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FoxO1-mediated inhibition of STAT1 alleviates tubulointerstitial fibrosis and tubule apoptosis in diabetic kidney disease.

ABSTRACT: BACKGROUND:Tubulointerstitial fibrosis (TIF) plays an important role in the progression of diabetic kidney disease (DKD). Forkhead box O1 (FoxO1) is involved in the regulation of metabolism and cell apoptosis, but its function in renal TIF induced by DKD is less well understood. METHODS:Human kidney biopsies with DKD and normal controls were used to detect apoptosis and TIF induced by diabetes. A mouse model with kidney-specific overexpression of Pax2-3aFoxO1 was established to further investigate the functions of FoxO1 in vivo. The in vitro roles of FoxO1 were analyzed in HK-2 cells with 3aFoxO1-knockin (3aFoxO1-KI) or FoxO1-knockdown (FoxO1-KD) via CRISPR/Cas9. Western blot, immunohistochemistry, and chromatin immunoprecipitation were used to explore the underlying mechanisms. FINDINGS:In this study, DKD patients had increased renal TIF and apoptosis. In vivo study showed that kidney-specific overexpression of Pax2-3aFoxO1 significantly reduced the expression of p-STAT1 with resultant renal functional impairment, retarding renal TIF and apoptosis in diabetic mice. Meanwhile, We observed that FoxO1-KD in HK-2 cells aggravated the expression of p-STAT1, leading to activation of epithelial-to-mesenchymal transition (EMT) and intrinsic apoptotic pathway. Conversely, EMT and apoptosis were significantly attenuated in HK-2 cells with 3aFoxO1-KI under hyperglycemic conditions. INTERPRETATION:Taken together, these data suggest that the protection role of FoxO1 against renal TIF and apoptosis in DKD is likely in part to target STAT1 signaling, which may be a promising strategy for long-term treatment of DKD. FUND: This work was supported by grants from the National Natural Science Foundation of China (grant numbers: 81570746 and 81770812).

SUBMITTER: Huang F

PROVIDER: S-EPMC6838438 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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FoxO1-mediated inhibition of STAT1 alleviates tubulointerstitial fibrosis and tubule apoptosis in diabetic kidney disease.

Huang Fengjuan F Wang Qingzhu Q Guo Feng F Zhao Yanyan Y Ji Linlin L An Tingting T Song Yi Y Liu Yang Y He Yanyan Y Qin Guijun G Qin Guijun G

EBioMedicine 20191016

<h4>Background</h4>Tubulointerstitial fibrosis (TIF) plays an important role in the progression of diabetic kidney disease (DKD). Forkhead box O1 (FoxO1) is involved in the regulation of metabolism and cell apoptosis, but its function in renal TIF induced by DKD is less well understood.<h4>Methods</h4>Human kidney biopsies with DKD and normal controls were used to detect apoptosis and TIF induced by diabetes. A mouse model with kidney-specific overexpression of Pax2-3aFoxO1 was established to fu ...[more]

PMID: 31629675

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Project description:Rationale: The rennin-angiotensin-aldosterone system (RAAS) plays a critical role in the pathogenesis of diabetic cardiomyopathy, but the role of a member of RAAS, angiotensin IV (Ang IV), in this disease and its underlying mechanism are unclear. This study was aimed to clarify the effects of Ang IV and its downstream mediator forkhead box protein O1 (FoxO1) on diabetic cardiomyopathy. Methods: In vivo, diabetic mice were treated with low-, medium- and high-dose Ang IV, AT4R antagonist divalinal, FoxO1 inhibitor AS1842856 (AS), or their combinations. In vitro, H9C2 cardiomyocytes and cardiac fibroblasts were treated with different concentrations of glucose, low-, medium- and high-dose Ang IV, divalinal, FoxO1-overexpression plasmid (FoxO1-OE), AS, or their combinations. Results: Ang IV treatment dose-dependently attenuated left ventricular dysfunction, fibrosis, and myocyte apoptosis in diabetic mice. Besides, enhanced autophagy and FoxO1 protein expression by diabetes were dose-dependently suppressed by Ang IV treatment. However, these cardioprotective effects of Ang IV were completely abolished by divalinal administration. Bioinformatics analysis revealed that the differentially expressed genes were enriched in autophagy, apoptosis, and FoxO signaling pathways among control, diabetes, and diabetes+high-dose Ang IV groups. Similar to Ang IV, AS treatment ameliorated diabetic cardiomyopathy in mice. In vitro, high glucose stimulation increased collagen expression, apoptosis, overactive autophagy flux and FoxO1 nuclear translocation in cardiomyocytes, and upregulated collagen and FoxO1 expression in cardiac fibroblasts, which were substantially attenuated by Ang IV treatment. However, these protective effects of Ang IV were completely blocked by the use of divalinal or FoxO1-OE, and these detrimental effects were reversed by the additional administration of AS. Conclusions: Ang IV treatment dose-dependently attenuated left ventricular dysfunction and remodeling in a mouse model of diabetic cardiomyopathy, and the mechanisms involved stimulation of AT4R and suppression of FoxO1-mediated fibrosis, apoptosis, and overactive autophagy.

