley rats were divided into four groups: control group, oxonic acid potassium (OA) model group, OA + SZF group, and OA + Allopurinol group. We evaluated the mediating effects of SZF on renal mitochondrial reactive oxygen species (ROS) and oxidative stress (OS) products, protein expression of NLRP3-ASC-caspase-1 axis, and downstream inflammatory factors IL-1ß and IL-18 after 7 weeks of animals feeding. RESULT: SZF alleviated OA-induced hyperuricemia and inhibited OS in hyperuricemic rats (P < 0.05). SZF effectively suppressed the expression of gene and protein of the NLRP3-ASC-caspase-1 axis through accommodating the ROS-TXNIP pathway (P < 0.05). CONCLUSION: Our data suggest that SZF alleviates renal tubular injury and inflammation infiltration by inhibiting NLRP3 inflammasome activation triggered by mitochondrial ROS in the kidneys of hyperuricemic lab rats.