BRCC36 regulates ß-catenin ubiquitination to alleviate vascular calcification in chronic kidney disease.

BACKGROUND: The prevalence of vascular calcification (VC) in chronic kidney disease (CKD) patients remains substantial, but currently, there are no effective pharmaceutical therapies available. BRCA1/BRCA2-containing complex subunit 36 (BRCC36) has been implicated in osteoblast osteogenic conversion; however, its specific role in VC remains to be fully elucidated. The aim of this study was to investigate the role and underlying mechanisms of BRCC36 in VC. METHODS: The association between BRCC36 expression and VC was examined in radial arteries from patients with CKD, high-adenine-induced CKD mice, and vascular smooth muscle cells (VSMCs). Western blotting, real-time polymerase chain reaction, immunofluorescence, and immunohistochemistry were used to analyse gene expression. Gain- and loss-of-function experiments were performed to comprehensively investigate the effects of BRCC36 on VC. Coimmunoprecipitation and TOPFlash luciferase assays were utilized to further investigate the regulatory effects of BRCC36 on the Wnt/ß-catenin pathway. RESULTS: BRCC36 expression was downregulated in human calcified radial arteries, calcified aortas from CKD mice, and calcified VSMCs. VSMC-specific BRCC36 overexpression alleviated calcium deposition in the vasculature, whereas BRCC36 depletion aggravated VC progression. Furthermore, BRCC36 inhibited the osteogenic differentiation of VSMCs in vitro. Rescue experiments revealed that BRCC36 exerts the protective effects on VC partly by regulating the Wnt/ß-catenin signalling pathway. Mechanistically, BRCC36 inhibited the Wnt/ß-catenin pathway by decreasing the K63-linked ubiquitination of ß-catenin. Additionally, pioglitazone attenuated VC partly through upregulating BRCC36 expression. CONCLUSIONS: Our research results emphasize the critical role of the BRCC36-ß-catenin axis in VC, suggesting that BRCC36 or ß-catenin may be promising therapeutic targets to prevent the progression of VC in CKD patients.

BRCC36 prevents vascular calcification in chronic kidney disease through the ß-catenin signalling pathway.

Vascular calcification (VC) is a strong predictor of cardiovascular mortality and overall mortality in patients with chronic kidney disease (CKD); however, the molecular mechanisms underlying VC have yet to be elucidated. Here, we report the role of the deubiquitinating enzyme BRCC36 in the process of VC in CKD. We established an in vitro VC model of vascular smooth muscle cells (VSMCs) and an adenine-induced CKD mouse model. The expression of BRCC36 was significantly decreased in both the in vivo and in vitro VC models. Alizarin red staining and calcium content assays showed that BRCC36 overexpression reduced calcium deposition in the presence of calcifying medium, while the contractile protein α-smooth muscle actin (α-SMA) was upregulated and phosphorylated ß-catenin was downregulated. Cell immunofluorescence showed that BRCC36 overexpression also reduced the expression of phosphorylated ß-catenin in the nucleus in the presence of calcifying medium. In addition, coimmunoprecipitation showed that BRCC36 can bind to ß-catenin. These results suggest that BRCC36 can interact with ß-catenin, the main effector protein of the Wnt/ß-catenin pathway, inhibiting the phosphorylation of ß-catenin and negatively regulating the cell signalling pathway, thereby inhibiting VC. This may provide new insights into the molecular mechanisms of VC in the context of CKD.

The role of AMP-activated protein kinase α1-mediated endoplasmic reticulum stress in alleviating the toxic effect of uremic toxin indoxyl sulfate on vascular endothelial cells by Klotho.

Recently, the Klotho protein (Klotho) has received substantial attention as protective factor against cardiovascular complications of chronic kidney disease (CKD). However, the direct effect and mechanism of Klotho on endothelial cells injury are not well-known. In this study, we incubated human vein umbilical endothelial cells (HUVECs) with uremic toxin indoxyl sulfate (IS) to mimic CKD internal environment and investigated the direct effect of Klotho on the HUVECs injury induced by IS and to explore the mechanism in this process. We found IS inhibited cell viability, increased endoplasmic reticulum stress, and mediated apoptosis of HUVECs. Treatment with Klotho significantly attenuated IS-induced above effects. Furthermore, Klotho alleviated the IS toxic effect on HUVECs via promoting AMP-activated protein kinase (AMPK) α1 phosphorylation instead of directly upregulating AMPKα1, which could be partly blocked by AMPK pathway inhibitor-Compound C. In addition, Klotho also inhibited intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression induced by IS. Altogether, these results indicated that Klotho can protect HUVECs from IS-induced injury by alleviating AMPKα1-mediated endoplasmic reticulum stress.

