Mesenchymal stem cells cultured in serum-free medium ameliorate experimental peritoneal fibrosis.

BACKGROUND: Mesenchymal stem cells (MSCs) provide potential treatments for peritoneal fibrosis. However, MSCs cultured in media containing serum bring risks of infection and other problems. In this study, we compared the effect of human MSCs in serum-free medium (SF-MSCs) on peritoneal fibrosis with that of MSCs cultured in medium containing 10% fetal bovine serum (10%MSCs). METHODS: Peritoneal fibrosis was induced by intraperitoneally injecting 0.1% chlorhexidine gluconate (CG). SF-MSCs or 10%MSCs were intraperitoneally administered 30 min after the CG injection. Ten days after the CG and MSC injections, we performed histological analyses and peritoneal equilibrium testing. In the in vitro experiments, we used transforming growth factor (TGF)-ß1-stimulated human peritoneal mesothelial cells incubated in conditioned medium from MSCs to examine whether the SF-MSCs showed enhanced ability to produce antifibrotic humoral factors. RESULTS: Histological staining showed that the SF-MSCs significantly suppressed CG-induced cell accumulation and thickening compared with that of the 10%MSCs. Additionally, the SF-MSCs significantly inhibited mesenchymal cell expression, extracellular matrix protein deposition and inflammatory cell infiltration. Peritoneal equilibration testing showed that compared with administering 10%MSCs, administering SF-MSCs significantly reduced the functional impairments of the peritoneal membrane. The in vitro experiments showed that although the conditioned medium from MSCs suppressed TGF-ß1 signaling, the suppression did not significantly differ between the SF-MSCs and 10%MSCs. CONCLUSIONS: Serum-free culture conditions can enhance the antifibrotic abilities of MSCs by suppressing inflammation. Administering ex vivo expanded SF-MSCs may be a potential therapy for preventing peritoneal fibrotic progression.

Utility of glomerular Gd-IgA1 staining for indistinguishable cases of IgA nephropathy or Alport syndrome.

BACKGROUND: Pathological findings in Alport syndrome frequently show mesangial proliferation and sometimes incidental IgA deposition, in addition to unique glomerular basement membrane (GBM) changes including thin basement membrane and/or lamellation. However, similar GBM abnormalities are also often observed in IgA nephropathy. Both diseases are also known to show hematuria, proteinuria, and sometimes macrohematuria when associated with viral infection. Therefore, it can be difficult to make a differential diagnosis, even based on clinical and pathological findings. Some recent articles demonstrated that galactose-deficient IgA1 (Gd-IgA1)-specific monoclonal antibody (KM55) could potentially enable incidental IgA deposition to be distinguished from IgA nephropathy. METHODS: We performed comprehensive gene screening and glomerular Gd-IgA1 and type IV collagen α5 chain immunostaining for five cases with both IgA deposition and GBM changes to confirm that Gd-IgA1 can help to distinguish these two diseases. RESULTS: Four of the cases were genetically diagnosed with Alport syndrome (Cases 1-4) and one was IgA nephropathy with massive GBM changes, which had a negative gene test result (Case 5). In Cases 1-4, glomerular Gd-IgA1 deposition was not detected, although there was positivity for IgA in the mesangial area. In Case 5, glomerular Gd-IgA1 deposition was observed. CONCLUSION: Gd-IgA1 expression analysis could clearly differentiate these two disorders. This approach can be applied to identify these two diseases showing identical clinical and pathological findings.

Different risk factors are associated with vascular access patency after construction and percutaneous transluminal angioplasty in patients starting hemodialysis.

