The complement factor 5a receptor 1 has a pathogenic role in chronic inflammation and renal fibrosis in a murine model of chronic pyelonephritis
Authors: Naheed Choudhry, Ke Li, Ting Zhang, Kun-Yi Wu, Yun Song, Conrad A. Farrar, Na Wang, Cheng-Fei Liu, Qi Peng, Weiju Wu, Steven H. Sacks, Wuding Zhou
Journal: Kidney International (2016)

Complement factor 5a (C5a) interaction with its receptor (C5aR1) contributes to the pathogenesis of inflammatory diseases, including acute kidney injury. However, its role in chronic inflammation, particularly in pathogen-associated disorders, is largely unknown. Here we tested whether the development of chronic inflammation and renal fibrosis is dependent on C5aR1 in a murine model of chronic pyelonephritis. C5aR1-deficient (C5aR1-/-) mice showed a significant reduction in bacterial load, tubule injury and tubulointerstitial fibrosis in the kidneys following infection, compared with C5aR1-sufficient mice. This was associated with reduced renal leukocyte infiltration specifically for the population of Ly6Chi proinflammatory monocytes/macrophages and reduced intrarenal gene expression of key proinflammatory and profibrogenic factors in C5aR1-/- mice following infection. Antagonizing C5aR1 decreased renal bacterial load, tissue inflammation and tubulointerstitial fibrosis. Ex vivo and in vitro studies showed that under infection conditions, C5a/C5aR1 interaction upregulated the production of proinflammatory and profibrogenic factors by renal tubular epithelial cells and monocytes/macrophages, whereas the phagocytic function of monocytes/macrophages was down-regulated. Thus, C5aR1-dependent bacterial colonization of the tubular epithelium, C5a/C5aR1-mediated upregulation of local inflammatory responses to uropathogenic E. coli and impairment of phagocytic function of phagocytes contribute to persistent bacterial colonization of the kidney, chronic renal inflammation and subsequent tubulointerstitial fibrosis. Complement factor 5a (C5a) interaction with its receptor (C5aR1) contributes to the pathogenesis of inflammatory diseases, including acute kidney injury. However, its role in chronic inflammation, particularly in pathogen-associated disorders, is largely unknown. Here we tested whether the development of chronic inflammation and renal fibrosis is dependent on C5aR1 in a murine model of chronic pyelonephritis. C5aR1-deficient (C5aR1-/-) mice showed a significant reduction in bacterial load, tubule injury and tubulointerstitial fibrosis in the kidneys following infection, compared with C5aR1-sufficient mice. This was associated with reduced renal leukocyte infiltration specifically for the population of Ly6Chi proinflammatory monocytes/macrophages and reduced intrarenal gene expression of key proinflammatory and profibrogenic factors in C5aR1-/- mice following infection. Antagonizing C5aR1 decreased renal bacterial load, tissue inflammation and tubulointerstitial fibrosis. Ex vivo and in vitro studies showed that under infection conditions, C5a/C5aR1 interaction upregulated the production of proinflammatory and profibrogenic factors by renal tubular epithelial cells and monocytes/macrophages, whereas the phagocytic function of monocytes/macrophages was down-regulated. Thus, C5aR1-dependent bacterial colonization of the tubular epithelium, C5a/C5aR1-mediated upregulation of local inflammatory responses to uropathogenic E. coli and impairment of phagocytic function of phagocytes contribute to persistent bacterial colonization of the kidney, chronic renal inflammation and subsequent tubulointerstitial fibrosis. Urinary tract infections (UTIs) remain among the most common human infectious diseases worldwide (∼150–250 million cases globally per year).1Zowawi H.M. Harris P.N. Roberts M.J. et al.The emerging threat of multidrug-resistant gram-negative bacteria in urology.Nat Rev Urol. 2015; 12: 570-584Crossref PubMed Scopus (233) Google Scholar, 2Ronald A.R. Nicolle L.E. Stamm E. et al.Urinary tract infection in adults: research priorities and strategies.Int J Antimicrob Agents. 