Rong Tang

Role of Fosinopril and Valsartan on Klotho Gene Expression Induced by Angiotensin II in Rat Renal Tubular Epithelial Cells.

Background/Aims: Klotho gene, a new anti-aging gene, is mainly expressed in the kidney tubules. Several studies have found the relationship between klotho and emergence and development of renal diseases. This study set out to explore the role of fosinopril (Fos) and valsartan (Val) on klotho expression induced by angiotensin II (Ang II) in rat renal tubular epithelial cells (NRK-52E). Methods: NRK-52E cells were divided into five groups according to the treatment of Ang II, Fos and Val. Transforming growth factor-β1 (TGF-β1), p38, phospho-p38 (p-p38), p53, and Sp1 protein expression were determined by immunohistochemical and Western blotting analysis. Klotho expression was detected by reverse transcription-polymerase chain reaction and Western blotting analysis. Results: Ang II upregulated TGF-β1, p-p38 and p53 expression, and inhibited Sp1 and klotho expression in NRK-52E cells. After the intervention of Fos and/or Val, TGF-β1, p-p38 and p53 expression were downregulated, Sp1 and klotho expression were upregulated. TGF-β1 and p53, Sp1 and klotho expression exhibited a positive linear correlation, respectively. Conclusion: We conclude that Fos and Val have a protective role in Ang II-induced renal damage, and it may be through mechanism of inhibiting TGF-β1, p-p38 and p53 expression, thus upregulating Sp1 and klotho expression.

Fosinopril and Losartan Regulate Klotho Gene and Nicotinamide Adenine Dinucleotide Phosphate Oxidase Expression in Kidneys of Spontaneously Hypertensive Rats

Background/Aims: Klotho, a newly identified antiaging gene, predominantly detected in the kidney, has pleiotropic protective effects on kidney diseases. Several studies have confirmed the association between Klotho and oxidative stress. The present studies were performed to explore effects of fosinopril (Fos) and losartan (Los) on Klotho and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression in kidneys of spontaneously hypertensive rats (SHR). Methods: Twenty-four male 22-week-old SHR were randomly divided into three groups: model group, Fos group and Los group. Wistar-Kyoto rats were taken as control. After 8 weeks, urinary N-acetyl-β-D-glucosaminidase (NAGase), 24 h urinary protein (Upro), serum creatinine (Scr), blood urea nitrogen (BUN) and renal pathological changes were detected. Renal mRNA and protein expression of Klotho and three subunits of NADPH oxidase protein expression were evaluated. Results: As compared to the model group, NAGase, Upro, Scr and BUN were decreased; the typical renal pathological damage was relieved in the Fos or Los group. Fos or Los inhibited the reduction of Klotho expression, and reduced the elevation of NADPH oxidase expression. Conclusion: Abnormal expression of Klotho and NADPH oxidase plays important roles in progression of hypertensive renal damage. Fos and Los can increase Klotho expression, and inhibit NADPH oxidase expression, which may be one of the mechanisms of their protective effects in hypertensive renal damage.

Spironolactone Rescues Dot1a-Af9-Mediated Repression of Endothelin-1 and Improves Kidney Injury in Streptozotocin-Induced Diabetic Rats

The molecular mechanism linking aldosterone and endothelin-1 in the development of diabetic nephropathy has not been completely elucidated. Here, we provide evidence showing that streptozotocin-induced diabetic rats have significantly increased aldosterone and endothelin-1 in the kidney tissue and markedly decreased expression of Dot1a and Af9. Blocking aldosterone with spironolactone significantly reduced proteinuria, glomerulosclerosis, tubulointerstitial injury and endothelin-1 expression, and significantly increased Dot1a and Af9 expression. Increasing Dot1a and Af9 expression by spironolactone or by stable transfection led to impaired endothelin-1 expression in NRK-52 cells. In contrast, downregulation of Dot1a and Af9 by aldosterone in NRK-52E cells caused upregulation of endothelin-1. Genetic inactivation of Dot1l, which encodes Dot1a, in Aqp2-expressing principal cells of mouse kidney impaired association of Dot1a and H3 dimethyl K79 with the specific subregions of endothelin-1 promoter, and upregulates endothelin-1 mRNA and protein expression. Our data suggest that Dot1a and Af9 repress endothelin-1 in vitro and in vivo. Excessive aldosterone induces kidney injury, in part possibly by downregulating Dot1a and Af9, and thus relieving Dot1a-Af9-mediated repression to increase endothelin-1 transcription. Spironolactone ameliorates kidney injury in Streptozotocin-induced diabetic rats, possibly by restoring Dot1a-Af9-mediated repression to reduce endothelin-1 expression. Therefore, Dot1a and Af9 as aldosterone-downregulated targets are negative regulators of endothelin-1 transcription in vitro and in vivo, and may be considered as new potential therapeutic targets of kidney injury in diabetes.

