Noriaki Yorioka

Klotho Inhibits Transforming Growth Factor-β1 (TGF-β1) Signaling and Suppresses Renal Fibrosis and Cancer Metastasis in Mice

Fibrosis is a pathological process characterized by infiltration and proliferation of mesenchymal cells in interstitial space. A substantial portion of these cells is derived from residing non-epithelial and/or epithelial cells that have acquired the ability to migrate and proliferate. The mesenchymal transition is also observed in cancer cells to confer the ability to metastasize. Here, we show that renal fibrosis induced by unilateral ureteral obstruction and metastasis of human cancer xenografts are suppressed by administration of secreted Klotho protein to mice. Klotho is a single-pass transmembrane protein expressed in renal tubular epithelial cells. The extracellular domain of Klotho is secreted by ectodomain shedding. Secreted Klotho protein directly binds to the type-II TGF-β receptor and inhibits TGF-β1 binding to cell surface receptors, thereby inhibiting TGF-β1 signaling. Klotho suppresses TGF-β1-induced epithelial-to-mesenchymal transition (EMT) responses in cultured cells, including decreased epithelial marker expression, increased mesenchymal marker expression, and/or increased cell migration. In addition to TGF-β1 signaling, secreted Klotho has been shown to inhibit Wnt and IGF-1 signaling that can promote EMT. These results have raised the possibility that secreted Klotho may function as an endogenous anti-EMT factor by inhibiting multiple growth factor signaling pathways simultaneously.

PPAR- γ agonist attenuates renal interstitial fibrosis and inflammation through reduction of TGF- β

Thiazolidinediones (TZDs), synthetic peroxisome proliferator-activated receptor (PPAR)-γ ligands, have a central role in insulin sensitization and adipogenesis. It has been reported that TZDs exert protective effects in both diabetic and nondiabetic models of renal disease, although the exact mechanism is not well understood. In particular, only a few studies have reported the renoprotective effects of TZDs in nondiabetic models of tubulointerstitial fibrosis and inflammation. Therefore, we investigated the anti-fibrotic and anti-inflammatory effects of the TZD troglitazone in the mouse model of unilateral ureteral obstruction (UUO). C57BL/6J mice underwent UUO and were studied after 3 and 7 days. Animals were divided into three groups and received control vehicle, troglitazone (150 mg/kg per day) or troglitazone (300 mg/kg per day) by gavage. Kidneys were harvested for morphological, mRNA and protein analysis. Reverse-transcriptase–PCR was used to assess the expression of transforming growth factor-β1 (TGF-β1) and the TGF-β1 type I receptor (TGFβR-I). Protein expression was assessed by western blotting (TGFβR-I) and immunostaining (TGFβR-I, α-smooth muscle actin (α-SMA), type I collagen (collagen I), F4/80, and proliferating cell nuclear antigen (PCNA)). The expression of α-SMA, collagen I, and F4/80 was decreased in mice treated with troglitazone compared with the control group. The numbers of PCNA-positive interstitial cells were decreased in mice treated with troglitazone. TGF-β1 mRNA and TGFβR-I mRNA and protein expression were decreased in the group treated with troglitazone compared with the control group. The beneficial effects of troglitazone treatment were also dose dependent. PPAR-γ agonist significantly reduced TGF-β and attenuated renal interstitial fibrosis and inflammation in the model of UUO.

Assessment of renal fibrosis with diffusion-weighted MR imaging: Study with murine model of unilateral ureteral obstruction