2001; 17: 343-348Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 3Stamm W.E. Norrby S.R. Urinary tract infections: disease panorama and challenges.J Infect Dis. 2001; : S1-S4Crossref PubMed Scopus (426) Google Scholar UTIs present as a wide spectrum of diseases, including bladder infection (cystitis), kidney infection (pyelonephritis), and associated renal damage (e.g., renal fibrosis). Kidney infection is usually caused by ascending infections of the lower urinary tract. Recurrent or long-standing (chronic) kidney infections can result in renal tubulointerstitial fibrosis, which is a much more common situation in children or in patients with diabetes mellitus or urinary obstructions. Although antibiotics are available to treat the disease, a number of challenges remain, including frequent recurrence, persistence of infection, and the increasing risk for resistance to antibiotics.1Zowawi H.M. Harris P.N. Roberts M.J. et al.The emerging threat of multidrug-resistant gram-negative bacteria in urology.Nat Rev Urol. 2015; 12: 570-584Crossref PubMed Scopus (233) Google Scholar, 4Foxman B. The epidemiology of urinary tract infection.Nat Rev Urol. 2010; 7: 653-660Crossref PubMed Scopus (883) Google Scholar It is therefore imperative to improve our current understanding of the pathogenesis of UTIs and develop novel therapeutic strategies to improve current treatment. Uropathogenic Escherichia coli (UPEC) is the primary cause of UTIs, and most UPEC express a variety of fimbriae (e.g., P, type 1) that enable them to bind and invade uroepithelial cells.5Connell H. Hedlund M. Agace W. Svanborg C. Bacterial attachment to uro-epithelial cells: mechanisms and consequences.Adv Dent Res. 1997; 11: 50-58Crossref PubMed Scopus (34) Google Scholar Although innate immunity plays an essential role in the first line of host defense against pathogens, in UTIs most human UPEC strains are resistant to complement-mediated killing.6Taylor P.W. Bactericidal and bacteriolytic activity of serum against gram-negative bacteria.Microbiol Rev. 1983; 47: 46-83Crossref PubMed Google Scholar, 7Li K. Zhou W. Hong Y. et al.Synergy between type 1 fimbriae expression and C3 opsonisation increases internalisation of E. coli by human tubular epithelial cells.BMC Microbiol. 2009; 9: 64Crossref PubMed Scopus (23) Google Scholar Bacteria-mediated acute inflammatory responses can cause renal tissue inflammation and epithelium destruction, allowing bacteria to enter the underlying tissue,8Yadav M. Zhang J. Fischer H. et al.Inhibition of TIR domain signaling by TcpC: MyD88-dependent and independent effects on Escherichia coli virulence.PLoS Pathog. 2010; 6: e1001120Crossref PubMed Scopus (96) Google Scholar, 9Lane M.C. Mobley H.L. Role of P-fimbrial-mediated adherence in pyelonephritis and persistence of uropathogenic Escherichia coli (UPEC) in the mammalian kidney.Kidney Int. 2007; 72: 19-25Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 10Wei Y. Li K. Wang N. et al.Activation of endogenous anti-inflammatory mediator cyclic AMP attenuates acute pyelonephritis in mice induced by uropathogenic Escherichia coli.Am J Pathol. 2015; 185: 472-484Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar and persistent bacterial colonization and chronic inflammation can lead to tubular atrophy and tubulointerstitial fibrosis.11Goluszko P. Moseley S.L. Truong L.D. et al.Development of experimental model of chronic pyelonephritis with Escherichia coli O75:K5:H-bearing Dr fimbriae: mutation in the dra region prevented tubulointerstitial nephritis.J Clin Invest. 1997; 99: 1662-1672Crossref PubMed Scopus (110) Google Scholar C5a receptor 1 (C5aR1) is a 350–amino acid glycoprotein and member of the G-protein–coupled receptor superfamily of proteins that is expressed in myeloid cells (e.g., neutrophils and monocytes/macrophages [MO/MΦs]) and nonmyeloid cells, including renal tubular epithelial cells.12Peng Q. Li K. Sacks S.H. Zhou W. The role of anaphylatoxins C3a and C5a in regulating innate and adaptive immune responses.Inflamm Allergy Drug Targets. 