BACKGROUND: The objective of this multicenter, prospective observational study was to determine the factors related to patency rates after construction of vascular access (VA) and the first percutaneous transluminal angioplasty (PTA). METHODS: The 24-month primary and secondary patency rates after construction of a radiocephalic arteriovenous fistula (RC-AVF) and arteriovenous graft (AVG) were evaluated using the Kaplan-Meier method and log-rank test. The 12-month post-PTA patency rate was also investigated. A Cox proportional hazard model was used to identify clinical parameters associated with the primary patency rate and the post-PTA patency rate. RESULTS: A total of 611 patients were enrolled in the study. The primary patency rate after VA construction was lower in hemodialysis (HD) patients with an AVG than in those with an AVF. Aging (hazard ratio [HR], 1.02 per 1 year; p < 0.001), female sex (HR, 1.41; p = 0.03), diabetes mellitus (HR, 1.37; p = 0.03), low serum albumin (HR, 0.76 per 1-g/dL decrease; p = 0.02), and use of an erythropoietin-stimulating agent (HR, 1.62; p = 0.02) were risk factors for VA problems. The post-PTA patency rate was associated with aging (HR, 1.02; p < 0.001), diabetes mellitus (HR, 1.49; p = 0.02), polycystic kidney disease (HR, 2.14; p = 0.01), temporary catheter use for initiation of HD (HR, 1.60; p = 0.02), and period from VA construction to use (HR, 0.99; p = 0.04). CONCLUSION: Although a poor patency rate is commonly associated with advanced age and diabetes, different risk factors affect patency between VA construction and the first PTA.

Chronic kidney disease caused by maternally inherited diabetes and deafness: a case report.

Maternally inherited diabetes and deafness (MIDD) is a mitochondrial genetic disorder with variable clinical presentations, which can delay its diagnosis. Herein, we report the case of a 57-year-old Japanese man with MIDD who developed chronic kidney disease. He developed proteinuria long before his diabetes and deafness; at the age of 36 years, a renal biopsy showed minor glomerular abnormality and electron microscopy showed mild mitochondrial degeneration in the distal tubular epithelial cells. Twenty years later, a second renal biopsy showed nephrosclerosis with interstitial fibrosis and arteriolar hyaline thickening, despite the absence of hypertension and relatively good glycemic control. Granular swollen epithelial cells were found in the medullary collecting duct epithelium. Electron microscopy showed accumulating mitochondria in podocytes and tubular cells, leading to the diagnosis of MIDD. A muscle biopsy also showed ragged-red fibers, despite the absence of muscle weakness. Mitochondrial DNA analysis revealed an m.3243A > G mutation, and taurine supplementation was initiated. Our findings suggest that mitochondrial dysfunction is mainly associated with progressive renal damage.

High-normal albuminuria is strongly associated with incident chronic kidney disease in a nondiabetic population with normal range of albuminuria and normal kidney function.

BACKGROUND: Albuminuria and estimated glomerular filtration rate (eGFR) are clinically measured to evaluate the severity of chronic kidney disease (CKD). The aim of our study was to clarify the association between clinical parameters, including albuminuria and eGFR, and the risk of incident CKD in a nondiabetic population with normal range of albuminuria and eGFR. METHODS: A 10-year follow-up, retrospective cohort study involving 317 Japanese men (mean age, 42 years) with eGFR ≥ 90 mL/min/1.73 m2 and urine albumin-to-creatinine ratio (UACR) < 30 mg/gCr was performed. Participants were free of diabetes mellitus. Multivariate logistic regression approaches were used to assess independent predictors of the incidence of CKD. RESULTS: Twenty-nine (9%) participants developed CKD (eGFR < 60 mL/min/1.73 m2 and/or UACR ≥ 30 mg/gCr) through 10 years of follow-up. At the baseline examination, age, blood pressure, UACR, and eGFR were higher in participants who developed CKD than in those without CKD. After adjustment for confounders, high-normal albuminuria (P < 0.001) and hypertension (P = 0.045) were associated with an increased incidence of CKD. From receiver-operating characteristic curves, UACR ≥ 7.0 mg/gCr was defined as high-normal albuminuria. Logistic regression analysis also showed that, in addition to presence of hypertension, UACR ≥ 7.0 mg/gCr was identified as an independent risk of incident CKD within 10 years after adjustment for age, body mass index, smoking status, and dyslipidemia [UACR: odds ratio (OR) 17.36 (95% CI 6.16-48.93, P < 0.001)]. CONCLUSION: High-normal albuminuria and hypertension are associated with incident CKD in a nondiabetic population with normal-range UACR and eGFR.

High-normal albuminuria is associated with subclinical atherosclerosis in male population with estimated glomerular filtration rate ≥60 mL/min/1.73 m2: A cross-sectional study.

