Irisin ameliorates diabetic kidney disease by restoring autophagy in podocytes.

Many studies have highlighted the importance of moderate exercise. While it can attenuate diabetic kidney disease, its mechanism has remained unclear. The level of myokine irisin in plasma increases during exercise. We found that irisin was decreased in diabetic patients and was closely related to renal function, proteinuria, and podocyte autophagy injury. Muscle-specific overexpression of PGC-1α (mPGC-1α) in a mouse model is known to increase plasma irisin levels. The mPGC-1α mice were crossed with db/m mice to obtain db/db mPGC-1α+ mice in the present study. Compared to db/db mice without mPGC-1α, plasma irisin was increased, and albuminuria and glomerular pathological damage were both alleviated in db/db mPGC-1α+ mice. Impaired autophagy in podocytes was restored as well. Irisin inhibited the activation of the PI3K/AKT/mTOR signaling pathway in cultured human podocytes and improved damaged autophagy induced by high glucose levels. Then, db/db mice were treated with recombinant irisin, which had similar beneficial effects on the kidney as those in db/db mPGC-1α+ mice, with alleviated glomerular injury and albuminuria. Moreover, the autophagy in podocytes was also significantly restored. These results suggest that irisin secreted by skeletal muscles protects the kidney from diabetes mellitus damage. It also restores autophagy in podocytes by inhibiting the abnormal activation of the PI3K/AKT/mTOR signaling pathway. Thus, irisin may become a new drug for the prevention and treatment of diabetic nephropathy.

A retrospective cohort study on the pathology and outcomes of type 2 diabetic patients with renal involvement.

OBJECTIVE: To investigate the association of clinical and histological characteristics and the development of ESRD in T2DM patients with renal involvement. METHODS: We conducted a retrospective analysis of clinical and pathologic data from T2DM patients who underwent renal biopsy (n = 120). RESULTS: The mean age, duration of diabetes, and eGFR were 50.9 ± 11.2 years, 92.8 ± 41.3 months, 55.1 ± 42.3 mL/min/1.73 m2, respectively. Among these patients, 57 (47.5%) were diagnosed with diabetic nephropathy (DN), and 63 (52.5%) with non-diabetic renal disease (NDRD). The most common subtype of NDRD is membranous nephropathy. Compared with the NDRD group, the DN group had a longer duration of diabetes, worse renal function, and a higher proportion of diabetic retinopathy. Kaplan-Meier analysis showed that the 5-year renal survival rate of the DN group was only 41%, whereas that of the NDRD group was 84%. ESRD was defined as eGFR below 15 mL/min/1.73 m2. After multivariate adjustment, the risk of ESRD in DN patients was 3.81 times higher than that in NDRD patients. According to Glomerular Class, the 5-year renal survival rate of type IIA, IIB, III, and IV in the DN group was 88, 56, 28, and 15%, respectively. Kaplan-Meier analysis showed that there was a significant difference in renal survival among different glomerular classes or different interstitial fibrosis and tubular atrophy (IFTA) scores. But Cox proportional hazards analysis indicated that only IFTA score (HR 2.75, 95% CI 1.37-5.51, P = 0.001), but not the glomerular class (HR 1.21, 95% CI 0.73-2.00, P = 0.465), could predict renal outcome when adjusting for multivariate. CONCLUSION: The prognosis of DN patients is significantly worse than that of NDRD patients. Compared with glomerular lesions, tubulointerstitial lesions were associated with higher risk for renal death in DN patients.

The influence of cardiac valvular calcification on all-cause and cardiovascular mortality in maintenance hemodialysis patients.