2009; 8: 236-246Crossref PubMed Scopus (106) Google Scholar The well-known ligand for C5aR1 is C5a (also called an anaphylatoxin), which is a 74–amino acid glycopolypeptide fragment generated during complement activation by cleavage of complement C5. The interaction of C5aR1 with C5a mediates a broad spectrum of proinflammatory reactions, such as an increase in vascular permeability, recruitment of leukocytes to sites of injury or infection, generation of cytotoxic oxygen radicals (by granulocytes), and generation of proinflammatory mediators (by myeloid and nonmyeloid cells). A large body of research has demonstrated that C5a/C5aR1 signaling contributes to the pathogenesis of a wide range of inflammatory pathologies, including renal disorders.12Peng Q. Li K. Sacks S.H. Zhou W. The role of anaphylatoxins C3a and C5a in regulating innate and adaptive immune responses.Inflamm Allergy Drug Targets. 2009; 8: 236-246Crossref PubMed Scopus (106) Google Scholar, 13Klos A. Tenner A.J. Johswich K.O. et al.The role of the anaphylatoxins in health and disease.Mol Immunol. 2009; 46: 2753-2766Crossref PubMed Scopus (478) Google Scholar, 14Li K. Zhou W. Anaphylatoxins in organ transplantation.Semin Immunol. 2013; 25: 20-28Crossref PubMed Scopus (14) Google Scholar Furthermore, there is compelling evidence from sepsis studies indicating that C5a/C5aR1 signaling can provide counterregulatory effects in host defense through impairment of innate immune cell function and induce excessive inflammatory responses.15Ward P.A. The dark side of C5a in sepsis.Nat Rev Immunol. 2004; 4: 133-142Crossref PubMed Scopus (342) Google Scholar Pathogenic roles for C5a/C5aR1 signaling have also been reported in a number of other animal models of infectious disease, such as malaria, acute pneumococcal otitis media, and gram-negative bacteremia.16Patel S.N. Berghout J. Lovegrove F.E. et al.C5 deficiency and C5a or C5aR blockade protects against cerebral malaria.J Exp Med. 2008; 205: 1133-1143Crossref PubMed Scopus (85) Google Scholar, 17Tong H.H. Lambert G. Li Y.X. et al.Deletion of the complement C5a receptor alleviates the severity of acute pneumococcal otitis media following influenza A virus infection in mice.PLoS One. 2014; 9: e95160Crossref PubMed Scopus (7) Google Scholar, 18Hollmann T.J. Mueller-Ortiz S.L. Braun M.C. Wetsel R.A. Disruption of the C5a receptor gene increases resistance to acute gram-negative bacteremia and endotoxic shock: opposing roles of C3a and C5a.Mol Immunol. 2008; 45: 1907-1915Crossref PubMed Scopus (51) Google Scholar However, the roles for C5aR1 in chronic kidney disease, particularly under conditions of infection, are largely unknown. Given that (i) C5aR1 is expressed in renal resident and inflammatory cells and is up-regulated under pathological conditions,12Peng Q. Li K. Sacks S.H. Zhou W. The role of anaphylatoxins C3a and C5a in regulating innate and adaptive immune responses.Inflamm Allergy Drug Targets. 2009; 8: 236-246Crossref PubMed Scopus (106) Google Scholar, 19Peng Q. Li K. Smyth L.A. et al.C3a and C5a promote renal ischemia-reperfusion injury.J Am Soc Nephrol. 2012; 23: 1474-1485Crossref PubMed Scopus (149) Google Scholar, 20Gueler F. Rong S. Gwinner W. et al.Complement 5a receptor inhibition improves renal allograft survival.J Am Soc Nephrol. 2008; 19: 2302-2312Crossref PubMed Scopus (88) Google Scholar, 21Lewis E.C. Mizrahi M. Toledano M. et al.alpha1-Antitrypsin monotherapy induces immune tolerance during islet allograft transplantation in mice.Proc Natl Acad Sci U S A. 2008; 105: 16236-16241Crossref PubMed Scopus (161) Google Scholar, 22Abe K. Miyazaki M. Koji T. et al.Enhanced expression of complement C5a receptor mRNA in human diseased kidney assessed by in situ hybridization.Kidney Int. 2001; 60: 137-146Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar (ii) C5a/C5aR1 signaling is a strong driver of tissue inflammation,13Klos A. Tenner A.J. Johswich K.O. et al.The role of the anaphylatoxins in health and disease.Mol Immunol. 2009; 46: 2753-2766Crossref PubMed Scopus (478) Google Scholar, 14Li K. Zhou W. Anaphylatoxins in organ transplantation.Semin Immunol. 2013; 25: 20-28Crossref PubMed Scopus (14) Google Scholar and (iii) C5a/C5aR1 signaling has a negative impact on phagocyte function,23Huber-Lang M.S. Younkin E.M. Sarma J.V. et al.Complement-induced impairment of innate immunity during sepsis.J Immunol. 2002; 169: 3223-3231Crossref PubMed Scopus (150) Google Scholar, 24Wang M. Krauss J.L. Domon H. et al.Microbial hijacking of complement-toll-like receptor crosstalk.Sci Signal. 