The Protective Effects And Mechanisms Of Curcumin On Renal Tubulointerstitial Fibrosis

The pathogenesis of CKD is characterized by progressive loss of kidney function and relentless accumulation and deposition of extracellular matrix (ECM), leading to widespread tissue fibrosis. The deterioration of renal function is largely determined by the extent and severity of tubulointerstitial fibrosis(TIF) in many forms of renal disease, both in animal models and in patients. As population of the patients with end-stage renal disease(ESRD) is increasing at a rate of approximately 7% per year, the human and economic burden of CKD to the families and society is enormous. It is significant meaning that studying further the molecular mechanisms of TIF and searching effective therapeutic strategy to slow the progression of ESRD.Tubulointerstitial fibrosis is characterized by atrophy of tubule proliferation of myofibroblast and accumulation and deposition of ECM, the role of tubular epithelial-mesenchymal transformation(EMT) has been emphasized. There is increasing evidence that transforming growth factor-β1 (TGF-β1) plays an essential role in tubulointerstitial fibrosis process. Smad proteins are intracellular mediators of TGF-βsignaling. Upon ligand stimulation, receptor-regulated Smads (R-Smads) are phosphorylated by serine/threonine kinase receptors, form complexes with common Smad, and translocate into the nucleus, where they regulate the transcription of target genes together with other transcription factor.Chinese herbs have a long history in chronic kidney disease therapy and have some obvious superiority over western medicine. However, the mechanism of Chinese herb's therapeutic efficacy is not so clear due to the lag of pharmacological study,which influences the application of Chinese herb on CKD.To find some good Chinese herb for tubulointerstitial fibrosis and to reveal the mechanism of its therapeutic effect is important in Chinese herb exploitation.Curcumin is a phenol pigment which is abstracted from the curcuma rhizome of curcuma genus in ginger family. It is the main component of curcuma and is known to have many effects, such as hypolipidemic, antioxidant, scavenging free radicals, anti-inflammatory, anti-tumor, anti-microbial, and so on. However, it is not clear whether curcumin may ameliorate the progression of tubulointerstitial fibrosis. The purpose of our study is to explore the effects and mechanisms of curcumin on renal lesions and try to find a new way of prevention and treatment of tubulointerstitial fibrosis.Part One:The effect of curcumin on renal lesions in unilateral ureteral obstruction (UUO) ratsObjective:To study the effect of curcumin on renal lesions following unilateral ureteral obstruction in rats. Method:The animal model of renal disease was induced by unilateral ureteral obstruction in male SD rats. They were divided into three groups: The sham, UUO, and UUO receiving curcumin treatment daily. In UUO group unilateral ureteral obstruction was achieved by ligating the left ureter with silk through a left ateral incision. Curcumin group was injected with 50mg/Kg of curcumin intraperitoneally every day after UUO; And dimathyl sulfoxide(DMSO) was given intraperitoneally daily for Sham-operated and UUO group. Rats were killed at 3,7,14 days after operation(for each group, n=5),and the levels of blood serum albumin, creatinine, urea nitrogen and urinary protein excretion of 24h were examined at each time point. Obstructed kidneys were harvested and partial renal tissues were fixed in 4% formaldehyde and embedded with Parafin. HE and Masson trichrome were used to examine the area of Pathological change, The de novo expression of a-smooth muscle actin(a-SMA) were assessed by immunohistochemistry method. Results:Compared with the UUO group, the degree of swelling and hydrocele of the obstructive kidney in curcumin group was milder and the color was redder. There was no significant difference in the level of albumin, BUN,Scr and urinary protein excretion of 24h of all groups. The degree of tubular damage and the number of inflammatory cells in treatment group was much lower than that in UUO group at each time point(P<0.05). The de novo expression of SMA expression increased in time-dependent manner in both UUO group and treatment group, while it was significantly lower in curcumin group at every time point(P<0.05). Conclusion:Curcumin could attenuate the degree of renal tissue injury and the expression of SMA in UUO model.Part Two:The effects of curcumin on the expression of a-SMA and PAI-1 in the human proximal tubule cells (HK-2)Objective:To explore the effect of curcumin on the expression of a-SMA and PAI-1 in HK-2 cells. Method:HK-2 cells were cultured with DMEM/F12 including 10% fetal bovine serum. Cells were divided in different groups:control group, TGF-β(5ng/ml)group, TGF-β+curcumin group(different concentration or different duration). Total RNA and protein were abstracted after stimulation, RT-PCR, Western-blot and ELISA were performed to test changes of a-SMA and PAI-1 level. Result:Low concentration curcumin (<20μmol/L) led to no effects on the morphology or the viability of HK-2 cells. The expression of PAI-1 and a-SMA mRNA increased 3.01 and 2.87 times after treatment with 5ng/ml TGF-β1 for 8h. Curcumin dose-dependently blocked the TGF-β1 effects, and dose of 10μmol/L curcumin showed maximal inhibition effect(P＜0.05). Curcumin could block for up to 24 h, whereas the maximal inhibition effect occurred at 8h. Western Blot and ELISA results showed that the protein expression of PAI-1 and a-SMA were downregulated dose-and time-dependently by curcumin treatment. Conclusion:TGF-β1 upregulated the expression a-SMA and PAI-1 both in mRNA and protein level,while curcumin could inhibit the TGF-β1 profibrotic action in a time-and dose-dependent manner and showed a potent anti-fibrotic effect by influencing the anti-fibrosis gene in HK-2 cells.Part Three:The effect of curcumin on TGF-β1/Smad signaling pathway in HK-2 cellsObjective:To investigate the role of curcumin in modulating TGF-β1/Smad signaling Pathway in HK-2 and explore it's underlying mechanisms. Method:Western Blot was performed to investigate the expressions of Smad2 and Smad3 phosphorylation induced by TGF-β1 with/without curcumin in different concentrations for different times. We also observed the effect of curcumin on nuclear accumulation of R-Smads and the effect of protein phosphatase inhibitor (Microcystin-LR)on Smad2 phosphorylation. Result: Western blot analyses revealed that Smad2 and Smad3 were phosphorylated at 15 min after 5ng/ml TGF-β1 stimulation. Smad2 and Smad3 were predominantly localized in the cytosolic fraction of the untreated HK-2 cells, after incubation with TGF-β1,they were translocated to the nuclear fraction. However, curcumin could inhibited TGF-β1-induced Smad2 phosphorylation (but not Smad3)and Smad2/3 nuclear translocation significantly at the 10μmol/L concentration.Microcystin-LR, a specific protein phosphatase inhibitor, could partly reverse the inhibition effect of curcumin on Smad2 phosphorylation.Conclusion:Our study indicate that curcumin might be a potent antifibrotic drug by reducing the phosphorylation of Smad2 and disturbing the nuclear translocation of Smad-2 and-3, while Smad3 phosphorylation is not a mechanism involved in the inhibitory action of curcumin. The effect of curcumin on smad2 phosphorylation may be mediated, at least in part, by its serinethreonine protein phosphatases inducing effect.