Resident Dendritic Cells Prevent Postischemic Acute Renal Failure by Help of Single Ig IL-1 Receptor-Related Protein

Maciej Lech, Alejandro Avila-Ferrufino, Ramanjaneyulu Allam, Stephan Segerer, Alexander Khandoga, Fritz Krombach, Cecilia Garlanda, Alberto Mantovani and Hans-Joachim Anders
J Immunol September 15, 2009, 183 (6) 4109-4118; DOI: https://doi.org/10.4049/jimmunol.0900118

Abstract

Ischemia-reperfusion (IR) triggers tissue injury by activating innate immunity, for example, via TLR2 and TLR4. Surprisingly, TLR signaling in intrinsic renal cells predominates in comparison to intrarenal myeloid cells in the postischemic kidney. We hypothesized that immune cell activation is specifically suppressed in the postischemic kidney, for example, by single Ig IL-1-related receptor (SIGIRR). SIGIRR deficiency aggravated postischemic acute renal failure in association with increased renal CXCL2/MIP2, CCL2/MCP-1, and IL-6 mRNA expression 24 h after IR. Consistent with this finding interstitial neutrophil and macrophage counts were increased and tubular cell necrosis was aggravated in Sigirr-deficient vs wild-type IR kidneys. In vivo microscopy revealed increased leukocyte transmigration in the postischemic microvasculature of Sigirr-deficient mice. IL-6 and CXCL2/MIP2 release was much higher in Sigirr-deficient renal myeloid cells but not in Sigirr-deficient tubular epithelial cells after transient hypoxic culture conditions. Renal IR studies with chimeric mice confirmed this finding, as lack of SIGIRR in myeloid cells largely reproduced the phenotype of renal IR injury seen in Sigirr−/− mice. Additionally, clodronate depletion of dendritic cells prevented the aggravated renal failure in Sigirr−/− mice. Thus, loss of function mutations in the SIGIRR gene predispose to acute renal failure because SIGIRR prevents overshooting tissue injury by suppressing the postischemic activation of intrarenal myeloid cells.

Acute renal ischemia can cause renal failure and occurs and impairs outcome in the various types of shock or during kidney transplantation. It is becoming increasingly evident that ischemia-reperfusion (IR)4 activates innate immune elements that enhance the subsequent tissue injury (1). How does ischemia translate into innate immune activation? Recent data suggest that this process involves a group of germline-encoded pathogen-recognition receptors that are highly conserved among species from Drosophila to humans, that is, the TLRs (2). TLRs recognize molecular motifs shared by different groups of microorganisms (3). TLRs are transmembrane receptors in outer or endosomal membranes of immune cells, such as macrophages, dendritic cells, neutrophils, B cells, and NK cells. TLRs are also expressed in nonimmune cells, including kidney tubular epithelial cells and mesangial cells (4). TLR signaling involves either of the two cytoplasmic adaptor molecules MyD88 or Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) that facilitate the activation of MAPK, NF-κB, or IFN-related factors (3). Hence, in infected tissues TLR activation fosters the production of proinflammatory cytokines, chemokines, and other soluble mediators that set up local inflammation. Interestingly, damaged tissues seem to release endogenous molecules that can activate TLRs just like microbes (5, 6). This mechanism contributes to various types of sterile inflammation, including that being associated with IR injury (IRI). For example, Tlr4- and/or Tlr2-deficient mice are protected from IRI of the liver (7), the heart (8, 9), the brain (10), and the kidney (11, 12). Although the network of intrarenal myeloid cells is considered to significantly contribute to danger signaling in the kidney (13), studies with Tlr2- and Tlr4-chimeric mice propose that TLR2 and TLR4 signaling in renal parenchymal cells predominantly contributes to renal IRI (11, 12). We therefore hypothesized that inhibitory factors might suppress TLR signaling specifically in intrarenal myeloid cells during IR.

