Bo Young Nam

Circulating α-klotho levels in CKD and relationship to progression.

BACKGROUND α-Klotho is reported to have protective effects against kidney injury, and its renal expression is decreased in many experimental models of kidney disease. However, circulating α-klotho levels in human chronic kidney disease (CKD) and the relationship to progression are unknown. STUDY DESIGN Post hoc analysis of a prospective cohort study. SETTING & PARTICIPANTS 243 of 301 participants from a CKD cohort at our institution between January 2006 and December 2011 were eligible for the study. PREDICTOR Baseline α-klotho levels. OUTCOMES Primary outcome was the composite of doubling of baseline serum creatinine concentration, end-stage renal disease, or death. End-stage renal disease was defined as onset of treatment by renal replacement therapy. MEASUREMENTS Serum α-klotho and fibroblast growth factor 23 (FGF-23) were measured using enzyme-linked immunosorbent assay. RESULTS Lower serum α-klotho levels were associated with more severe CKD stage in the cross-sectional analysis of the baseline data (P for trend < 0.001). In the adjusted multivariable linear regression model, log(α-klotho) was associated independently with estimated glomerular filtration rate (β = 0.154; P = 0.001). Cox regression analysis showed that baseline α-klotho level independently predicted the composite outcome after adjustment for age, diabetes, blood pressure, estimated glomerular filtration rate, proteinuria, parathyroid hormone level, and FGF-23 level (HR per 10-pg/mL increase, 0.96; 95% CI, 0.94-0.98; P < 0.001). When patients were categorized into 2 groups according to baseline median α-klotho value, 43 (35.2%) patients with α-klotho levels ≤396.3 pg/mL reached the primary composite outcome compared with 19 (15.7%) with α-klotho levels >396.3 pg/mL (HR, 2.03; 95% CI, 1.07-3.85; P = 0.03). LIMITATIONS Uncontrolled dietary phosphorus intake and use of frozen samples. CONCLUSIONS This observational study showed that low circulating α-klotho levels were associated with adverse kidney disease outcome, suggesting that α-klotho is a novel biomarker for CKD progression. More data from larger prospective longitudinal studies are required to validate our findings.

Irisin, a novel myokine is an independent predictor for sarcopenia and carotid atherosclerosis in dialysis patients

OBJECTIVE In end-stage renal disease, deleterious effect of sarcopenia on cardiovascular disease has been explained mainly by chronic inflammation. However, evidence emerged that skeletal muscles mediate their protective effect against sarcopenia by secreting myokines. Therefore, we sought to investigate the effect of irisin, a recently introduced myokine, on the association between sarcopenia and cardiovascular disease in peritoneal dialysis (PD) patients. METHODS Serum irisin concentrations were assessed by enzyme-linked immunosorbent assay in 102 prevalent PD patients and 35 age- and sex-matched controls. To determine sarcopenia and cardiovascular disease, anthropometric indices including mid-arm muscle circumference (MAMC) and carotid intima-media thickness (cIMT) were measured. RESULTS Serum irisin concentrations were significantly lower in PD patients than in controls (184.2 ± 88.0 vs. 457.2 ± 105.5 ng/mL, P < 0.001). In PD patients, univariate linear regression analysis showed that serum irisin was positively correlated with MAMC and thigh circumference, but negatively correlated with residual renal function and cIMT. Multivariate analysis revealed that MAMC (per 1 cm increase, B = 8.78, 95% confidence interval [CI] = 0.77-16.79, P = 0.03) had an independent association with serum irisin. In addition, serum irisin was a significant independent predictor for carotid atherosclerosis even after adjustment for high-sensitivity C-reactive protein in PD patients (per 1 g/mL increase, odds ratio = 0.990, 95% CI = 0.982-0.997, P = 0.007). CONCLUSION This study demonstrated that serum irisin was significantly associated with sarcopenia and carotid atherosclerosis in PD patients. Additional studies to provide a confirmation and examine possible mechanisms are warranted.

DECREASED CIRCULATING KLOTHO LEVELS IN PATIENTS UNDERGOING DIALYSIS AND RELATIONSHIP TO OXIDATIVE STRESS AND INFLAMMATION