2010; 3: ra11Crossref PubMed Scopus (166) Google Scholar together with the pathological features of chronic kidney infection (i.e., persistent bacterial colonization, tissue inflammation, and tubulointerstitial fibrosis),11Goluszko P. Moseley S.L. Truong L.D. et al.Development of experimental model of chronic pyelonephritis with Escherichia coli O75:K5:H-bearing Dr fimbriae: mutation in the dra region prevented tubulointerstitial nephritis.J Clin Invest. 1997; 99: 1662-1672Crossref PubMed Scopus (110) Google Scholar, 25Zhou M, Magi-Galluzzi C. Genitourinary Pathology. 2nd ed.: Elsevier Health Sciences Division; 2015. Foundations in Diagnostic Pathology.Google Scholar we hypothesized that C5aR1 may play a pathogenic role in chronic kidney infection. To test this hypothesis, we used a well-established murine model of chronic pyelonephritis induced by the UPEC strain IH11128 and C5aR1-deficient (C5aR1-/-) mice, as well as a C5aR1 antagonist, to determine the role of C5aR1 in chronic kidney infection (i.e., bacterial load, tissue inflammation, and tubulointerstitial fibrosis). We also investigated the cellular basis of the C5a/C5aR1 axis, which contributes to the pathogenesis of chronic kidney infection, by examining the influence of C5aR1 on cellular infiltration of the kidney following renal infection. In addition, we measured the effects of C5a/C5aR1 on the production of proinflammatory and profibrogenic factors by primary cultured renal tubular epithelial cells (RTECs) and MO/MΦs in response to bacterial stimulation and assessed the impact of C5a/C5aR1 on the phagocytic function of MO/MΦs. Our data demonstrate that following infection, early and persistent bacterial colonization, renal inflammation, and tubulointerstitial fibrosis are dependent on C5aR1, suggesting that C5aR1 facilities the pathogenesis of chronic kidney infection by enhancement of bacterial colonization of tubular epithelium, promotion of local inflammatory responses, and impairment of phagocytic function of MO/MΦs. Previous studies in a murine model of chronic kidney infection induced by IH11128 have shown that bacterial colonization of the kidney can be detected at 1 to 2 days after infection and persist for months.11Goluszko P. Moseley S.L. Truong L.D. et al.Development of experimental model of chronic pyelonephritis with Escherichia coli O75:K5:H-bearing Dr fimbriae: mutation in the dra region prevented tubulointerstitial nephritis.J Clin Invest. 1997; 99: 1662-1672Crossref PubMed Scopus (110) Google Scholar We therefore assessed bacterial load in the kidney and bladder of wild-type (WT) and C5aR1-/- mice at three stages of infection, namely, early (day 2), intermediate (day 14), and late (day 56) after infection, by counting bacterial colony-forming units (CFUs) recovered from kidney or bladder tissue samples on agar plates.26Hagberg L. Engberg I. Freter R. et al.Ascending, unobstructed urinary tract infection in mice caused by pyelonephritogenic Escherichia coli of human origin.Infect Immun. 1983; 40: 273-283PubMed Google Scholar After bacterial inoculation into the bladder (postinfection), bacterial colonies in the kidney and bladder peaked at day 1 to day 2; afterwords, there was a trend. However, C5aR1-/- mice had significantly lower bacterial colony counts in the kidneys at all time points after infection (day 2, day 4, and day 56) and in the bladder at day 2 and day 14 after infection compared with WT mice (Figure 1a and b). We also performed fluorescence microscopy analysis of bacterial colonies in infected kidney tissues from WT and C5aR1-/- mice at day 2 after inoculation with fluorescence-labeled bacteria. Consistent with the results of the agar plate assay, bacterial colonies in the renal tubular epithelium were significantly lower in C5aR1-/- mice compared with in WT mice (Figure 1c and d). Collectively, these data demonstrate that C5aR1 deficiency reduces bacterial load in the kidney and bladder. The UPEC strain IH11128 used in this study lacks expression of P fimbriae; therefore, they do not cause severe tissue destruction. However, they do mediate the chronic inflammatory process within the renal parenchyma, leading to progressive tissue injury.11Goluszko P. Moseley S.L. Truong L.D. et al.Development of experimental model of chronic pyelonephritis with Escherichia coli O75:K5:H-bearing Dr fimbriae: mutation in the dra region prevented tubulointerstitial nephritis.J Clin Invest. 