Metabolome-wide Mendelian randomization reveals causal effects of betaine and N-acetylornithine on impairment of renal function.

Background: Chronic kidney disease (CKD) is a common public health problem, which is characterized as impairment of renal function. The associations between blood metabolites and renal function remained unclear. This study aimed to assess the causal effect of various circulation metabolites on renal function based on metabolomics. Methods: We performed a two-sample Mendelian randomization (MR) analysis to estimate the causality of genetically determined metabolites on renal function. A genome-wide association study (GWAS) of 486 metabolites was used as the exposure, while summary-level data for creatinine-based estimated glomerular filtration rate (eGFR) or CKD occurrence were set the outcomes. Inverse variance weighted (IVW) was used for primary causality analysis and other methods including weight median, MR-egger, and MR-PRESSO were applied as complementary analysis. Cochran Q test, MR-Egger intercept test, MR-PRESSO global test and leave-one-out analysis were used for sensitivity analysis. For the identified metabolites, reverse MR analysis, linkage disequilibrium score (LDSC) regression and multivariable MR (MVMR) analysis were performed for further evaluation. The causality of the identified metabolites on renal function was further validated using GWAS data for cystatin-C-based eGFR. All statistical analyses were performed in R software. Results: In this MR analysis, a total of 44 suggestive associations corresponding to 34 known metabolites were observed. After complementary analysis and sensitivity analysis, robust causative associations between two metabolites (betaine and N-acetylornithine) and renal function were identified. Reverse MR analysis showed no causal effects of renal function on betaine and N-acetylornithine. MVMR analysis revealed that genetically predicted betaine and N-acetylornithine could directly influence independently of each other. The causal effects of betaine and N-acetylornithine were also found on cystatin-C-based eGFR. Conclusion: Our study provided evidence to support the causal effects of betaine and N-acetylornithine on renal function. These findings required further investigations to conduct mechanism exploration and drug target selection of these identified metabolites.

The correlation between serum growth differentiation factor 15 and malnutrition in patients with end-stage kidney disease on hemodialysis.

OBJECTIVE: Malnutrition commonly occurs in patients undergoing maintenance hemodialysis. Early detection of malnutrition could allow early interventions to prevent later complications. At present, there are not many biomarkers with high predictive value of end-stage kidney disease (ESKD)-related malnutrition, especially for early malnutrition in hemodialysis patients, which needs more in-depth research. Therefore, we performed a cross-sectional study on 97 patients to identify biomarkers for malnutrition in hemodialysis patients. RESEARCH METHODS & PROCEDURES: 7-point subjective global assessment (SGA) was applied to evaluate the nutritional status of patients on hemodialysis. Serum levels of growth differentiation factor 15 (GDF15), albumin, pre-albumin, c-reactive protein (CRP), tumor necrosis factor alpha (TNF-α), hemoglobin, low density lipoprotein-cholesterol, and high density lipoprotein-cholesterol were detected before hemodialysis. Logistic analysis and linear regression were used to analyze the association between GDF15 levels and the SGA score after adjustment for basic characteristics and laboratory findings. RESULTS: Among the 97 patients on hemodialysis, 51 had malnutrition (SGA < 6). There was no difference between the malnourished and well nourished (SGA ≥ 6) groups for dialysis duration, cholesterol, CRP, TNF-α, and hemoglobin. The malnutrition group had significantly lower grip strength (p < 0.05). GDF15 levels correlated negatively with the SGA score after adjustment for possible confounding factors [rho (male) = -0.312, rho(female)= -0.437;P(male) = 0.0181, P(female) = 0.005], and might contribute to the malnutritional status, the AUCs of GDF15 for malnutrition was 0.697 (p = 0.011) in male and 0.828 (p < 0.001) in female. CONCLUSIONS: GDF15 is associated with malnutrition according to the SGA score in patients with ESKD on hemodialysis, suggesting that GDF15 might be involved in the pathogenesis of malnutrition patients with ESKD in this setting. Furthermore, GDF15 is likely to be a potential diagnostic biomarker for malnutrition according to the SGA score.

Gut microbiota dysbiosis and protein energy wasting in patients on hemodialysis: an observational longitudinal study.

