The Role And Mechanism Of C-Src Signaling Pathway In Diabetic Nephropathy

Objectives : Diabetes mellitus(DM) is a chronic metabolic disorder characterized by hyperglycemia in the context of insulin resistance(IR) and insulin secretion deficiency due to β-cell dysfunction. The global prevalence of type 2 diabetes mellitus(T2DM) is continuously rising, being held responsible for about 90% of all the 347 million diabetes cases worldwide. DM can cause serious microvascular and macrovascular complications, such as diabetic nephropathy(DN), diabetic retinopathy, diabetic neuropathy, ischemic heart disease and cerebrovascular disease. Therefore, the development of safe and effective drugs for DM is imperative, for which the molecular level target therapy represents a promising approach.As a major microvascular complication of DM and the leading cause of end-stage renal disease, DN affects about one third of patients with type 1 or T2 DM. For T2 DM specifically, prevalence rate ranges from 6.6% to 18.51%, as reported in China. Growing evidence has shown that tubulointerstitial injury is also an important feature of DN and a better predictor of renal dysfunction than glomerular damage. Several studies with substantial specimen counts have indicated that the apoptosis of renal tubular epithelial cells(RTEC) is frequently observed in kidney sections from humans, mice and rats with DM, suggesting that the apoptosis of RTEC is a hallmark of DN and a reliable indicator of disease progression. Moreover, accumulating evidences indicated that hyperglycemia could induce RTEC injury and in turn lead to renal dysfunction. However, the mechanism of how hyperglycemia causes RTEC injury remains poorly understood.c-Src is a member of the Src tyrosine kinase family that is involved in many cellular events such as mitosis, cell growth and tumorigenesis. c-Src is phosphorylated and activated in response to a variety of external stimuli, and subsequently mediates intracellular signal transduction by phosphorylating tyrosine residues of numerous cellular cytosolic, nuclear and membrane proteins. Specifically, its activity is reportedly enhanced in the kidneys of diabetic animals, glomerular mesangial cells and podocytes exposed to high glucose(HG). Therefore, c-Src is now widely acknowledged as an important regulators implicated in DN. However, the exact role of c-Src signaling pathway on the injury of RTEC and pathogenesis of DN still remains to be defined.In the present study, we investigate the correlation between HG-stimulated c-Src signaling pathway activation and the renal injury. In vivo, using c-Src inhibitor PP2, we will explore the impact c-Src signaling pathways on renal dysfunction and cellular apoptosis. In vitro, we will investigate the role and molecular mechanisms of c-Src signaling pathway in apoptosis of renal tubular epithelial cells treated with HG. This study will shed light on effective therapeutic targets for the precision management of DN.Methods:1 The role of c-Src signaling pathway in renal injury of diabetic db/db miceMale BKS db/db mice in C57BL6 background and non-diabetic littermate control db/m mice were purchased from the Model Animal Research Center of Nanjing University. According to the guidelines of National Institute of Health, all experimental animals were housed in the specific-pathogen-free(SPF) conditions with clean sawdust bedding. All procedures involving mice were reviewed and approved by the Ethics Committee of Hebei Medical University. At Eight weeks of age, the mice were randomly divided into three groups: control group(db/m mice, n=10), diabetes group(db/db mice, n=10), and PP2 group(db/db mice treated with PP2, n =10). The PP2 group received 2 mg/kg PP2 dissolved in DMSO every other day by i.p. injection, as previously described. Mice in the control group or diabetes group received the same amount of normal saline. At the age of 16 weeks, the animals were individually housed in metabolic cages for 24 h urine collection. At the end of the experiments, the mice were fasted for 6 h before collecting blood samples. The plasma and urinary supernatants were frozen and stored at-80℃ until further biochemical analysis. Subsequently, the animals were sacrificed and the kidneys were harvested. Partial renal cortices were fixed in 4% glutaraldehyde for electron microscopic observation. Partial renal tissures were fixed in 4% neutral formalin for histochemical and immunohistochemical staining. The total proteins of the renal cortical tissues were extracted, and the expression of phospho-Src, total c-Src, phospho-p38 MAPK, total p38 MAPK, Cleaved caspase-3, Bax, Bcl-2 and β-actin protein was analyzed by Western blotting, respectively.2 The role of c-Src/p38 MAPK pathway in HK-2 cells apoptosis induced by HGThe HK-2 cells were obtained from American Type Culture Collection(ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium(DMEM) supplemented with 10% fetal bovine serum, 