OBJECTIVE: To investigate the effect of cardiac valve calcification (CVC) on all-cause and cardiovascular mortality in maintenance hemodialysis (MHD) patients. METHODS: A retrospective cohort study was conducted in 183 long-term hemodialysis patients with complete follow-up data from January 1, 2012, to December 30, 2015. The baseline data between CVC and non-CVC groups were compared. Kaplan-Meier method was used to analyze all-cause and cardiovascular mortality. The effect of CVC on prognosis was analyzed using the Cox proportional hazard regression model and subgroup analysis. RESULTS: Among 183 patients under hemodialysis, 104 (56.8%) were males, with an average age of 56.1 ± 17.0 years and 68 (37.2%) were complicated with valvular calcification. The median follow-up period was 30.8 months. All-cause and cardiovascular mortality were 50% vs. 14.8% and 25% vs. 7.0% in the CVC and non-CVC groups, respectively (P < 0.05). Kaplan-Meier indicated that differences in all-cause and cardiovascular mortality were statistically significant between the two groups (P < 0.001). Cox regression analysis showed that CVC significantly increased all-cause (hazards ratio [HR] 2.161 [1.083-4.315]) and cardiovascular mortality (3.435 [1.222-9.651]) after adjusting for multiple factors. Meanwhile, CVC also increases the incidence of new-onset cardiovascular events. Subgroup analysis revealed that all-cause and cardiovascular mortality were significantly higher in patients with aortic valve calcification (AVC) than in patients with mitral valve calcification (MVC). Multivariate calibration showed that AVC increased the risk of cardiovascular death (HR 5.486 [1.802-16.702]) (P < 0.05), whereas MVC did not. By further comparing the echocardiographic data of the two groups, the incidence of LVH and pulmonary hypertension in the AVC group was significantly higher than that in the MVC group. CONCLUSION: Valve calcification increases the risk of all-cause and cardiovascular mortality in MHD patients, also new-onset cardiovascular events, and aortic valve calcification contributes more to the risk of cardiovascular mortality.

Sirt3 modulates fatty acid oxidation and attenuates cisplatin-induced AKI in mice.

Fatty acid oxidation (FAO) dysfunction is one of the important mechanisms of renal fibrosis. Sirtuin 3 (Sirt3) has been confirmed to alleviate acute kidney injury (AKI) by improving mitochondrial function and participate in the regulation of FAO in other disease models. However, it is not clear whether Sirt3 is involved in regulating FAO to improve the prognosis of AKI induced by cisplatin. Here, using a murine model of cisplatin-induced AKI, we revealed that there were significantly FAO dysfunction and extensive lipid deposition in the mice with AKI. Metabolomics analysis suggested reprogrammed energy metabolism and decreased ATP production. In addition, fatty acid deposition can increase reactive oxygen species (ROS) production and induce apoptosis. Our data suggested that Sirt3 deletion aggravated FAO dysfunction, resulting in increased apoptosis of kidney tissues and aggravated renal injury. The activation of Sirt3 by honokiol could improve FAO and renal function and reduced fatty acid deposition in wide-type mice, but not Sirt3-defective mice. We concluded that Sirt3 may regulate FAO by deacetylating liver kinase B1 and activating AMP-activated protein kinase. Also, the activation of Sirt3 by honokiol increased ATP production as well as reduced ROS and lipid peroxidation through improving mitochondrial function. Collectively, these results provide new evidence that Sirt3 is protective against AKI. Enhancing Sirt3 to improve FAO may be a potential strategy to prevent kidney injury in the future.

Circular RNA expression profiles in cisplatin-induced acute kidney injury in mice.

Aim: This study was carried out to identify the expression profile and role of circRNAs in cisplatin-induced acute kidney injury (AKI). Materials & methods: In this study, an AKI model was established in cisplatin-treated mice, and the expression of circRNAs was profiled by next-generation sequencing. The differential expression levels of selected circRNAs were determined by quantitative real-time polymerase chain reaction. Bioinformatics analysis was conducted to predict the functions. Results: In total, 368 circRNAs were detected to be differentially expressed in response to cisplatin treatment. Bioinformatics analysis indicated that the parental genes of the differentially expressed circRNAs were predominantly implicated in the cell and cell part, cellular process and cancer pathways. Conclusion: CircRNAs might be differentially expressed in AKI, which are potentially involved in pathophysiology of cisplatin-induced nephrotoxicity.

Activation of the Nrf2-ARE Pathway Ameliorates Hyperglycemia-Mediated Mitochondrial Dysfunction in Podocytes Partly Through Sirt1.