PURPOSE To test, in a murine model of unilateral ureteral obstruction (UUO), whether the magnetic resonance (MR) imaging-derived apparent diffusion coefficient (ADC) changes during the progression of renal fibrosis and correlates with the histopathologic changes observed in renal fibrogenesis. MATERIALS AND METHODS This study was approved by the institutional animal care and use committee. A UUO was created in each of 14 mice. In five mice, longitudinal diffusion-weighted (DW) imaging was performed before the UUO (day 0) and on days 3 and 7 after the UUO and was followed by histopathologic analysis. The nine remaining mice were examined with cross-sectional studies on days 0 (n = 4) and 3 (n = 5). ADCs were measured with a spin-echo echo-planar sequence at five b values ranging from 350 to 1200 sec/mm(2). Differences in ADC among the time points and between the sides were assessed by using Tukey-Kramer and Student t tests, respectively. ADC was correlated with cell density and alpha-smooth muscle actin (alpha-SMA, a marker of myofibroblasts) expression at linear regression analysis. RESULTS Histopathologic examination revealed typical renal fibrosis on the side with UUO. The ADC decreased over time on the UUO side, from (1.02 +/- 0.06 [standard deviation]) x 10(-3) mm(2)/sec on day 0 to (0.70 +/- 0.08) x 10(-3) mm(2)/sec on day 3 (P < .001) and (0.57 +/- 0.10) x 10(-3) mm(2)/sec on day 7 (P < .001). The percentage change in ADC was greater on the UUO side than on the contralateral side on days 3 (29% +/- 9, P = .05) and 7 (44% +/- 11, P < .01). ADC correlated with both increased cell density and increased alpha-SMA expression (P < .001 for both correlations). CONCLUSION An ADC decrease in renal fibrosis is associated with an increased number of cells, including fibroblasts. ADC has the potential to serve as a sensitive noninvasive biomarker of renal fibrosis.

A-Kinase Anchoring Protein AKAP220 Binds to Glycogen Synthase Kinase-3β (GSK-3β) and Mediates Protein Kinase A-dependent Inhibition of GSK-3β

Glycogen synthase kinase-3 (GSK-3) is regulated by various extracellular ligands and phosphorylates many substrates, thereby regulating cellular functions. Using yeast two-hybrid screening, we found that GSK-3β binds to AKAP220, which is known to act as an A-kinase anchoring protein. GSK-3β formed a complex with AKAP220 in intact cells at the endogenous level. Cyclic AMP-dependent protein kinase (PKA) and type 1 protein phosphatase (PP1) were also detected in this complex, suggesting that AKAP220, GSK-3β, PKA, and PP1 form a quaternary complex. It has been reported that PKA phosphorylates GSK-3β, thereby decreasing its activity. When COS cells were treated with dibutyryl cyclic AMP to activate PKA, the activity of GSK-3β bound to AKAP220 decreased more markedly than the total GSK-3β activity. Calyculin A, a protein phosphatase inhibitor, also inhibited the activity of GSK-3β bound to AKAP220 more strongly than the total GSK-3β activity. These results suggest that PKA and PP1 regulate the activity of GSK-3β efficiently by forming a complex with AKAP220.

Effect of a carbonaceous oral adsorbent on the progression of CKD: a multicenter, randomized, controlled trial.

BACKGROUND The carbonaceous oral adsorbent AST-120 slows the deterioration of kidney function in patients with advanced chronic kidney disease (CKD). However, information about AST-120 in patients with less severe stages of CKD is lacking. STUDY DESIGN Randomized controlled trial. SETTING & PARTICIPANTS 75 medical facilities, 460 patients with CKD with serum creatinine (sCr) concentrations less than 5.0 mg/dL (not undergoing dialysis). INTERVENTION Random assignment to either a low-protein diet and antihypertensive medication in the control group or that treatment combined with AST-120 (6 g/d). OUTCOMES & MEASUREMENTS Composite primary end point: doubling of sCr level, increase in sCr level to 6.0 mg/dL or more, need for dialysis or transplantation, or death. SECONDARY OUTCOMES adverse events and changes in estimated creatinine clearance (CCr) rate, proteinuria (protein in milligrams per day), and quality of life. RESULTS Mean sCr level was 2.66 mg/dL and estimated CCr was 22.4 mL/min in both groups. During 56 weeks, numbers of primary end-point events (43 for control versus 42 for AST-120) and event-free survival (P = 0.9) did not differ between groups. Gastrointestinal adverse events were less common in the control group than the AST-120 group (2 versus 32 events). Estimated CCr decreased more in the control group than in the AST-120 group (-15% per year versus -12% per year, relative to the baseline value; [corrected] P = 0.001). Median proteinuria changed from protein of 1,162 to 1,167 mg/d in the control group versus 1,102 to 906 mg/d in the AST-120 group (P = 0.2). LIMITATION Infrequent primary end-point events. CONCLUSION AST-120 did not substantially slow the progression of kidney disease in patients with moderate to severe CKD during 1 year.

