Huijuan Mao

The roles of oxidative stress, endoplasmic reticulum stress, and autophagy in aldosterone/mineralocorticoid receptor-induced podocyte injury.

Podocytes play an important role in the pathogenesis and progression of glomerulosclerosis. Recent studies indicate that aldosterone/mineralocorticoid receptor (MR) is a major contributor of chronic kidney disease (CKD) progression. Aldosterone/MR induces glomerular podocyte injury, causing the disruption of the glomerular filtration barrier and proteinuria. The present study investigated the mechanisms by which aldosterone/MR mediated podocyte injury, focusing on the involvement of oxidative stress, endoplasmic reticulum (ER) stress, and autophagy. We observed that aldosterone/MR induced ER stress and podocyte injury both in vivo and in vitro. Blockade of ER stress significantly reduced aldosterone/MR-induced podocyte injury. In addition, we found that ER stress-induced podocyte injury was mediated by CCAAT/enhancer-binding protein (C/EBP) homologous protein (Chop). Interestingly, autophagy was also enhanced by aldosterone/MR. Pharmacological inhibition of autophagy resulted in increased apoptosis. Inhibition of ER stress significantly reduced aldosterone/MR-induced autophagy. In addition, the activation of ER stress increased the formation of autophagy, which protected podocytes from apoptosis. Moreover, we observed that the addition of ROS scavenger, N-acetyl cystein (NAC), blocked both ER stress and autophagy by aldosterone/MR. Collectively, these results suggest that oxidant stress-mediated aldosterone/MR-induced podocyte injury via activating ER stress, which then triggers both Chop-dependent apoptosis and autophagy to cope with the injury. These findings may guide us to therapeutic strategies for glomerular diseases.

P53 Contributes to Cisplatin Induced Renal Oxidative Damage via Regulating P66shc and MnSOD.

Background/Aims: Cisplatin is widely used to treat malignancies. However, its major limitation is the development of dose-dependent nephrotoxicity. The precise mechanisms of cisplatin-induced kidney damage remain unclear. Previous study demonstrated the central role of mitochondrial ROS (mtROS) in the pathogenesis of cisplatin nephrotoxicity. The purpose of this study was to explore the mechanism of mtROS regulation in cisplatin nephrotoxicity. Methods: p53, MnSOD and p66shc were detected at mRNA and protein levels by qPCR and western blot in HK2 cells. mtROS levels were determined by DCFDA and MitoSOX staining. Cell viability and cell apoptosis were accessed by CCK-8 assay, TUNEL assay and flow cytometry, respectivesly. siRNAs were used to knock down p53 and p66shc expression and subsequent changes were observed. In vivo assays using a mouse model of cisplatin-induced acute kidney injury were used to validate the in vitro results. Results: In HK2 cells, cisplatin exposure decreased the MnSOD and increased the expression of p53 and p66shc. MnTBAP, a MnSOD mimic, blocked cisplatin-induced the generation of mtROS and cell injury. P66shc and p53 siRNAs rendered renal cells resistant to cisplatin-induced mtROS production and cell death. Furthermore, knockdown of p53 restored MnSOD and inhibiting p66shc. Consistent with these results, we revealed that p53 inhibitor reduced cisplatin-induced oxidative stress and apoptosis by regulating MnSOD and p66shc in the kidney of cisplatin-treated mice. Conclusion: Our study identifies activation of p53 signalling as a potential strategy for reducing the nephrotoxicity associated with cisplatin treatments and, as a result, broadens the therapeutic window of this chemotherapeutic agent.

Plasma FGF23 levels and heart rate variability in patients with stage 5 CKD

SummaryFibroblast growth factor 23(FGF23) is a bone-derived hormone which regulates mineral homeostasis but may also have a role in cardiovascular disease. Here, we found that higher plasma FGF23 was independently associated with decreased heart rate variability in stage 5 CKD patients and parathyroidectomy may reverse these abnormal indicators.IntroductionLower heart rate variability (HRV) in patients with chronic kidney disease (CKD) compared with healthy controls is associated with increased risk of cardiovascular disease (CVD). Higher levels of plasma FGF23 also predict higher risk of CVD. Here, we aimed to evaluate the relationship between plasma FGF23 levels and HRV in patients with stage 5 CKD and to investigate longitudinal changes of them together with the correlation between their changes in two severe secondary hyperparathyroidism (SHPT) subgroups with successful parathyroidectomy (PTX) and persistent SHPT.MethodsThis cross-sectional study included 100 stage 5 CKD patients, 78 controls, and a prospective study in two PTX subgroups classified as successful PTX (n = 24) and persistent SHPT (n = 4) follow-up. Blood examination and 24-h Holter monitoring for HRV were measured.ResultsMost HRV indices were lower in stage 5 CKD patients than in healthy controls, and plasma FGF23 levels were higher. In multivariate stepwise regression models, levels of plasma FGF23 and serum parathyroid hormone (PTH) were correlated with HRV. The successful PTX subgroup had significant improvements over baseline in HRV indices. Persistent SHPT subgroup had numerically similar changes in HRV indices. However, plasma FGF23 levels decreased in both subgroups.ConclusionsPlasma FGF23 levels were higher in CKD patients than in controls, much higher in patients with severe SHPT. FGF23 was independently associated with decreased HRV in stage 5 CKD. Successful PTX may reverse these abnormal indicators and contribute to decreases in the risk of cardiovascular disease.