Project description:BackgroundRenal 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.MethodsMouse 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-β 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.ResultsWe observed that SDMA (from 0.01 to 10 µM) dose-dependently inhibited the expression of pro-fibrotic markers in TGF-β 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-β stimulated HK2 cells. Furthermore, blockage of STAT4 attenuated the anti-fibrotic effect of SDMA in TGF-β stimulated HK2 cells. Conversely, overexpression of STAT4 reversed the anti-fibrotic effect of SDMA in TGF-β stimulated HK2 cells.ConclusionTaken together, our study indicates that renal SDMA ameliorates renal tubulointerstitial fibrosis through inhibition of STAT4.

Project description:It is well known that rennin-angiotensin-aldosterone system (RAAS) plays a critical role in the development of diabetic cardiomyopathy. The present study was aimed to clarify the role of a novel member of RAAS, angiotensin IV (Ang IV) and its downstream mediator forkhead box protein O1 (FoxO1), in the pathogenesis of diabetic cardiomyopathy. In vivo, diabetic mice were treated with low-, medium- and high-dose Ang IV, AT4R antagonist divalinal, FoxO1 inhibitor AS1842856 (AS), or their combinations. In vitro, cardiomyocytes and cardiofibroblasts were treated with different concentrations of glucose, low-, medium- and high-dose Ang IV, divalinal, FoxO1-overexpression plasmid (FoxO1-OE), AS or their combinations. The results showed that Ang IV treatment dose-dependently attenuated left ventricular dysfunction, fibrosis, and myocyte apoptosis in diabetic mice. Besides, enhanced autophagy and FoxO1 protein expression by diabetes were dose-dependently suppressed by Ang IV treatment. However, these cardioprotective effects of Ang IV were completely abolished by divalinal administration. Bioinformatics analysis revealed that the differentially expressed genes were enriched in autophagy, apoptosis, and FoxO signaling pathways among control, diabetes, and diabetes+high-dose Ang IV groups. Similar to Ang IV, AS treatment ameliorated diabetic cardiomyopathy in mice. In vitro, high glucose stimulation increased collagen expression, apoptosis, overactive autophagy flux and FoxO1 nuclear translocation in cardiomyocytes, and upregulated collagen and FoxO1 expression in cardiofibroblasts, which were substantially attenuated by Ang IV treatment. However, these protective effects of Ang IV were completely blocked by the use of divalinal or FoxO1-OE, and these detrimental effects were reversed by the additional administration of AS. In summary, Ang IV treatment dose-dependently attenuated left ventricular dysfunction and remodeling in a mouse model of diabetic cardiomyopathy, and the mechanisms involved stimulation of AT4R and suppression of FoxO1-mediated fibrosis, apoptosis, and overactive autophagy.

Dataset Information

FoxO1-mediated inhibition of STAT1 alleviates tubulointerstitial fibrosis and tubule apoptosis in diabetic kidney disease.

Publications

FoxO1-mediated inhibition of STAT1 alleviates tubulointerstitial fibrosis and tubule apoptosis in diabetic kidney disease.

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