BACKGROUND: Low-grade albuminuria has been considered a predictor of cardiovascular mortality. We investigated the relationship between high-normal albuminuria and subclinical atherosclerosis in non-diabetic men with estimated glomerular filtration rate (eGFR) ≥60 mL/min/1.73 m2. METHODS: In this cross-sectional study, 1,756 men with eGFR ≥60 mL/min/1.73 m2 and urine albumin-to-creatinine ratio (UACR) <30 mg/g, who attended general health checkups between April 2012 and March 2015, underwent blood sampling, urinalysis, and carotid ultrasonography. We excluded the subjects who were diabetic and/or received an anti-hypertensive drug. Carotid intima-media thickness (IMT) and the number of focal atheromatous plaques were used as indicators of subclinical atherosclerosis. Multiple linear regression analysis was performed to identify clinical factors associated with carotid IMT. Poisson regression analysis was used to assess the determinants of the carotid plaque number. RESULTS: Median UACR was 4.8 mg/g (interquartile range, 3.6-6.9 mg/g). Compared with subjects with low-normal UACR (<10.0 mg/g), subjects with high-normal UACR (10.0-29.8 mg/g) had greater IMT and higher carotid plaque number. High-normal UACR was independently associated with thickened IMT in the model adjusted for conventional cardiovascular disease risk factors. Moreover, participants with high-normal UACR were also more likely to be associated with increased plaque count (prevalence ratio: 1.06; 95% confidence interval: 1.01-1.14) after adjustment for conventional cardiovascular disease risk factors. CONCLUSIONS: Our results indicate that high-normal albuminuria is associated with both carotid IMT and plaque formation in the non-diabetic male population with eGFR ≥60 mL/min/1.73 m2.

Serum-Free Medium Enhances the Immunosuppressive and Antifibrotic Abilities of Mesenchymal Stem Cells Utilized in Experimental Renal Fibrosis.

Serum used in culture medium brings risks of immune reactions or infections and thus may hinder using ex vivo expanded mesenchymal stem cells (MSCs) for medical treatment. Here, we cultured MSCs in a serum-free medium (SF-MSCs) and in a medium containing 10% fetal bovine serum (10%MSCs) and investigated their effects on inflammation and fibrosis. MSC-conditioned medium suppressed transforming growth factor-ß1-induced phosphorylation of Smad2 in HK-2 cells, with no significant difference between the two MSCs. This finding suggests that the direct antifibrotic effect of SF-MSCs is similar to that of 10%MSCs. However, immunohistochemistry revealed that renal fibrosis induced by unilateral ureteral obstruction in rats was more significantly ameliorated by the administration of SF-MSCs than by that of 10%MSCs. Coculture of MSCs and monocytic THP-1 cell-derived macrophages using a Transwell system showed that SF-MSCs significantly induced polarization from the proinflammatory M1 to the immunosuppressive M2 phenotype macrophages, suggesting that SF-MSCs strongly suppress the persistence of inflammation. Furthermore, the gene expression of tumor necrosis factor-α-induced protein 6 (TSG-6), which inhibits the recruitment of inflammatory cells, was higher in SF-MSCs than in 10%MSCs, and TSG-6 knockdown in SF-MSCs attenuated the anti-inflammatory responses in unilateral ureteral obstruction rats. These findings imply that SF culture conditions can enhance the immunosuppressive and antifibrotic abilities of MSCs and the administration of ex vivo expanded SF-MSCs has the potential to be a useful therapy for preventing the progression of renal fibrosis. Stem Cells Translational Medicine 2018;7:893-905.

Dec1 and CLOCK Regulate Na+/K+-ATPase ß1 Subunit Expression and Blood Pressure.