1997; 99: 1662-1672Crossref PubMed Scopus (110) Google Scholar We therefore assessed the renal histopathology of infected WT and C5aR1-/- mice at different stages of infection. Renal histopathological changes, including cellular infiltration, tubular damage, and interstitial inflammation, were observed at all time points after infection. Tubular atrophy and interstitial inflammation became more apparent at the later time points. The changes were predominantly located within the corticomedullary junction but were also observed in other areas (e.g., the outer cortex and inner medulla). On the basis of these changes, we performed histological scoring of periodic acid–Schiff– and hematoxylin and eosin–stained kidney sections from individual mice in the WT and C5aR1-/- groups. Compared with WT mice, C5aR1-/- mice exhibited attenuated renal histopathological lesions at all time points (days 2, 14, and 56) (Figure 2a and b). We also assessed renal function in WT and C5aR1-/- mice after infection by measuring blood urea nitrogen (BUN) levels. Blood urea nitrogen was not significantly elevated in all infected mice when compared with normal mice, but there was a trend toward increased blood urea nitrogen in WT mice at the late time point after infection (Figure 2c). These data demonstrate that renal histopathological lesions and late functional impairment were reduced in C5aR1-/- mice after infection. Cellular infiltration was further analyzed by flow cytometry and immunohistochemistry at early and intermediate time points after infection, as described previously.10Wei Y. Li K. Wang N. et al.Activation of endogenous anti-inflammatory mediator cyclic AMP attenuates acute pyelonephritis in mice induced by uropathogenic Escherichia coli.Am J Pathol. 2015; 185: 472-484Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar Flow cytometry analysis of renal cell suspensions showed that when compared with WT, C5aR1-/- kidneys had lower numbers of leukocytes (CD45+) at day 2 after infection and a lower proportion of MO/MΦs (Ly6G-CD11b+) within CD45+ cells at day 14 after infection (Figure 3a–c). In addition, compared with WT, C5aR1-/- kidneys had a lower Ly6Chi population and reduced ratios of Ly6Chi to Ly6Clo populations within the MO/MΦs (Ly6G-CD11b+) compartment at both day 2 and day 14 after infection (Figure 3a, d, and e). However, C5aR1-/- kidneys had a similar proportion of neutrophils (Ly6G+) within CD45+ cells compared with WT kidneys at day 2 and day 14 after infection (Figure 3a and f). Immunohistochemistry showed that when compared with WT, C5aR1-/- kidneys had lower numbers of CD45+ cells (day 2 after infection) and F4/80+ cells (day 14 after infection) than WT kidneys (Figure 3g–3i), which is in agreement with the results of flow cytometry analysis. Collectively, these results indicate that C5aR1 deficiency not only caused a general reduction in cellular infiltration and MO/MΦ accumulation but also specifically reduced inflammatory monocytes' infiltration of the kidney in response to renal infection. However, C5aR1 deficiency did not cause a reduction of neutrophil infiltrate at the time points studied in this model.Figure 3C5a receptor (C5aR) deficiency influences the extent and phenotype of cellular infiltrates in the kidneys following renal infection. Renal inflammatory cell infiltration was analyzed in infected wild-type (WT) and C5aR1-/- mice at days 2 and 14 after infection by flow cytometry. (a) Stepwise gating strategy used in flow cytometric analysis of leukocytes, neutrophils, monocytes/macrophages (MO/MΦs), and Ly6chi MO/MΦs in kidney tissues. (b–f) Quantification of leukocytes (CD45+), MO/MΦ (Ly6G-CD11b+), Ly6chi population, and ratio of Ly6chi to Ly6clo populations within the Ly6G-CD11b+ compartment and neutrophils (Ly6G+), respectively. Each dot represents an individual mouse. Data were analyzed by Student's t test (n = 5 mice per group). **P < 0.05. ***P < 0.005. (g–i) Immunohistochemistry. (g) Representative images of CD45- and F4/80-stained kidney sections from infected WT and C5aR1-/- mice (n = 4 mice per group). Arrows show positively stained cells. Bar = 100 μm. (h,i) Quantification of CD45+ and F4/80+ cells. Data were analyzed by Student's t test (40–50 viewing fields [0.04 mm2 per field] from 4 mice per group). ***P < 0.001. A representative of 2 independent experiments is shown.View Large Image Figure ViewerDownload (PPT) Persistent tissue inflammation and fibrogenesis play an important role in the progression of chronic kidney disease. We therefore used semiquantitative reverse transcriptase polymerase chain reaction to assess renal tissue inflammation and fibrogenesis of WT and C5aR1-/- mice following renal infection. Gene expression of proinflammatory cytokines (tumor necrosis factor-α [TNF-α] and interleukin-1β [IL-1β]), chemokines (keratinocyte-derived protein chemokine [KC], monocyte chemoattactant protein-1 [MCP-1]), chemokine receptor (C-C motif chemokine receptor 2 [CCR2]), and profibrogenic factors (transforming growth factor-β [TFG-β] and platelet-derived growth factor [PDGF]) was significantly reduced in C5aR1-/- mice compared with in WT mice at all time points studied (Figure 4a–4c). In contrast, gene expression of antifibrogenic factor (hepatocyte growth factor) was significantly higher in C5aR1-/- mice than in WT mice at day 2 and day 14 after infection (Figure 4d). Collectively, these results indicate that absence of C5aR1 attenuates renal tissue inflammation and fibrogenesis following renal infection. To further investigate the impact of C5aR1 on the development of renal fibrosis, we assessed the extent of collagen deposition and extracellular matrix production in kidneys from WT and C5aR1-/- mice following renal infection. Sirius red staining was performed on kidney sections at day 14 and day 56 after infection. Compared with WT kidneys, C5aR1-/- kidneys exhibited a significant reduction of Sirius red staining (Figure 5a and b). We also examined gene expression of several major extracellular matrix and cytoskeletal proteins in kidneys from WT and C5aR1-/- mice at day 14 and day 56 after infection using semiquantitative reverse transcriptase polymerase chain reaction. Intrarenal expression of mRNA encoding for extracellular matrix proteins (collagen I and fibronectin) and cytoskeleton proteins (α-smooth muscle and vimentin) was significantly reduced in C5aR1-/- mice compared with in WT mice. In contrast, C5aR1-/- mice had significantly higher levels of mRNA for collagen IV (which is required for renal tubular epithelial cell structural and functional integrity27Timpl R. Structure and biological activity of basement membrane proteins.Eur J Biochem. 1989; 180: 487-502Crossref PubMed Scopus (811) Google Scholar) than did WT mice (Figure 5c). Taken together, these findings indicate that C5aR1 deficiency protects mice from renal fibrosis and parenchymal loss following renal infection. In addition to studying C5aR1-/- mice, we used a well-known C5aR1 antagonist (PMX53)28March D.R. Proctor L.M. Stoermer M.J. et al.Potent cyclic antagonists of the complement C5a receptor on human polymorphonuclear leukocytes. Relationships between structures and activity.Mol Pharmacol. 2004; 65: 868-879Crossref PubMed Scopus (87) Google Scholar to assess whether this approach can curtail renal infection, tissue inflammation, and fibrosis. WT mice were administered PMX53 or control agent daily for 13 days. Consistent with observations made in C5aR1-/- mice, renal histopathology, tissue inflammation/fibrogenesis, collagen deposition, and bacterial load were significantly reduced in the kidneys of PMX53-treated mice compared with in the control group at day 14 after infection (Figure 6a–e). These results further confirmed the pathogenic role for C5aR1 in this model and also suggest a therapeutic potential for targeting C5aR1 in chronic kidney infection. To further investigate whether the major effects of C5aR1 on chronic inflammation and renal fibrosis are dependent on the initial impact of C5aR1 or the subsequent responses, we performed an additional set of in vivo experiments. WT mice were given PMX53 or control agent at the following different stages of infection: (i) starting at day 0 (2 hours before the inoculation) and continuing up to day 2 (early administration) and (ii) starting at day 3 after the inoculation and continuing up to day 13 (late administration). Early bacterial colonization was assessed at day 3 after infection following early administration. Mice that received PMX53 exhibited significantly fewer CFUs in the