Myricetin induces M2 macrophage polarization to alleviate renal tubulointerstitial fibrosis in diabetic nephropathy via PI3K/Akt pathway

BACKGROUND Development of end-stage renal disease is predominantly attributed to diabetic nephropathy(DN).Previous studies have indicated that myricetin possesses the potential to mitigate the pathological alterations observed in renal tissue.Never-theless,the precise molecular mechanism through which myricetin influences the progression of DN remains uncertain.AIM To investigate the effects of myricetin on DN and explore its potential therapeutic mechanism.METHODS Db/db mice were administered myricetin intragastrically on a daily basis at doses of 50 mg/kg or 100 mg/kg for a duration of 12 wk.Subsequently,blood and urine indexes were assessed,along with examination of renal tissue pathology.Kidney morphology and fibrosis were evaluated using various staining techniques including hematoxylin and eosin,periodic acid–Schiff,Masson’s trichrome,and Sirius-red.Additionally,high-glucose culturing was conducted on the RAW 264.7 cell line,treated with 25 mM myricetin or co-administered with the PI3K/Akt inhibitor LY294002 for a period of 24 h.In both in vivo and in vitro settings,quantification of inflammation factor levels was conducted using western blotting,real-time qPCR and ELISA.RESULTS In db/db mice,administration of myricetin led to a mitigating effect on DN-induced renal dysfunction and fibrosis.Notably,we observed a significant reduction in expressions of the kidney injury markers kidney injury molecule-1 and neutrophil gelatinase associated lipocalin,along with a decrease in expressions of inflammatory cytokine-related factors.Furthermore,myricetin treatment effectively inhibited the up-regulation of tumor necrosis factor-alpha,interleukin-6,and interluekin-1βinduced by high glucose in RAW 264.7 cells.Additionally,myricetin modulated the M1-type polarization of the RAW 264.7 cells.Molecular docking and bioinformatic analyses revealed Akt as the target of myricetin.The protective effect of myricetin was nullified upon blocking the polarization of RAW 264.7 via inhibition of PI3K/Akt activation using LY294002.CONCLUSION This study demonstrated that myricetin effectively mitigates kidney injury in DN mice through the regulation of macrophage polarization via the PI3K/Akt signaling pathway.

Expression of Connective Tissue Growth Factor in Renal Tubulointerstitial Fibrosis in Rats and Its Pathogenic Role

Summary： In order to explore the role of connective tissue growth factor （CTGF） in the pathogenesis of renal tubulointerstitial fibrosis, 48 Wistar rats were randomly divided into sham-operated and unilateral ureteral obstruction （UUO） group. On the postoperative day 1, 3, 7 and 14, the rats were killed and the kidneys were removed. The renal tubulointerstitial injury index was evaluated according to the MASSON staining. The mRNA levels of CTGF, transforming growth factor β1 （TGF-β1）. collagen [ （col I ）, and plasminogen activator inhibitor-1 （PAI 1） were detected using rexerse transcriptional-polymerase chain reaction （RT PCR）. Immunohistochemistry was performed to evaluale the protein expression of the above factors, and the relations among them were analyzed. Quantitative expression of CTGF protein in the kidneys was also assessed using Western blot. The results showed that TGF-β1 mRNA level was increased at first day after UUO, followed by a marked elevation of CTGF mRNA level, which began to increase 3 days after UUO （P〈0.01）. With the progression of the disease, the mRNA expression of CTGF, col I and PAI-1 was increased progressively. Immunohistochemistry revealed that the CTGF protein expression was significantly increased in fibrotic areas and tubular epithelial cells 3 days after UUO. On the post-UUO day 7, the protein level of CTGF was positively related to the renal tubulointerstitial injury index （r =0.62, P〈0.01）, the expression of TGF-β1 （r=0.85, P〈0.01）, colI （r=0.78, P〈0.01）, and PAI-1（r=0.76, P〈0.01）. Upon Western blot analysis, CTGF protein expression began to increase 3 days after UUO, and appeared progressively throughout the time course （P〈0.01, as compared with sham-operated group）. It is concluded that CTGF can be induced by TGF-β and mediate various profibrotic actions of this cytokine, such as increasing extracellular matrix （ECM） synthesis and decreasing ECM degradation. The increased expression of CTGF may play a crucial role in the development and progression of tubulointerstitial fibrosis.

