Microthrombotic Renal Vascular Lesions Are Associated to Increased Renal Inflammatory Infiltration in Murine Lupus Nephritis

Abstract

Background: Vascular microthrombotic lesions in lupus nephritis with or without antiphospholipid antibodies may relate to worse renal outcomes. Whether microthrombotic lesions are a consequence of renal inflammation or independently contribute to renal damage is unclear. Our aim was to investigate the relationship between microthrombotic renal vascular lesions and nephritis progression in MRL/lpr mice. Methods: MRL/lpr mice were analyzed for the presence of renal microvascular, glomerular and tubulointerstitial lesions and the effect of anti-aggregation (aspirin or clopidogrel) and dexamethasone on renal clinical and pathological manifestations was evaluated. Intravascular platelet aggregates (CD41), peri- (F4/80), and intraglomerular (Mac-2) macrophage infiltration, and C3 deposition were quantified by immunohistochemistry. Renal function was assessed by measuring proteinuria, and serum levels of creatinine and albumin. Anti-dsDNA and anti-cardiolipin antibodies, and thromboxane B2 levels were quantified by ELISA. Results: Frequency of microthrombotic renal lesions in MRL/lpr mice was high and was associated with immune-mediated renal damage. Proteinuria positively correlated with glomerular macrophage infiltration and was higher in mice with proliferative glomerular lesions. All mice had detectable anti-dsDNA and anti-cardiolipin IgG, regardless the presence of microthrombosis. Proteinuria and glomerular macrophage infiltration were significantly reduced in all treatment groups. Dexamethasone and platelet anti-aggregation similarly reduced glomerular damage and inflammation, but only platelet anti-aggregation significantly reduced anti-cardiolipin antibodies, renal complement deposition and thromboxane B2 levels. Conclusions: Platelet anti-aggregation reduced renal inflammatory damage, renal complement deposition, anti-cardiolipin antibodies, and thromboxane B2 levels and in MRL/lpr mice, suggesting that platelet activation has a pathogenic effect on immune-mediated nephritis. Our results point to MRL/lpr mice with lupus nephritis as an appropriate model to analyze the potential impact of anti-thrombotic intervention on renal inflammation.

Keywords: complement; inflammation; lupus nephritis; macrophages; microthrombosis; platelets.

Figure 1

Representative images of immunohistochemical detection in kidney biopsies of MRL/ lpr mice with LN. Immunolabeling detection of F4/80 periglomerular macrophagic deposits, Mac-2 intraglomerular macrophagic infiltration, CD41+ platelet aggregates, and C3 complement deposition in MRL/ lpr mice with LN. Bar 50 μm.

Figure 2

Positive associations between renal function measurement, vascular and immunohistochemical parameters in MRL/ lpr mice with LN. (A) Positive correlation between Mac-2 glomerular infiltration and degree of proteinuria in MRL/lpr mice with LN. Values obtained using the parametric Pearson test, ^**p ≤ 0.01. (B) Increased % of glomerular C3 deposition in mice with presence of microthrombotic lesions, measured as TMA, ^**p ≤ 0.01.

Figure 3

Effect of anti-inflammatories and anti-aggregants in MRL/ lpr mice with LN. (A) IHQ staining of Mac-2 (left), F4/80 (center), and C3 (right) in MRL/ lpr mice treated with Dexamethasone (Dexa), Aspirin (ASA), and Clopidogrel (Clop), compared to control group mice (PBS). (B) Anti-inflammatory and anti-aggregant effect in MRL/ lpr with LN in glomerular and periglomerular macrophagic deposits, determined as percentage of Mac-2 and F4/80, respectively, and glomerular complement deposition quantified as percentage of C3 area. Statistical analysis performed using U-Mann Whitney t-test for two independent samples or one-way ANOVA followed by Dunn's multiple comparison test, as needed. ^*p ≤ 0.05; ^**p ≤ 0.01.

Figure 4

Effect of treatment with anti-inflammatory and anti-aggregation agents in proteinuria and serum parameters in MRL/ lpr mice. (A) Levels of proteinuria (mg) in MRL/ lpr mice. Serum creatinin (mg/dl) levels (B), IgG anti-dsDNA (U/ml), and IgG anti-cardiolipin (U/ml) antibodies (C–D), and serum levels of TxB2 (pg/ml) (E) in MRL/ lpr mice. Statistical analysis performed using one-way ANOVA followed by Dunn's multiple comparison test, as needed. ^*p ≤ 0.05; ^**p ≤ 0.01.