Toshiki Doi

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.

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.

Inhibition of SET Domain–Containing Lysine Methyltransferase 7/9 Ameliorates Renal Fibrosis

TGF-β1 activity results in methylation of lysine 4 of histone H3 (H3K4) through SET domain-containing lysine methyltransferase 7/9 (SET7/9) induction, which is important for the transcriptional activation of fibrotic genes in vitro. However, in vivo studies utilizing an experimental model of renal fibrosis are required to develop therapeutic interventions that target SET7/9. In this study, we investigated the signaling pathway of TGF-β1-induced SET7/9 expression and whether inhibition of SET7/9 suppresses renal fibrosis in unilateral ureteral obstruction (UUO) mice and kidney cell lines. Among the SET family, SET7/9 was upregulated on days 3 and 7 in UUO mice, and the upregulation was suppressed by TGF-β1 neutralizing antibody. TGF-β1 induced SET7/9 expression via Smad3 in normal rat kidney (NRK)-52E cells. In human kidney biopsy specimens from patients diagnosed with IgA nephropathy and membranous nephropathy, SET7/9 expression was positively correlated with the degree of interstitial fibrosis (r=0.59, P=0.001 in patients with IgA nephropathy; and r=0.58, P<0.05 in patients with membranous nephropathy). In addition, small interfering RNA-mediated knockdown of SET7/9 expression significantly attenuated renal fibrosis in UUO mice. Sinefungin, an inhibitor of SET7/9, also suppressed the expression of mesenchymal markers and extracellular matrix proteins and inhibited H3K4 mono-methylation (H3K4me1) in kidneys of UUO mice. Moreover, sinefungin had an inhibitory effect on TGF-β1-induced α-smooth muscle actin expression and H3K4me1 in both NRK-52E and NRK-49F cells. In conclusion, sinefungin, a SET7/9 inhibitor, ameliorates renal fibrosis by inhibiting H3K4me1 and may be a candidate therapeutic agent.

Inhibition of H3K9 histone methyltransferase G9a attenuates renal fibrosis and retains klotho expression

H3K9 methyltransferase G9a is reportedly induced by transforming growth factor-β1 (TGF-β1) and has an important role in the development of epithelial-mesenchymal transposition in cancer cells. Since the transcriptional activity of the Klotho gene is regulated by H3K9 modification, we investigated the effects of G9a on renal fibrosis and klotho expression. G9a levels were significantly upregulated by day 7 in the kidneys of unilateral ureteral-obstructed mice, but this was inhibited by TGF-β1-neutralizing antibody. Administration of G9a small interfering RNA not only decreased α-smooth muscle actin and fibronectin but also increased klotho expression in the ureteral-obstructed mice. Similarly, intraperitoneal injection of BIX01294, a specific inhibitor of G9a, showed beneficial effects on renal fibrosis and klotho expression with decreased monomethylation of H3K9 (me1). In in vitro experiments, BIX01294 also inhibited TGF-β1-induced fibrotic changes and klotho downregulation along with suppressed H3K9me1. In human kidney biopsy specimens, areas of G9a immunostaining correlated positively with H3K9me1 levels, as well as fibrotic markers, but correlated negatively with klotho expression. Thus, TGF-β1-induced G9a has an important role in the progression of renal fibrosis and reduced klotho expression through H3K9me1.

Optical waveguides on silicon chips

Optical waveguides consisting of a SiOxNy (0≤x,y≤1) core and SiO2 cladding layers have been fabricated using Si LSI technologies. Low‐pressure chemical vapor deposition (CVD) at 750 °C and remote‐plasma CVD at 300 °C have been employed for the deposition of core materials. The propagation loss of ridge type waveguides depends on the size (thickness and width), refractive index and surface roughness of the core. A minimum loss of 1.5 dB/cm has been obtained. Micromirrors made from substrate‐Si, poly‐Si or Al, which change the light propagation direction, have been fabricated using wet‐chemical or chemical dry etching. The light bending efficiency ranges from 60% to 80% depending on the material. Simulations and measurements of the propagation losses of branched and curved waveguides have been carried out. It is shown that the simulated results are in good qualitative agreement with the experimental data for both branched and curved waveguides. Single‐chip integration of light emitting diodes (LEDs), microm...

