Inhibition of c-Src/p38 MAPK pathway ameliorates renal tubular epithelial cells apoptosis in db/db mice

Highlights

• Inhibition of c-Src activity improves metabolic parameters and prevents renal cell apoptosis in diabetic db/db mice.

• Inhibition of c-Src activity inhibits phosphorylation of c-Src and p38 MAPK in the kidneys of diabetic db/db mice.

• Inhibition of c-Src activity prevents HG-induced c-Src and p38 MAPK activation in HK-2 cells.

• Inhibition of p38 MAPK activity prevents HG-induced c-Src activation and expression of PPARγ and CHOP in HK-2 cells.

• Inhibition of c-Src or p38 MAPK activity prevents HG-induced apoptosis of HK-2 cells.

Abstract

Renal tubular epithelial cells (RTEC) apoptosis, which plays a key role in the pathogenesis and progression of diabetic nephropathy (DN), is believed to be contributive to the hyperglycemia-induced kidney failure, though the exact mechanisms remain elusive. In this study, we investigated how inhibition of c-Src/p38 MAPK pathway would affect RTEC apoptosis. The c-Src inhibitor PP2 i.p. administered every other day for 8 weeks to diabetic db/db mice significantly reduced their kidney weights, daily urinary volumes, blood glucose, blood urea nitrogen, serum creatinine, triglyceride and urine albumin excretion, whereas deactivation of c-Src and p38 MAPK were also observed, along with decreases in both Bax/Bcl-2 ratio and cleaved caspase-3 level in the kidneys. In vitro, exposure of HK-2 cells (a human RTEC line), to high glucose (HG) promoted phosphorylation of c-Src and p38 MAPK, and subsequently, as revealed by western blotting, TUNEL assay and flow cytometry, increased cell death, which can be inhibited by PP2. Especially, a specific p38 MAPK inhibitor, SB203580, that both attenuated HG-induced c-Src activation and abrogated the expression of PPARγ and CHOP, also reduced apoptosis. Taken together, PP2 inhibits c-Src and therefore reduces apoptosis in RTEC, which at least in part, is due to suppressed p38 MAPK activation in diabetic kidney.

Introduction

As a major microvascular complication of diabetes mellitus (DM) and the leading cause of end-stage renal disease (Barnett, 2006, Foundation, 2012), DN affects about one third of patients with type 1 or type 2 DM (Reutens and Atkins, 2011). For type 2 DM specifically, prevalence rate ranges from 6.6% to 18.51%, as reported in China (Lu et al., 2009, Xu et al., 2012). Although DN is always characterized by glomerular hypertrophy, mesangial cell proliferation, glomerular sclerosis and interstitial fibrosis, glomerular basement membrane (GBM) thickening, growing evidence has shown that tubulointerstitial injury is also a important feature of DN and a better predictor of renal dysfunction than glomerular damage (Gilbert and Cooper, 1999). Indeed, the renal tubular cell injuries, i.e. tubular hypertrophy, reduced organic ion transport and tubular atrophy, have been recognized a key to the pathogenesis and progression of DN. Several studies with substantial specimen counts have indicated that the apoptosis of 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 (Brezniceanu et al., 2010). Moreover, accumulating evidences indicated that hyperglycemia could induce RTEC injury and in turn lead to renal dysfunction (Abdo et al., 2013, Chacko et al., 2010, Shi et al., 2015). 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 (Shalloway et al., 1992, Summy and Gallick, 2003). To maintain normal cell proliferation, differentiation and apoptosis, the activity of c-Src has to be tightly regulated, wherein two phosphorylation sites, Tyrosine 416 (Thr416) and Tyrosine 527 (Th527), are crucial. The phosphorylation at Thr416 activates c-Src, whereas that at Th527 suppresses it (Irtegun et al., 2013). Therefore, the Thr416 phosphorylation level is a common indicator of c-Src activation. 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 (Bacarizo et al., 2014). Specifically, c-Src and serine/threonine protein kinase p38 MAPK have been demonstrated to enhance each other's activity in a positive feedback loop, wherein c-Src stimulates p38 MAPK phosphorylation (Cheng et al., 2010, Lin et al., 2015), and p38 MAPK in turn promotes c-Src autophosphorylation (Mugabe et al., 2010, Yaghini et al., 2007).

As a member of the MAPK family (also including at least JNK, ERK1/2 and ERK5), p38 MAPK is specifically activated by phosphorylation in response to a variety of stress stimuli. Several studies have implicated a critical role of the p38 MAPK in pathological conditions including nephropathy. Indeed, increased phosphorylation of p38 MAPK has been observed in renal biopsy specimens from different types of glomerulonephritis patients, and the phosphorylation level of p38 MAPK correlates with renal dysfunction and histopathology (Stambe et al., 2004). In addition, the same research team also reported that administration of a specific p38 MAPK inhibitor, NPC 31145, prevented renal function loss and substantially reduced proteinuria (Stambe et al., 2003). Mechanistically, inhibition of p38 MAPK activity can markedly decrease the secretion of interleukin-1β, tumor necrosis factor-α and monocyte chemoattractant protein-1, and therefore ameliorate renal injury in mice (Furuichi et al., 2002). Collectively, these findings strongly suggest that the p38 MAPK pathway may be closely associated with renal injury and dysfunction. However, the exact role of c-Src/p38 MAPK 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 p38 MAPK activation and the c-Src pathway, and evaluated the effects of PP2 treatment on renal function and RTEC apoptosis in diabetic db/db mice, aiming to unravel its working mechanisms and to evaluate its clinical potential.