Blood pressure shows a circadian rhythm, and recent studies have suggested the involvement of a molecular clock system in its control. In the clock system, the CLOCK (circadian locomotor output cycles kaput):BMAL1 (brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein-1) heterodimer enhances promoter activity of clock genes, and DEC1 (BHLHE40/STRA13/SHARP-2) represses CLOCK/BMAL1-enhanced promoter activity through competition for binding to the clock element, CACGTG E-box. However, the molecular mechanisms by which this system regulates blood pressure remain unclear. Here, we show that DEC1 suppressed the expression of ATP1B1, which encodes the ß1 subunit of the Na+/K+-ATPase and elevated blood pressure. Using chromatin immunoprecipitation and chromatin immunoprecipitation-on-chip analyses, we found that DEC1 and CLOCK bound to E-boxes in the ATP1B1 promoter. Luciferase assays revealed that CLOCK:BMAL1 heterodimer enhanced transcription from the ATP1B1 promoter, whereas DEC1 suppressed this transactivation. Accordingly, Atp1b1 mRNA and protein levels in mouse kidney, aorta, and heart showed a circadian rhythm that was antiphasic to the blood pressure rhythm. Furthermore, Dec1-deficient mice showed enhanced Atp1b1 expression in these tissues and reduced blood pressure. In contrast, Clock-mutant mice showed reduced Atp1b1 expression and elevated blood pressure. Our results raise the possibility that transcriptional regulation of Atp1b1 by DEC1 and CLOCK:BMAL1 contributes to blood pressure.

Deficiency of the basic helix-loop-helix transcription factor DEC1 prevents obesity induced by a high-fat diet in mice.

Obesity is a major public health problem in developed countries resulting from increased food intake and decreased energy consumption and usually associated with abnormal lipid metabolism. Here, we show that DEC1, a basic helix-loop-helix transcription factor, plays an important role in the regulation of lipid consumption in mouse brown adipose tissue (BAT), which is the major site of thermogenesis. Homozygous Dec1 deletion attenuated high-fat-diet-induced obesity, adipocyte hypertrophy, fat volume and hepatic steatosis. Furthermore, DEC1 deficiency increased body temperature during daytime and enhanced the expression of uncoupler protein 1, a key factor of thermogenesis, and various lipolysis-related genes in interscapular BAT. In vitro experiments suggested that DEC1 suppresses the expression of various lipolysis-related genes induced by the heterodimer of peroxisome proliferator-activated receptor γ and retinoid X receptor α (RXRα) through direct binding to RXRα. These observations suggest that enhanced lipolysis in BAT caused by DEC1 deficiency leads to an increase in lipid consumption, thereby decreasing lipid accumulation in adipose tissues and the liver. Thus, DEC1 may serve as an energy-saving factor that suppresses lipid consumption, which may be relevant to managing obesity.

Inhibition of the H3K4 methyltransferase SET7/9 ameliorates peritoneal fibrosis.

Transforming growth factor-ß1 (TGF-ß1) is a major mediator of peritoneal fibrosis and reportedly affects expression of the H3K4 methyltransferase, SET7/9. SET7/9-induced H3K4 mono-methylation (H3K4me1) critically activates transcription of fibrosis-related genes. In this study, we examined the effect of SET7/9 inhibition on peritoneal fibrosis in mice and in human peritoneal mesothelial cells (HPMCs). We also examined SET7/9 expression in nonadherent cells isolated from the effluent of peritoneal dialysis (PD) patients. Murine peritoneal fibrosis was induced by intraperitoneal injection of methylglyoxal (MGO) into male C57/BL6 mice over 21 days. Sinefungin, a SET7/9 inhibitor, was administered subcutaneously just before MGO injection (10 mg/kg). SET7/9 expression was elevated in both MGO-injected mice and nonadherent cells isolated from the effluent of PD patients. SET7/9 expression was positively correlated with dialysate/plasma ratio of creatinine in PD patients. Sinefungin was shown immunohistochemically to suppress expression of mesenchymal cells and collagen deposition, accompanied by decreased H3K4me1 levels. Peritoneal equilibration tests showed that sinefungin attenuated the urea nitrogen transport rate from plasma and the glucose absorption rate from the dialysate. In vitro, sinefungin suppressed TGF-ß1-induced expression of fibrotic markers and inhibited H3K4me1. These findings suggest that inhibiting the H3K4 methyltransferase SET7/9 ameliorates peritoneal fibrosis.