Effects and Mechanism of Irbesartan on Tubulointerstitial fibrosis in 5/6 Nephrectomized Rats

Tubulointerstitial fibrosis(TIF)is a common pathological feature of end-stage kidney disease.Previous studies showed that upregulation of TGFβ1 notably contributed to the chronic renal injury and irbesartan halted the development of TIF in rats with 5/6 renal mass reduction.This study was to investigate the effects of irbesartan on chronic TIF and the mechanism involved TGFβ1 in the rodent model of chronic renal failure involving 5/6 nephrectomy.The results showed that irbesartan significantly attenuated th...

Epithelial to mesenchymal transition in the progression of tubulointerstitial fibrosis

Objective To review the mechanisms of epithelial to mesenchymal transition （EMT） and its role in the progression of tubulointerstitial fibrosis. Data sources The data used in this review were obtained mainly from the studies of EMT reported from 2000-2006. Study selection Relevant articles on studies of EMT in tubulointerstitial fibrosis were selected. Data were mainly extracted from the 45 articles listed in the reference section of this review. Results The process of EMT has gained wide recognition as candidate mechanism in progression of chronic fibrotic disorders. New markers were identified and facilitate the observation of EMT. EMT is regulated by many factors through activation of kinase-dependent signaling cascades. Recent findings suggest that EMT is a reversible process, which can be controlled by factors for their epithelial inducing activities. Conclusion Remarkable progresses of EMT research have been made recently. Preventing or reversing EMT is a promising strategy against renal fibrosis.

Yishen Jiangzhuo Granules(益肾降浊冲剂) Affect Tubulointerstitial Fibrosis via A Mitochondrion-Mediated Apoptotic Pathway

Objective： To investigate the effect of Yishen Jiangzhuo Granules （益肾降浊冲剂 , YSJZG） on mitochondrial injury and regeneration and renal tubular epithelial cell apoptosis in chronic renal failure （CRF） rats and explore its mechanism from molecular pathology, gene, protein levels, and relative pathway. Methods： The CRF rat model was established using 5/6 nephrectomy. Sixty rats were randomly divided into six groups： sham-operation group, model （CRF） group, Niaoduqing Granules （尿毒清颗粒）-treated group [5 g/（kg.day）], low-, moderate-, and high-dose [L-YSJZG, M-YSJZG, H-YSJZG at 3, 6, and 9 g/（kg-day）] YSJZG-treated group （n=10 each）. The levels of serum creatinine （Scr）, blood urea nitrogen （BUN）, and 24-h urine protein were assessed after 10 weeks of treatment. The tubulointerstitial injury and collagen deposition were evaluated using periodic acid-schiff stain and Masson staining. Renal tubular epithelial cell apoptosis was assessed using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay, mitochondrial injury was observed using an electron microscope, and superoxide dismutase （SOD）, glutathione （GSH） and malondialdehyde （MDA） levels were assessed using chromometry. Transforming growth factor- 1 β（TGF-β 1） expression was assessed using immunohistochemistry. The expressions of Bax, Bcl-2, peroxisome proliferator-activated receptor γ coactivator-1 α （PGC-1α）, mitochondrial transcription factor A （Tfam）, mitogen-activated protein kinases （MAPK） phosphorylation were evaluated by Western blot. Results： YSJZG decreased the 24-h urine protein, BUN, Scr, remnant kidney weight-to-body weight ratio, renal tubular injury, deposition of collagen, and the apoptosis of renal tubular epithelial cells in a dose-dependent manner. YSJZG dose-dependently restored the number and structure of mitochondria and the expression of Tfam and PCG-1 α, up-regulated the expression of Bcl-2, and inhibited the expression of Bax. YSJZG also dose-dependently inhibited TGF- 13 1 expression, increased SOD and GSH activity, decreased the MDA level, and inhibited p38MAPK and pERK1/2 phosphorylation （all P〈0.01）. Conclusion： YSJZG improved the renal function in rats with CRF and inhibited the progression of tubulointerstitial fibrosis by dose-dependently alleviating mitochondrial injury, restoring the expression of Tfam and PCG-1α , and inhibiting renal tubular epithelial cell apoptosis through inhibiting activation of reactive oxygen species-MAPK signaling.

