Shigehiro Doi

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.

Ultra-short echo time (UTE) MR imaging of the lung: Comparison between normal and emphysematous lungs in mutant mice

To investigate the utility of ultra‐short echo time (UTE) sequence as pulmonary MRI to detect non‐uniform disruption of lung architecture that is typical of emphysema.

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.

Endothelial nitric oxide synthase intron 4 polymorphism influences the progression of renal disease

Background/Aim: Nitric oxide is a potent regulator of intrarenal hemodynamics and may influence the renal function. We investigated whether polymorphism of intron 4 of the endothelial constitutive nitric oxide synthase (ecNOS) gene is related to the progression of chronic renal failure. Methods: Polymorphism of ecNOS intron 4 was studied in 1,005 hemodialysis patients (710 with nondiabetic nephropathy and 295 with diabetic nephropathy) and was compared with the findings in 189 healthy subjects. ecNOS genotypes were determined by the polymerase chain reaction, followed by agarose gel electrophoresis. Results: The frequencies of ecNOS4a/a, ecNOS4a/b, and ecNOS4b/b genotypes were, respectively, 0% (0/189), 13.8% (26/189), and 86.2% (163/189) in the control group; 1.7% (12/710), 22.1% (157/710), and 76.2% (541/710) in the nondiabetic nephropathy group, and 1.0% (3/295), 22.7% (67/295), and 76.3% (225/295) in the diabetic nephropathy group. The frequency of ecNOS4a (ecNOSa/a and ecNOSa/b) was significantly higher in both the nondiabetic group and in the diabetic group than in the controls (p = 0.0025 and p = 0.0438, respectively). Conclusion: There was a significantly higher frequency of the a allele of intron 4 in both nondiabetic and diabetic hemodialysis patients, so the polymorphism of intron 4 of the ecNOS gene may have a wide influence on the progression of renal disease.

Activation of signal transducer and activator of transcription 3 correlates with cell proliferation and renal injury in human glomerulonephritis

BACKGROUND Signal transducer and activator of transcription (STAT) 3 plays an important role in the regulation of cell proliferation. However, the mechanism of STAT3 activation in human glomerulonephritis is unclear. METHODS STAT3 activation was determined using immunohistochemistry for phosphorylated STAT3 (p-STAT3) in normal human kidney and various types of glomerulonephritis. We also identified the cell exhibiting activated p-STAT3 expression in human glomerulonephritis and correlated STAT3 activation with renal function and histologic injury. RESULTS p-STAT3 staining was identified in glomeruli and some tubules in normal human kidney. p-STAT3 positive glomerular cells were significantly increased in lupus nephritis, IgA nephropathy and vasculitis compared with normal kidney. p-STAT3 positive tubulointerstitial cells were significantly increased in IgA nephropathy and vasculitis compared with normal kidney. Glomerular and tubulointerstitial p-STAT3 staining was significantly decreased after steroid therapy. There was a significant correlation between the number of p-STAT3 positive cells and the number of PCNA positive glomerular and tubulointerstitial cells in all cases of glomerulonephritis. Furthermore, renal function inversely correlated with the number of p-STAT3 positive glomerular and tubulointerstitial cells in all cases of glomerulonephritis. CONCLUSIONS The present study has identified STAT3 activation in normal human kidney and a marked increase in STAT3 activation in many forms of glomerulonephritis. The correlation of STAT3 activation with clinical and histologic parameters suggests that this pathway plays an important role in the pathogenesis of kidney disease. Furthermore, localization of STAT3 activation to individual cell types suggests that this pathway may play a pivotal role in promoting renal inflammation and fibrosis.

Protective Effects of Peroxisome Proliferator-activated Receptor γ Ligand on Apoptosis and Hepatocyte Growth Factor Induction in Renal Ischemia-reperfusion Injury

Background. Renal ischemia-reperfusion injury affects the long-term outcome of renal graft survival. Thiazolidinediones (TZDs), synthetic peroxisome proliferator-activated receptor (PPAR)-&ggr; ligands, have been shown to exert therapeutic effects upon renal ischemia-reperfusion injury far beyond their use as insulin sensitizers. It has also been reported that hepatocyte growth factor (HGF) has a beneficial effect on renal ischemia-reperfusion injury and that TZDs induce increased HGF mRNA expression and protein secretion. We investigated the effect of troglitazone, one of the TZDs, in a rat model of renal ischemia-reperfusion injury. Methods. A 45-minute period of warm renal ischemia was induced by bilateral clamping at 37°C with rats being sacrificed before the onset of ischemia and at 2, 4, 6, and 12 hr after reperfusion. The expression of PPAR-&ggr; was measured by reverse-transcriptase polymerase chain reaction (RT-PCR) and western blotting while the production of HGF was investigated by RT-PCR and immunohistochemistry. The effect of troglitazone treatment on the level of apoptosis was determined by staining for cleaved caspase-3 and single-stranded DNA (ssDNA). Results. The numbers of cleaved caspase-3 and ssDNA positive cells were decreased in rats treated with troglitazone. The production of HGF mRNA and protein was most intense at 4 hr. The expression of PPAR-&ggr; and HGF was increased in the group treated with troglitazone compared with the control group. Conclusions. Pretreatment of rats with the PPAR-&ggr; ligand troglitazone decreased apoptotic cell death in renal ischemia-reperfusion injury as a result of the induction of HGF.

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.