Several factors control TLR signaling but their cell type-specific expression patterns and functional roles in disease remain poorly described (14). Single Ig IL-1-related Receptor (SIGIRR), also known as Toll-IL-1 receptor 8 (TIR8), is a member and inhibitor of the TLR/IL-R family that is highly expressed in the kidney (15, 16, 17). Its small extracellular domain does not bind ligands, and its intracellular domain cannot activate NF-κB because it lacks two essential amino acids (Ser447 and Tyr536) in the highly conserved TIR domain (16). SIGIRR inhibits TLRs by modulating their homo- and heterodimer formation and/or their interaction with other components of the outside-in signaling complex (18). Pathogen challenge or damaging the intestinal epithelial barrier surfaces in Sigirr-deficient mice results in severe immunity-mediated tissue damage (17, 19, 20, 21, 22). This was referred to enhanced LPS signaling in dendritic cells and intestinal epithelia. In the kidney SIGIRR is expressed by tubular epithelial cells and by intrarenal myeloid cells; however, we recently described that SIGIRR specifically suppresses LPS signaling in intrarenal APCs (23). We hypothesized that SIGIRR represents (one of) the factor(s) that specifically controls the activation of intrarenal myeloid cells during renal IRI by suppressing TLR signaling and thereby limiting postischemic renal tissue damage.

Materials and Methods

Animal studies

Tir8/Sigirr-deficient mice were generated as previously described, genotyped, and backcrossed to the C57BL/6J strain (F6) (15). Mice were housed in groups of five in filter top cages with unlimited access to food and water. Cages, nest lets, food, and water were sterilized by autoclaving before use. All experimental procedures were approved by the local government authorities. Chimeric mice were generated by lethally irradiating 6-wk-old recipient mice with 9 Gy of total-body irradiation (GC40-107; MDS Nordion). Within 6 h, 4.5 × 106 bone marrow cells either from congenic or allogenic donor mice were transplanted by i.v. injection in a total volume of 200 μl of normal saline. Complete engraftment of donor bone marrow was allowed for 8 wk, and the ablation of recipient hematopoietic cells and full reconstitution of donor marrow (>95%) was confirmed by flow cytometry for H-2k in spleens from five sentinel mice (H-2b). Cl2MDP (clodronate) was a gift from Roche Diagnostics and was incorporated into liposomes as previously described (24). Adult mice were injected i.v. with 200 μl of clodronate or control liposomes on days −3 and −2 before the IR procedure as described (25).

Induction of renal IRI

Groups of mice (n = 6) were anesthetized as described (26) before both renal pedicles or only the left renal pedicle was clamped for 45 min with a microaneurysm clamp via flank incision (Medicon). Body temperature was maintained throughout the procedure by placing the mice on a 37°C heating pad. After clamp removal the kidney was inspected for restoration of bloodflow evidenced by returning to its original color before closing the wound with standard sutures (27). To maintain fluid balance, all mice were supplemented with 1 ml of saline administered s.c. Mice were sacrificed 1, 5, and 10 days after the procedure, and IRI and contralateral (sham) kidneys were divided up to be either snap-frozen in liquid nitrogen or fixed in 10% buffered formalin.

Histological evaluation

Kidneys were embedded in paraffin, and periodic acid-Schiff (PAS) stains and immunostaining on 2-μm sections were performed as described (28). Postischemic tubular injury was scored by assessing the percentage of tubules in the corticomedullary junction that displayed cell necrosis, loss of the brush border, cast formation, and tubular dilatation as follows (27): 0, none; 1, ≤10%; 2, 11–25%; 3, 26–45%; 4, 46–75%; and 5, >76%. For immunostaining the following primary Abs were used: rat anti-Mac2 (1/50; Cedarlane Laboratories), rat anti-mouse neutrophils (1/50; Serotec), anti-Ccl2/Mcp-1 (1/1000; Santa Cruz Biotechnology). To count interstitial cells, 10 cortical high-power fields (×200) were analyzed. All assessments were performed by two blinded observers (A.A.-F. and H.J.A.).

RNA preparation and real-time quantitative (TaqMan) RT-PCR

Reverse transcription and real-time RT-PCR from total renal RNA was prepared as described (23). A SYBR Green dye detection system was used for quantitative real-time PCR on a LightCycler 480 (Roche). Gene-specific primers (300 nM; Metabion) were used as listed in Table I⇓. Controls consisting of double-distilled H2O were negative for target and housekeeper genes.

View this table:
Table I.