Drp1-dependent mitophagy protects against cisplatin-induced apoptosis of renal tubular epithelial cells by improving mitochondrial function

Cisplatin chemotherapy often causes acute kidney injury (AKI) in cancer patients. There is increasing evidence that mitochondrial dysfunction plays an important role in cisplatin-induced nephrotoxicity. Degradation of damaged mitochondria is carried out by mitophagy. Although mitophagy is considered of particular importance in protecting against AKI, little is known of the precise role of mitophagy and its molecular mechanisms during cisplatin-induced nephrotoxicity. Also, evidence that activation of mitophagy improved mitochondrial function is lacking. Furthermore, several evidences have shown that mitochondrial fission coordinates with mitophagy. The aim of this study was to investigate whether activation of mitophagy protects against mitochondrial dysfunction and renal proximal tubular cells injury during cisplatin treatment. The effect of mitochondrial fission on mitophagy was also investigated. In cultured human renal proximal tubular cells, we observed that 3-methyladenine, a pharmacological inhibitor of autophagy, blocked mitophagy and exacerbated cisplatin-induced mitochondrial dysfunction and cells injury. In contrast, autophagy activator rapamycin enhanced mitophagy and protected against the harmful effects of cisplatin on mitochondrial function and cells viability. Suppression of mitochondrial fission by knockdown of its main regulator dynamin-related protein-1 (Drp1) decreased cisplatin-induced mitophagy. Meanwhile, Drp1 suppression protected against cisplatin-induced cells injury by inhibiting mitochondrial dysfunction. Our results provide evidence that Drp1-depedent mitophagy has potential as renoprotective targets for the treatment of cisplatin-induced AKI.

Diagnostic Accuracy Study of Intraoperative and Perioperative Serum Intact PTH Level for Successful Parathyroidectomy in 501 Secondary Hyperparathyroidism Patients

Parathyroidectomy (PTX) is an effective treatment for severe secondary hyperparathyroidism (SHPT); however, persistent SHPT may occur because of supernumerary and ectopic parathyroids. Here a diagnostic accuracy study of intraoperative and perioperative serum intact parathyroid hormone (iPTH) was performed to predict successful surgery in 501 patients, who received total PTX + autotransplantation without thymectomy. Serum iPTH values before incision (io-iPTH0), 10 and 20 min after removing the last parathyroid (io-iPTH10, io-iPTH20), and the first and fourth day after PTX (D1-iPTH, D4-iPTH) were recoded. Patients whose serum iPTH was >50 pg/mL at the first postoperative week were followed up within six months. Successful PTX was defined if iPTH was <300 pg/mL, on the contrary, persistent SHPT was regarded. There were 86.4% patients underwent successful PTX, 9.8% remained as persistent SHPT and 3.8% were undetermined. Intraoperative serum iPTH demonstrated no significant differences in two subgroups with or without chronic hepatitis. Receiver operating characteristic (ROC) curves showed that >88.9% of io-iPTH20% could predict successful PTX (area under the curve [AUC] 0.909, sensitivity 78.6%, specificity 88.5%), thereby avoiding unnecessary exploration to reduce operative complications. D4-iPTH >147.4 pg/mL could predict persistent SHPT (AUC 0.998, sensitivity 100%, specificity 99.5%), so that medical intervention or reoperation start timely.

Association of Increased Serum Leptin with Ameliorated Anemia and Malnutrition in Stage 5 Chronic Kidney Disease Patients after Parathyroidectomy

Leptin is an adipokine that regulates various metabolism, but its association with secondary hyperparathyroidism (SHPT), a clinical manifestation of chronic kidney disease-mineral and bone disorder (CKD-MBD), remains obscure. Parathyroidectomy (PTX) is recommended for severe SHPT patients. Here, the associations between circulating leptin and clinical characteristics in CKD patients were investigated. Effects of PTX on leptin production were analyzed in vivo and in vitro. Controls and CKD patients had approximate serum leptin levels in that a larger proportion of CKD patients with body mass index (BMI) <23 kg/m2. Serum leptin was related to anemia, albumin, and bone metabolism disorders in CKD patients. Lower intact parathyroid hormone (PTH) was related with higher leptin in PTX patients group. Severe SHPT inhibited uremia-enhanced leptin production in 3T3-L1 adipocytes, which was attenuated after PTX. High levels of PTH were found to reduce Akt phosphorylation and leptin production in vitro but high levels of calcium and phosphorus were not. Successful PTX was found to improve anemia and malnutrition in severe SHPT patients, and this was correlated with increased circulating leptin levels via up-regulated Akt signaling in adipocytes. These findings indicated the therapeutic potential of leptin and related target pathway for improving survival and quality of life in CKD.

Parathyroidectomy Increases Heart Rate Variability and Leptin Levels in Patients with Stage 5 Chronic Kidney Disease.

