Activation of Src kinase in platelet-derived growth factor-B-dependent tubular regeneration after acute ischemic renal injury

Abstract

We previously reported that the platelet-derived growth factor B-chain (PDGF-B)/PDGF receptor (PDGFR) axis is involved in tubular regeneration after ischemia/reperfusion injury of the kidney. In the present study, we examined the activation of Src tyrosine kinase, a crucially important signaling molecule for PDGFR, and assessed the role of Src in PDGF-B-dependent renal tubular regeneration afterischemia/reperfusion injury. Immunoblot using clone 28, a monoclonal antibody specific for the active form of Src kinases, demonstrated increased active Src expression in the injured rat kidney 6 hours after reperfusion with peak activation at 12 hours. In vitro kinase assay confirmed increased Src activity that concurred with PDGFR-beta activation as detected by the increment of receptor-phosphorylated tyrosine. Immunohistochemistry using clone 28 demonstrated that active Src was preferentially expressed in the S3 segment of the proximal tubule in reperfused kidney, where it is not normally expressed. This enhanced expression of active Src was co-localized with the increased PDGFR expression in the tubular cells that were undergoing cell proliferation cycle. Trapidil administration suppressed Src and PDGFR-beta activation in the reperfused kidney and resulted in deteriorated renal function. These findings suggest that active Src participates in PDGF-B-dependent regeneration of tubular cells from acute ischemic injury.

Figure 1.

Western blot analysis for Src kinase expression in the OSOM of the rat kidney after ischemia/reperfusion. A: Detection of the active form of Src kinase with clone 28. Representative results of three independent experiments are shown. Clone 28 detected a 60-kd protein that was found to correspond to the active form of Src kinase. B: Quantification of the signal intensities noted in independent experiments is summarized for the active form of Src kinase. Data are expressed as the mean ± SD (n = 3). Significant differences between the kidneys of sham-operated and injured rats are indicated by asterisks (*, P < 0.05; **, P < 0.01).

Figure 2.

The effect of inhibition of the PDGF-B/PDGFR axis on the expression of active Src kinase by Trapidil treatment in the OSOM of the reperfused ischemic kidney, as determined by Western blot analysis. A: A representative immunoblot analysis of active Src kinase expression 12 hours after insult. B: The mean results of three independent experiments are shown (mean ± SD, n = 3).

Figure 3.

In vitro Src kinase assay in the OSOM of the rat kidney after ischemia/reperfusion. A: The result of the assay was detected as a 45-kd protein of enolase. Representative results of three independent experiments are shown. The band in lanes 2 and 3 were derived from the two different injured rats at 12 hours after reperfusion without treatment of Trapidil. The band in lane 4 corresponded to Trapidil-treated rats with ischemic injury at 12 hours after reperfusion. B: Quantification of the signal intensities noted in independent experiments is summarized for the active form of Src kinase. Data are expressed as the mean ± SD (n = 3). I/R, ischemia/reperfusion injury: T, Trapidil.

Figure 4.

PDGFR-β activity was examined by immunoprecipitating the receptor and immunoblotting with antibody against phosphotyrosine. A: The phosphorylated PDGFR-β was detected. Representative results of three independent experiments are shown. Middle band was derived from the nontreated injured rats at 12 hours after reperfusion. Right band corresponded to PDGFR-β activity in the kidney of Trapidil-treated rats with ischemic injury at 12 hours after reperfusion. B: Quantification of the signal intensities noted in independent experiments is summarized for phosphorylated PDGFR-β expression. Data are expressed as the mean ± SD (n = 3). I/R, ischemia/reperfusion injury: T, Trapidil.

Figure 5.

A–F: Tissue distribution of the active form of Src kinase in the normal rat kidney. Expression of the active form of Src kinase was detected in the cytoplasm of the distal tubuli, the thick ascending limb, and the collecting ducts. However, the S3 segment of the proximal tubule did not stain positively for active Src kinase. The convoluted proximal tubule of the cortex was also completely negative for active Src kinase (A). The proximal tubule of the S3 segment in the outer stripe of the outer medulla was also negative (B). The thick ascending limb was positive in the inner stripe of the outer medulla (C). Strong immunoreactivity was observed in the collecting duct of the inner medulla (D). E: Tissue distribution of the active form of Src kinase 12 hours after reperfusion of the kidney after acute ischemic injury. The cytoplasm of injured PTCs in the S3 segment showed strong immunoreactivity for active Src kinase (arrowheads). F: The active Src expression in Trapidil-treated injured kidney at 12 hours after reperfusion was also localized in the S3 segments of PTCs of the OSOM (arrowheads). Note that the distribution was identical as seen in the postischemic kidneys without Trapidil treatment (E), however, the staining was weaker (F). CX, cortex; OS, outer stripe of the outer medulla; IS, inner stripe of the outer medulla; IM, inner medulla; G, glomerulus; DT, distal tubule; TAL, thick ascending limb; CD, collecting duct. A representative immunohistochemical staining from six independent experiments is shown. Original magnifications: × 100 (A–D); ×200 (E and F).

Figure 6.

Immunofluorescence staining for both active Src and PDGFR-β 12 hours after ischemia/reperfusion. PDGFR-β expression (green, A) and active Src kinase expression (red, B) were observed in the S3 segments of tubular cells. Superimposition of the active Src and PDGFR-β images produced by immunofluorescence staining showed yellow fluorescence in the cytoplasm of many cells, indicating co-localization of PDGFR-β and active Src kinase (C).

Figure 7.

Staining for both active Src kinase and PCNA 48 hours after ischemia/reperfusion. Note that tubular cells in the S3 segment showed concomitant expression of PCNA (dark brown in color) in their nuclei and active Src kinase (light brown in color) in their cytoplasm (A; arrowheads). In the kidneys of sham-operated rats, tubular cells in the S3 segment did stain positively for neither active Src kinase nor PCNA (B). Original magnifications: ×200 (A); ×100 (B).

Figure 8.

The effect of inhibition of the PDGF-B/PDGFR axis on cellular proliferation in the OSOM of the reperfused ischemic kidney. Note that fewer PTCs in the S3 segment were positive for PCNA in the reperfused ischemic kidney after treatment with Trapidil (A), as compared to reperfused kidney but not treated by Trapidil (B). None of the cells were positively stained for PCNA in normal control kidney (C). Original magnifications: ×40 (A, B, C) or (A–C).