BACKGROUND/AIMS: Previously we have shown that activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-antioxidant response element (ARE) attenuated hyperglycemia-induced damage in podocytes, but the molecular mechanism remains unknown. METHODS: Tert-butylhydroquinone (t-BHQ) and small interfering RNAs (siRNAs) were used to regulate Nrf2 expression, while nicotinamide and siRNAs were used to regulate sirtuin 1 (Sirt1) activity and expression, respectively. Mitochondrial superoxide, membrane potential and ATP levels were measured to assess changes in mitochondrial function. Nephrin and synaptopodin expression were measured by western blot analysis. Human podocytes and db/db diabetic mice were used in this study. RESULTS: t-BHQ pretreatment of human podocytes exposed to high glucose (HG) alleviated mitochondrial dysfunction, enhanced the expression of Sirt1, nephrin and synaptopodin and lowered BSA permeability compared with podocytes exposed to HG without t-BHQ pretreatment (p< 0.05). Human podocytes exposed to HG had more severe mitochondrial dysfunction, lower expression of Sirt1, synaptopodin and nephrin and higher BSA permeability than podocytes exposed to HG when Nrf2 expression was downregulated by siRNAs (p< 0.05). The protection provided by activation of the Nrf-ARE pathway in podocytes exposed to HG was partially diminished when Sirt1 expression or activity was decreased by siRNAs or inhibitor compared with podocytes exposed to HG and pretreated with t-BHQ (p< 0.05). When nicotinamide and t-BHQ were both administered to db/db mice, we observed higher levels of urinary albumin/creatinine, lower nephrin and synaptopodin expression, more severe mesangial matrix deposition, collagen deposition on pathological slides and mitochondrial structural damage in podocytes compared to db/db mice treated only with t-BHQ. CONCLUSIONS: Our findings suggest that crosstalk between Sirt1 and the Nrf2-ARE anti-oxidative pathway forms a positive feedback loop and that protection provided by t-BHQ activation of the Nrf2-ARE pathway in db/db mice is partly dependent on Sirt1.

Activation of Sirtuin 3 by Silybin Attenuates Mitochondrial Dysfunction in Cisplatin-induced Acute Kidney Injury.

Silybin is a secondary metabolite isolated from the seeds of blessed milk thistle (Silybum marianum) that has anti-inflammatory, antioxidative, antifibrotic, and antitumor properties. Here, we showed that silybin protected against cisplatin-induced acute kidney injury (AKI) by improving mitochondrial function through the regulation of sirtuin 3 (SIRT3) expression. Male SV129 and SIRT3 knockout (KO) mice were administered a single intraperitoneal (i.p.) injection of cisplatin with or without treatment with silybin. Moreover, cultured HK2 cells were used to evaluate mitochondrial morphology and function. Our data suggested that silybin enhanced SIRT3 expression after cisplatin administration both in vivo and in vitro. Silybin treatment improved mitochondrial function and bioenergetics in wild-type, but not SIRT3-defective, cells and mice. Moreover, we demonstrated that silybin markedly attenuated cisplatin-induced AKI and tubular cell apoptosis and improved cell regeneration in a SIRT3-dependent manner. Collectively, these results suggest that silybin is a pharmacological activator of SIRT3 capable of protecting against cisplatin-induced tubular cell apoptosis and AKI by improving mitochondrial function. Thus, silybin could serve as a potential clinical renoprotective adjuvant treatment in cisplatin chemotherapy.

Poor sleep quality is responsible for the nondipper pattern in hypertensive but not in normotensive chronic kidney disease patients.

AIM: This study was designed to evaluate the relationship between sleep quality and hypertension and to determine if there was an association between nondipper blood pressure (BP) and sleep quality in chronic kidney disease (CKD) patients. METHODS: A total of 775 pre-dialysis CKD patients (314 normal BP patients, 461 hypertension patients) defined as dippers or nondippers by ambulatory BP monitoring were recruited for this study. Demographics and clinical correlates were collected, including body mass index, estimated glomerular filtration rate (eGFR) and other measures. Sleep quality was measured using the Pittsburgh Sleep Quality Index (PSQI). RESULTS: A total of 185 (58.9%) patients with normal BP and 341 (74.0%) hypertensive patients had a nondipper BP pattern. The hypertension group had a higher prevalence of the nondipper BP pattern, smoking, alcohol intake and diabetes mellitus (DM) and lower eGFR levels and poorer sleep quality than the normal BP group. Patients with the nondipper BP pattern had lower haemoglobin, worse renal function and poorer sleep quality when compared with hypertensive CKD patients with the dipping BP pattern. PSQI scores were significantly associated with the rate of nocturnal BP decline (P < 0.05) in the hypertension group but not in the normal BP group. Poor sleep quality was an independent factor affecting BP pattern in hypertensive CKD patients using multivariate linear and logistic regression analyses. There was no association between sleep quality and hypertension in CKD patients after multivariate logistic regression analyses. CONCLUSION: Poor sleep quality, which is commonly observed in pre-dialysis CKD patients, is an independent associated factor of the nondipper BP pattern in hypertensive CKD patients. No association was found between poor sleep and nondipper BP in normotensive patients.

