C5aR1 promotes acute pyelonephritis induced by uropathogenic E. coli
Ke Li, Kun-Yi Wu, Weiju Wu, Na Wang, Ting Zhang, Naheed Choudhry, Yun Song, Conrad A. Farrar, Liang Ma, Lin-lin Wei, Zhao-Yang Duan, Xia Dong, En-Qi Liu, Zong-Fang Li, Steven H. Sacks, Wuding Zhou
Ke Li, Kun-Yi Wu, Weiju Wu, Na Wang, Ting Zhang, Naheed Choudhry, Yun Song, Conrad A. Farrar, Liang Ma, Lin-lin Wei, Zhao-Yang Duan, Xia Dong, En-Qi Liu, Zong-Fang Li, Steven H. Sacks, Wuding Zhou
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Research Article Nephrology

C5aR1 promotes acute pyelonephritis induced by uropathogenic E. coli

Abstract

C5a receptor 1 (C5aR1) is a G protein–coupled receptor for C5a and also an N-linked glycosylated protein. In addition to myeloid cells, C5aR1 is expressed on epithelial cells. In this study, we examined the role of C5aR1 in bacterial adhesion/colonization of renal tubular epithelium and addressed the underlying mechanisms of this role. We show that acute kidney infection was significantly reduced in mice with genetic deletion or through pharmacologic inhibition of C5aR1 following bladder inoculation with uropathogenic E. coli (UPEC). This was associated with reduced expression of terminal α-mannosyl residues (Man; a ligand for type 1 fimbriae of E. coli) on the luminal surface of renal tubular epithelium and reduction of early UPEC colonization in these mice. Confocal microscopy demonstrated that UPEC bind to Man on the luminal surface of renal tubular epithelium. In vitro analyses showed that C5a stimulation enhances Man expression in renal tubular epithelial cells and subsequent bacterial adhesion, which, at least in part, is dependent on TNF-α driven by C5aR1-mediated intracellular signaling. Our findings demonstrate a previously unknown pathogenic role for C5aR1 in acute pyelonephritis, proposing a potentially novel mechanism by which C5a/C5aR1 signaling mediates upregulation of carbohydrate ligands on renal tubules to facilitate UPEC adhesion.

Authors

Ke Li, Kun-Yi Wu, Weiju Wu, Na Wang, Ting Zhang, Naheed Choudhry, Yun Song, Conrad A. Farrar, Liang Ma, Lin-lin Wei, Zhao-Yang Duan, Xia Dong, En-Qi Liu, Zong-Fang Li, Steven H. Sacks, Wuding Zhou

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Figure 3

C5aR1 is required for mannosyl residue expression in renal tubules and bacterial colonization of renal tract epithelium (in vivo).

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C5aR1 is required for mannosyl residue expression in renal tubules and b...
(A) Representative images of mannosyl residue (Man) expression detected by fluorescein-labeled Galanthus nivalis lectin (GNL) (green) in kidney sections of WT and C5aR1–/– mice (i.e., noninfected and infected with J96 for 6 hours). Scale bars: 100 μm. (B) Quantification of Man expression in renal tubules corresponding to the WT and C5aR1–/– mice in A. Data were analyzed by 1-way ANOVA with Tukey’s multiple comparisons test (n = 9 viewing fields from 3 [normal] or 4 [infected] mice/group). ***P < 0.001. (C) Representative fluorescence microscopy images of kidney sections of WT and C5aR1–/– mice at 6 hours postinoculation (hpi) of TRITC-labeled J96 (red), proximal tubular marker (L-fucose) detected by Lotus tetragonolobus lectin (LTL) (green), and nuclear marker DAPI (blue) are shown, demonstrating early bacterial colonization of renal tubular epithelium. Boxed regions correspond to the bottom images. Arrows indicate bacterial colonies. Scale bars: 50 μm (top) and 25 μm (bottom). (D) Quantification of bacterial colonies in renal tabular epithelium corresponding to the WT and C5aR1–/– mice in C. Data were analyzed by unpaired 2-tailed Student’s t test (n = 60 viewing fields from 8 mice/group, under ×200 magnification). ***P < 0.001. (E) Bacterial loads in kidney tissues corresponding to the WT and C5aR1–/– mice in C were also determined by bacterial plate count assay. Data were analyzed by Mann-Whitney test. **P < 0.01. In A–E, a representative of 2 experiments is shown. (F) Fluorescence microscopy images of binding of bacteria (red) to Man (green) at the luminal surface of renal tubular epithelium of WT mice following the inoculation of TRITC-labeled J96 (6 hpi). Scale bars: 10 μm. (G) Fluorescence microscopy images demonstrating the colocalization of C5aR1 (red) and Man (green) in renal tubules of WT mice. Scale bars: 20 μm. (H) C5aR1 immunofluorescence microscopy images of the renal tubule of WT mice at 6 hpi of TRITC-labeled J96, demonstrating the association of C5aR1 (green) and bacteria (red) in renal tubular epithelial cells. Scale bars: 10 μm. (F–H) A representative of 3 experiments is shown.