1% streptomycin-penicillin mixture in a 95% air and 5% CO2 atmosphere at 37℃. The culture mediumwas replaced every two days. After reaching 70-80% confluence, the cells were washed once with serum-free DMEM medium, and then growth-arrested in serum-free DMEM medium for 24 h to synchronize the cell growth. The cells were randomly divided into 7 group: normal glucose group(5.6 mmol/L glucose, NG), normal glucose plus mannitol group(5.6 mmol/L glucose+24.5 mmol/L mannitol, M), normal glucose plus PP2 group(5.6 mmol/L glucose+2 μmol/L PP2, NG+PP2), normal glucose plus SB203580 group(5.6 mmol/L glucose+10 μmol/L SB203580, NG+SB203580), high glucose group(30 mmol/L glucose, HG), high glucose plus PP2 group(30 mmol/L glucose+2 μmol/L PP2,HG+PP2) and high glucose plus SB203580 group(30 mmol/L glucose+10 μmol/L SB203580,HG+SB203580). PP2 and SB203580 were added to the culture medium 2 h prior to high glucose. The cells were cultured for 48 h, and HK-2 cells were subsequently harvested. The total proteins of HK-2 cells were extracted, and the expression of phospho-Src, total c-Src, phospho-p38 MAPK, total p38 MAPK, PPARγ, CHOP, Cleaved caspase-3, Bax, Bcl-2 and β-actin protein was analyzed by Western blotting, respectively. HG-induced c-Src and p38 MAPK activation in HK-2 cells were analyzed by cellular immunofluorescence. Apoptosis of HK-2 cells were also determined by TUNEL assay and flow cytometry analysis, respectively.3 The role of c-Src/EGFR pathway in HK-2 cells apoptosis induced by high glucose.The HK-2 cells were obtained from American Type Culture Collection(ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium(DMEM) supplemented with 10% fetal bovine serum, 1% streptomycin-penicillin mixture in a 95% air and 5% CO2 atmosphere at 37℃. The culture mediumwas replaced every two days. After reaching 70-80% confluence, the cells werewashed once with serum-free DMEM medium, and then growth-arrested in serum-free DMEM medium for 24 h to synchronize the cell growth. The cells were randomly divided into 4 group: normal glucose group(5.6 mmol/L glucose, NG), normal glucose plus mannitol group(5.6 mmol/L glucose+24.5 mmol/L mannitol, M), high glucose group(30 mmol/L glucose, HG) high glucose plus AG1478 group(30 mmol/L glucose+0.2 mmol/L AG1478,HG+ AG1478). AG1478 was added to the culture medium 2 h prior to HG. The cells were cultured for 48 h, and HK-2 cells were subsequently harvested. The total proteins of HK-2 cells were extracted, and the expression of phospho-EGFR, total EGFR, GRP78, CHOP, Cleaved caspase-3, Bax, Bcl-2 and β-actin protein was analyzed by Western blotting, respectively. Apoptosis of HK-2 cells were also determined by flow cytometry analysis. The viability of HK-2 cells was analyzed by MTT analysis.Results:1 The role of c-Src signaling pathway in renal injury of diabetic db/db mice(1) Body weights, food intake, kidney weights and daily urinary volumes were significantly higher in diabetic group than in control group(P<0.05 or 0.01) but not kidney weights/ body weights ratio(P<0.01). Treatment of db/db mice with PP2 attenuated the increases in kidney weights, kidney weights/ body weights ratio and daily urinary volumes associated with diabetes(P<0.05 or 0.01). However, the treatment with PP2 significantly increased db/db mice body weight and food intake(P<0.01).(2) All db/db mice displayed increased blood glucose levels compared with db/m mice(P<0.01). Incidentally, the db/db mice treated with PP2 displayed slightly decreased blood glucose levels(P<0.05). Blood urea nitrogen, serum creatinine, triglyceride and urine albumin excretion were significantly higher in the db/db group than in db/m group(P<0.05 or 0.01), which were fully recovered to the db/m level by PP2(P<0.05 or 0.01).(3) As examined by immunohistochemical staining, the expression level of the c-Src activation marker phospho-Src, was found to be notably upregulated in the renal proximal tubules of db/db mice, which could be markedly ameliorated by PP2 treatment.(4) As examined by Western blotting, the phospho-Src/total-c-Src ratio in db/db mice, which was significantly higher than that in db/m mice(P<0.05), could be restored by PP2 treatment(P<0.05). Moreover, the higher phospho-p38/total-p38 ratio in db/db mice(P<0.05) could also be decreased by PP2(P<0.01).(5) The Bax/Bcl-2 ratio and caspase-3 cleavage were markedly increased in db/db mice than in the db/m mice(P<0.01), indicating enhanced intrinsic apoptosis, whereas PP2 treatment significantly reversed these changes(P<0.01).2 The role of c-Src/p38 MAPK pathway in HK-2 cells apoptosis induced by high glucose(1) HK-2 cells incubated with HG showed a time-dependent increase of c-Src phosphorylation at Tyr416, which was most prominent in the first 12 h, and sustained to at least 48 h(P<0.05 or 0.01). Moreover, the phospho-p38/total-p38 ratio was also increased in HK-2 cells incubated with high concentrations of glucose, in a time dependent manner(P<0.05 or 0.01).