Ling Nie

Klotho Protects Against Indoxyl Sulphate-Induced Myocardial Hypertrophy

Left ventricular hypertrophy (LVH) is a common complication in patients with CKD and an independent risk factor for death. Changes in the levels of uremic solutes or Klotho have been reported to be related to CKD, whereas the relationships between these factors and CKD-associated LVH remain unclear. Here, we investigated the interaction between Klotho and indoxyl sulfate (IS), a typical uremic solute, in CKD-associated LVH. In a survey of 86 patients with CKD, a negative relationship was found between serum levels of IS and Klotho (r=-0.59, P<0.001). Furthermore, serum levels of IS and Klotho were independently associated with LVH (for IS: r=0.69, P<0.001; for Klotho: r=-0.49, P<0.001). In normal mice, intraperitoneal injection of IS for 8 weeks induced LVH accompanied by substantial downregulation of renal Klotho. Notably, IS-induced LVH was more severe in heterozygous Klotho-deficient (kl/+) mice. In vitro, treatment with Klotho strongly inhibited IS-induced cardiomyocyte hypertrophy by blocking oxidative stress and inhibiting p38 and extracellular signal-regulated protein kinase 1/2 signaling pathways. In a mouse model of CKD-associated LVH, the renal expression of Klotho was lower and the level of serum IS was higher than in healthy controls. Moreover, treatment of CKD mice with Klotho protein significantly restrained the development of LVH. Taken together, these results suggest that Klotho is an endogenous protector against IS-induced LVH, and the imbalance between Klotho and IS may contribute to the development of LVH in CKD.

Klotho suppresses renal tubulo-interstitial fibrosis by controlling basic fibroblast growth factor-2 signalling.

Increased basic fibroblast growth factor‐2 (FGF2) and reduced Klotho have both been reported to be closely associated with renal fibrosis. However, the relationship between Klotho and FGF2 remains unclear. We demonstrate that FGF2 induced tubulo‐epithelial plasticity in cultured HK‐2 cells, accompanied by a reduction in Klotho expression, whereas recombinant Klotho protein could inhibit the action of FGF2. The FGF2 effects required extracellular signal‐regulated protein kinase 1/2 activation, which was suppressed by Klotho. Moreover, Klotho also restrained FGF2‐induced fibroblast proliferation and activation. The inhibitory effect of Klotho on the activity of FGF2 was likely due to its potent ability to compete with FGF2 binding to FGF receptor 1. Unilateral ureteral obstruction (UUO)‐induced renal fibrosis was associated with an increase in FGF2 and a reduction in Klotho expression in wild‐type mice, whereas FGF2–/– mice largely preserved Klotho expression and developed only mild renal fibrosis after obstructive injury. Furthermore, administration of Klotho protein in UUO mice significantly reduced renal fibrosis, concomitant with a marked suppression of FGF2 production and signalling. These studies demonstrate a feedback loop between Klotho depletion and FGF2 activation in renal fibrosis. Our results also suggest that Klotho treatment reduces renal fibrosis, at least in part, by inhibiting FGF2 signalling. Copyright

Amelioration of uremic toxin indoxyl sulfate-induced endothelial cell dysfunction by Klotho protein.

Indoxyl sulfate (IS), a common kind of uremic toxin, is considered as a risk factor for aggravating endothelial function in CKD patients due to its oxidative activity. The anti-aging protein Klotho, which is produced by the kidneys and down-regulated in uremic conditions, has the ability to resist oxidative stress. Here, we carried out an in vitro study to investigate the deleterious effects of IS on endothelial cells and the protective role of Klotho protein. The cultured human umbilical vein endothelial cells (HUVECs) were incubated with IS in the presence or absence of Klotho protein. The release of reactive oxygen species (ROS) and the expression of monocyte chemoattractant protein-1 (MCP-1) were enhanced while the cell viability and production of nitric oxide (NO) were inhibited by IS in a concentration-dependent manner. Meanwhile, the phosphorylation of p38MAPK and the nuclear translocation of NF-κB were increased in HUVECs treated with IS. Pretreatment with Klotho protein resulted in remarkable increase of cell viability and decrease of ROS production in IS-treated HUVECs. Like ROS scavenger, N-acetyl-l-cysteine (NAC), Klotho protein could inhibit the IS-induced activations of p38MAPK and NF-κB. Moreover, Klotho protein could also attenuate IS-induced reduction of NO production and up-regulation of MCP-1 expression. These results suggest that IS can damage the functions of endothelial cells. Klotho protein has the ability to ameliorate the IS-induced endothelial dysfunction, which may be partly through inhibiting the ROS/p38MAPK and downstream NF-κB signaling pathways.

Indoxyl sulfate induces oxidative stress and hypertrophy in cardiomyocytes by inhibiting the AMPK/UCP2 signaling pathway.

