Absence of the Lectin Activation Pathway of Complement Ameliorates Proteinuria-Induced Renal Injury

Abstract

Proteinuria is an adverse prognostic feature in renal diseases. In proteinuric nephropathies, filtered proteins exert an injurious effect on the renal tubulointerstitium, resulting in inflammation and fibrosis. In the present study, we assessed to what extent complement activation via the lectin pathway may contribute to renal injury in response to proteinuria-related stress in proximal tubular cells. We used the well-established mouse model of protein overload proteinuria (POP) to assess the effect of lectin pathway inhibition on renal injury and fibrotic changes characteristic of proteinuric nephropathy. To this end, we compared experimental outcomes in wild type mice with MASP-2-deficient mice or wild type mice treated with MASP-2 inhibitor to block lectin pathway functional activity. Multiple markers of renal injury were assessed including renal function, proteinuria, macrophage infiltration, and cytokine release profiles. Both MASP-2-deficient and MASP-2 inhibitor-treated wild type mice exhibited renoprotection from proteinuria with significantly less tubulointerstitial injury when compared to isotype control antibody treated mice. This indicates that therapeutic targeting of MASP-2 in proteinuric nephropathies may offer a useful strategy in the clinical management of proteinuria associated pathologies in a variety of different underlying renal diseases.

Keywords: MASP; MBL-associated serine proteases; kidney; lectin pathway; nephropathy; proteinuria.

Figure 1

Urine and serum parameters in mice with POP. WT and MASP-2^−/− mice were either left untreated (control) or subject to POP as described. (A) Serum total protein concentrations in control mice and mice with POP; (B) 24 h urine protein excretion in mice with POP; (C) serum creatinine levels in control mice and mice with POP. In all graphs, individual symbols represent values from individual animals measured in duplicate. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 by one-way ANOVA with Bonferroni post-hoc test for multiple comparisons.

Figure 2

Histological and ultrastructural appearance of kidneys from control mice and mice with POP. Kidneys from mice were sectioned and stained with H&E or prepared for electron microscopy (EM). (A) Represents H&E stained sections from untreated WT. (B) Represents H&E stained sections from untreated MASP-2-deficient mice. (C) Depicts H&E stained sections from proteinuric WT mice, whereas (D) depicts H&E stained sections from proteinuric MASP-2-deficient mice. (E) Represents sections examined by EM from proteinuric WT mice. (F) Represents sections examined by EM from proteinuric MASP-2-deficient mice. In H&E sections scale bar = 100 μm and in electron micrographs scale bar = 5 μm.

Figure 3

Macrophage infiltration in kidneys of mice with POP. Photomicrographs represent kidneys from animals with POP stained for macrophages. (A) Non-proteinuric wild type (WT) controls; (B) non-proteinuric MASP-2^−/− controls; (C) proteinuric WT mice with POP; (D) proteinuric MASP-2^−/− mice with POP; (E) negative control lacking primary antibody in a section from non-proteinuric WT mice Scale bar = 100 μm. Graph depicts quantification of macrophage infiltration in POP mice as described in Methods. Each data point represents values from an individual animal. ^*p < 0.05, by one-way ANOVA with Bonferroni post-hoc test for multiple comparisons.

Figure 4

Cell apoptosis in kidneys of mice with POP. Apoptotic cells in mouse kidneys were identified by TUNEL staining as per Methods. (A) Wild type (WT) control mice; (B) proteinuric WT mice with POP; (C) MASP-2^−/− control mice; (D) proteinuric MASP-2^−/− mice with POP. Arrows indicate examples of apoptotic cells, scale bar = 100 μm. Graph depicts quantification of apoptotic cells with each data point derived from an individual animal. ^***p < 0.0001 by one-way ANOVA with Bonferroni post-hoc test for multiple comparisons.

Figure 5

Investigation of mouse kidneys for inflammatory and pro-fibrotic mediators. IHC was performed for (A–E) TGFβ1; (F–J) TNFα; and (K–O) IL-6, in (A,F,K) depict wild type (WT) control mice; (B,G,L) depict MASP-2^−/− control mice; (C,H,M) depict WT mice with POP; and (D,I,N) depict MASP-2^−/− mice with POP; (E,J,O) depict negative controls where sections from WT mice lack primary antibody. Scale bar = 100 μm. Quantification of IHC staining is shown in graphs alongside their relevant IHC panels. Each data point is derived from an individual animal. ^*p < 0.05, ^**p < 0.01 by one-way ANOVA with Bonferroni post-hoc test for multiple comparisons.

Figure 6

Deposition of C3c as a measure of serum lectin pathway activity, serum creatinine levels, urine protein excretion, and histopathology in control and antibody treated POP WT mice. (A,B) Depict residual serum LP activity in each group of animals measured as deposition of C3c by ELISA using (A) BSA blocking buffer; (B) skimmed milk blocking buffer. Graphs (C,D) represent 24 h urine protein excretion and serum creatinine, respectively, in proteinuric animals injected with saline, isotype control antibodies, or HG4 antibodies as labeled. Each data point depicts serum analysis from an individual animal. ^***p < 0.0001 by one-way ANOVA with Bonferroni post-hoc test for multiple comparisons. (E–H) Kidneys from mice were sectioned and stained with H&E. (E) WT control, (F) saline injected proteinuric WT mice; (G) isotype control antibody injected proteinuric WT mice; (H) HG4 injected proteinuric WT mice. Scale bar = 100 μm.

Figure 7

Tubulointerstitial macrophage infiltration, apoptosis and TGFβ1 expression in antibody treated and control WT mice with POP. Macrophage infiltration (A–D scale bar = 100 μm), cell apoptosis (E–H scale bar = 50 μm), and TGFβ1 protein expression (I–L scale bar = 100 μm) were assessed and quantified according to Methods. (A,E,I) Saline injected POP mice; (B,F,J) isotype control injected POP mice and; (C,G,K) HG4 antibody injected POP mice; (D,H,L) negative control sections from control WT mice lacking primary antibody or anti-digoxigenin antibody in the case of apoptosis detection. Graphs depict quantification of macrophage staining, numbers of apoptotic cells, or quantification of TGFβ1 staining. Each data point relates to an individual animal. For macrophage and apoptosis quantification ^*p < 0.01, and for TGFβ1 quantification ^*p < 0.05 both by one-way ANOVA with Bonferroni post-hoc test for multiple comparisons.