Jujuboside A ameliorates tubulointerstitial fibrosis in diabetic mice through down-regulating the YY1/TGF-β1 signaling pathway

Diabetic nephropathy(DN)is one of the most common complications of diabetes mellitus,which is characterized in renal tubulointerstitial fibrosis(TIF).The current study was designed to investigate the protective effect of Jujuboside A(Ju A)on TIF in type 2 diabetes(T2DM)mice,and explore its underlying anti-fibrosis mechanism.A mouse T2DM model was established using high fat diet(HFD)feeding combined with intraperitoneal injection of streptozotocin(STZ).Then,diabetic mice were treated with Ju A(10,20 and 40 mg·kg^(−1)·d^(−1),i.g.)for 12 weeks.Results showed that administration of Ju A not only down-regulated fasting blood glucose(FBG)levels,but also improved hyperlipidemia and renal function in diabetic mice.Moreover,the reduced ECM accumulation was observed in the renal cortex of Ju A treated diabetic mice,while the TIF progression was also attenuated by Ju A through blocking the epithelial-to-mesenchymal transition(EMT)of renal tubular epithelial cells(RTECs).Further mechanism studies showed that Ju A treatment effectively down-regulated the protein expression and subsequent nuclear translocation of Yin Yang 1(YY1)in the renal cortex of diabetic mice,and reduced the levels of transforming growth factor-β1(TGF-β1)in the serum and renal cortex of Ju A treated mice.According to in vitro studies,the up-regulated YY1/TGF-β1 signaling pathway was restored by Ju A in high glucose(HG)cultured HK-2 cells.Taken together,these findings demonstrated that Ju A can ameliorate the TIF of DN through down-regulating the YY1/TGF-β1 signaling pathway.

Evidence-Based Therapy May Improve Outcome in Glomerulonephritis—A Prospective Field Survey

Introduction and aims: Although glomerulonephritis is rare in the general population it is the second most important cause for end-stage renal failure. The therapy of glomerulonephritis is guided by a limited number of individual clinical trials and treatment recommendations are based on meta-analysis and Cochrane Systematic Reviews. The impact of such therapy standards on the prognosis of glomerulonephritis is not known. Methods: Between October 2002 and December 2008 patients with abnormal urine findings and/or decreasing renal function of unknown cause were referred for renal biopsy. In a collaboration of out-patient nephrologists with a major teaching hospital, all patients received treatment recommendations according to evidence-based therapy guidelines based on Cochrane Systematic Reviews. Patient charts were systematically reviewed and patients were re-examined for follow-up until November 2009. Cox Regression analysis was performed to identify independent prognostic factors. Results: Two hundred patients with primary or secondary glomerulonephritis were identified. Complete follow-up data were available from 196 patients with 324 therapeutic interventions. The mean follow-up was 2.8 ± 2.0 years. Among all patients, 37% remained unchanged ill, 13% died, 17% had progressing renal disease, while 19% had a complete and 14% a partial remission. Proteinuria declined in primary glomerulonephritis (5.0 ± 5.4 g/d to 2.1 ± 3.4 g/d, p Conclusions: In a multivariate model of standardised glomerulonephritis therapy the presence of tubulointerstitial fibrosis was associated with death or progresssive renal disease, while prednisolone-based therapy regimens and intensified nephrological follow-up resulted in a significant delay of endstage-renal failure. This result should direct future health care policies because glomerulonephritis accounts for nearly 20% of the dialysis population.