Tocilizumab histologically improved AA renal amyloidosis in a patient with multicentric Castleman disease: A case report
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Multicentric Castleman disease (MCD) is a rare systemic lymphoproliferative disorder and is infrequently associated with renal complications that include amyloid A (AA) amyloidosis. Although it has been reported that patients with MCD and amyloidosis usually have a poor prognosis, recently, tocilizumab, a humanized anti-interleukin-6 receptor antibody, has emerged as an effective and specific treatment for AA amyloidosis secondary to chronic inflammatory disorders. Here we report a case of an MCD patient with secondary AA renal amyloidosis who was successfully treated with tocilizumab. The patient was initially referred to nephrology specialists because of a decline in renal function and proteinuria. Percutaneous renal biopsy revealed the presence of Congo red-positive amorphous depositions and AA protein-positive areas in glomeruli, vessel walls, and interstitium, confirming a diagnosis of renal AA amyloidosis secondary to MCD. At 1 year after starting tocilizumab treatment, a second renal biopsy showed the clearance of amyloid deposits in the interstitium. These observations suggest that tocilizumab may be an effective therapy for AA amyloidosis secondary to MCD.
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pH after the first session of direct hemoperfusion with polymyxin B-immobilized fibers predicts mortality in patients with sepsis and septic shock.

BACKGROUND: The definition of sepsis was updated to sepsis-3 in February 2016. Currently, direct hemoperfusion therapy using the polymyxin B-immobilized fiber cartridge (PMX-DHP) is widely performed to treat sepsis and septic shock. However, the prognostic factors of PMX-DHPs in patients with sepsis using the new definition are unclear. We retrospectively assessed prognostic factors in patients who had received PMX-DHP therapy for sepsis and septic shock. METHODS: We included 71 patients with severe infection who underwent PMX-DHP treatment from January 2006 to August 2015 in this study. Participants were re-evaluated according to the criteria of sepsis-3. The patients were divided into two groups based on having survived (n = 59) or not survived (n = 12) for 28 days after PMX-DHP treatment. Clinical data before and after PMX-DHP treatment were compared between the two groups. RESULTS: Non-survivors showed a lower Glasgow Coma Scale (GCS) score before PMX-DHP treatment compared with 28-day survivors [12 (6-14) vs 14 (12-15), P < 0.01]. Furthermore, pH after the first PMX-DHP session was significantly lower in non-survivors than in survivors (7.28 ± 0.23 vs 7.39 ± 0.06, P = 0.03). Multivariate logistic regression analysis showed that only lower pH after the first PMX-DHP session was a predictor of 28-day mortality independent of age, sex, GCS score, and mean arterial pressure (odds ratio per pH of 0.01, 0.93; 95% confidence interval, 0.83-0.99; P = 0.02). CONCLUSION: The pH after the first PMX-DHP session is an independent risk factor for mortality in patients receiving PMX-DHP for sepsis and septic shock.

High-normal albuminuria and incident chronic kidney disease in a male nondiabetic population.

BACKGROUND: High-normal albuminuria is an important risk factor for incident chronic kidney disease in diabetic populations, in contrast to an uncertain association in nondiabetic populations. This study aimed to reveal the relationship between high-normal albuminuria and incident chronic kidney disease in a Japanese nondiabetic population. METHODS: A 10-year follow-up retrospective cohort study was performed involving 1378 Japanese men (mean age 44 ± 5.3 years) without chronic kidney disease and diabetes mellitus. Chronic kidney disease was diagnosed as either estimated glomerular filtration rate < 60 mL/min/1.73 m2 or a urine albumin-to-creatinine ratio ≥ 30 mg/g. RESULTS: At baseline, age, estimated glomerular filtration rate, and the presence of hematuria, hypertension, and dyslipidemia were independently associated with the albumin-to-creatinine ratio. Among the 1378 participants, 185 (13.4%) fulfilled diagnostic criteria for chronic kidney disease over the 10-year follow-up period. Median annual estimated glomerular filtration rate decline showed a deterioration with increasing quartiles of baseline albumin-to-creatinine ratio (P = 0.004). Participants who had a baseline albumin-to-creatinine ratio in the highest quartile (5.9-28.9 mg/g) were more likely to develop micro- or macroalbuminuria (odds ratio: 16.23, 95% confidence interval 6.56-54.03), chronic kidney disease (odds ratio: 2.48, 95% confidence interval 1.64-3.82), and hypertension (odds ratio 2.06, 95% confidence interval 1.30-3.31), but not diabetes mellitus compared with those who had an albumin-to-creatinine ratio in the lowest quartile (1.3-3.6 mg/g) after adjustment for potential confounders. CONCLUSIONS: High-normal albuminuria was associated with incident chronic kidney disease in this Japanese nondiabetic male population.

