Genetic loci modulate macrophage activity and glomerular damage in experimental glomerulonephritis

Abstract

The Wistar Kyoto (WKY) rat is uniquely susceptible to experimentally induced crescentic glomerulonephritis. Two major quantitative trait loci (QTLs) on chromosomes 13 (Crgn1) and 16 (Crgn2) with logarithm of odds >8, as well as five other loci (Crgn3 through 7), largely explain this genetic susceptibility. To understand further the effects of Crgn1 and Crgn2, we generated a double-congenic strain by introgressing these loci from glomerulonephritis-resistant Lewis rats onto the WKY genetic background. Induction of nephrotoxic nephritis in the double-congenic rats (WKY.LCrgn1,2) produced markedly fewer glomerular crescents, reduced macrophage infiltration, and decreased expression of glomerular TNF-alpha and inducible nitric oxide synthase expression compared with control animals. Bone marrow and kidney transplantation studies between parental and WKY.LCrgn1,2 strains, together with in vitro experiments, demonstrated that Crgn1 and Crgn2 contribute exclusively to circulating cell-related glomerular injury by regulating macrophage infiltration and activation. The residual genetic susceptibility to crescentic glomerulonephritis in WKY.LCrgn1,2 rats associated with macrophage activity (especially with enhanced metalloelastase expression) rather than macrophage infiltration. Taken together, these results demonstrate that a genetic influence on macrophage activation, rather than number, determines glomerular damage in immune-mediated glomerulonephritis.

Figure 1.

Genetic map showing the transferred chromosomal segments in congenic lines. Map distances are based on the SHRSP × BN genetic map (http://rgd.mcw.edu/) and are in cM. ■, Chromosomal region transferred from the donor LEW strain (designated as LL); □, chromosomal region transferred from the recipient WKY strain (designated as WW); ▩, recombination zone. Microsatellite markers D13Rat86 and D16Rat78 are underlined and represent the peak of linkage for Crgn1 and Crgn2, respectively.

Figure 2.

NTN-related phenotypes and macrophage activation in single- and double-congenic lines (WKY, n = 6 rats; all congenics, n = 8 rats per strain). (A) Percentage of glomerular crescents in single-congenic (WKY.LCrgn1, WKY.LCrgn2) and double-congenic (WKY.LCrgn1,2) rats in comparison with parental WKY rats. (B) Macrophage infiltration assessed by quantitative measurement of percentage of ED-1⁺ area per glomerular cross-section (gcs). (C) Proteinuria levels measured in WKY and single- and double-congenic lines. (D) Histology showing a crescentic glomerulus in a WKY rat and a mildly hypercellular glomerulus in a WKY.LCrgn1,2 rat (hematoxylin and eosin). ED-1 immunohistochemistry demonstrates extensive glomerular monocyte and macrophage infiltration in WKY, whereas WKY.LCrgn1,2 rats display reduced staining. (E and F) Glomerular TNF-α (E) and iNOS (F) expression as assessed by qRT-PCR 10 days after injection of NTS (n = 6 rats per strain). (G) Macrophage activation was assessed in WKY, LEW, and congenic BMDMs by Fc receptor–mediated phagocytosis and oxidization (n = 5 rats per strain, without NTN induction). BMDMs are stimulated with Fc oxyBURST, and the WKY rat shows significantly more activation than all of the other strains at all time points (P < 0.001; error bars, SEM). (H) Sandwich ELISA for secretion of TNF-α in LPS-stimulated (100 ng/ml) parental and single- and double-congenic BMDMs (n = 6 rats per strain). **P < 0.01, *P < 0.05 versus WKY. Magnification, ×200 in D.

Figure 3.

Crgn1 and Crgn2 control the circulating cell-related glomerular inflammation in the WKY rat. (A) Percentage of glomerular crescents in parental and WKY.LCrgn1,2 rats 10 days after the injection of NTS with or without BM transplantation. When BM is transferred from WKY.LCrgn1,2 to WKY rats (WKY.LCrgn1,2 → WKY, n = 8 rats), the reduction in crescent formation is not significantly different from WKY.LCrgn1,2 animals that do not receive a transplant (P = 0.28, n = 8 rats) 10 days after NTN induction. All rats show significant reduction (P < 0.01) in percentage of glomerular crescents compared with WKY, and WKY.LCrgn1,2 → WKY rats show significantly increased percentage of glomerular crescents (P < 0.01) when compared with LEW → WKY rats (n = 8 rats are used in all groups). (B) Kidneys from WKY. LCrgn1,2 rats transplanted into the WKY recipients (n = 5 rats) do not show any reduction in the glomerular crescent formation. (C and D) Mesangial cell MCP-1 production assessed in cell supernatants by sandwich ELISA (C) and cell layers by qRT-PCR (D) in parental (WKY, LEW), single-congenic (WKY.LCrgn1, WKY.LCrgn2), and double-congenic lines (WKY.LCrgn1,2) 24 hours after TNF-α (2 ng/ml) stimulation. Mesangial MCP-1 quantities are the result of five independent experiments and expressed as relative quantities compared with WKY rats. *P < 0.05, **P < 0.01 versus WKY; ns, nonsignificant compared with WKY.

Figure 4.

Macrophage activation (and not infiltration) controls genetic susceptibility to Crgn encoded by loci outside of Crgn1 and Crgn2. (A) Number of ED-1⁺ cells per glomerular cross-section (gcs) in parental and WKY.LCrgn1,2 rats 10 days after the injection of NTS with or without BM transplantation. Although WKY.LCrgn1,2 → WKY and LEW → WKY rats develop 52 and 12% glomerular crescents, respectively (P < 0.01; Figure 3), ED-1⁺ cells per gcs are similar (P = 0.32) in the two groups. The ED-1⁺ cells per gcs are significantly reduced (P < 0.05) in all groups when compared with WKY rats (n = 8 rats in all groups). (B) WKY and WKY.LCrgn1,2 BMDMs express significantly higher levels of MMP-12 when compared with LEW BMDMs (n = 6 rats per strain), indicating the genetically determined macrophage activation status in primary macrophages. WKY.LCrgn1,2 BMDMs also show increased MMP-12 expression when compared with WKY ones. **P < 0.01 versus WKY.

Figure 5.

Glomerular MMP-12 expression correlates with the severity of crescent formation following BM transplantation. (A) MMP-12 expression is assessed by qRT-PCR in cultured glomeruli 10 days after the injection of NTS and after BM transplantation (n = 8 rats in each group). (B) Linear regression analysis shows positive correlation (R² = 0.61, P < 0.01) with the percentage of glomerular crescents shown in Figure 3A. **P < 0.01 versus LEW → WKY; ns, nonsignificant compared with LEW → WKY. LEW=>WKY, LEW BM transplanted to WKY; WKY.LCrgn1,2=> WKY, double congenic BM transplanted to WKY; LEW=>WKY.LCrgn1,2, LEW BM transplanted to double congenic.