Primers used for real-time RT-PCR

Western blot

Western blot for SIGIRR protein was performed as previously described in detail (23). In brief, kidney protein extractions were incubated in 2× loading buffer for 30 min at 65°C, resolved by 12% SDS-PAGE, and transferred to an Immobilon-P membrane (Millipore). After blocking with 1% Western blocking solution (Roche), the filter was incubated with a goat polyclonal anti-SIGIRR Ab (1/1000; R&D Systems) overnight in 0.5% Western blocking solution (Roche). Immune complexes were visualized using a peroxidase-conjugated donkey anti-goat IgG Ab (1/10,000; Dianova, Hamburg, Germany) for 1 h and processed for detection by ECL (Amersham Pharmacia Biotech Europe).

In vitro studies

Primary tubular epithelial cells were isolated from 6-wk-old C57BL/6 wild-type or Sigirr-deficient mice. Kidney cell suspensions were prepared by applying the mashed kidney onto 30-μm pre-separation filters (Miltenyi Biotec). The cell suspension was incubated for 2 h at 37°C, and the nonadherent tubular epithelial cells were harvested and applied to culture dishes coated with collagen I (Sigma-Aldrich) in predefined K1 medium as described (23). Resident myeloid cells were isolated from renal cell suspension by magnetic bead isolation using a rat monoclonal anti-mouse/human-CD11b Ab as previously described (23). Renal myeloid cells were plated (3 × 105 cells/ml) in 10% FCS/1% penicillin-streptavidin/RPMI 1640 medium supplemented with 0.5 ng/ml recombinant murine GM-CSF (ImmunoTools) and grown until they were 70–80% confluent. For hypoxia experiments, cells were kept without FCS and 1 μg/ml ultrapure LPS (InvivoGen) in a Modula incubator chamber (Billups-Rothenberg) flushed with 100% N2 as previously described (29). After 3 h of hypoxic culture, half of the supernatants were removed and reperfusion was simulated by adding fresh culture medium containing 20% FCS. Supernatants were collected after another 24 h. IL-6 levels were determined by ELISA (R&D Systems) according to the manufacturer’s instructions.

In vivo microscopy on ischemic cremaster muscles

The surgical procedure to induce IR in cremaster muscle has been described previously (30). In brief, the right cremaster muscle was exposed in anesthetized mice and muscle ischemia was induced by clamping all supplying vessels using a vascular clamp. After 30 min the vascular clamp was removed and reperfusion was restored for 120 min. The setup for intravital microscopy was centered arround an Olympus BX 50 upright microscope (Olympus Microscopy) equipped for stroboscopic fluorescence epi-illumination microscopy. Microscopic images were recorded with an analog black-and-white CCD video camera (Cohu 4920). Centerline bloodflow velocity was measured by injecting green fluorescent microspheres (2 μm in diameter; Molecular Probes) via an arterial catheter. Beads that were flowing freely along the vessels’ centerline were used to determine bloodflow velocity. Emigrated cells were counted in regions of interest covering 75 μm on both sides of a vessel over a 100-μm vessel length. From measured vessel diameters and centerline blood flow velocity, apparent wall shear stress was calculated, assuming a parabolic flow velocity profile over the vessel cross-section. Paraffin-fixed muscle sections were stained with rat anti-mouse Ly6G or CD45 (BD Biosciences) and counterstained with Mayer’s hemalaun. The numbers of interstitial Ly6G- and CD45-positive cells were quantified by light microscopy (magnification ×400) on two sections (15 ± 5 observation fields per section) from six animals per experimental group in a blinded manner.

Statistical analysis

Data were expressed as means ± SEM. Data from wild-type and Sigirr-deficient mice were compared with ANOVA on ranks followed by the Student-Newman-Keuls test using SigmaStat Software (Jandel Scientific). A t test was used for direct comparisons between wild-type and Sigirr-deficient cells per mouse. Data are expressed as mean values ± SEM. A p value of <0.05 indicated statistical significance.