Mizoribine Ameliorates Renal Injury and Hypertension along with the Attenuation of Renal Caspase-1 Expression in Aldosterone-Salt-Treated Rats

Aldosterone-salt treatment induces not only hypertension but also extensive inflammation that contributes to fibrosis in the rat kidney. However, the mechanism underlying aldosterone-salt-induced renal inflammation remains unclear. Pyroptosis has recently been identified as a new type of cell death that is accompanied by the activation of inflammatory cytokines. We hypothesized that aldosterone-salt treatment could induce inflammation through pyroptosis and that mizoribine, an effective immunosuppressant, would ameliorate the renal inflammation that would otherwise cause renal fibrosis. Ten days after recovery from left uninephrectomy, rats were given drinking water with 1% sodium chloride. The animals were divided into three groups (n = 7 per group): (1) vehicle infusion group, (2) aldosterone infusion group, or (3) aldosterone infusion plus oral mizoribine group. Aldosterone-salt treatment increased the expression of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 and caspase-1, and also increased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells. However, the oral administration of mizoribine attenuated these alterations. Furthermore, mizoribine inhibited hypertension and renal fibrosis, and also attenuated the aldosterone-induced expression of serum/glucocorticoid-regulated kinase and α epithelial sodium channel. These results suggest that caspase-1 activation plays an important role in the development of inflammation induced by aldosterone-salt treatment and that it functions as an anti-inflammatory strategy that protects against renal injury and hypertension.

The Serum Lipids Levels may be Underestimated in Patients on Hemodialysis

OBJECTIVE Although lipid disorders are a well-known risk factor for cardiovascular disease (CVD) in the general population, the optimal management with lipid-lowering therapy to reduce CVD risks and mortality in hemodialysis (HD) patients remains controversial. In the clinical setting, dyslipidemia can be diagnosed based on the detection of elevated lipid concentrations at the beginning of HD. This study investigated changes in the levels of serum lipids during a single HD session. METHODS The serum total cholesterol, triglyceride and high-density lipoprotein (HDL) cholesterol levels were measured in 31 HD patients at zero, two and four hours after the beginning of a single HD session. The data were analyzed using the Wilcoxon signed-rank test, a linear mixed model and Spearmans rank correlation analysis. RESULTS The serum total cholesterol, HDL cholesterol and non-HDL cholesterol levels increased significantly during the HD session. Even after the lipid parameters were corrected for changes in the total protein level, the total cholesterol and HDL cholesterol levels increased, whereas the non-HDL cholesterol levels did not change significantly. The percentage change in the serum levels of these lipid fractions correlated strongly with the percentage change in the ultrafiltration volume per body weight. In contrast, the serum triglyceride levels were decreased significantly at two hours compared with the levels noted at the beginning of HD and gradually increased at four hours. CONCLUSION The serum lipid levels are influenced significantly by HD treatment and ultrafiltration. Evaluating the degree of dyslipidemia at the beginning of a HD session may therefore underestimate the levels of serum lipids in HD patients with a large amount of weight gain, thus resulting in the use of insufficient lipid-lowering therapy.

Expression of age-related factors during the development of renal damage in patients with IgA nephropathy

BackgroundChronic kidney disease patients share clinical and pathological features with the general aging population. Increased oxidative DNA damage, accumulation of cell cycle-arrested cells and decreased Klotho expression are assumed to be age-related factors that are reportedly linked to kidney disease. This study sought to determine the association between these age-related factors and renal damage in patients with IgA nephropathy (IgAN).MethodsWe performed a cross-sectional analysis of 71 patients who were diagnosed with IgAN by renal biopsy. Expression of 8-hydroxydeoxyguanosine (8-OHdG, a marker of oxidative DNA damage), p16 (a marker of cell cycle-arrest) and Klotho (an anti-aging protein) were evaluated by immunohistochemical staining of renal biopsy samples. We correlated the changes in expression of these markers with Lee’s pathologic grades and the Oxford classification. We also investigated the independent association between these markers and interstitial fibrosis using multiple linear regression analysis.Results8-OHdG and p16 increased but Klotho decreased with progression of pathologic grade. Expression of 8-OHdG and p16 increased with the deterioration of mesangial hypercellularity and segmental glomerulosclerosis. In addition, p16 increased but Klotho decreased with progression of tubular atrophy/interstitial fibrosis. In univariate regression analysis, age, body mass index, systolic blood pressure, urinary protein excretion and expression of 8-OHdG, p16 and Klotho showed significant correlations with interstitial fibrosis. Multivariable regression analyses revealed that aging, increased renal expression of p16 and decreased expression of Klotho were independently correlated with interstitial fibrosis.ConclusionsThe age-related factors might play important roles in the development of IgAN.

