Scavenger Receptor Class B Type I Deficiency Induces Iron Overload and Ferroptosis in Renal Tubular Epithelial Cells via Hypoxia-Inducible Factor-1α/Transferrin Receptor 1 Signaling Pathway.

Aims: Scavenger receptor class B type I (SRBI) promotes cell cholesterol efflux and the clearance of plasma cholesterol. Thus, SRBI deficiency causes abnormal cholesterol metabolism and hyperlipidemia. Studies have suggested that ferroptosis is involved in lipotoxicity; however, whether SRBI deficiency could induce ferroptosis remains to be investigated. Results: We knocked down or knocked out SRBI in renal HK-2 cells and C57BL/6 mice to determine the expression levels of ferroptosis-related regulators. Our results demonstrated that SRBI deficiency upregulates transferrin receptor 1 (TFR1) expression and downregulates ferroportin expression, which induces iron overload and subsequent ferroptosis in renal tubular epithelial cells. TFR1 is known to be regulated by hypoxia-inducible factor-1α (HIF-1α). Next, we investigated whether SRBI deletion affected HIF-1α. SRBI deletion upregulated the mRNA and protein expression of HIF-1α, and promoted its translocation to the nucleus. To determine whether HIF-1α plays a key role in SRBI-deficiency-induced ferroptosis, we used HIF-1α inhibitor and siHIF-1α in HK-2 cells, and found that downregulation of HIF-1α prevented SRBI-silencing-induced TFR1 upregulation and iron overload, and eventually reduced ferroptosis. The underlying mechanism of HIF-1α activation was explored next, and the results showed that SRBI knockout or knockdown may upregulate the expression of HIF-1α, and promote HIF-1α translocation from the cytoplasm into the nucleus via the PKC-ß/NF-κB signaling pathway. Innovation and Conclusion: Our study showed, for the first time, that SRBI deficiency induces iron overload and subsequent ferroptosis via the HIF-1α/TFR1 pathway.

Computed tomography-determined skeletal muscle density predicts 3-year mortality in initial-dialysis patients in China.

BACKGROUND: Skeletal muscle mass and quality assessed by computed tomography (CT) images of the third lumbar vertebra (L3) level have been established as risk factors for poor clinical outcomes in several illnesses, but the relevance for dialysis patients is unclear. A few studies have suggested a correlation between CT-determined skeletal muscle mass and quality at the first lumbar vertebra (L1) level and adverse outcomes. Generally, chest CT does not reach beyond L1. We aimed to determine whether opportunistic CT scan (chest CT)-determined skeletal muscle mass and quality at L1 are associated with mortality in initial-dialysis patients. METHODS: This 3-year multicentric retrospective study included initial-dialysis patients from four centres between 2014 and 2017 in China. Unenhanced CT images of the L1 and L3 levels were obtained to assess skeletal muscle mass [by skeletal muscle index, (SMI), cm2 /m2 ] and quality [by skeletal muscle density (SMD), HU]. Skeletal muscle measures at L1 were compared with those at L3. The sex-specific optimal cutoff values of L1 SMI and L1 SMD were determined in relation to all-cause mortality. The outcomes were all-cause death and cardiac death. Cox regression models were applied to investigate the risk factors for death. RESULTS: A total of 485 patients were enrolled, of whom 257 had both L1 and L3 images. Pearson's correlation coefficient between L1 and L3 SMI was 0.84 (P < 0.001), and that between L1 and L3 SMD was 0.90 (P < 0.001). No significant association between L1 SMI and mortality was observed (P > 0.05). Low L1 SMD (n = 280, 57.73%) was diagnosed based on the optimal cutoff value (<39.56 HU for males and <33.06 HU for females). Multivariate regression analysis revealed that the low L1 SMD group had higher risks of all-cause death (hazard ratio 1.80; 95% confidence interval 1.05-3.11, P = 0.034) and cardiac death (hazard ratio 3.74; 95% confidence interval 1.43-9.79, P = 0.007). CONCLUSIONS: In initial-dialysis patients, there is high agreement between the L1 and L3 measures for SMI and SMD. Low SMD measured at L1, but not low SMI, is an independent predictor of both all-cause death and cardiac death.

Empagliflozin attenuates the renal tubular ferroptosis in diabetic kidney disease through AMPK/NRF2 pathway.