Results

SIGIRR is expressed in the kidney during IRI

To determine whether SIGIRR is expressed in the renal ischemia, we first measured SIGIRR mRNA levels in IRI and sham kidneys by real-time PCR. Groups of Sigirr−/− and Sigirr+/+ mice underwent unilateral clamping of the renal artery for 45 min, and IRI and control kidneys were harvested at 1, 5, and 10 days after surgery. Consistent with our previous studies, kidneys from 6-wk-old C57BL/6 mice expressed SIGIRR mRNA. Surgery significantly induced renal SIGIRR mRNA levels at day 1 in both sham and IR kidneys; at day 5 this was only significant for sham kidneys (Fig. 1⇓). At day 10 all renal SIGIRR mRNA levels declined to baseline levels (Fig. 1⇓B). At the protein level SIGIRR expression was not found to be much regulated in postischemic as well as contralateral kidneys (Fig. 1⇓B). A statistically significant inducation was noted in contralateral kidneys at day 1 postsurgery and a reduction in IR kidneys was noted at day 10 (Fig. 1⇓B). We conclude that SIGIRR is constitutively expressed in mouse kidneys.

FIGURE 1.
FIGURE 1.

Renal Sigirr expression. A, Total RNA was extracted from kidneys of 6-wk-old healthy C57BL/6 mice or ischemic and contralateral kidneys of C57BL/6 mice at different time intervals after surgery (n = 5–6). TIR8/SIGIRR mRNA expression levels were determined by real-time RT-PCR and expressed as mean of the ratio Sigirr/18S rRNA ± SD; ∗, p < 0.05 vs baseline. B, SIGIRR protein expression was determined by Western blot analysis. Proteins were prepared from kidneys of untouched C57BL/6 Sigirr+/+ or Sigirr−/− mice as indicated. Additionally, kidneys were obtained from Sigirr+/+ mice 1, 5, or 10 days after IR or sham surgery. Upper panel shows means ± SEM of densitometric analysis of three blots of separate biological samples. Wild-type is set as 1. The lower panel shows one representative blot.

Lack of SIGIRR aggravates renal IRI

To determine the functional role of SIGIRR in acute renal failure we first induced bilateral renal IR in Sigirr+/+ and Sigirr−/− mice (n = 5–6), respectively. In wild-type mice serum creatinine levels increased from 0.4 ± 0.2 mg/dl at baseline to 1.4 ± 0.2 mg/dl within 24 h. In Sigirr−/− mice serum creatinine levels increased from 0.4 ± 0.1 to 2.9 ± 0.7 mg/dl (p < 0.05 vs wild-type), indicating that lack of SIGIRR aggravated acute renal failure after bilateral renal IR. Twenty-four hour survival was 100% in both groups of mice. Lack of SIGIRR enhanced IRI also after unilateral IR as evidenced by the composite score of widespread tubular necrosis, loss of the brush border, cast formation, and tubular dilatation at the corticomedullary junction in the IR kidney (Fig. 2⇓). The difference of IRI between Sigirr−/− and Sigirr+/+ mice was evident at days 1, 5, and 10 postsurgery. In contrast, contralateral kidneys were neither affected by the surgical procedure nor by the SIGIRR genotype. Thus, SIGIRR is required to prevent inappropriate kidney damage and acute renal failure after IR.

FIGURE 2.
FIGURE 2.

IR-triggered tubular injury in Sigirr−/− and Sigirr+/+ mice. Postischemic and contralateral kidneys were obtained from all mice at various time intervals as indicated. A, PAS stains from paraffin-embedded kidneys from wild-type mice (upper panel) and Sigirr-deficient mice (lower panel) are shown at a magnification of ×100. B, Semiquantitative morphometry of tubular injury is shown as means ± SEM from five to six mice of each group. ∗, p < 0.05 vs wild-type of the respective time point.