Deubiquitinase inhibitor PR-619 reduces Smad4 expression and suppresses renal fibrosis in mice with unilateral ureteral obstruction

Deubiquitinating enzymes (DUBs) remove ubiquitin from their substrates and, together with ubiquitin ligases, play an important role in the regulation of protein expression. Although transforming growth factor (TGF)-β1-Smad signaling is a central pathway of renal fibrosis, the role of DUBs in the expression of TGF-β receptors and Smads during the development of renal fibrosis remains unknown. In this study, we investigated whether PR-619, a pan-DUB inhibitor, suppresses fibrosis in mice with unilateral ureteral obstruction (UUO) and TGF-β1-stimulated normal rat kidney (NRK)-49F cells, a rat renal fibroblast cell line. Either the vehicle (dimethyl sulfoxide) or PR-619 (100 μg) was intraperitoneally administered to mice after UUO induction once a day for 7 days. Administration of PR-619 attenuated renal fibrosis with downregulation of mesenchymal markers, extracellular matrix proteins, matrix metalloproteinases, apoptosis, macrophage infiltration, and the TGF-β1 mRNA level in UUO mice. Although type I TGF-β receptor (TGF-βRI), Smad2, Smad3, and Smad4 protein expression levels were markedly increased in mice with UUO, administration of PR-619 suppressed only Smad4 expression but not TGF-βRI, Smad2, or Smad3 expression. PR-619 also had an inhibitory effect on TGF-β1-induced α-smooth muscle actin expression and reduced Smad4 levels in NRK-49F cells. Our results indicate that PR-619 ameliorates renal fibrosis, which is accompanied by the reduction of Smad4 expression.

Na+-Cl− cotransporter-mediated chloride uptake contributes to hypertension and renal damage in aldosterone-infused rats

Recently, in addition to epithelial sodium channel alpha-subunit (αENaC), the thiazide-sensitive sodium-chloride cotransporter (NCC) and pendrin, also known as sodium-independent chloride/iodide transporter, were reported to be activated by aldosterone. Here, we investigated whether chloride (Cl-) is responsible for hypertension, inflammation, and renal damage in aldosterone-infused rats. Following left nephrectomy, 8-wk-old male Sprague-Dawley rats were allocated into four groups: 1) drinking 1.0% sodium chloride solution with aldosterone infusion (Aldo/NaCl rats); 2) drinking 1.44% sodium bicarbonate solution with aldosterone infusion (Aldo/NaHCO3 rats); 3) drinking distilled water with aldosterone infusion (Aldo/water rats); and 4) drinking distilled water without aldosterone infusion (sham rats). Additionally, heminephrectomized rats with aldosterone infusion were fed a 0.26% NaCl diet (control); 8.0% NaCl diet (high-Na/high-Cl); or a 4.0% NaCl 6.67% sodium citrate diet (high-Na/half-Cl). Last, Aldo/NaCl rats were treated with or without hydrochlorothiazide. Blood pressure in the Aldo/NaCl rats was significantly higher than in the Aldo/NaHCO3 rats, which was associated with the increased expression of NCC. Expression of markers of inflammation (CD3, CD68, interleukin-17A) and fibrosis (α-smooth muscle actin, collagen 1) were also increased in Aldo/NaCl rats. Similarly, aldosterone-infused rats fed a high-Na/half-Cl diet had lower blood pressure than those fed a high-Na/high-Cl diet, with a reduction of phosphorylated NCC, but not αENaC and pendrin. NCC inhibition with hydrochlorothiazide attenuated interleukin-17A protein expression along with the phosphorylation of NCC in Aldo/NaCl rats. These findings suggest that NCC-mediated Cl- uptake plays important roles in the development of aldosterone-induced hypertension and renal injury.