Background: Protein energy wasting (PEW) is common in patients on hemodialysis, and its development may involve gut microbial dysbiosis. However, the exact relationship between the composition of different flora and the development of PEW remains unclear. Methods: This is an observational longitudinal study on 115 patients undergoing hemodialysis who were followed up for 1 year. All the patients were evaluated at baseline, and different microbiota compositions were determined. After a 1 year follow-up period, the correlations between clinical parameter variations and the relative abundance of different gut flora were assessed using Spearman correlation. Moreover, the associations of the abundance of different gut microbiota with decrease in lean tissue mass and the development of PEW were analyzed using ROC curve and logistical regression analyses. Results: We found that the relative abundances of Actinobacteria and Bifidobacteriaceae were significantly lower in patients with PEW than in those who did not develop PEW (p < 0.05). The abundance of Actinobacteria and Bifidobacteriaceae correlated positively with variations in serum albumin levels (r = 0.213, p = 0.035 and r = 0.214, p = 0.034, respectively), lean tissue mass (r = 0.296, p = 0.007 and r = 0.238, p = 0.002, respectively), and lean tissue index (r = 0.377, p < 0.001 and r = 0.419, p < 0.001, respectively). The area under the ROC curve or AUC values of Actinobacteria and Bifidobacteriaceae for the prediction of lean tissue mass decrease ranged from 0.676 to 0.708 (p < 0.05). Thus, decrease in the abundance of Actinobacteria and Bifidobacteriaceae may be associated with decrease in lean tissue mass and the occurrence of PEW. Conclusion: The present findings imply Actinobacteria and Bifidobacteriaceae may be potential markers for predicting skeletal muscle mass decrease and PEW development in patients on hemodialysis.

Autophagosome protects proximal tubular cells from aldosterone-induced senescence through improving oxidative stress.

Aldosterone exerts an enormous function on proximal tubular cells (PTC) senescence, which is a common pathomechanism contributing to renal dysfunction. Numerous studies have shown that oxidative stress is deeply involved in the pathophysiologic processes of chronic kidney diseases. The study aims to investigate whether autophagy could regulate the process of senescence through oxidative stress in PTC both in vivo and ex vivo. Our results suggested that aldosterone treatment increased the senescence and oxidative stress as evidenced by increased percent of SA-ß-Gal positive cells, reactive oxygen species level, expression of NADPH oxidase 4 (NOX4) rather than NOX2, and the up-regulation of p21 in cultured PTC. Furthermore, the alternation of the expression of p62 and LC3-II/LC3-I demonstrated that aldosterone treatment remarkably influenced autophagic flux. NOX4 siRNA treatment or autophagy induction with rapamycin reduced the oxidative stress and senescence in aldosterone-induced PTC. On the contrary, inhibition of autophagy with chloroquine worsened these changes. Similar results were further confirmed in vivo. Our results suggested that autophagy may become a realistic therapeutic strategy against aldosterone-induced PTC injury via improving oxidative stress.

Curcumin attenuates angiotensin II-induced podocyte injury and apoptosis by inhibiting endoplasmic reticulum stress.

Podocytes are an important component of the glomerular filtration barrier in the kidneys. The dysfunction and apoptosis of podocytes are important factors that can lead to the progression of chronic kidney disease (CKD). In CKD, angiotensin II is continuously elevated in circulation and is considered to have key roles in inducing podocyte injury and apoptosis. Curcumin is a hydrophobic polyphenolic compound extracted from turmeric. Increasing evidence demonstrates that curcumin has a protective effect on the kidneys in CKD. However, the mechanisms mediating this protective effect remain unclear. The aim of this study was to explore whether curcumin could protect against angiotensin II-induced injury and apoptosis of podocytes. We performed western blotting, immunofluorescence, phalloidin staining, and terminal deoxynucleotidyl transferase nick-end labeling staining to observe the expression level of podocyte-specific proteins, apoptosis-related proteins, and the arrangement of F-actin. We found that curcumin could reverse angiotensin II-induced podocyte injury and apoptosis in a dose-dependent manner. In addition, curcumin dose-dependently attenuated a pro-apoptotic pathway, activated by angiotensin II-induced endoplasmic reticulum stress. Conversely, the protective effects of curcumin were impaired upon addition of tunicamycin, an activator of endoplasmic reticulum stress. Thus, we speculate that curcumin protects against angiotensin II-induced podocyte injury and apoptosis, at least partly by inhibiting endoplasmic reticulum stress.

The Chronic Adverse Effect of Chloroquine on Kidney in Rats through an Autophagy Dependent and Independent Pathways.