SIGIRR suppresses early IL-6, CCL2/MCP-1, and CXCL2/MIP2 expression in renal IRI

It is known that SIGIRR controls bacterial LPS- or lipopetide-induced expression of cytokines and chemokines (17); hence, lack of SIGIRR might aggravate renal IRI in association with increased local cytokine and chemokine expression. We therefore determined the renal mRNA levels of IL-6, CCL2/MCP-1, and CXCL2/MIP2. Lack of SIGIRR was associated with significantly higher mRNA levels of IL-6, CCL/MCP-1, and CXCL2/MIP2 only at day 1 after renal artery clamping (Fig. 3⇓, A and B). We sought to validate the increased production of proinflammatory mediators on the protein level and performed immunostaining for CCL2/MCP-1. CCL2/MCP-1-specific staining signals were strongest on renal sections at 5 days postsurgery and localized to tubular epithelial cells as well as to interstitial cells (Fig. 3⇓C). Quantification of CCL2/MCP-1-positive interstitial cells indicated higher numbers in IR kidneys of Sigirr-deficient mice at all time points as compared with wild-type mice, while low numbers of these cells were found in contralateral kidneys of both genotypes (Fig. 3⇓C). Thus, SIGIRR suppresses IL-6, CCL2/MCP-1, and CXCL2/MIP2 expression after IR.

FIGURE 3.
FIGURE 3.

Renal cytokine and chemokine mRNA expression after IRI. Total RNA was extracted from ischemic and contralateral kidneys of C57BL/6 mice at different time intervals after IR (n = 5–6). Messenger RNA expression levels were determined for the cytokine IL-6 (A) or the chemokines CXCL2/MIP2 and CCL2/MCP-1 (B) by real-time RT-PCR. Data are expressed as mean of the ratio vs the respective 18S rRNA level ± SEM; ∗, p < 0.05 vs wild-type. C, CCL2/MCP-1 immunostaining was performed on paraffin-embedded renal sections obtained 5 days postsurgery from mice of both genotypes as indicated. Interstitial cells (indicated by arrows) show positive staining signals in Sigirr−/− mice only. Original magnification ×400 and ×1000 in the lowest panel. Quantification of interstitial CCL2/MCP-1 positive cells from Sigirr−/− mice (filled bars) and wild-type mice (open bars) all time points is shown as means ± SEM from five to six mice of each group. ∗, p < 0.05 vs wild-type of the respective time point.

Lack of SIGIRR enhances interstitial leukocyte recruitment after IRI

Increased renal CCL2/MCP-1 and CXCL2/MIP2 expression should support additional recruitment of neutrophils and macrophages in renal IRI. In fact, immunostaining revealed increased numbers of neutrophils in the renal interstitial compartment 24 h after renal artery clamping in Sigirr-deficient as compared with wild-type mice, while neutrophil counts remained unaffected at later time points or in contralateral kidneys (Fig. 4⇓, A and B). Furthermore, renal IRI was associated with interstitial macrophage infiltrates evident from day 5 after renal artery clamping. Consistent with its effect on interstitial neutrophil counts, lack of SIGIRR was associated with increased numbers of interstitial macrophages at days 5 and 10 in Sigirr-deficient mice in IRI (Figs. 4⇓, A and C). Interestingly, at day 5 a significant increase of interstitial macrophages was also noted in contralateral kidneys (Fig. 4⇓C).

FIGURE 4.
FIGURE 4.

Renal leukocyte recruitment after IRI. Postischemic and contralateral kidneys were obtained from all mice at various time intervals as indicated. A, Immunostaining for neutrophils was performed on renal sections from wild-type mice (upper panel) and Sigirr-deficient mice (lower panel) as described in Materials and Methods (original magnification, ×100). Neutrophil (B) and macrophage (C) counts from 10 high-power fields from each section are shown as means ± SEM from five to six mice of each group. ∗, p < 0.05 vs wild-type of the respective time point.

Lack of SIGIRR enhances postischemic transendothelial leukocyte migration

The coincident increase of CCL2/MCP-1 and CXCL2/MIP2 mRNA expression and leukocyte numbers in Sigirr-deficient IRI kidneys might be a result of increased leukocye migration through activated endothelia. To test this hypothesis we quantified transendothelial leukocyte migration along postcapillary venules 30 and 120 min after inducing IRI in cremaster muscles of male wild-type and Sigirr-deficient mice using in vivo microscopy. Vessel diameter, centerline blood flow velocity, and wall shear rate were routinely measured and did not differ among the experimental groups (Table II⇓). As compared with baseline levels, IR significantly increased leukocyte transmigration at 30 and 120 min as compared with sham-operated mice (Fig. 5⇓, A and B). Lack of SIGIRR was associated with a significant increase of transmigrated leukocytes into the interstitial compartment at 120 min after IR as compared with wild-type mice (Fig. 5⇓B). Consistent with our renal IRI study M. cremaster immunostaining revealed that neutrophils are the predominant immune cell type recruiting to muscle at this early postischemic time point (80–90% of all interstitial CD45+ cells; Fig. 5⇓C). These data show that SIGIRR specifically suppresses transendothelial leukocyte migration in IRI.