As a typical protein-bound uremic toxin, indoxyl sulfate is considered to be able to induce cardiomyocytes hypertrophy by promoting oxidative stress in chronic kidney disease (CKD). Uncoupling protein 2 (UCP2), a member of the uncoupling protein family, may protect cardiomyocytes from oxidative stress by suppressing mitochondrial reactive oxygen species (ROS). In the present study, we aimed to determine whether UCP2 was involved in indoxyl sulfate-induced cardiomyocytes hypertrophy. We demonstrated that indoxyl sulfate could increase the ROS levels in a time and dose-dependent manner in cultured neonatal rat cardiomyocytes. Significant increases in [(3)H]-leucine incorporation, cell volume, and the mRNA expression levels of atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and beta myosin heavy chain (β-MHC) were detected in cardiomyocytes after treatement with indoxyl sulfate at the concentration of 500μM for 48h, accompanied by a decreased expression of UCP2. In contrast, cardiomyocytes transfected with the lentiviral vector carrying UCP2 gene were resistant to indoxyl sulfate-induced ROS production and cell hypertrophy. Additionally, indoxyl sulfate-induced UCP2 reduction was correlated with the inhibition of AMP-activated protein kinase (AMPK) activity, while pretreatment with AICAR, an AMPK activator, effectively attenuated indoxyl sulfate-induced UCP2 down-regulation and hypertrophy in cardiomyocytes. Taken together, these results suggest that indoxyl sulfate-induced cardiomyocytes hypertrophy was partly due to the inhibition of AMPK/UCP2 signaling and the enhancement of oxidative stress.

Resveratrol inhibits renal interstitial fibrosis in diabetic nephropathy by regulating AMPK/NOX4/ROS pathway.

Renal interstitial fibrosis is a major pathologic feature of diabetic nephropathy, while the pathogenesis and therapeutic interventions of diabetic renal interstitial fibrosis are not well established. In this study, we first demonstrated that high glucose could induce renal fibroblast (NRK-49F) cell proliferation and activation to myofibroblasts, accompanied by a significant increase in the intracellular levels of reactive oxygen species (ROS) derived from nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4). ROS-mediated ERK1/2 activation was found to play a crucial role in high glucose-induced fibroblast proliferation and activation. Resveratrol, like the NOX4-targeting small interfering RNA (siRNA), markedly inhibited high glucose-induced fibroblast proliferation and activation by reducing NOX4-derived ROS production. It was then revealed that the increase in the expression of NOX4 induced by high glucose was due to the inactivation of AMP-activated protein kinase (AMPK), which could be reversed by resveratrol. Further in vivo investigation demonstrated that resveratrol treatment significantly attenuated renal fibrosis in db/db mice, accompanied by an evident increase in phospho-AMPK and decrease in NOX4. In summary, our results suggest that high glucose can directly promote renal fibroblasts proliferation and activation in a ROS-dependent manner, and resveratrol is a potential therapeutic agent against diabetic renal fibrosis via regulation of AMPK/NOX4/ROS signaling.Key messageResveratrol inhibits high glucose-induced NRK cell activation by decreasing NOX4-derived ROS.Resveratrol inhibits high glucose-induced NOX4 expression in NRK cells via activation of AMPK.ROS-activated ERK1/2 signaling is involved in high glucose-induced NRK cell activation.Resveratrol attenuated renal fibrosis in db/db mice via regulation of AMPK/NOX4/ROS signaling.

Computational prediction and experimental assessment of an HLA-A*0201-restricted cytotoxic T lymphocyte epitope from neutral endopeptidase

Neutral endopeptidase (NEP) is the first target antigen identified on podocytes in human membranous nephropathy (MN). Cytotoxic T lymphocytes (CTLs) are considered essential for glomerular destruction in MN model. The aim of this study was to show that the CTL epitopes of NEP could be used to design more effective and better tolerated therapies. The CTL epitopes of NEP were screened using the long-distance prediction system SYFPEITHI and the Bioinformatics and Molecular Analysis Section of the MHC Peptide Binding Predictions program. Peptides were synthesized and immunoreactivity was assessed by peptide-MHC-binding affinity assay, cytotoxicity assay and HLA-A2.1/Kb transgenic mice immunization. Five candidates were identified according to the high scores generated by the computer predicting system. Peptide NEP375–383 (FIMDLVSSL), which up-regulated HLA-A2.1 molecular expression, showed a high affinity to HLA-A2.1, whereas NEP268–276, NEP297––305 and NEP492–500 (QLALEMNKV, MLLYNKMRL and KLNNEYLEL) showed a moderate affinity and NEP559–567 (ILQPPFFSA) only had a low affinity. Cytotoxicity assay further showed that NEP268–276 and NEP375–383 could induce NEP-specific CTL responses in vitro. Unexpectedly, we found that a single CTL epitope, NEP375–383, could induce proteinuria and glomerular injury in HLA-A2.1/Kb transgenic mice in vivo. HLA-A*0201-restricted CTL epitope NEP375–383 can serve as a potential candidate for designing MN vaccine.