(2) Compared with NG groups, exposure of HK-2 cells to HG for 48 h significantly increased phosphorylation of c-Src at Tyr416(P<0.05), which was significantly inhibited by PP2(P<0.05). It also markedly reduced the HG-induced p38 MAPK phosphorylation(P<0.05). Moreover, the immunofluorescence microscopy observation is consistent with the Western blotting results. As an osmotic control, mannitol had no effect on activation of c-Src and p38 MAPK.(3) Compared with NG groups, exposure of HK-2 cells to HG for 48 h significantly increased phosphorylation of c-Src at Tyr416(P<0.01), whereas SB203580 treatment significantly prevented HG-induced c-Src phosphorylation(P<0.05). In addition, we further examined the role of SB203580 on two downstream targets of PPARγ and CHOP, in HK-2 cells incubated with high concentrations of glucose. Exposure of HK-2 cells to HG for 48 h significantly increased the expression of PPARγ and CHOP(P<0.05), which could be significantly attenuated by SB203580(P<0.05 or 0.01). As an osmotic control, mannitol showed no such effect.(4) Exposure of HK-2 cells to HG significantly increased the Bax/Bcl-2 ratio and caspase-3 cleavage(P<0.05), which was markedly attenuated by PP2 and SB203580(P<0.05). As an osmotic control, mannitol had no such effect. Apoptosis of HK-2 cells were also determined by TUNEL assay and flow cytometry analysis, respectively. HG induced more HK-2 cell apoptosis than did normal glucose(P<0.01), which could be ameliorated by either PP2 or SB203580(P<0.05), as was confirmed by the Annexin V-FITC and PI staining.(5) In addition, we further assessed whether a combination of PP2 and SB203580 can have an additive/synergistic effect on the HG-induced apoptosis in HK-2 cells. Although this combination led to a mild increase of Bax/Bcl-2 ratio and Cleaved caspase-3 expression compared with PP2 or SB203580 alone, the differences did not reach statistical significance.3 The role of c-Src/EGFR pathway in HK-2 cells apoptosis induced by high glucose(1) HK-2 cells incubated with HG showed a time-dependent increase of EGFR phosphorylation at Tyr1068, which was most prominent in the first 6 h, and sustained to at least 48 h(P<0.01).(2) Compared with NG groups, exposure of HK-2 cells to HG for 48 h significantly increased phosphorylation of EGFR at Tyr1068(P<0.01), which was significantly inhibited by PP2(P<0.01). As an osmotic control, mannitol had no effect on activation of EGFR.(3) Compared with NG groups, exposure of HK-2 cells to HG for 48 h significantly increased phosphorylation of EGFR at Tyr1068(P<0.01), which was significantly inhibited by AG1478(P<0.05). As an osmotic control, mannitol had no effect on activation of EGFR.(4) In addition, we further examined the role of AG1478 on two downstream targets of GRP78 and CHOP in HK-2 cells incubated with high concentrations of glucose. Exposure of HK-2 cells to HG for 48 h significantly increased the expression of GRP78 and CHOP(P<0.01), which could be significantly attenuated by AG1478(P<0.05). As an osmotic control, mannitol showed no such effect.(5) Exposure of HK-2 cells to HG significantly increased the Bax/Bcl-2 ratio and caspase-3 cleavage(P<0.01), which was markedly attenuated by AG1478(P<0.05 or 0.01). As an osmotic control, mannitol had no such effect. Apoptosis of HK-2 cells were also determined by flow cytometry analysis. HG induced more HK-2 cell apoptosis than did normal glucose, which could be ameliorated by AG1478.(6) In addition, we further examined the role of AG1478 on cellular viability of HK-2 cells analyzed by MTT. Exposure of HK-2 cells to HG significantly decreased the cellular viability of HK-2 cells(P<0.01), which was markedly improved by AG1478(P<0.05). As an osmotic control, mannitol had no such effect.Conclusions:1 In vivo, inhibition of c-Src activity by PP2 significantly improves metabolic parameters and preserves renal function in diabetic db/db mice. PP2 treatment can not only attenuate HG-induced c-Src and p38 MAPK activation, but also disease the cellular apoptosis in type 2 diabetic kidneys.2 In vitro, HK-2 cells incubated with HG showed a time-dependent increase of c-Src phosphorylation at Tyr416 and p38 MAPK phosphorylation at Thr180/Tyr182. As an upstream regulator, inhibition of c-Src attenuated HG-induced p38 MAPK activation. Inhibition of p38 MAPK not only attenuated HG-induced c-Src activation, but also abrogated the expression of PPARγ and CHOP in HK-2 cells. Inhibition of c-Src/p38 MAPK signaling pathway significantly inhibited HG-induced HK-2 cells apoptosis.3 In vitro, HK-2 cells incubated with HG showed a time-dependent increase of EGFR phosphorylation at Tyr1068. As an upstream regulator, inhibition of c-Src attenuated HG-induced EGFR activation. Inhibition of EGFR attenuated HG-induced expression of GRP78 and CHOP in HK-2 cells. Inhibition of c-Src/EGFR signaling pathway not only significantly inhibited HG-induced HK-2 cells apoptosis, but also markedly improved cellular viability of HK-2 cells.