♦ Introduction: It has been reported that klotho deficiency is associated with oxidative stress and inflammation in experimental kidney disease models. Patients with endstage renal disease (ESRD) are particularly characterized by increased oxidative stress and inflammation. However, little is known about the relationship between these features and klotho in patients with ESRD. ♦ Methods: We conducted a single-center, cross-sectional study of 78 patients receiving peritoneal dialysis (PD). Serum concentrations of klotho, high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), and 8-isoprostane were measured by enzyme-linked immunosorbent assay. To define factors independently associated with klotho, we determined Spearman’s correlation coefficients for between co-variates and conducted multiple linear regression analyses. ♦ Results: Patients were classified by median concentration of klotho. In patients with klotho levels > 329.6 pg/mL, serum 8-isoprostane and IL-6 levels were significantly higher than in those with klotho levels < 329.6 pg/mL. In correlation analyses, log 8-isoprostane (γ = –0.310, p = 0.006) and log IL-6 (γ = –0.343, p = 0.002) were inversely correlated with log klotho. After adjustment for age, gender, mean arterial pressure, log intact parathyroid hormone, and log IL-6, log 8-isoprostane was independently associated with log klotho (β = –0.158, p = 0.040). However, the significant relationship between klotho and IL-6 was not seen in an adjusted model. ♦ Conclusions: This study showed that circulating klotho levels were significantly associated with 8-isoprostane levels in patients undergoing PD, suggesting a potential link between klotho deficiency and enhanced oxidative stress in ESRD patients.

The monocyte chemoattractant protein-1 (MCP-1)/CCR2 system is involved in peritoneal dialysis-related epithelial–mesenchymal transition of peritoneal mesothelial cells

Epithelial–mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) has a role in the process of peritoneal fibrosis (PF), a serious complication in peritoneal dialysis (PD) patients. Even though monocyte chemoattractant protein-1 (MCP-1) was demonstrated to directly increase extracellular matrix (ECM) synthesis, the role of the MCP-1/CCR2 system in PD-related EMT and ECM synthesis in cultured human PMCs (HPMCs) and in an animal model of PD has never been elucidated. In vitro, HPMCs were exposed to 5.6 mM glucose (NG), NG+MCP-1 (10 ng/ml) (NG+MCP-1), or 100 mM glucose (HG) with or without CCR2 inhibitor (RS102895) (CCR2i) or a dominant-negative mutant MCP-1-expressing lentivirus (LV-mMCP-1). In vivo, PD catheters were inserted into 60 Sprague-Dawley rats, and saline (Control, C) (N=30) or 4.25% PD solution (PD) (N=30) was infused for 4 weeks. Twenty rats from each group were treated with empty LV or LV-mMCP-1 intraperitoneally. Snail, E-cadherin, α-smooth muscle actin (α-SMA), and fibronectin protein expression in HPMCs and the peritoneum was evaluated by western blot analysis. Compared with NG cells, Snail, α-SMA, and fibronectin expression was significantly increased, while E-cadherin expression was significantly decreased in HPMCs exposed to HG and NG+MCP-1, and these changes were significantly abrogated by CCR2i (P<0.05). In addition, MCP-1-induced EMT was significantly attenuated by anti-TGF-β1 antibody. In PD rats, Snail and fibronectin expression was significantly increased in the peritoneum, whereas the ratios of E-cadherin/α-SMA protein expression were significantly decreased (P<0.05). The thickness of the peritoneum and the intensity of Masson’s trichrome staining in the peritoneum were also significantly higher in PD rats than in C rats (P<0.05). These changes in PD rats were significantly abrogated by LV-mMCP-1. These findings suggest that the MCP-1/CCR2 system is directly involved in PD-related EMT and ECM synthesis and that this is mediated, at least in part, via TGF-β1.

Translationally controlled tumour protein is associated with podocyte hypertrophy in a mouse model of type 1 diabetes

Aims/hypothesisTranslationally controlled tumour protein (TCTP) is thought to be involved in cell growth by regulating mTOR complex 1 (mTORC1) signalling. As diabetes characteristically induces podocyte hypertrophy and mTORC1 has been implicated in this process, TCTP may have a role in the pathogenesis of diabetes-induced podocyte hypertrophy.MethodsWe investigated the effects and molecular mechanisms of TCTP in diabetic mice and in high glucose-stimulated cultured podocytes. To characterise the role of TCTP, we conducted lentivirus-mediated gene silencing of TCTP both in vivo and in vitro.ResultsGlomerular production of TCTP was significantly higher in streptozotocin induced-diabetic DBA/2J mice than in control animals. Double-immunofluorescence staining for TCTP and synaptopodin revealed that podocyte was the principal cell responsible for this increase. TCTP knockdown attenuated the activation of mTORC1 downstream effectors and the overproduction of cyclin-dependent kinase inhibitors (CKIs) in diabetic glomeruli, along with a reduction in proteinuria and a decrease in the sizes of podocytes as well as glomeruli. In addition, knockdown of TCTP in db/db mice prevented the development of diabetic nephropathy, as indicated by the amelioration of proteinuria, mesangial expansion, podocytopenia and glomerulosclerosis. In accordance with the in vivo data, TCTP inhibition abrogated high glucose-induced hypertrophy in cultured podocytes, which was accompanied by the downregulation of mTORC1 effectors and CKIs.Conclusions/interpretationThese findings suggest that TCTP might play an important role in the process of podocyte hypertrophy under diabetic conditions via the regulation of mTORC1 activity and the induction of cell-cycle arrest.