Correlation between Serum Lipid Levels and Measured Glomerular Filtration Rate in Chinese Patients with Chronic Kidney Disease.

INTRODUCTION: Dyslipidemia is often detected in patients with chronic kidney disease (CKD). Previous studies of the relationship between lipid profiles and kidney function have yielded variable results. We aimed to investigate the correlation between serum lipid levels and kidney function evaluated by measured glomerular filtration rate (mGFR) in Chinese patients with CKD. METHODS: A cross-sectional study was conducted on 2036 Chinese CKD patients who had mGFR. Linear regression analysis was performed to evaluate the correlation between different serum lipid levels and mGFR, while logistic regression analysis was used to investigate the association between CKD stages and the risk of different types of dyslipidemia. RESULTS: The mean age was 55 years and the mean mGFR was 63 mL/min/1.73m2. After adjusting for some confounders (age, gender, body mass index, a history of diabetes, fasting glucose, a history of hypertension, systolic blood pressure, diastolic blood pressure, smoking status, hemoglobin, serum potassium, serum albumin, and serum uric acid), serum triglyceride level showed a negative correlation with mGFR (ß = -0.006, P = 0.006) in linear regression analysis, and CKD stages were positively related to the risk of hypertriglyceridemia (odds ratios were 1.329, 1.868, 2.514 and P were 0.046, < 0.001, < 0.001 for CKD stage 2, 3, 4/5, respectively) in logistic regression anlysis. CONCLUSIONS: Serum triglyceride level is independently association with mGFR. Patients with reduced kidney function are more likely to have higher serum triglyceride levels. Further longitudinal, multicenter and well-conducted studies are needed to provide more evidence.

The ambulatory arterial stiffness index and target-organ damage in Chinese patients with chronic kidney disease.

BACKGROUND: The ambulatory arterial stiffness index (AASI) can be used to predict cardiovascular morbidity and mortality in hypertensive patients. However, data on AASI in Chinese patients with chronic kidney disease (CKD) is not available. METHODS: This cross-sectional study enrolled 583 CKD patients. Univariate and multivariate analyses were used to evaluate the relationship between AASI and renal function and parameters of cardiovascular injury. RESULTS: Patients with a higher AASI had a higher systolic blood pressure, a lower estimated glomerular filtration rate (eGFR), a higher serum cystatin C, a higher left ventricular mass index (LVMI) and carotid intima-media thickness (cIMT). Univariate analyses showed that AASI was positively correlated with serum cystatin C (r=0.296, P < 0.001), serum creatinine (r=0.182, P < 0.001), and LVMI (r = 0.205, P < 0.001) and negatively correlated with the eGFR (r = -0.200, P < 0.001). Multivariate analyses revealed that serum cystatin C, eGFR, serum creatinine and LVMI were independently correlated with AASI. CONCLUSIONS: These data suggest that AASI was closely correlated with renal function and parameters of cardiovascular injury in Chinese CKD patients. Good quality, long-term, large longitudinal trials to validate the role of AASI in clinical practice for Chinese CKD patients.

Serum immunoglobulin A/C3 ratio predicts progression of immunoglobulin A nephropathy.