Renal tubular damage plays a key role in the pathogenesis of diabetic kidney disease (DKD), and one of the main pathological process associated with DKD in diabetic mice is the ferroptosis, a novel form of cell death caused by iron-dependent lipid peroxidation. Several researches suggested that empagliflozin may treat renal injury, but its effects on diabetic-related ferroptosis and underlying mechanisms were not fully elucidated. In this study, the influence of empagliflozin on renal injury was evaluated in vivo and in vitro in a mouse model and in high-glucose (HG) or Erastin-stimulated renal HK-2 cell line, respectively. Ferroptosis-related markers were assessed, including GSH, labile iron levels, and ferroptosis regulators by Western blot, qRT-PCR, immunohistochemistry, and immunofluorescence. The level of malondialdehyde (MDA) and the fluorescence intensity of BODIPY probe indicated the level of lipid peroxidation. It was demonstrated that solute carrier family 7, member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) were less expressed in renal biopsy samples from patients affected by DKD than in those from non-diabetic renal disease patients (NDRD), proving the ferroptosis of tubular epithelial cells in case of DKD. Furthermore, empagliflozin markedly decreased the ferroptosis impairment in DKD mice, as well as in HG model of HK-2 cells. Our investigations showed the ability of empagliflozin to suppress ferroptosis was partially countered by AMP-activated protein kinase (AMPK) inhibitor, which led to a reduction of the nuclear translocation of the antioxidant transcription factor NFE2-related factor 2 (NRF2) and downregulation of target genes such as GPX4, ferritin heavy chain 1 (FTH1), and SLC7A11, while AMPK agonists were responsible for the enhancement of the protective effects of empagliflozin. Taken together, our findings showed that empagliflozin may prevent the development of ferroptosis by promoting the AMPK-mediated NRF2 activation pathway, providing important insights for possible novel treatment approaches for DKD.

Ultrasound-assessed diaphragm dysfunction predicts clinical outcomes in hemodialysis patients.

Skeletal muscle atrophy is prevalent and remarkably increases the risk of cardiovascular (CV) events and mortality in hemodialysis (HD) patients. However, whether diaphragm dysfunction predicts clinical outcomes in HD patients is unknown. This was a prospective cohort study of 103 HD patients. After assessment of diaphragm function by ultrasonography and collection of other baseline data, a 36-month follow-up was then initiated. Participants were divided into diaphragm dysfunction (DD+) group and normal diaphragm function (DD-) group, according to cutoff value of thickening ratio (i.e. the change ratio of diaphragm thickness) at force respiration. The primary endpoint was the first nonfatal CV event or all-cause mortality. A secondary endpoint was less serious CV events (LSCEs, a composite of heart failure readmission, cardiac arrhythmia or myocardial ischemia needed pharmacological intervention in hospital). 98 patients were eligible to analysis and 57 (58.16%) were men. 28 of 44 patients(63.64%) in DD+ group and 23 of 54 patients (42.59%) in DD- group had at least one nonfatal CV event or death (p = 0.038). Compared to DD- group, DD+ group had significantly higher incidence of LSCEs (21 vs.14, p = 0.025) and shorter survival time (22.02 ± 12.98 months vs. 26.74 ± 12.59 months, p = 0.046). Kaplan-Meier analysis revealed significantly higher risks of primary endpoint (p = 0.039), and LSCEs (p = 0.040) in DD+ group. Multivariate hazard analysis showed that DD+ group had significantly higher risk of primary endpoint [hazard ratio (HR) 1.59; 95% confident interval (CI) 1.54-1.63], and LSCEs (HR 1.47; 95%CI 1.40-1.55). Ultrasound-assessed diaphragm dysfunction predicts clinical outcomes in HD patients.Trial registration: This study was registered with Chinese Clinical Trials Registry ( www.chictr.org.cn ) as ChiCTR1800016500 on Jun 05, 2018.

Regulator of calcineurin 1 deletion attenuates mitochondrial dysfunction and apoptosis in acute kidney injury through JNK/Mff signaling pathway.