Apoptosis occurs differentially according to glomerular size in diabetic kidney disease

BACKGROUND Apoptosis, which is involved in the process of mesangial cell and podocyte loss in diabetic nephropathy, is known to be regulated by protein kinase B/Akt (Akt). A number of studies have therefore investigated the activity of Akt under diabetic conditions, but the results have not been consistent. In this study, we hypothesized that apoptosis may occur differentially and that Akt may be differentially activated according to glomerular size in diabetic kidney disease. METHODS Fifty male Sprague-Dawley rats were injected intraperitoneally with diluent (C, n = 25) or streptozotocin (DM, n = 25). After 3 months, glomeruli were isolated using sieves with pore sizes of 250, 150, 125 and 75 μm and then classified into large glomeruli (on the 125-μm sieve, LG) and small glomeruli (on the 75-μm sieve, SG) groups. Western blot analyses for phospho-Akt, apoptosis-related molecules (Bax, Bcl-2, active fragments of Caspase-3 and phospho-p53) and cyclin-dependent kinase inhibitors were performed. CONCLUSIONS The numbers of total cells and podocytes in isolated glomeruli were determined using transmission electron microscopy. Akt phosphorylation was significantly decreased in DM-LG, while it was significantly increased in DM-SG (P < 0.05). The ratio of Bax/Bcl-2 protein expression and active fragments of Caspase-3 and phospho-p53 protein expression were significantly increased in DM-LG compared to DM-SG and C-SG (P < 0.001 and P < 0.01, respectively). In contrast, the expression of p27(Kip1) and p21(Cip1) was significantly increased in DM-SG compared to DM-LG and C-SG (P < 0.05). The numbers of total glomerular cells and podocytes were significantly decreased in DM-LG (P < 0.05). In conclusion, these data show differential expression of Akt activity and apoptosis-related molecules according to glomerular size in diabetic nephropathy, suggesting that apoptosis may be more operative in more hypertrophic glomeruli, resulting in fewer glomerular cells and podocytes in diabetic nephropathy.

Gamma Linolenic Acid Exerts Anti-Inflammatory and Anti-Fibrotic Effects in Diabetic Nephropathy

Purpose This study was undertaken to investigate the effects of gamma linolenic acid (GLA) on inflammation and extracellular matrix (ECM) synthesis in mesangial and tubular epithelial cells under diabetic conditions. Materials and Methods Sprague-Dawley rats were intraperitoneally injected with either a diluent [n=16, control (C)] or streptozotocin [n=16, diabetes (DM)], and eight rats each from the control and diabetic groups were treated with evening primrose oil by gavage for three months. Rat mesangial cells and NRK-52E cells were exposed to medium containing 5.6 mM glucose and 30 mM glucose (HG), with or without GLA (10 or 100 µM). Intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), and fibronectin (FN) mRNA and protein expression levels were evaluated. Results Twenty-four-hour urinary albumin excretion was significantly increased in DM compared to C rats, and GLA treatment significantly reduced albuminuria in DM rats. ICAM-1, MCP-1, FN mRNA and protein expression levels were significantly higher in DM than in C kidneys, and these increases were significantly abrogated by GLA treatment. In vitro, GLA significantly inhibited increases in MCP-1 mRNA expression and protein levels under high glucose conditions in HG-stimulated mesangial and tubular epithelial cells (p<0.05, respectively). ICAM-1 and FN expression showed a similar pattern to the expression of MCP-1. Conclusion GLA attenuates not only inflammation by inhibiting enhanced MCP-1 and ICAM-1 expression, but also ECM accumulation in diabetic nephropathy.

