Role of PDGF B-chain and PDGF receptors in rat tubular regeneration after acute injury

Abstract

Various polypeptide growth factors are generally considered to be involved in the regulation of the nephrogenic process both after acute renal injury and during renal development. Because platelet-derived growth factor B-chain (PDGF-B) has been reported to be expressed in immature tubulus of the developing kidney, PDGF-B could play a role in the process of tubulogenesis. We examined the expression of PDGF-B and PDGF receptors alpha and beta and their localization in kidneys after ischemia/reperfusion injury. The mRNA expressions of PDGF-B, PDGFR-alpha, and PDGFR-beta were enhanced after injury. In the immunohistochemical analysis and/or in situ hybridization, PDGF-B and PDGFR-alpha, beta were expressed after reperfusion in the S3 segment of the proximal tubuli, where they were not expressed normally. The expressions of proliferating cell nuclear antigen and vimentin were concomitantly observed with PDGF-B and PDGFRs in the tubular cells of injured S3 segment at 48 hours after injury. Next, the inhibition of the PDGF-B/PDGFRs axis with either Trapidil or Ki6896, which was found to inhibit the phosphorylation of PDGFR-beta selectively, resulted in a rise of serum creatinine, higher mortality rate, abnormal regenerating process, and suppressed proliferation of tubular epithelial cells. These findings suggest that the PDGF-B/PDGFRs axis is involved in the proliferation of injured tubular cells and plays an important role in the regeneration of tubular cells from acute ischemic injury.

Figure 1.

Northern analysis for PDGF-B and PDGFRs in rat kidney after ischemia/reperfusion. Representative result of independent experiments are shown in A. The means and SE bars of the signal intensities from independent experiments are summarized for PDGF-B (B), PDGFR-β (C), and PDGFR-α (D). Data are expressed as mean ± SE (n = 3–7). Significant difference between control and injured rat groups is indicated by asterisks (*, P < 0.05; **, P < 0.01)

Figure 2.

Immunohistochemistry for PDGF-B in rat kidney. In the control kidney (A; ×50), immunoreactive products were recognized in the convoluted proximal tubuli and distal tubuli. However, the S3 segment of the proximal tubuli was completely negative for PDGF-B. In kidneys at 6 hours after ischemia/reperfusion (B; ×100), some cells of the S3 segment were positive for PDGF-B. The TAL adjacent to the injured tubuli showed strong immunoreactivities. In kidneys at 48 hours (C; ×100), injured proximal tubuli were positive. MR, Medullary ray; S3, the S3 segment of the proximal tubuli; TAL, the thick ascending limb.

Figure 3.

In situ hybridization for expression of PDGF-B mRNA in rat kidney after ischemia/reperfusion. Strong mRNA signals were detected in the injured tubuli of the outer stripe of the outer medulla, and moderate signals were observed in the tubuli adjacent to the injured tubuli (A; ×50). Signals of PDGF-B were not observed in the preparation hybridized with sense probe (B; ×50).

Figure 4.

Immunohistochemistry for PDGFR-β in the normal kidney. Immunoreactivities for PDGFR-β were observed in the glomerulus, the distal tubuli, the TAL, and the collecting ducts. The convoluted proximal tubuli in the cortex were completely negative (A; ×100). The proximal tubuli of the S3 segment in the outer stripe of the outer medulla were also negative (B; ×50). The TAL was positive in the inner stripe of the outer medulla (C; ×100). The strong immunoreactivity was observed in the collecting ducts of the inner medulla (D; ×100). Cx, cortex; OS, outer stripe of the outer medulla; IS, inner stripe of the outer medulla; IM, inner medulla; G, glomerulus; DT, distal tubule; CD, collecting duct.

Figure 5.

Immunohistochemistry for PDGFR-β in the injured tubuli after ischemia/reperfusion. In low power view (×15) of kidneys 6 hours after ischemia/reperfusion (A), immunoreactive products for PDGFR-β were observed in some of the proximal tubuli of the S3 segments both in the medullary ray and in the outer stripe of the outer medulla. In high power view (B; ×100), immunoreactivities for PDGFR-β appeared in some cells of the S3 segment of the proximal tubuli. In low power view (×15) of kidneys 48 hours after ischemia/reperfusion (C), intense signals were observed in most of the proximal tubuli of the S3 segments both in the medullary ray and in the outer stripe of the outer medulla. In high power view (D; ×100), strong immunoreactivities were recognized in the whole cytoplasm of proximal tubular cells in the S3 segments. MR, medullary ray; OS, outer stripe of the outer medulla.

Figure 6.

Immunohistochemistry for PDGF-B/PDGFRs, PCNA, and vimentin in rat kidney after ischemia/reperfusion. In kidneys 48 hours after ischemia/reperfusion, expression of PDGF-B (A; ×100) and PCNA (B; ×100;) was concomitantly observed in the injured tubular cells of the S3 segment. In the S3 segment, PDGFR-β (C; ×150) was observed in accordance with PCNA expression (D; ×150). The immunoreactivity for vimentin (E; ×150) was observed in the tubular cells where PDGFR-β immunoreactivity was strong, although tubular cells with weak immunoreactivity for PDGFR-β were negative for vimentin. At 48 hours after ischemia/reperfusion, PDGFR-α (F; ×100) was observed in accordance with PCNA expression (G; ×100) in the S3 segment.

Figure 7.

The effect of the inhibition of the PDGF/PDGFRs axis on the concentrations of serum creatinine in rats with ischemia/reperfusion injury. A: Serum creatinine values in injured rats treated with Trapidil on day 1. B: Time course of serum creatinine values in injured rats treated with Ki 6896. (*, P < 0.05; **, P < 0.01). ARF, acute renal failure due to ischemia/reperfusion.

Figure 8.

The effect of the inhibition of PDGF/PDGFRs axis on morphology in rats with ischemia/reperfusion injury at day 6. In HE staining, hypertrophic tubular epithelial cells are clustered and obliterate tubular lumina in rats treated with Ki 6896 (A; ×25, B; ×100). Tubular lamina were well preserved in vehicle groups, although they included papillary regenerating epithelial cells and cellular debris (C; ×25, D; ×50). PCNA-positive cells were scattered within single-layered tubule-lining epithelial cells at low frequency, mainly in S3 segment of Ki 6896 group (E; ×50). In contrast, numerous PCNA-positive cells were distributed within the surface-lining epithelial cells frequently, not only in S3 but also cortical area in vehicle group. Furthermore, PCNA-positive cells was often clustered in multilayered concentric form in the regenerating tubules of vehicle group (F; ×50).