BACKGROUND: Chloroquine (CQ), a classic autophagy inhibitor, is used clinically for malaria prophylaxis and pulmonary hypertension treatment. The adverse effects of CQ on morphological and functional changes in the kidney were investigated in the current study due to CQ accumulation in the kidney. METHODS: Twelve male Sprague-Dawley rats were randomly divided into 2 groups for 4 weeks: group 1, control (n = 6); and group 2, CQ administration group (50 mg-1·kg per day ip; n = 6). Serum aldosterone and vasopressin were measured by radioimmunoassay. Immunofluorescence was used to colocalize Tunel with aquaporin 1, aquaporin 2 (AQP2), and Tamm-Horsfall protein. Expression of AQP2 and mineralocorticoid (MR) was detected by western blot and immunohistochemistry. RESULTS: In the present study, 4 weeks of CQ administration were shown to induce severe kidney injury, including glomerular sclerosis and tubular cells apoptosis, especially distal tubular cells. Decreased expression of LC3II/I and p-AKT was demonstrated in CQ-treated rats. Glomerular and proximal tubule injury were associated with impaired autophagy flux, and distal tubule injury may be associated with downregulated cyclic adenosine monophosphate (cAMP)/PKA/AKT signaling. Both MR and AQP2, which are mainly located in the distal tubule and collecting duct, were significantly reduced in CQ-treated rats, thus leading to increased exosomal secretion of AQP2 in urine. Additionally, chronic CQ administration increased aldosterone and vasopressin levels in serum, but lowered the blood pressure, glomerular filtration rate, and urine concentration. CONCLUSIONS: CQ administration damages glomerular, proximal tubule autophagy, and severe distal tubular cells apoptosis by inhibiting cAMP/PKA/AKT signaling.

Epidermal growth factor receptor mimotope alleviates renal fibrosis in murine unilateral ureteral obstruction model.

Macrophages have been recognized as a vital factor that can promote renal fibrosis. Previously we reported that the EGFR mimotope could alleviate the macrophage infiltration in the Sjögren's syndrome-like animal model. In current study, we sought to observe whether the active immunization induced by the EGFR mimotope could ameliorate renal fibrosis in the murine Unilateral Ureteral Obstruction (UUO) model. A series of experiments showed the EGFR mimotope immunization could ameliorate renal fibrosis, reduce the expressions of fibronectin, α-SMA and collagen I and alleviate the infiltrations of F4/80+ macrophages in UUO model. Meanwhile, the EGFR mimotope immunization could inhibit the EGFR downstream signaling. Additionally, the frequency of and F4/80+CD9+/FAS+ macrophages significantly increased in spleen after the EGFR mimotope immunization. These evidence suggested that the EGFR mimotope could alleviate renal fibrosis by both inhibiting EGFR signaling and promoting macrophages apoptosis.

Ghrelin attenuates UUO-induced renal fibrosis via attenuation of Nlrp3 inflammasome and endoplasmic reticulum stress.

Background/aims: All chronic kidney disease (CKD) can eventually develop into renal fibrosis. We explored the renoprotective effects of a gastric peptide, ghrelin, and investigated whether endoplasmic reticulum stress (ERS) and the NLR family pyrin domain-containing 3 (NLRP3) inflammasome mediate the protective effect of ghrelin in unilateral ureteral obstruction (UUO). METHODS: Male C57BL/6J mice were divided into vehicle- or ghrelin-treated sham-operated groups and vehicle- or ghrelin-treated UUO groups. The kidneys were harvested on postoperative day 14. Renal fibrosis was evaluated by periodic acid-Schiff, Masson trichrome, and immunohistochemical (IHC) staining. To assess renal fibrosis, α-smooth muscle actin and type I collagen were detected. NLRP3 inflammasome and ERS activation were also detected via western blotting. The effect of ghrelin on cultured renal cells was further confirmed in HK-2 cells. RESULTS: Compared with the sham mice, UUO mice developed obvious renal fibrosis; pathological and IHC staining showed increased matrix accumulation and elevated ERS, NLRP3 inflammasome was activated both in vivo and in vitro. Ghrelin significantly attenuated collagen fibril accumulation and apoptosis by reducing NLRP3 inflammasome activation and ERS in obstructed kidneys. CONCLUSIONS: Ghrelin may attenuate UUO-induced renal fibrosis by inhibiting the NLRP3 inflammasome and ERS in vivo. Therefore, ghrelin might be an effective strategy for preventing CKD.