Hemolytic Streptococcus may exacerbate kidney damage in IgA nephropathy through CCL20 response to the effect of Th17 cells

Background The exacerbation of IgA nephropathy (IgAN) is related to respiratory tract infection with hemolytic streptococcus (HS), but the mechanism is unknown. In this study we investigated the role of chemokine ligand 20 (CCL20) in response to the effect of T helper 17 (Th17) cells in the pathogenesis of IgAN associated with HS. Methods Thirty mice were randomly divided into five groups: control mice (control), IgAN mice (IgAN), HS-infected IgAN mice (HS-IgAN), CCL20-treated IgAN mice (CCL20-IgAN), and CCL20-treated HS infected IgAN mice (CCL20-HS-IgAN). IgAN mice were induced with lipopolysaccharide, carbon tetrachloride and bovine serum albumin. Then the mice were sensitized with CCL20 antibody and infected with alpha-hemolytic streptococcus (α-HS) isolated from tonsils in sequence. Urine Albumin-Creatinine ratio and sediments were measured. The pathological changes in kidney and lung tissues were observed under microscopy. Th17 cells and regulatory T cells (Tregs) in kidneys were tested by flow cytometry. CCL20, IL-17A, IL-6 and IL-21 in the kidneys were detected by ELISA. Results The IgAN mice had albuminuria and microscopic hematuria, renal mesangial proliferation, IgA deposition, high electron dense deposition in glomerular mesangial region, decreased frequency of Tregs, increased frequency of Th17 and Th17-Treg ratio. Furthermore, Th17-related cytokines CCL20, IL-17A, IL-6 and IL-21 were all increased in the kidneys of IgAN mice. Compared with IgAN mice, the manifestations in HS-IgAN mice were more severe, but alleviated in CCL20-treated groups. Conclusion α-HS may exacerbate kidney damage in IgAN through CCL20 response to the effect of Th17 cells.

Smad7 gene transfer attenuates angiogenesis in peritoneal dialysis rats

Transforming growth factor‐β (TGF‐β) has been shown to play a role in peritoneal angiogenesis associated with peritoneal dialysis (PD). The present study investigated whether blockade of TGF‐β signalling with Smad7 has a therapeutic effect on PD induced‐peritoneal angiogenesis.

Inhibition of Rho-kinase alleviates peritoneal fibrosis and angiogenesis in a rat model of peritoneal dialysis

Abstract Background/Aims: The present study investigated whether Rho-kinase inhibition had a therapeutic role on the pathogenesis of peritoneal fibrosis and angiogenesis. Methods: A rat model of peritoneal dialysis was induced by a daily intraperitoneal infusion of 4.25% Dianeal. Those rats were treated with Rho-kinase inhibitor, fasudil. Immunofluorescence, Western blot and RT-PCR were used to detect the expression of TGF-β1, Collagen I, αSMA and VEGF in each group. Microvessel density (MVD) was measured by immunohistochemistry. Rho-kinase activity was determined by western immunoblotting. Results: Rho-kinase was activated in the peritoneum of the PD group, which was inhibited by fasudil. Compared with PD group, the mRNA and protein expressions of TGF-β1, αSMA and Collagen I were significantly downregulated in fasudil treatment groups in a dose-dependent manner, and the expression of VEGF and peritoneal MVD was also significantly downregulated in fasudil treatment groups in a dose-dependent manner. Conclusion: The Rho-kinase was activated in the peritoneum of the peritoneal dialysis rats, and the inhibition of Rho-kinase by fasudil can remarkably decrease peritoneal fibrosis and angiogenesis.