Inhibition of H3K9 methyltransferase G9a ameliorates methylglyoxal-induced peritoneal fibrosis.

Activity of H3K9 histone methyltransferase G9a is reportedly induced by transforming growth factor-ß1 (TGF-ß1) and plays an important role in the progression of cancer and fibrosis. In this study, we investigated whether inhibition of G9a-mediated H3K9 methylation attenuates peritoneal fibrosis in mice and human peritoneal mesothelial cells (HPMCs). Nonadherent cells of peritoneal dialysis (PD) patients were isolated from PD effluent to examine expression of G9a. Peritoneal fibrosis was induced by peritoneal injection of methylglyoxal (MGO) in male C57/B6 mice for 3 weeks. BIX01294, a G9a inhibitor, was administered by subcutaneous injection. Effects of BIX01294 on MGO-induced pathological and functional changes in mice were evaluated by immunohistochemistry and a peritoneal equilibration test. HPMCs were isolated from human omentum, and the inhibitory effect of BIX01294 on TGF-ß1-induced fibrotic changes was investigated in the HPMCs by western blotting. G9a was upregulated in nonadherent cells of human PD effluent, the peritoneum of MGO-injected mice, and TGF-ß1-stimulated HPMCs. BIX01294 significantly reduced the submesothelial zone thickness and cell density in MGO-injected mice. Immunohistochemical staining revealed that BIX01294 treatment decreased not only mono-methylation of H3K9 (H3K9me1), but also the number of mesenchymal cells, accumulation of collagen, and infiltration of monocytes. In addition to the pathological changes, BIX01294 reduced the level of TGF-ß1 in peritoneal fluid and improved peritoneal functions. Furthermore, BIX01294 inhibited TGF-ß1-induced fibrotic changes along with suppression of H3K9me1 in HPMCs. Therefore, inhibition of H3K9 methyltransferase G9a suppresses peritoneal fibrosis through a reduction of H3K9me1.

Valproic acid attenuates renal fibrosis through the induction of autophagy.

BACKGROUND: Renal fibrosis is a common pathological feature of the progression of chronic kidney disease. Although valproic acid (VPA) has been recently shown to induce autophagy, the effect of VPA-induced autophagy on renal fibrosis remains unknown. We, therefore, investigated whether VPA-induced autophagy suppresses renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO). METHODS: Male C57BL/6 mice were divided into five groups (n = 8 per group): (1) sham group; (2) vehicle group; (3) VPA-treated group; (4) 3-methyladenine (3-MA; autophagy inhibitor)-treated group; and (5) VPA plus 3-MA-treated group. Mice underwent UUO and the kidneys were studied after 5 days. We also investigated the effect of VPA-induced autophagy on α-smooth muscle actin (α-SMA) in transforming growth factor (TGF)-ß1-stimulated rat kidney fibroblasts and epithelial cells. RESULTS: VPA attenuated renal fibrosis and induced autophagy in UUO mice, while 3-MA increased renal fibrosis and suppressed autophagy. In addition, the anti-fibrotic effect of VPA was diminished by 3-MA in UUO mice. In rat kidney fibroblasts and epithelial cells, VPA suppressed TGF-ß1-stimulated α-SMA expression and induced autophagy. In contrast, 3-MA enhanced α-SMA expression while inhibiting autophagy. Furthermore, the combined use of VPA and 3-MA treatments increased the expression of α-SMA compared with VPA treatment alone in TGF-ß1-stimulated rat kidney fibroblasts and epithelial cells, which was accompanied by the inhibition of autophagy. CONCLUSION: These findings suggest that VPA may be a candidate drug for the treatment of renal fibrosis through the induction of autophagy.