1,25-Dihydroxyvitamin D3 Attenuates Angiotensin II-Induced Renal Injury by Inhibiting Mitochondrial Dysfunction and Autophagy.

BACKGROUND/AIMS: A recent study has shown that 1,25-dihydroxyvitamin D3 (1,25-D3), the active form of vitamin D, can ameliorate renal dysfunction. In this study, we aimed to determine the role of 1,25-D3 in angiotensin (Ang II)-induced renal injury and investigate the underlying mechanisms involved. METHODS: C57BL/6J mice were treated with Ang II and/or 1,25-D3 (or saline as the control) for 2 weeks. Renal injury was evaluated using transmission electron microscopy and periodic acid-Schiff reagent and Masson's trichrome staining. The pro-fibrotic and pro-inflammatory factors were assessed using real-time PCR. The renal apoptotic pathway was evaluated with TUNEL staining and western blot. Mitochondrial dysfunction (MtD) was determined using real-time PCR and electron microscopy. The activation of autophagy was detected using western blot. RESULTS: In the Ang II-infused mice, expanded mesangial regions, tubulointerstitial fibrosis, and foot process fusion were observed; the levels of the pro-fibrotic and pro-inflammatory cytokines and MtD were also increased when compared with the control group. However, we found that administration of 1,25-D3 significantly improved renal function and MtD and reduced the pro-fibrotic and pro-inflammatory cytokine levels. Furthermore, 1,25-D3 significantly inhibited Ang II-induced autophagy dysfunction (determined by inhibition of Beclin-1 activation and reduction of the LC3-II/LC3-I ratio). CONCLUSION: Our findings suggest that 1,25-D3 may attenuate Ang II-induced renal injury by improving MtD and modulating autophagy. 1,25-D3 may be a new therapeutic for the treatment of CKD.

Glucagon-like peptide-1 analog prevents obesity-related glomerulopathy by inhibiting excessive autophagy in podocytes.

To investigate the role of glucagon-like peptide-1 analog (GLP-1) in high-fat diet-induced obesity-related glomerulopathy (ORG). Male C57BL/6 mice fed a high-fat diet for 12 wk were treated with GLP-1 (200 µg/kg) or 0.9% saline for 4 wk. Fasting blood glucose and insulin and the expression of podocin, nephrin, phosphoinositide 3-kinase (PI3K), glucose transporter type (Glut4), and microtubule-associated protein 1A/1B-light chain 3 (LC3) were assayed. Glomerular morphology and podocyte foot structure were evaluated by periodic acid-Schiff staining and electron microscopy. Podocytes were treated with 150 nM GLP-1 and incubated with 400 µM palmitic acid (PA) for 12 h. The effect on autophagy was assessed by podocyte-specific Glut4 siRNA. Insulin resistance and autophagy were assayed by immunofluorescence and Western blotting. The high-fat diet resulted in weight gain, ectopic glomerular lipid accumulation, increased insulin resistance, and fusion of podophyte foot processes. The decreased translocation of Glut4 to the plasma membrane and excess autophagy seen in mice fed a high-fat diet and in PA-treated cultured podocytes were attenuated by GLP-1. Podocyte-specific Glut4 siRNA promoted autophagy, and rapamycin-enhanced autophagy worsened the podocyte injury caused by PA. Excess autophagy in podocytes was induced by inhibition of Glut4 translocation to the plasma membrane and was involved in the pathology of ORG. GLP-1 restored insulin sensitivity and ameliorated renal injury by decreasing the level of autophagy.

Protective Effects of Glucagon-Like Peptide-1 Analog on Renal Tubular Injury in Mice on High-Fat Diet.

