| Background and objective:Chronic kidney disease(CKD)is a groupfor heterogeneous disorders affecting kidney structure and function presenting as a growing public health problem worldwide.Regardless of the initiating insults or contributing risk factors,the common pathological features in patients with CKD is inflammation,tubular atrophy,and renal interstitial fibrosis.Renal fibrosis is the final common manifestation of chronic kidney disease that leads to end-stage renal disease.However,the pathogenesis and the initial molecular events leading to chronic renal fibrosis and eventually chronic renal failure remain to be fully understood.Recent studies have shown that angiotensin ?(Ang ?)contributes to renal fibrosis by upregulating profibrotic factors and inducing accumulation of extracellular matrix proteins.There are emerging evidences suggest that an aberrant activation of PI3Ky signaling may play an important role in regulating profibrotic phenotypes.In this study,we investigate whether a blockade of PI3Ky signaling exerts any beneficial effect on alleviating kidney injury and renal fibrosis by using an Ang ?-induced renal damage mouse model.Methods:1.Experiments in vivo:(1)A mouse model of angiotensin?(Ang ?)-induced renal damage was established by uninephectomy(UNX)on C57BL/6J mice and a continuous Ang II infusion(1.5 ?g/kg/min)via subcutaneous osmotic mini-pumps.(2)Mice were randomly divided into 3 groups,operated control group,Ang II only group,Ang II with AS605240 group.After 24 hours of the model construction finished,the mice in Ang ?/AS605240 group were orally administered(via gavage)with AS605240 at 50mg/(kg-d)and the rest of mice were given normal saline with the same volume.(3)Data were collected after 4 weeks of animal model establishment.Peripheral blood were collected from ocular enucleation,and used for detecting the levels of Scr,BUN.(4)After 4 weeks of gavage,all the mice in each group were sacrificed and perfused with PBS and the kidney tissues were retrieved for total RNA and protein isolations,as well as for histological analysis.(5)The renal injury and fibrosis were observed and detected by hematoxylin and eosin staining.Interstitial collagen deposition were detected by Masson and Pircosirius Red staining.Fibronectin?Colla??-SMA in kidney were marked and stained by antibodies.All the data were measured by using software Image Pro Plus 6.0,the positive data were calculated through the ratio of positive area to renal vertical section area.All the statistics were calculated by software SPSS 12.0.(6)The protein expression of fibronectin?Col1a??-SMA in kidney were also detected by Western Blot.The mRNA expression of IL-1??IL-6?TGF-?1?TNF-? were detected by PCR.All the statistics were calculated by software SPSS 12.0.2.Experiments in vitro:(1)Select one appropriate working concentration of Ang ? and AS605240 for the following experiments by crystal violet staining.(2)Ang ? stimulates NIH-3T3 cells for 0?24?48 hours.Crystal violet staining and reading was used to detect cell proliferation.Real-time PCR was applied to measure the mRNA level of fibronectin?Col1a??-SMA?CTGF.Western Blot was applied to detect the protein level of Akt/p-Akt.Results:1.Results in vivo:(1)Inhibition of PI3Ky signaling decreased the levels of Scr,BUN and improves renal functions in a mouse model of Ang ?-induced renal injury.(2)According to the results of H&E staining,the degree of renal injury was mitigated by PI3K? inhibitor AS605240.(3)Masson and Pircosirius Red staining revealed that Ang ? treatment led to a significant increase in interstitial collagen deposition in the kidney,compared with the Ang ?+AS605240 group(p<0.05).(4)Our immunohistochemical results indicated that Ang ? treatment led to a significant increase in the expression of interstitial fibronectin,type ?collagen and ?-SMA in the mouse kidney.However,the expression of fibronectin,type ? collagen and ?-SMA was significantly down-regulated by AS605240.Results of Western Blot were consistent with the IHC staining results.(4)The Ang ?-upregulated expression of IL-6,TNF-?,IL-1?,and TGF-?1 was significantly attenuated in the mice co-treated with Ang ?and AS605240.2.Results in vitro:(1)Crystal violet staining assay showed that Ang-? was able to stimulate NIH-3T3 cell proliferation at 24h and 48h time points,which was effectively blocked by PI3K? inhibitor AS605240.(2)Ang ? up-regulated the mRNA expression of myofibroblast marker?-SMA and fibrosis-related genes fibronectin,CTGF and Colla in NIH-3T3 cells in vitro.However,the Ang ?-induced expression of the above genes was effectively suppressed by PI3K? inhibitor AS605240.(3)Subconfluent NIH3T3 cells were serum-starved for 24h,and then treated with Ang ? and/or AS605240,or DMSO control for 48 hrs.The Western Blot results showed that the total Akt protein levels were similar among the three groups,the phosphorylated Akt(p-Akt)level was significantly increased in Ang ?-stimulated cells,which was effectively blocked by PI3K? inhibitor AS605240.Conclusions:1.Uninephectomy and Ang ? treatment accelerated the progression renal injury and fibrosis in C57BL/6J mice.2.PI3K? inhibitor AS605240 effectively mitigates Ang ?-induced increases in serum creatinine and blood urea nitrogen,renal interstitial collagen deposition,the accumulation of ECM proteins and the activation of myofibroblasts and the expression of fibrosis-related genes in vivo.3.We also demonstrated that Ang ?-upregulated expression of IL-6,TNF-?,IL-1? and TGF-?1 is significantly attenuated in the mice treated with AS605240.4.In addition,AS605240 effectively inhibits Ang ?-induced cell proliferation,fibrosis-related genes expression and phosphorylation of Akt in fibroblast cells.5.Taken together,our results demonstrate that PI3K? may function as a critical mediator of Ang ?-induced renal injury and fibrosis.It is thus conceivable that targeted inhibition of PI3K? signaling may constitute a novel therapeutic approach to the clinical management of renal fibrosis,renal hypertension and/or CKD. |
PDF Full Text Request