Linagliptin Ameliorates Methylglyoxal-Induced Peritoneal Fibrosis in Mice.

Recent studies have reported increases of methylglyoxal (MGO) in peritoneal dialysis patients, and that MGO-mediated inflammation plays an important role in the development of peritoneal fibrosis through production of transforming growth factor-ß1 (TGF-ß1). Linagliptin, a dipeptidyl peptidase-4 inhibitor, exerts anti-inflammatory effects independent of blood glucose levels. In this study, we examined whether linagliptin suppresses MGO-induced peritoneal fibrosis in mice. Male C57/BL6 mice were divided into three groups: control, MGO injection plus saline, and MGO injection plus linagliptin (n = 6 per group). Peritoneal fibrosis was induced by daily intraperitoneal injection of saline containing 40 mmol/L MGO for 21 days. Saline was administered intraperitoneally to the control group. Linagliptin (10 mg/kg) or saline were administrated by once-daily oral gavage from 3 weeks before starting MGO injections. Immunohistochemical staining revealed that linagliptin suppressed expression of α-smooth muscle actin and fibroblast-specific protein-1, deposition of type I and III collagen, and macrophage (F4/80) infiltration. Peritoneal equilibration testing showed improved peritoneal functions in mice treated with linagliptin. Peritoneal injection of MGO increased plasma levels of glucagon-like peptide-1 (GLP-1) in mice, and a further increase was observed in linagliptin-treated mice. Although MGO increased plasma glucose levels, linagliptin did not decrease plasma glucose levels. Moreover, linagliptin reduced the TGF-ß1 concentration in the peritoneal fluid of MGO-treated mice. GLP-1 receptor (GLP-1R) was expressed in monocytes/macrophages and linagliptin suppressed GLP-1R expression in MGO-injected mice. These results suggest that oral administration of linagliptin ameliorates MGO-induced peritoneal fibrosis.

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

H3K9 methyltransferase G9a is reportedly induced by transforming growth factor-ß1 (TGF-ß1) 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-ß1-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-ß1-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-ß1-induced G9a has an important role in the progression of renal fibrosis and reduced klotho expression through H3K9me1.

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

TGF-ß1 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-ß1-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-ß1 neutralizing antibody. TGF-ß1 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-ß1-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.

High glucose promotes TGF-ß1 production by inducing FOS expression in human peritoneal mesothelial cells.

BACKGROUND: High glucose (HG) induces production of transforming growth factor-beta1 (TGF-ß1), but the mechanism remains elusive. The aim of this study was to determine the gene(s) involved in HG-induced TGF-ß1 production in human peritoneal mesothelial cells (HPMCs). METHODS: Microarray analysis was performed following a 3-h preincubation of HPMCs in 4 or 0.1% glucose medium. Transcriptional genes were selected using Gene Ontology analysis for biological processes, including regulation of transcription and DNA-dependent. The effects of small interfering RNA (siRNA) treatments on the up-regulation of TGF-ß1 mRNA were assessed by quantitative real-time polymerase chain reaction (qPCR). Finally, enzyme-linked immunosorbent assay (ELISA) was performed to determine which gene(s) contribute to the production of TGF-ß1 protein in the medium. RESULTS: Microarray analysis revealed that the expression of 51 genes increased by more than 3-fold. Gene ontology analysis identified 13 genes for further study. qPCR confirmed mRNA amplification for 9 of the 13 genes. Furthermore, HG-induced up-regulation of TGF-ß1 mRNA was attenuated by the siRNA of 4 genes: MDS1 and EVI1 complex locus (MECOM), FBJ murine osteosarcoma viral oncogene homolog B (FOSB), FBJ murine osteosarcoma viral oncogene homolog (FOS) and activating transcription factor 3 (ATF3). ELISA showed that siRNA treatment of FOS, but not MECOM, FOSB or ATF3, suppressed the increase of TGF-ß1 protein in the medium. CONCLUSIONS: FOS is a downstream effector of HG stimulation in HPMCs that contributes to TGF-ß1 production, suggesting that blocking FOS expression may be a therapeutic target for peritoneal fibrosis.