The RIFLE versus AKIN classification for incidence and mortality of acute kidney injury in critical ill patients: A meta-analysis

The sensitivity and accuracy of the Risk/Injury/Failure/Loss/End-stage (RIFLE) versus acute kidney injury Network (AKIN) criteria for acute kidney injury (AKI) in critically ill patients remains uncertain. Therefore, we performed a systematic review and meta-analysis to investigate the incidence and prognostic value of the RIFLE versus AKIN criteria for AKI in critically ill patients. Literatures were identified by searching Medline, Embase, PubMed, and China National Knowledge Infrastructure (CNKI) database. Nineteen studies with 171,889 participants were included. The pooled estimates of relative risk (RR) were analyzed. We found that the RIFLE and AKIN criteria is different for the incidence of AKI in intensive care unit (ICU) patients (P = 0.02, RR = 0.88), while not for cardiac surgery patients (P = 0.30, RR = 0.93). For AKI-related hospital mortality, the AKIN criteria did not show a better ability in predicting hospital mortality in either ICU (P = 0.19, RR = 1.01) or cardiac surgery patients (P = 0.61, RR = 0.98) compared to RIFLE criteria. Our findings supported that the AKIN criteria can identify more patients in classifying AKI compared to RIFLE criteria, but not showing a better ability in predicting hospital mortality. Moreover, both RIFLE and AKIN criteria for AKI in cardiac surgery patients had better predictive ability compared with the ICU patients.

Lack of Association between Interleukin-10 Gene Polymorphisms and Graft Rejection Risk in Kidney Transplantation Recipients: A Meta-Analysis.

Background Interleukin-10 (IL-10) is an important immunomodulatory cytokine. Several studies focused the association between IL-10 promoter gene polymorphisms and graft rejection risk in kidney transplantation recipients. However, the results of these studies remain inconclusive. The aim of this study was to conduct a meta-analysis to further assess the associations. Methods The PubMed, Embase, and Ovid Medline databases were searched. Two independent authors extracted data, and the effects were estimated from an odds ratio (OR) with 95% confidence intervals (CIs). Subgroup and sensitivity analyses identified sources of heterogeneity. Results A total of 16 studies including 595 rejection patients and 1239 stable graft patients were included in order to study the IL-10 -1082 (rs1800896 G/A), -819 (rs1800871 C/T), -592 (rs1800872 C/A) and IL-10 (-1082,-819,-592) polymorphisms. The -1082 G/A polymorphism was not associated with an increased graft rejection risk (OR = 1.03; 95%CI, 0.85–1.25, P = 0.74 for GA+AA vs. GG model). Moreover, all of the -819 C/T (OR = 1.06, 95%CI, 0.79–1.42, P = 0.70 for TA+TT vs. CC model), -592 C/A (OR = 1.10, 95% CI, 0.85–1.42, P = 0.47 for AC+AA vs. CC model) and IL-10 (-1082,-819,-592) polymorphisms (OR = 1.00, 95%CI, 0.79–1.27, P = 0.98 for I+L vs. H model) did not increase the graft rejection risk. In addition, we also performed subgroup analysis by ethnic group (mainly in Europeans or Asians) and rejection type (acute or chronic). There was also lack of evidence of a significant association between the IL-10 gene polymorphism and graft rejection risk. The present meta-analysis indicated that the IL-10 gene polymorphism was not associated with graft rejection risk in kidney transplantation recipients. Conclusion This meta-analysis found evidence that the IL-10 polymorphism does not increase the risk of graft rejection in kidney transplantation recipients. Further chronic rejection and other ethnic population studies are needed to confirm our results.

High phosphate-induced downregulation of PPARγ contributes to CKD-associated vascular calcification

Medial arterial calcification associated with hyperphosphatemia is a main cause of cardiovascular mortality in patients with chronic kidney disease (CKD), but the mechanisms underlying high phosphate-induced vascular calcification remain largely unknown. Here, we observed a significant decrease in the expression of peroxisome proliferator-activated receptor-gamma (PPARγ) in calcified arteries both in CKD patients and in a mouse model of CKD with hyperphosphatemia. In vitro, high phosphate treatment led to a decreased expression of PPARγ in mouse vascular smooth muscle cells (VMSCs), accompanied by apparent osteogenic differentiation and calcification. Pretreatment with PPARγ agonist rosiglitazone significantly reversed high phosphate-induced VSMCs calcification. Further investigation showed that methyl-CpG binding protein 2 (Mecp2)-mediated epigenetic repression was involved in high phosphate-induced PPARγ downregulation. Moreover, the expression of Klotho that has the ability to inhibit vascular calcification by regulating phosphate uptake decreased with the PPARγ reduction in VSMCs after high phosphate treatment, and rosiglitazone failed to inhibit high phosphate-induced calcification in VSMCs with knockdown of Klotho or in aortic rings from Klotho-deficient (kl/kl) mice. Finally, an in vivo study demonstrated that oral administration of rosiglitazone could increase Klotho expression and protect against high phosphate-induced vascular calcification in CKD mice. These findings suggest that the inhibition of PPARγ expression may contribute to the pathogenesis of high phosphate-induced vascular calcification, which may provide a new therapeutic target for vascular calcification in CKD patients.