AIM: The serum immunoglobulin A (IgA)/C3 ratio has been shown to be a good predictor of histological lesions and prognosis for patients with IgA nephropathy (IgAN) in Japanese. But its validity in the Chinese population is unclear. We sought to explore the long-term outcomes of IgAN, its clinical and histopathological predictors in Chinese patients. In particular, the role of serum IgA/C3 ratio in the course of IgAN was addressed. METHODS: A total of 217 biopsy-diagnosed IgAN patients were recruited into this prospective cohort with a mean follow-up of 36 months (25-75th percentile, 27-48). Sociodemographics, serum IgA/C3 level, other clinical examinations and Lee's histological grade were measured. The patients with a decline of estimated glomerular filtration rate (eGFR) > 50% or developing end-stage renal disease (ESRD) were defined as progression. RESULTS: A total of 21 patients was found to progress (9.7%). In multivariate analysis, renal end point of IgAN was significantly predicted by proteinuria ≥1 g/day (relative risk (RR) = 2.65, 95% confidence interval (CI) 1.01-7.68), hypertension (RR = 3.15, 95% CI 1.07-9.29), higher Lee's histological grade (RR = 4.67, 95% CI 1.43-15.25) and serum IgA/C3 ratio ≥ 3.32 (RR = 4.31, 95% CI 1.33-13.96). CONCLUSION: A proportion of patients with IgAN developed end stage renal disease in a Chinese group. In addition to some traditional risk factors, we also confirmed that IgA/C3 ratio is a useful predictor of poor outcomes of IgAN in Chinese patients.

Advanced glycation end-products activate the renin-angiotensin system through the RAGE/PI3-K signaling pathway in podocytes.

PURPOSE: The purpose of this study was to investigate the effects of advanced glycation end-products (AGEs) on the components of the renin-angiotensin system (RAS) in podocytes and to understand the mechanism of these effects. METHODS: Immortalized mouse podocytes were exposed to various concentrations of AGEs for different time intervals. The expression levels of angiotensinogen (AGT), angiotensin II type 1 and 2 receptors (AT1R and AT2R) and renin were examined by real-time PCR and western blot; the receptor for AGEs (RAGE) and both Akt and phosphorylated Akt were examined by western blot; levels of angiotensin II (Ang II) were assayed by ELISA, and the activity of angiotensin-converting enzyme (ACE) was evaluated by measuring the production of hippuric acid in vitro. RESULTS: Treatment with AGEs resulted in significant increases in the expression of AGT (62%, P=0.002) and AT1R (59%, P=0.01). Moreover, Ang II levels increased significantly in both cell lysates (70%, P=0.018) and conditioned media (65%, P=0.01). ACE activity was also significantly higher in cell lysates (68% , P= 0.035) and conditioned media (65%, P=0.023). There were no changes in renin or AT2R expression (P > 0.05). AGEs did increase the expression of RAGE by 50% (P=0.012) and the phosphorylation of Akt by 100% (P=0.001). When podocytes were pretreated with anti-RAGE antibody (50 µg/ml) or the phosphoinositide 3-kinase (PI3-K) inhibitor, LY294002 (10 µM), the AGEs-induced increases in AGT and AT1R expression were reduced. Likewise, Ang II levels and ACE activity decreased significantly. CONCLUSION: AGEs activate the RAS in podocytes through the RAGE-PI3-K/Akt-dependent pathway and lead to an increase in podocyte apoptosis.

Mesangial medium from IgA nephropathy patients induces podocyte epithelial-to-mesenchymal transition through activation of the phosphatidyl inositol-3-kinase/Akt signaling pathway.

BACKGROUND: Podocyte injury plays an important role in glomerulosclerosis in IgA nephropathy (IgAN). Eepithelial-to-mesenchymal transition (EMT) caused by different factors is the main reason for podocyte damage. This study hypothesized that conditioned mesangial medium may induced EMT process of podocytes and thereby lead to glomerular injury or sclerosis. MATERIALS AND METHODS: Podocytes were incubated in medium from mesangial cells incubated with aggregated IgA1(aIgA1) isolated from IgAN patients. Wortmannin were used to inhibit phosphatidylinositol-3-kinase (PI3-K) in podocytes. RESULTS: Western blot analysis, real-time PCR and confocal fluorescent microscopy demonstrated that reduced expression of P-Cadherin, Zonula occludens-1 (ZO-1) and podocin, increased expression of fibroblast -specific protein (FSP-1), α-smooth muscle action(α-SMA) and desmin in podocytes exposed to medium from mesangial cells incubated with aIgA1 isolated from IgAN patients compared with podocytes cultured in RPMI 1640 medium containing 0.5% fetal bovine serum ( FBS) (p<0.05). Mesangial medium resulted in a greater albumin influx across the podocyte monolayer (p<0.05). Phosphorylation of Akt increased with this medium, as indicated by an increase in the p-Akt/Akt ratio. Treatment with wortmannin partly restored the changes in epithelial and mesenchymal markers and albumin influx. IgAN patients with massive proteinuria showed remarkable α-SMA and FSP-1 expression in podocytes. CONCLUSION: Our findings indicated that mesangial medium from cells incubated with aIgA1 isolated from IgAN patients induced EMT in podocytes and the PI3-K/ Akt-signaling pathway was involved in the process.