Ischemia-reperfusion (I/R) induced acute kidney injury (AKI), characterized by excessive mitochondrial damage and cell apoptosis, remains a clinical challenge. Recent studies suggest that regulator of calcineurin 1 (RCAN1) regulates mitochondrial function in different cell types, but the underlying mechanisms require further investigation. Herein, we aim to explore whether RCAN1 involves in mitochondrial dysfunction in AKI and the exact mechanism. In present study, AKI was induced by I/R and cisplatin in RCAN1flox/flox mice and mice with renal tubular epithelial cells (TECs)-specific deletion of RCAN1. The role of RCAN1 in hypoxia-reoxygenation (HR) and cisplatin-induced injury in human renal proximal tubule epithelial cell line HK-2 was also examined by overexpression and knockdown of RCAN1. Mitochondrial function was assessed by transmission electron microscopy, JC-1 staining, MitoSOX staining, ATP production, mitochondrial fission and mitophagy. Apoptosis was detected by TUNEL assay, Annexin V-FITC staining and Western blotting analysis of apoptosis-related proteins. It was found that protein expression of RCAN1 was markedly upregulated in I/R- or cisplatin-induced AKI mouse models, as well as in HR models in HK-2 cells. RCAN1 deficiency significantly reduced kidney damage, mitochondrial dysfunction, and cell apoptosis, whereas RCAN1 overexpression led to the opposite phenotypes. Our in-depth mechanistic exploration demonstrated that RCAN1 increases the phosphorylation of mitochondrial fission factor (Mff) by binding to downstream c-Jun N-terminal kinase (JNK), then promotes dynamin related protein 1 (Drp1) migration to mitochondria, ultimately leads to excessive mitochondrial fission of renal TECs. In conclusion, our study suggests that RCAN1 could induce mitochondrial dysfunction and apoptosis by activating the downstream JNK/Mff signaling pathway. RCAN1 may be a potential therapeutic target for conferring protection against I/R- or cisplatin-AKI.

Paraoxonase 1 Ameliorates Renal Lipotoxicity by Activating Lipophagy and Inhibiting Pyroptosis.

Several studies in recent years have shown that lipid overload causes lipotoxic damage to the kidney, and oxidative stress, inflammation, and autophagic arrest are all important mechanisms of renal lipotoxicity. However, effective measures with therapeutic effects on renal lipotoxicity are limited. The present study indicated the protective effect of the paraoxonase 1 (PON1) against renal lipotoxicity in high-fat diet-fed scavenger receptor class B type I-deficient (SR-BI-/-) mice. The results showed that SR-BI-/- mice exhibited significant renal pathologic characteristics, such as oxidative stress, inflammation, and fibrosis, under a normal chow diet, and were accompanied by dyslipidemia and reduced plasma PON1 activity and renal PON1 levels. PON1 overexpression significantly attenuated the above pathologic changes in the kidneys of SR-BI-/- mice fed with a high-fat diet. Mechanistically, PON1 may ameliorate renal oxidative stress by reducing reactive oxygen species production, reduce renal lipid accumulation by inhibiting AKT/mechanistic target of rapamycin kinase pathway to activate lipophagy, and reduce the occurrence of inflammation and cell death by inhibiting Nod-like receptor family protein 3 inflammasome-mediated pyroptosis. The present study is the first to show that PON1 overexpression can effectively alleviate renal lipotoxicity injury, and PON1 may be a promising therapeutic strategy for the treatment of renal lipotoxicity-related diseases.

Regulators of calcineurin 1 deficiency attenuates tubulointerstitial fibrosis through improving mitochondrial fitness.

Renal fibrosis is the common pathological process of various chronic kidney diseases (CKD). Recent studies indicate that mitochondrial fragmentation is closely associated with renal fibrosis in CKD. However, the molecular mechanisms leading to mitochondrial fragmentation remain to be elucidated. The present study investigated the role of regulators of calcineurin 1 (RCAN1) in mitochondrial fission and renal interstitial fibrosis using conditional knockout mice in which RCAN1 was genetically deleted in tubular epithelial cells (TECs). TEC-specific deletion of RCAN1 attenuated tubulointerstitial fibrosis and epithelial to mesenchymal transition (EMT)-like phenotype change after unilateral ureteral obstruction (UUO) and ischemia reperfusion injury (IRI) through suppressing TGF-ß1/Smad3 signaling pathway. TEC-specific deletion of RCAN1 also reduced the tubular apoptosis after UUO by inhibiting cytochrome c/caspase-9 pathway. Ultrastructure analysis revealed a marked decrease in mitochondrial fragmentation in TECs of RCAN1-deficient mice in experimental CKD models. The expression of mitochondrial profission proteins dynamin-related protein 1 (Drp1) and mitochondrial fission factor (Mff) was also downregulated in obstructed kidney of TEC-specific RCAN1-deficient mice. Furthermore, TEC-specific deletion of RCAN1 attenuated the dysfunctional tubular autophagy by regulating PINK1/Parkin-induced mitophagy in CKD. RCAN1 knockdown and knockout similarly improved the mitochondrial quality control in HK-2 cells and primary cultured mouse tubular cells stimulated by TGF-ß1. Put together, our data indicated that RCAN1 plays an important role in the progression of tubulointerstitial fibrosis through regulating the mitochondrial quality. Therefore, targeting RCAN1 may provide a potential therapeutic approach in CKD.