Platelets are relevant mediators of renal injury induced by primary endothelial lesions

Abstract

Several studies have suggested a prominent (pro)inflammatory and harmful role of platelets in renal disease, and newer work has also demonstrated platelet release of proangiogenic factors. In the present study, we investigated the role of platelets in a mouse model of selective endothelial cell injury using either platelet depletion or the pharmacological P2Y12 receptor blocker clopidogrel as an interventional strategy. The concanavalin A/anti-concanavalin A model was induced in left kidneys of C57bl/6J wild-type mice after initial platelet depletion or platelet-inhibiting therapy using clopidogrel. FACS analysis of glycoprotein IIb/IIIa/P-selectin double-positive platelets and platelet-derived microparticles demonstrated relevant platelet activation after the induction of selective endothelial injury in mice. Enhanced platelet activation persisted for 5 days after disease induction and was accompanied by increased amounts of circulating platelet-derived microparticles as potential mediators of a prolonged procoagulant state. By immunohistochemistry, we detected significantly reduced glomerular injury in platelet-depleted mice compared with control mice. In parallel, we also saw reduced endothelial loss and a consequently reduced repair response as indicated by diminished proliferative activity. The P2Y12 receptor blocker clopidogrel demonstrated efficacy in limiting platelet activation and subsequent endothelial injury in this mouse model of renal microvascular injury. In conclusion, platelets are relevant mediators of renal injury induced by primary endothelial lesions early on, as demonstrated by platelet depletion as well as platelet inhibition via the P2Y12 receptor. While strategies to prevent platelet-endothelial interactions have shown protective effects, the contribution of platelets during renal regeneration remains unknown.

Keywords: clopidogrel; platelet microparticles; platelets; renal microvascular endothelial injury; thrombotic microangiopathy.

Fig. 1.

Platelet activation and generation of platelet-derived microparticles (PMPs) after selective endothelial cell (EC) injury. A and B: in sham-operated mice (A) as well as in mice with selective EC injury (B), FACS analysis detected three distinct populations consisting of microparticles (MPs), platelets (PLs), and immune complexes (ICs). d, day. C: the numer of P-selectin positive, CD41-positive platelets was significantly higher in both groups undergoing EC injury compared with the sham control group, whereas no differences could be detected between the groups with and without clopidogrel (CLOPI) treatment (data table). D: in parallel, large amounts of microparticles were generated in mice after the induction of selective EC injury (data table), without differences between clopidogrel-treated and untreated mice. ***P < 0.001 and **P < 0.001 by two-way ANOVA.

Fig. 2.

Reduced glomerular thrombus formation due to reduced injury after platelet depletion. A: staining for glycoprotein (GP)Ibα in a control (Ctrl) mouse 24 h after disease induction. B: platelet-derived thrombi and fibrin-positive glomeruli were assessed after acid fuchsin orange G (AFOG) staining. C and D: the deposition of platelets (C) and fibrin-rich thrombi (D) in glomeruli of platelet-depleted (PLT depl) animals compared with PBS-injected control animals was reduced. **P < 0.01.

Fig. 3.

Reduced capillary rarefaction and necessity of repair after platelet depletion. A: renal tissue sections were double stained with anti-mouse endothelial cell antigen (MECA)-32 and anti-PCNA antibodies to identify proliferating ECs. A control mouse 24 h after disease induction is shown. B: endothelial rarefaction was reduced in platelet-depleted mice. C: proliferating ECs were assessed as PCNA-positive/MECA-32-positive cells. D: proliferative response of tubulointerstitial cells was evaluated after PCNA staining. **P < 0.01; #f < 0.05. n.s., Not significant.

Fig. 4.

Clopidogrel treatment protects from glomerular injury and reduces the inflammatory response. A and B: for the evaluation of glomerular injury, we used Periodic acid-Schiff-stained tissue sections (A) and a semiquantitative scoring system from 0 to 4 representing the degree of injury (B). C: fibrin thrombus formation was evaluated after AFOG staining. D: monocytes/macrophages were evaluated after staining for F4/80. *P < 0.05 and ***P < 0.001; #P < 0.05 by Mann-Whitney U-test.

Fig. 5.

Clopidogrel treatment protects renal capillaries. A and B: staining of CD31 (A) was used to evaluate glomerular capillary rarefaction (B). C: tubulointerstitial capillary rarefaction was assessed after staining for MECA-32 as described in methods. D–F: glomerular (D), tubulointerstitial (E), and EC proliferation (MECA-32-positive/PCNA-positive cells) were evaluated after double staining for MECA-32 and PCNA (F). *P < 0.05 and **P < 0.01; ##P < 0.01 by by Mann-Whitney U-test.