Effects of fosinopril and losartan on renal Klotho expression and oxidative stress in spontaneously hypertensive rats.

OBJECTIVE To explore effects of fosinopril and losartan on renal Klotho expression and oxidative stress in spontaneously hypertensive rats (SHR) and the mechanisms underlying the protection against renal damage. METHODS Fifteen male SHRs (22 weeks old) were randomly divided into 3 groups (n=5 in each group): a SHR group, a fosinopril group [10 mg/(kg.d)], and a losartan group [50 mg/(kg.d)]. Age-matched Wistar-Kyoto (WKY) rats were chosen for a control group. Eight weeks later, tail arterial pressure, 24 hours urinary protein (Upro),urinary N-acetyl-β-D-glucosaminidase (NAGase) were measured. Renal pathological changes were examined under light microscopy by HE staining. The renal mRNA and protein expression of Klotho were determined by RT-PCR, immunohistochemical staining or Western blot. The levels of total antioxidant capacity (TAOC), malondialdehyde (MDA), Cu/Zn superoxide dismutase (Cu/Zn-SOD), Mn superoxide dismutase (Mn-SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were determined. RESULTS The typical pathological characteristics of hypertensive renal damage were observed in the kidney of the SHR group.Compared with the SHR group, the systolic pressure, Upro, and urinary NAGase, the content of MDA and renal pathological damage was reduced while the renal Klotho expression and activities of TAOC, Cu/Zn-SOD, CAT, and GSH-Px were increased (P<0.05 or P<0.01) in the fosinopril or losartan group. There was no significant difference in renal Mn-SOD level among the 4 groups (P>0.05). CONCLUSION Fosinopril and losartan can exert protection against hypertensive renal damage through upregulating Klotho expression as well as reducing oxidative stress.

Losartan and Dexamethasone may inhibit chemotaxis to reduce the infiltration of Th22 cells in IgA nephropathy.

Abstract Angiotensin II is considered a major profibrotic factor that is involved in tissue remodeling processes, as the inhibition of Angiotensin II can halt renal inflammatory processes. Dexamethasone, an important anti‐inflammatory and immunosuppressive agent, has been widely used to treat renal disease for decades. In this study, we explored the frequency of Th22 cells in a mouse model of IgA nephropathy and compared the possible effects of Losartan and Dexamethasone on Th22 cells. The experiments were performed using 6‐week‐old BALB/c female mice in an established IgA nephropathy model. The mice were randomly separated into 4 groups, which were administered Losartan (30 mg/kg/d) or Dexamethasone (10 mg/kg/d) and subjected to IgA nephropathy or the normal control treatment for 1 month. The frequency of Th22 cells was measured via flow cytometry, and the relative pathological changes in renal morphology were measured with different pathological staining methods. Immunohistochemistry was performed to verify the expression of CCR10 and CCL27, which is specialized receptor on Th22 cells and its corresponding chemokine, respectively. The concentrations of CCL27 and IL‐22 in renal tissue homogenates and sera were detected using ELISAs. Losartan and Dexamethasone differentially decreased the frequency of Th22 cells after 1 month, and mesangial cell proliferation was also improved. Moreover, the expression of CCR10, CCL27 and IL‐22 was reduced by treatment with either drug. However, significant differences between Losartan and Dexamethasone were not observed. Based on these findings, Losartan and Dexamethasone may suppress inflammatory responses by inhibiting the chemotaxis of Th22 cells in IgA nephropathy. HighlightsLosartan and Dexamethasone may reduce the abundance of Th22 cells in IgAN.CCR10, CCL27 and IL‐22 expression was comparably reduced by Losartan and Dexamethasone.Losartan and Dexamethasone may suppress inflammatory responses by inhibiting chemokine pathways in Th22 cells.