Effect of Sanqi Oral Liquid on the Expressions of CD4＋, CD8＋ and CD68＋Cells in 5/6 Nephrectomized Rats with Chronic Renal Failure~

Objective： To explore the mechanisms of Chinese herbal medicine Sanqi Oral Liquid , composed of Astragalus membranaceus and Panpax notoginseng, in alleviating renal injury by observing its effect on the expressions of CD4＋, CD8＋ and CD68＋ cells in 5/6 nephrectomized rats with chronic renal failure. Methods： A total of 102 SD rats were randomly divided into six groups： three treatment groups were administrated with high, medium and low dosage of Sanqi Oral Liquid respectively by gavage; a normal group, a 5/6 nephrectomized model group, and a group treated with coated aldehyde oxygenstarch were used as controls. Following oral administration of Sanqi Oral Liquid for 12 weeks, the general condition and renal pathological changes were observed, and the renal function, platelet count （PLT） and the expressions of CD4~, CD8＊ and CD68~ cells were determined for each group. Results： There were proliferation of mesangial matrix, renaltubularnecrosis and obvious tubulointerstitial fibrosis in the model group, and they were much milder in the treatment groups. Compared with the model group, the amounts of blood urea nitrogen （BUN）, serum creatinine （Scr） and PLT in the treatment groups decreased （P〈0.05 for all）; and in the group administrated of medium dosage of Sanqi Oral Liquid, the expression of CD4＋ cells was up-regulated and those of CD8＋ and CD68＋ cells were down-regulated （P〈0.05 for all）, leading to an increased ratio of CD4＋/CD8＋ （P〈0.01）. Conclusion： Sanqi Oral Liquid has a significant effect on regulating lymphocyte subsets, reducing the infiltration of macrophages in renal tissues and alleviating tubulointerstitial fibrosis, and this may be one of mechanisms of Sanqi Oral Liquid in delaying the progression of chronic kidney diseases.

Inhibition of the Tubular Epithelial-to-Mesenchymal Transition in vivo and in vitro by the Uremic Clearance Granule(尿毒清颗粒)

Objective： To investigate the effect of the Uremic Clearance Granule （UCG, 尿毒清颗粒）, a Chinese patent medicine, on tubular epithelial-to-mesenchymal transition （EMT） in a unilateral ureteral obstruction （UUO） model in vivo and transforming growth factor （TGF）- 131 induced EMT of HK-2 cells in vitro. Methods： In vivo study, 50 Sprague Dawley rats were divided into three groups： a sham operation group （n=10）, a UUO group （n=20）, and a UUO with UCG treatment group （n=20）. The UCG was given at a dose of 4.5 g/kg body weight per day by gavage after surgery. In vitro study, HK-2 cells were cultured in 10% fetal bovine serum （FBS）, 10% healthy rat serum, 10% FBS and TGF-13 1 （10 ng/mL）, 10% healthy rat serum and TGF-13 1, or 10% rat serum containing the uremic clearance granule and TGF-13 1. The expression of the epithelial marker E-cadherin and the mesenchymal markers vimentin and e^-smooth muscle actin （ot-SMA） in kidney tissues and HK-2 cells were investigated by Western blot analysis and immunofluorescence staining. Results： The rats of the UUO group showed obvious tubulointerstitial fibrosis, compared with the sham operation group rats. Tubulointerstitial fibrosis score was reduced by 17.5%± 1.1% at day 7 and by 20.0%_＋ 1.2% at day 14 in the UCG-treated group, compared with the UUO group. The UCG could maintained expression of E-cadherin and suppressed expression of vimentin and α-SMA in kidney tissues of UUO rats at days 7 and 14, as determined by Western blot analysis and immunofluorescence staining. Rat serum containing the UCG partially inhibited TGF- β 1-induced fibroblast phenotype of HK-2 cells and maintained the epithelial morphology of HK-2 cells in vitro. This occurred partially through a reduction of vimentin expression and an increase of E-cadherin expression. Conclusion： These results suggest that the UCG prevents tubular EMT and may be a promising agent for treating tubulointerstitial fibrosis.