Background: In chronic kidney disease (CKD) patients, decreased heart rate variability (HRV) reflects impaired cardiac automatic nervous function and high risk of cardiovascular disease (CVD). Lower HRV in patients with severe secondary hyperparathyroidism (SHPT), a clinical manifestation of CKD-mineral and bone disorder (CKD-MBD), could be reversed by parathyroidectomy (PTX). It has been proved that leptin interacts with the autonomic nervous function. However, the associations between leptin and HRV in CKD patients and their longitudinal changes in SHPT patients after PTX are still unknown. Methods: This was a cross-sectional study including 141 stage 5 CKD patients, and a prospective study in 36 severe SHPT patients with PTX. HRV was measured by Holter and serum leptin was measured by ELISA. Serum leptin levels were adjusted for body mass index (BMI) and transformed using natural logarithm (lnleptin/BMI). Results: With a gradient of lnleptin/BMI across quartiles from Q1 to Q4 in CKD patients, HRV indices showed no differences among quartiles. Patients in Q1 group had higher mean 24 h heart rates, and lower ln(very low frequency) (lnVLF) than other quartiles, although there were no statistically significant difference. In multivariate stepwise regression, serum leptin/BMI was an independent predictor for low frequency/high frequency. HRV indices and lnleptin/BMI levels were increased in severe SHPT patients after PTX. Compared to other quartiles, SHPT patients in Q1 group had larger improvement of lnVLF after PTX. Conclusion: Circulating leptin levels may be a novel treatment target to reduce CVD risk in advanced CKD-MBD patients.

Low-Site Peritoneal Catheter Implantation Decreases Tip Migration and Omental Wrapping

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Effect of secondary hyperparathyroidism serum on endothelial cells and intervention with Klotho.

The aim of the present study was to investigate the effect of the serum of patients with secondary hyperparathyroidism (SHPT) on endothelial cells and to examine the protective effect and the possible mechanism of Klotho. A total of three types of mixed serum from 15 patients with SHPT scheduled for parathyroidectomy, 10 chronic kidney disease (CKD) patients at stage 5 without SHPT and 15 healthy volunteers were collected. Initially, human umbilical vein endothelial cells (HUVECs) were incubated in vitro with the three types of serum and levels of proliferation were compared by assessing viable cell numbers with cell counting kit-8 (CCK-8). Subsequently, HUVECs were divided into three groups: Control group (healthy serum medium), SHPT group (SHPT serum) and Klotho treatment group (SHPT serum and Klotho). The proliferative and apoptotic levels of endothelial cells were evaluated by CCK-8 and flow cytometry, respectively. The levels of extracellular signal-regulated kinase (ERK1/2) and phosphorylated forms of ERK1/2 (p-ERK1/2) were detected using western blotting (with or without ERK1/2 inhibitor PD98059). The synthesis of nitric oxide (NO) was measured using the nitrate reduction method. The proliferation of HUVECs was inhibited by the serum from SHPT patients and CKD-5 patients without SHPT and the inhibitory effects of the SHPT serum were the most marked (P<0.05). Inhibition of HUVEC proliferation by SHPT serum occurred in a concentration-dependent manner within a specific range (5-20%; P<0.05) and also in a time-dependent manner within 6-24 h. Proliferation was partly restored and apoptosis was inhibited when 50-100 ng/ml Klotho was added into 10% SHPT serum (P<0.05). At the same time, the expression of p-ERK1/2 was upregulated, which may be inhibited by PD98059. The synthesis of NO was decreased in the SHPT group (P<0.05) and increased following treatment with Klotho (P<0.05). The results of the present study indicated that the proliferation of HUVECs was inhibited by the serum from SHPT patients. Klotho may partly antagonize this effect due to its inhibition of HUVEC apoptosis and upregulation of p-ERK1/2.

The Role and Mechanism of α-Klotho in the Calcification of Rat Aortic Vascular Smooth Muscle Cells

Objective. To investigate the role and possible mechanism of α-Klotho in the calcification and the osteogenic transition of cultured VSMCs. Methods. VSMCs were cultured in vitro and divided into 5 groups, each using a different medium: (1) control; (2) β-GP; (3) β-GP + Klotho; (4) β-GP + LiCl; (5) β-GP + Klotho + LiCl. Calcium deposits were visualized using Alizarin Red S staining. The calcium concentrations were determined by the o-cresolphthalein complexone method. BMP2, Runx2 and β-catenin levels were estimated by western blotting, and the level of α-SMA was determined by using immunofluorescence at day 12. Results. β-GP induced an increase in the expression of BMP2, Runx2, and β-catenin. The calcium content increased, and the expression of α-SMA decreased. Alizarin Red S staining was positive under the high phosphorus conditions. BMP2, Runx2, and β-catenin levels and the calcium content decreased when the cells were cultured with rmKlotho; however, the levels of each were upregulated after treatment with the LiCl. Conclusions. Klotho can ameliorate the calcification and osteogenic transition of VSMCs induced by β-GP. The mechanism of Klotho in preventing calcification in VSMCs may be partially mediated by the inhibition of the Wnt/β-catenin signaling pathway.