DEC1 Modulates the Circadian Phase of Clock Gene Expression

ABSTRACT DEC1 suppresses CLOCK/BMAL1-enhanced promoter activity, but its role in the circadian system of mammals remains unclear. Here we examined the effect of Dec1 overexpression or deficiency on circadian gene expression triggered with 50% serum. Overexpression of Dec1 delayed the phase of clock genes such as Dec1, Dec2, Per1, and Dbp that contain E boxes in their regulatory regions, whereas it had little effect on the circadian phase of Per2 and Cry1 carrying CACGTT E′ boxes. In contrast, Dec1 deficiency advanced the phase of the E-box-containing clock genes but not that of the E′-box-containing clock genes. Accordingly, DEC1 showed strong binding and transrepression on the E box, but not on the E′ box, in chromatin immunoprecipitation, electrophoretic mobility shift, and luciferase reporter assays. Dec1−/− mice showed behavioral rhythms with slightly but significantly longer circadian periods under conditions of constant darkness and faster reentrainment to a 6-h phase-advanced shift of a light-dark cycle. Knockdown of Dec2 with small interfering RNA advanced the phase of Dec1 and Dbp expression, and double knockdown of Dec1 and Dec2 had much stronger effects on the expression of the E-box-containing clock genes. These findings suggest that DEC1, along with DEC2, plays a role in the finer regulation and robustness of the molecular clock.

Mesenchymal stem cells ameliorate experimental peritoneal fibrosis by suppressing inflammation and inhibiting TGF-β1 signaling

Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have regenerative capability and exert paracrine actions on damaged tissues. Since peritoneal fibrosis is a serious complication of peritoneal dialysis, we tested whether MSCs suppress this using a chlorhexidine gluconate model in rats. Although MSCs isolated from green fluorescent protein–positive rats were detected for only 3 days following their injection, immunohistochemical staining showed that MSCs suppressed the expression of mesenchymal cells, their effects on the deposition of extracellular matrix proteins, and the infiltration of macrophages for 14 days. Moreover, MSCs reduced the functional impairment of the peritoneal membrane. Cocultures of MSCs and human peritoneal mesothelial cells using a Transwell system indicated that the beneficial effects of MSCs on the glucose-induced upregulation of transforming growth factor-β1(TGF-β1) and fibronectin mRNA expression in the human cells were likely due to paracrine actions. Preincubation in MSC-conditioned medium suppressed TGF-β1-induced epithelial-to-mesenchymal transition, α-smooth muscle actin, and the decrease in zonula occludens-1 in cultured human peritoneal mesothelial cells. Although bone morphogenic protein 7 was not detected, MSCs secreted hepatocyte growth factor and a neutralizing antibody to this inhibited TGF-β1 signaling. Thus, our findings imply that MSCs ameliorate experimental peritoneal fibrosis by suppressing inflammation and TGF-β1 signaling in a paracrine manner.

Effects of icodextrin on glycemic and lipid profiles in diabetic patients undergoing peritoneal dialysis.

Aim: Icodextrin reduces glucose absorption from the peritoneal dialysate. We conducted this prospective, open-labeled, multicenter study to determine the effects of icodextrin on glycemic and lipid parameters in diabetic patients undergoing continuous ambulatory peritoneal dialysis (PD) or automated PD. Methods: Patients were recruited from 15 institutions in Japan, and a total of 51 patients (15 women and 36 men, mean age: 59 ± 10 years, median duration of PD: 13 months) were enrolled. The patients were administered an overnight or daytime dwell of 1.5 or 2.0 l of 7.5% icodextrin-containing solution. At baseline and 3, 6, 9 and 12 months after the start of icodextrin, nonfasting blood was drawn for measurement of glycated hemoglobin (HbA1C) and serum lipids. Results: During icodextrin treatment, there was no change in overall HbA1C levels compared to baseline values; however, for those with baseline HbA1C ≧6.5% (n = 22), significant decreases in HbA1C were observed. Mean total/LDL cholesterol and triglycerides were decreased significantly during icodextrin treatment, with greater decreases for patients with baseline total cholesterol ≧220 mg/dl, LDL cholesterol ≧120 mg/dl or triglycerides ≧150 mg/dl. HDL cholesterol did not differ at any time point; however, values for patients with baseline HDL cholesterol <40 mg/dl tended to increase with marginal significance. Conclusions: In the current study, switching from glucose-containing dialysis solution to icodextrin resulted in improved lipid profiles and possibly a favorable metabolic profile, particularly in patients with poor glycemic control. These hypotheses remain to be proven in controlled clinical trials.