Valsartan Inhibited the Accumulation of Dendritic Cells in Rat Fibrotic Renal Tissue

To observe the accumulation of dendritic cells （DCs） in rat remnant kidney and its contribution to tubulointerstitial fibrosis, under influence of valsartan on DCs, a rat remnant kidney model was established by subtotal nephrectomy. Four experimental groups were included： normal, sham, model （SNx） and the group treated with Valsartan （SNxV）. Rats were killed at week 1, 4 and 12, respectively. CD1a^＋CD80^＋ DCs were assayed by double immunostaining method and the images were analyzed with Axioplan 2 microscopy. The expressions of P-selectin, TGF-β1, α-SMA, collagen Ⅲ and fibronectin were analyzed by immunohistochemistry or semi- quantitative RT-PCR, and the level of tubulointerstitial firosis （TIF） was scored. CD1a^＋CD80^＋ DCs were gradually increased among renal tubules, interstitium and vessels, especially in interstitium, and the number of DCs in model group at week 12 was much more than that in model groups at week 1 or 4. The expressions of P-selectin, TGF-β1, α-SMA, collagen Ⅲ and fibronectin in tubulointerstitial areas and the degree of TIF were increased substantially in model group at week 12. The accumulation of DCs in interstitium was well associated with the loss of renal function and the progression of tubulointerstitial fbrosis. Valsartan treatment inhibited the local accumulation of DCs and attenuated renal tubulointerstitial damage. The local DCs accumulation was related to tubulointerstitial fibrosis and renal dysfunction following renal ablation. Blockade to angiotensin II might be a potent way to attenuate renal immuno-inflammatory injury. Cellular ＆ Molecular Immunology. 2006;3（3）：213-220.

Role of connective tissue growth factor in experimental radiation nephropathy in rats

Background Connective tissue growth factor （CTGF） is a potent fibrogenic cytokine which has been associated with progressive glomerulosclerosis and tubulointerstitial fibrosis. We investigated the role of CTGF on the progression of a rat model of radiation nephropathy. Methods The model of radiation nephropathy in rats was established as follows： control group （n=12）, underwent only laparotomy; irradiated group （n=-20）, underwent a laparotomy, then the rats were subjected to a single dose 25 Gy X-ray to the kidneys. The rats were followed up one, three, six and nine months after renal exposure to radiation. Results Renal dysfunction was noted early in irradiated rats. Histological analysis showed focal glomerular sclerotic lesions at an early stage after irradiation. Radiation-induced glomerular and tubulointerstitial injuries were particularly severe the sixth month after irradiation as compared to the control group （P 〈0.01）. By immunohistochemistry, increased expression of CTGF was noted in the irradiated kidneys, which began to increase from the first month after irradiation, and remained significantly higher at the sixth and ninth month after irradiation （P 〈0.01）. Upon Western blot analysis CTGF protein expression showed an increase in the radiation treated kidneys compared with the control rats. The expression of CTGF closely correlated with the progression of radiation nephropathy. The expression of α-smooth muscle actin, vimentin, type Ⅲ collagen and type Ⅳ collagen was also high in the irradiated kidney as compared to control rat kidneys （P 〈0.05）, and was most severe at the sixth and ninth month after irradiation （P 〈0.01）. By double immunostaining, CTGF expressing cells were found to be α-SMA-positive myofibroblasts and vimentin-positive tubular epithelial cells. Glomerular expression of CTGF closely correlated with the glomerular expression of a-SMA （r=0.628, P 〈0.01）, vimentin （r=0.462, P 〈0.05） and accumulation of type IV collagen （r=0.584, P 〈0.01） in the irradiated kidney. Similarly, the expression of CTGF was positively correlated with the expression of α-SMA （r=0.613, P 〈0.01）, vimentin （r=0.629, P 〈0.01）, deposition of type Ⅲ collagen （r=0.741, P 〈0.001） and type Ⅳ collagen （r=0.799, P 〈0.0001） in the tubulointerstitium of the irradiated kidney. Finally the expression of CTGF after the irradiation of the kidney positively correlated with the levels of blood urea nitrogen and serum creatinine. Conclusion Overexpression of CTGF may play an important role in the development and progression of glomerulosclerosis and tubulointerstitial fibrosis in radiation nephropathy.