Podocyte hypertrophy precedes apoptosis under experimental diabetic conditions

Podocyte hypertrophy and apoptosis are two hallmarks of diabetic glomeruli, but the sequence in which these processes occur remains a matter of debate. Here we investigated the effects of inhibiting hypertrophy on apoptosis, and vice versa, in both podocytes and glomeruli, under diabetic conditions. Hypertrophy and apoptosis were inhibited using an epidermal growth factor receptor inhibitor (PKI 166) and a pan-caspase inhibitor (zAsp-DCB), respectively. We observed significant increases in the protein expression of p27, p21, phospho-eukaryotic elongation factor 4E-binding protein 1, and phospho-p70 S6 ribosomal protein kinase, in both cultured podocytes exposed to high-glucose (HG) medium, and streptozotocin-induced diabetes mellitus (DM) rat glomeruli. These increases were significantly inhibited by PKI 166, but not by zAsp-DCB. In addition, the amount of protein per cell, the relative cell size, and the glomerular volume were all significantly increased under diabetic conditions, and these changes were also blocked by treatment with PKI 166, but not zAsp-DCB. Increased protein expression of cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase, together with increased Bax/Bcl-2 ratios, were also observed in HG-stimulated podocytes and DM glomeruli. Treatment with either zAsp-DCB or PKI 166 resulted in a significant attenuation of these effects. Both PKI 166 and zAsp-DCB also inhibited the increase in number of apoptotic cells, as assessed by Hoechst 33342 staining and TUNEL assay. Under diabetic conditions, inhibition of podocyte hypertrophy results in attenuated apoptosis, whereas blocking apoptosis has no effect on podocyte hypertrophy, suggesting that podocyte hypertrophy precedes apoptosis.

(The) effect of statin on epithelial-mesenchymal transition in peritoneal mesothelial cells

Background Statins have recently been highlighted for their pleiotropic actions distinct from cholesterol-lowering effects. Despite this interest, it is currently unknown whether statin therapy inhibits peritoneal dialysis (PD)-related epithelial-mesenchymal transition (EMT). Methods In vitro, human peritoneal mesothelial cells (HPMCs) were exposed to 5.6 mM glucose (NG) or 100 mM glucose (HG) with or without simvastatin (1 µM). In vivo, PD catheters were inserted into 32 Sprague-Dawley rats, and saline (C, n = 16) or 4.25% peritoneal dialysis fluid (PDF) (PD, n = 16) was infused for 4 weeks. Eight rats from each group were treated with 5 mg/kg/day of simvastatin intraperitoneally. Changes in the protein expression of EMT markers such as E-cadherin, α-SMA, Snail, and fibronectin in HPMCs and the peritoneum were evaluated by Western blot analysis and immunofluorescence or immunohistochemical staining. We also explored whether activation of the mevalonate pathway and its downstream small GTPases were involved in dialysis-related peritoneal EMT and could be inhibited by statin treatment. Results Compared to NG cells, E-cadherin expression was significantly decreased, while α-SMA, Snail, and fibronectin expression were significantly increased in HPMCs exposed to HG, and these changes were abrogated by simvastatin (p<0.05). In addition, the cobblestone-like appearance of normal HPMCs was converted into a fibroblast-like morphology after HG treatment, which was reversed by simvastatin. These EMT-like changes were also observed in HPMCs treated with geranyl-geranyl pyrophosphate (5 µM). HG significantly increased the protein expression of RhoA and Rac1 in the membrane fractions, and these increases were ameliorated by simvastatin (p<0.05). In PD rats, E-cadherin in the peritoneum was significantly decreased, whereas α-SMA, Snail, and fibronectin expression were significantly increased (p<0.05) compared to C rats. The thickness of the mesothelial layer in the peritoneum were also significantly greater in PD rats than in C rats (p<0.05). These changes of the peritoneum in PD rats were significantly attenuated by simvastatin. Conclusion This study demonstrated that PD-related EMT was mediated via the mevalonate pathway, and statin treatment inhibited the EMT changes in HG-treated HPMCs and PDF-stimulated PD rats. These findings suggest that statins may be a promising therapeutic strategy for preservation of peritoneal membrane integrity in long-term PD patients.

PGC-1α Protects from Notch-Induced Kidney Fibrosis Development

Kidney fibrosis is the histologic manifestation of CKD. Sustained activation of developmental pathways, such as Notch, in tubule epithelial cells has been shown to have a key role in fibrosis development. The molecular mechanism of Notch-induced fibrosis, however, remains poorly understood. Here, we show that, that expression of peroxisomal proliferation g-coactivator (PGC-1α) and fatty acid oxidation-related genes are lower in mice expressing active Notch1 in tubular epithelial cells (Pax8-rtTA/ICN1) compared to littermate controls. Chromatin immunoprecipitation assays revealed that the Notch target gene Hes1 directly binds to the regulatory region of PGC-1α Compared with Pax8-rtTA/ICN1 transgenic animals, Pax8-rtTA/ICN1/Ppargc1a transgenic mice showed improvement of renal structural alterations (on histology) and molecular defect (expression of profibrotic genes). Overexpression of PGC-1α restored mitochondrial content and reversed the fatty acid oxidation defect induced by Notch overexpression in vitro in tubule cells. Furthermore, compared with Pax8-rtTA/ICN1 mice, Pax8-rtTA/ICN1/Ppargc1a mice exhibited improvement in renal fatty acid oxidation gene expression and apoptosis. Our results show that metabolic dysregulation has a key role in kidney fibrosis induced by sustained activation of the Notch developmental pathway and can be ameliorated by PGC-1α.