Effect of lipoproteins on cultured human mesangial cells

It was recently reported that low-density lipoprotein (LDL) promotes mesangial cell proliferation, and oxidized LDL is cytotoxic for mesangial cells. However, there have been few studies about the effects of other lipoproteins on mesangial cells. Accordingly, we investigated the effect of various lipoproteins on cultured human mesangial cells using 3H-thymidine (3H-TdR) incorporation and cell counting assays. We also investigated the levels of several cytokines in mesangial cell culture supernatants after stimulation by the lipoproteins. Addition of very-low-density lipoprotein (VLDL) at concentrations up to 100 micrograms/mL, intermediate-density lipoprotein (IDL) at up to 50 micrograms/mL, and LDL at up to 50 micrograms/mL induced the proliferation of cultured human mesangial cells, whereas cell growth was inhibited at higher concentrations. Oxidized LDL caused a concentration-dependent decrease of 3H-TdR incorporation. High-density lipoprotein (HDL) had no proliferative effective effect at any concentration. Exposure to VLDL, IDL, LDL, or a high concentration of HDL enhanced the secretion of interleukin-6, platelet-derived growth factor, and transforming growth factor-beta by mesangial cells, whereas tumor necrosis factor-alpha secretion was stimulated by oxidized LDL. These finding indicate that triglyceride (TG)-rich lipoproteins (VLDL and IDL) promote mesangial cell proliferation as well as LDL, whereas oxidized LDL has the reverse effect. These effects of lipoproteins may be related to modulation of various cytokines. Accordingly, TG-rich lipoproteins, LDL, and oxidized LDL may be involved in mesangial cell proliferation and injury in patients with mesangial proliferative glomerulonephritis.

Platelet-derived growth factor, interleukin (IL)-1 beta, IL-6R and tumor necrosis factor-alpha in IgA nephropathy. An immunohistochemical study.

We clarified the presence of platelet-derived growth factor (PDGF), interleukin (IL)-1 beta, IL-6, IL-6 receptor (IL-6R) and tumor necrosis factor-alpha (TNF-alpha) in renal biopsy specimens from 62 patients with IgA nephropathy, and discuss their relationship with mesangial cell proliferation, degree of histological damage and various clinical factors. Mesangial proliferation was determined histologically by PAS staining and the positive rate of proliferating cell nuclear antigen (PCNA). Renal biopsy specimens were stained using an enzyme-antibody method to determine the presence of cytokines and the receptor. PCNA-positive cells in the glomeruli significantly increased in patients positive for PDGF, IL-6, IL-6R and TNF-alpha. The degree of histological damage increased with the positive rates of PDGF, IL-6 or IL-6R and the number of PCNA-positive cells in the glomeruli. In the PDGF-A-positive patients, total urinary protein (TUP), urinary beta2-microglobulin (u-beta2-m) and systolic and diastolic blood pressure were significantly higher, and creatinine clearance (Ccr) was significantly lower than in the PDGF-A-negative patients. In the PDGF-B-positive patients, TUP, serum creatinine (s-Cr) and urinary and serum beta2-m increased significantly and Ccr decreased significantly. Il-1beta was not related to any clinical factors. In the IL-6-positive patients, TUP was significantly higher than in the IL-6-negative patients. In the IL-6R-positive patients, TUP, s-Cr, urinary beta2-m and systolic blood pressure were significantly higher than in the IL-6R-negative patients. In conclusion, PDGF, IL-6 and IL-6R may be closely related to mesangial cell proliferation, histological changes and deterioration of various clinical factors in patients with IgA nephropathy.