AIMS: The study aimed to investigate the renoprotective effect of glucagon-like peptide-1 (GLP-1) against renal tubular injury in C57BL/6 mice induced by a high-fat diet (HFD). METHODS: Twenty C57BL/6 mice were fed HFD for 12 weeks. Ten of these mice were treated with GLP-1 at 200 µg/kg subcutaneously twice daily for 4 weeks (HFDG group), and the other ten mice received vehicle only (HFD group). Ten mice fed standard rodent chow served as controls (Con group). Body weight, kidney weight, food intake, and systolic blood pressure were measured. The expression of endoplasmic reticulum stress (ERS) markers (BIP, p-eIF2α, ATF4, and CHOP) and apoptosis in the kidney were examined utilizing western blotting, immunohistochemistry and TUNEL, respectively. Angiotensin II and angiotensin II type 1 receptor (AT1R) were examined by ELISA. Human proximal tubule epithelial cells (HK2) were treated with GLP-1(150 nM) followed by treatment with palmitic acid (500 nM [PA]) for 24 h. HK2 cells treated with BSA were used as controls. The protein levels of ERS markers, apoptosis-associated protein, and AT1R were measured by western blotting. RESULTS: Increase of body weight, food intake, and systolic blood pressure was less pronounced in GLP-1 treated HFDG mice compared to HFD mice. The levels of ERS markers (BIP, p-eIF2α, ATF4, and CHOP) and apoptosis decreased following GLP-1 treatment in vivo and in vitro (p<0.05). Increased AT1R induced by HFD and PA were blocked with GLP-1 treatment. In contrast, the level of angiotensin II after GLP-1 treatment was not significantly different between the HFD and HFDG mice. CONCLUSION: The study indicated that saturated fatty acids induced ERS and apoptosis in the kidney and increased AT1R expression. GLP-1 treatment exerted renoprotective effects against saturated fatty acid-induced kidney tubular cell ERS and apoptosis together with inhibition of AT1R expression in vivo and in vitro.

Performance of the creatinine and cystatin C-based equations for estimation of GFR in Chinese patients with chronic kidney disease.

BACKGROUND: Currently, creatinine- or cystatin C-based glomerular filtration rate (GFR) estimation equation has been recommended to assess GFR in CKD patients. However, it is still obscure whether those equations performed consistently outstandingly in Chinese population. METHODS: The equations were validated in a population totaling 632 participants (mean age 61.6 ± 12.3 years). The estimated GFR (eGFR) calculated separately by six equations (C-MDRD, Ccys, Cscr-cys, CKD-EPIscr, CKD-EPIcys, and CKD-EPIscr-cys equations) was compared with the reference GFR (rGFR) measured by the 99mTc-DTPA renal dynamic imaging method. Participants were divided into age and rGFR specific subgroups. RESULTS: CKD-EPIscr-cys equation had a larger area under receiver operating characteristic curve (ROCAUC) and relative higher sensitivity (79.8 %) and specificity (93 %) to diagnose CKD. CKD-EPIscr-cys and CKD-EPIcys equations appeared to be more accurate with higher proportion of eGFR within 30 % of rGFR (P 30) value. Those two equations performed as well in older people as in the younger population. The CKD-EPIscr-cys equation acquired the highest P 30 (80.9 %) in subgroups with rGFR ≥60 mL/min/1.73 m2, while the CKD-EPIcys equation yielded the best performance in the rGFR <60 mL/min/1.73 m2 subgroup. CONCLUSION: CKD-EPIscr-cys formula had better capability to accurately evaluate GFR in the participants CKD stages 1-2 in Chinese ethnic. The application of the cystatin C-based equations may be the optimal one for patients of moderately to severely injured GFR. Considering the accuracy in the entire range of participants less ideally, the additional of the Chinese racial factor is assumed to be essential.

Endoplasmic Reticulum Chaperon Tauroursodeoxycholic Acid Attenuates Aldosterone-Infused Renal Injury.

Aldosterone (Aldo) is critically involved in the development of renal injury via the production of reactive oxygen species and inflammation. Endoplasmic reticulum (ER) stress is also evoked in Aldo-induced renal injury. In the present study, we investigated the role of ER stress in inflammation-mediated renal injury in Aldo-infused mice. C57BL/6J mice were randomized to receive treatment for 4 weeks as follows: vehicle infusion, Aldo infusion, vehicle infusion plus tauroursodeoxycholic acid (TUDCA), and Aldo infusion plus TUDCA. The effect of TUDCA on the Aldo-infused inflammatory response and renal injury was investigated using periodic acid-Schiff staining, real-time PCR, Western blot, and ELISA. We demonstrate that Aldo leads to impaired renal function and inhibition of ER stress via TUDCA attenuates renal fibrosis. This was indicated by decreased collagen I, collagen IV, fibronectin, and TGF-ß expression, as well as the downregulation of the expression of Nlrp3 inflammasome markers, Nlrp3, ASC, IL-1ß, and IL-18. This paper presents an important role for ER stress on the renal inflammatory response to Aldo. Additionally, the inhibition of ER stress by TUDCA negatively regulates the levels of these inflammatory molecules in the context of Aldo.