Proapoptotic effect of angiotensin II on renal tubular epithelial cells and protective effect of Cordyceps sinensis

OBJECTIVE To investigate the mechanism of the protective effect of Cordyceps sinensis (C. sinensis) on the apoptosis of cultured NRK-52E induced by angiotension II (AngII). METHODS NRK-52E cells were incubated with C. sinensis (0, 5, 10, 20, and 40 mg/L) and 10(-8) mol/ L AngII for 24, 48, 72 h. The optimal concentration of C. sinensis was selected. Either NRK-52E cells were incubated with different doses of AngII (0, 10(-12), 10(-10), 10(-8), and 10(-6) mol/L) for 24 h, or with 10(-8) mol/L AngII for 24, 48, and 72 h, to observe the effect of AngII on the apoptosis of NRK- 52E cells. The optimal concentration and time of AngII were selected. In another experiment cells were divided into 5 groups: a control, AngII (10(-8) mol/L), AngII (10(-8) mol/L)+ C. sinensis (40 mg/ L), Ang II (10(-8) mol/L)+ fosinopril (10(-5) mmol/L), and Ang II (10(-8) mol/L)+ fosinopril (10(-5) mol/ L)+C. sinensis (40 mg/L). MTT assay was used to test the changes in the proliferation of NRK-52E cultured with different concentration of C. sinensis for 24, 48, 72 h. The Annecxin V-FITC and PI stainings were applied to detect the apoptosis rate induced by AngII by flow cytometer (FCM) and to determine the eddects of C. sinensis. The activity of caspase-3 was assayed by spectrophotometry. RESULTS Certain concentrations of C. sinensis (10-40 mg/L) promoted the proliferation of NRK- 52E cells inhibited by AngII(P<0.05). AngII induced the apoptosis of NRK-52E in a dose and timedependent manner, accompanied with increased activity of caspase-3 (P<0.05). C. sinensis partially suppressed the apoptosis of NRK-52E induced by AngII, and declined the activity of caspase-3 (P<0.05). No significant difference was shown as between the fosinopril group and the fosinopril+C. sinensis group (P>0.05). CONCLUSION C. sinensis can suppress the apoptosis of NRK-52E by AngII, and the protective effect of C. sinensis may be inhibiting the activation of caspase-3 during the AngII-induced apoptosis of NRK-52E.

Aldosterone induces NRK‑52E cell apoptosis in acute kidney injury via rno‑miR‑203 hypermethylation and Kim-1 upregulation

Acute kidney injury (AKI) is characterized by an acute reduction in kidney function as identified by an increase in serum creatinine levels and reduction in urine output. Kidney injury molecule-1 (Kim-1) is a hallmark of kidney diseases, since it is typically non-detectable in the non-injured kidney, but upregulated and excreted in the urine during AKI. Aldosterone (Aldo) is a mediator of the renin-angiotensin-Aldo system with a pivotal role in the regulation of salt and extracellular fluid metabolism. In the present study, mice subjected to renal ischemia/reperfusion-induced AKI were investigated. The mice exhibited elevated levels of Aldo and angiotensin II, together with increased Kim-1 expression levels in renal tissue. Treatment of the mice with the Aldo receptor antagonist spironolactone decreased Kim-1 expression levels. These results suggest that Aldo may be associated with the expression of Kim-1 during AKI. However, the molecular mechanism underlying the role of Aldo in Kim-1 expression is unclear, and thus was investigated using NRK-52E cells. Aldo was found to induce the apoptosis of NRK-52E cells via the hypermethylation of rno-microRNA (miR)-203 and upregulation of Kim-1. In addition, luciferase reporter assays demonstrated that Kim-1 was a target gene of rno-miR-203 in NRK-52E cells. Furthermore, Aldo-induced NRK-52E cell apoptosis was reduced by treatment with pre-miR-203 and spironolactone to a greater extent when compared with either alone. The results may provide a promising diagnostic marker or novel therapeutic target for AKI.