How increased VEGF induces glomerular hyperpermeability: a potential signaling pathway of Rac1 activation.

Despite growing evidence for a pathogenic role of vascular endothelial growth factor (VEGF) in microvascular complications of diabetes, the underlying mechanism responsible for its detrimental effect remains unknown. In the current study, we hypothesized that some of the detrimental effects of VEGF on microvascular endothelial cells in the diabetic milieu stem from its aberrant signaling, which leads to perturbed tight junction assembly and increased endothelial permeability. Using an integrated in vitro approach, we investigated whether the effect of VEGF on endothelial cell permeability involves Rac1 GTPase activation and tight junction disassembly. Rac1 activity was detected by Western blotting in cell membrane protein as well as pull-down assay. The permeability of glomerular endothelial cells monolayer was detected as transendothelial electronic resistance. Then tyrosine phosphorylated occludin protein was detected by Western blotting after immunoprecipitation. N17Rac1 cells are obtained by transfection of glomerular endothelial cells with a dominant negative mutant of Rac1. The data obtained in this study indicate that activation of Rac1 GTPase contributes to VEGF-induced endothelial cell hyperpermeability. We also observed that Rac1 activation leads to increased endothelial permeability through tyrosine phosphorylation of occludin. Indeed, N17Rac1 cells dramatically attenuated the effect of VEGF on phospho-occludin and endothelial cell permeability. These results, when taken together, provide a framework for understanding the role of VEGF-induced Rac1/phospho-occludin pathway in the integrity of endothelial barrier function in the glomerulus.

Serum IgA1 from patients with IgA nephropathy up-regulates integrin-linked kinase synthesis and inhibits adhesive capacity in podocytes through indirect pathways.

PURPOSE: To investigate the influence of IgA1 isolated from IgA nephropathy (IgAN) patients on integrin-linked kinase (ILK) synthesis and adhesive capacity of podocytes through indirect pathways. METHODS: IgA1 was isolated from healthy control or IgAN patients' sera using jacalin affinity chromatography and S-200 chromatography. Podocytes were treated with medium from mesangial cells incubated with aggregated IgA1 (aIgA1, 100 microg/ml), in the presence or absence of valsartan (10(-5)M) or neutralizing antibodies of tumor necrosis factor-alpha (TNF-alpha, 50 ng/ml). Adhesive capacity of podocytes was assessed by cell counting manually and hexosaminidase assay. Real-time PCR and western blotting were used to detect the expression of ILK. RESULTS: Medium from mesangial cells incubated with aIgA1 from IgAN patients reduced podocyte adhesion to collagen compared with medium from mesangial cells incubated with control medium(RPMI-1640 with 0.5% FBS) (35.0+/-4.8% vs. 60.0+/-2.0%; P < 0.05). While medium from mesangial cells incubated with aIgA1 from IgAN patients upregulated ILK expression in podocytes at mRNA and protein levels compared with medium from mesangial cells without aIgA1 incubated (1.6-fold and 1.38-fold higher than control, respectively, P < 0.05). Defects in podocyte adhesion and up-regulation of ILK synthesis induced by medium from mesangial cells incubated with aIgA1 from IgAN patients can be partially reversed by the pre-treatment for 1 hour with valsartan(P < 0.05), while pre-treatment with neutralizing antibodies of TNF-alpha produced no protective effect on podocytes (P > 0.05). CONCLUSION: Serum IgA1 from IgAN patients may inhibit adhesive capacity and up-regulate ILK synthesis in podocytes through indirect pathways.