The role and mechanism of protein post­translational modification in vascular calcification (Review).

Vascular calcification is closely associated with morbidity and mortality in patients with chronic kidney disease, atherosclerosis and diabetes. In the past few decades, vascular calcification has been studied extensively and the findings have shown that the mechanism of vascular calcification is not merely a consequence of a high-phosphorus and high-calcium environment but also an active process characterized by abnormal calcium phosphate deposition on blood vessel walls that involves various molecular mechanisms. Recent advances in bioinformatics approaches have led to increasing recognition that aberrant post-translational modifications (PTMs) play important roles in vascular calcification. This review presents the latest progress in clarifying the roles of PTMs, such as ubiquitination, acetylation, carbamylation and glycosylation, as well as signaling pathways, such as the Wnt/ß-catenin pathway, in vascular calcification.

Damage of uremic myocardium by p-cresyl sulfate and the ameliorative effect of Klotho by regulating SIRT6 ubiquitination.

Uremic cardiomyopathy (UCM) is a common complication in patients with chronic kidney disease (CKD) and an important risk factor for death. P-Cresyl sulfate (PCS) is a damaging factor in UCM, and Klotho is a protective factor. However, the molecular mechanisms of Klotho and PCS in UCM and the relationship between PCS and Klotho are unclear. In vitro, Klotho treatment inhibited PCS-induced cardiomyocyte hypertrophy and apoptosis by blocking mTOR phosphorylation and inhibiting DNA double-strand breaks (DSBs), respectively. Moreover, PCS increased SIRT6 protein ubiquitination and downregulated SIRT6 protein expression, while Klotho inhibited SIRT6 protein ubiquitination and upregulated SIRT6 protein expression. In a mouse model of 5/6 nephrectomy (5/6Nx)-induced UCM, the expression of Klotho in the kidney and serum was decreased, and the expression of SIRT6 protein in myocardial tissues was lower. PCS further reduced Klotho and SIRT6 expression, aggravated heart structure and function abnormalities, and increased myocardial cell apoptosis in UCM mice. Administration of Klotho protein inhibited the downregulation of SIRT6 protein expression and improved cardiac structure and function. Furthermore, serum PCS level was associated with the left ventricular mass (LVM) and left ventricular mass index (LVMI) in hemodialysis patients. In conclusion, the uremic toxin PCS injures cardiomyocytes via mTOR phosphorylation and DSBs, and Klotho antagonizes the damaging effects of PCS. Moreover, the SIRT6 protein plays an important role in UCM, and Klotho suppresses SIRT6 ubiquitination induced by PCS, further improves cardiac structure and function in UCM and exerts protective effects.

Uraemic Cardiomyopathy in Different Mouse Models.

Uraemic cardiomyopathy (UCM) is one of the most common complications in chronic kidney disease (CKD). Our aim was to compare characteristics of various UCM mouse models. Mice were assigned to the following groups: the pole ligation group, 5/6 nephrectomy group (5/6Nx), uninephrectomy plus contralateral ischemia followed by reperfusion group (IR), adenine group, and sham group. Mice were sacrificed at 4, 8, and 16 weeks after surgery in the pole ligation, 5/6Nx, and IR groups, respectively. In the adenine group, mice were sacrificed at 16 weeks after the adenine diet. The structure and function of the heart and the expression of fibroblast growth factor 23 (FGF-23) and growth differentiation factor 15 (GDF-15) in hearts were assessed. The mortality in the 5/6 Nx group was significantly higher than that in the pole ligation, IR, and adenine groups. Echocardiogram and histological examination showed cardiac hypertrophy in the adenine,5/6Nx, ligation group, and IR group. In addition, cardiac fibrosis occurred in all CKD modeling groups. Interestingly, cardiac fibrosis was more serious in the IR and adenine groups. FGF-23 expression in sham mice was similar to that in modeling groups; however, the GDF-15 level was decreased in modeling groups. Our results suggest that the four models of UCM show different phenotypical features, molding time and mortality. GDF-15 expression in the hearts of UCM mice was downregulated compared with sham group mice.