kidney compared with mice that received control agent (Figure 6f). Chronic injury was assessed at day 14 after infection. Both early and late administration of PMX53 reduced renal fibrosis and tissue bacterial load, but early administration led to a more profound reduction compared with in the control group (Figure 6g–i). These results suggest that the initial impact of C5aR1 plays an important role in the limit of bacterial load and subsequent chronic inflammation and renal fibrosis.Figure 6Antagonizing C5a receptor (C5aR1) reduces renal inflammation and fibrosis following renal infection. (a–e) Wild-type mice were administered PMX53 or control (ctrl) agent daily for 13 days starting on the day of induction of renal infection. Assessment of chronic inflammation, renal fibrosis, and renal bacterial load was performed at day 14 after infection. (a) Representative images of periodic acid–Schiff (PAS)- or Sirius red (SR)-stained kidney sections taken at the corticomedullary junction. Arrows show tubular lesions or areas positively stained with Sirius red. Bar = 200 μm. (b) Histological scores in the mice illustrated in panel (a). Each dot represents an individual mouse. Data were analyzed by Student's t test (n = 5 mice per group). (c) Quantification of collagen deposition (Sirius red staining) in kidney sections of the mice in panel (a). Data were analyzed by Student's t test (20 to 30 viewing fields [1.92 mm2 per field] from 5 mice per group). (d) Bacterial loads in the kidneys. Each dot represents colony-forming units recovered from an individual mouse and is shown as average colony-forming units from 2 replicate agar plates. (e) Semiquantitative analysis of mRNA expression of extracellular matrix and cytoskeletal proteins in infected kidney tissue. Each dot represents an individual mouse and is shown as the mean of two replicate polymerase chain reaction results. (d,e) Data were analyzed by Student's t test (n = 5 mice per group). A representative of 2 independent experiments is shown. (f–i) Wild-type mice were given PMX53 or control agent at different stages of infection starting at day 0 (2 hours before the inoculation) and continuing up to day 2 (early administration) or starting at day 3 after the inoculation and continuing up to day 13 (late administration). (f) Colony-forming units recovered from the kidney of individual mice at day 3 after infection. Each dot represents an individual mouse. Data were analyzed by Student's t test (n = 5 mice per group). (g) Representative images of Sirius red–stained kidney sections., taken at the corticomedullary junction. Arrows show area positively stained with Sirius red. Bar = 100 μm. (h) Quantification of Sirius red staining in kidney sections of the mice in panel (g). Data were analyzed by Student's t test (20–35 viewing fields [1.92 mm2 per field] from 5–7 mice per group). (i) Colony-forming units recovered from the kidney of individual mice at day 14 after infection. Each dot represents an individual mouse. Data were analyzed by Student's t test (n = 7 mice per group). *P < 0.05, **P < 0.005, ***P < 0.001.View Large Image Figure ViewerDownload (PPT) Our in vivo data presented in this article suggest a critical role for C5aR1 in renal tissue inflammation and fibrogenesis following renal infection. To explore the cellular basis for the C5a/C5aR1 axis contributing to renal tissue inflammation and fibrogenesis, we used primary cell culture systems for RTECs and MO/MΦs and then used reverse transcriptase polymerase chain reaction to assess the effects of C5a/C5aR1 interactions on production of proinflammatory and profibrogenic factors by these cells in response to bacterial stimulation. Incubation with C5a alone led to only a small increase in proinflammatory and profibrogenic factor mRNA expression by RTECs and MO/MΦs. Incubation with heat-killed UPEC alone clearly increased mRNA expression of proinflammatory factors (i.e., tumor necrosis factor-α, interleukin-1β, monocyte chemoattactant protein-1, and keratinocyte-derived protein chemokine) by RTECs and MO/MΦs. In the presence of C5a, mRNA expression levels for those proinflammatory factors were further increased in RTECs and MO/MΦs compared with in the absence of C5a. Combined heat-killed UPEC and C5a treatment also increased