FIGURE 5.
FIGURE 5.

In vivo microscopy from postischemic cremaster muscles of wild-type and Sigirr-deficient mice. A, Representative reflected light oblique transillumination (RLOT) microscopic images (objective magnification, ×20) of postcapillary venules in the cremaster muscle of sham-operated wild-type mice, sham-operated Sigirr−/− mice, as well as of wild-type mice and Sigirr−/− mice after IR (30 min/120 min). Numbers of transmigrated leukocytes (arrows) were quantified at baseline conditions before ischemia as well as at 30 and 120 min of reperfusion. B, Panel shows results of leukocyte transmigration at representative time points. Data are expressed as mean ± SEM for n = 6 per group; ∗, p < 0.05 vs sham; #, p < 0.05 vs wild-type IR. C, Tissue samples were stained for the neutrophil marker Ly6G after 130 min of reperfusion. Microphotographs demonstrate extravascularly localized Ly6G+ neutrophils (pointed by arrows) from a sham-operated wild-type and Sigirr−/− mice, and wild-type and Sigirr−/− upon IR. Microscope magnification, ×400.

View this table:
Table II.

Hemodynamic parameters in C57BL/6 mice undergoing in vivo microscopya

Lack of SIGIRR increases IL-6, CCL2/MCP-1, and CXCL2/MIP2 release in postischemic renal CD11b+ cells but not in tubular epithelial cells

In acute kidney injury the intrarenal network of APCs and tubular epithelial cells both contribute to innate immune signaling (13, 23, 25, 26). We therefore questioned in which cell type SIGIRR regulates postischemic cytokine release. CD11b-positive myeloid cells as well as tubular epithelial cells were isolated from kidneys of wild-type and Sigirr-deficient mice and cultured in a 100% N2 atmosphere for 3 h. RNA was harvested 24 h after reoxygenation and fresh medium supply (Fig. 6⇓). The release of IL-6, CCL2/MCP-1, and CXCL2/MIP2 was significantly increased in postischemic renal myeloid cells but not in tubular epithelial cells from Sigirr-deficient cells (Fig. 6⇓). Thus, SIGIRR specifically suppresses postischemic cytokine and chemokine release in CD11b+ APCs.

FIGURE 6.
FIGURE 6.

In vitro studies with renal APCs and tubular epithelial cells. CD11b-positive APCs and tubular epithelial cells were prepared from Sigirr+/+ and Sigirr−/− mice and cultured under hypoxic conditions as described in Materials and Methods. Cell culture supernatants were harvested 24 h after reoxygenation and fresh medium supply for IL-6, CCL2/MCP-1, and CXCL2/MIP2 ELISA. Data represent means ± SEM from five independent cell isolations and each supernatant being analyzed in duplicates. ∗, p < 0.05 vs Sigirr+/+ cells. N.s., not significant.

Lack of SIGIRR in intrarenal immune cells determines the phenotype of Sigirr-deficient mice in renal IRI

The in vitro data suggested that the phenotype of Sigirr-deficient mice may largely depend on the role of SIGIRR in intrarenal APCs. To test this hypothesis we generated chimeric mice by transplanting 4.5 × 106 bone marrow cells from Sigirr-deficient or wild-type mice into lethally irradiated wild-type recipients. With this regimen transplanted bone marrows successfully reconstituted within 8 wk, showing >95% donor cells in spleens by flow cytometry. IRI was induced in all chimeric mice and tissues were harvested 24 h later. The assessment of IRI scores from kidney PAS stains revealed that recipients of Sigirr-deficient bone marrow almost completely replicated the extent of IRI and interstitial neutrophil counts at day 1 as seen in Sigirr-deficient mice (Fig. 7⇓A). Consistently, recipients of wild-type bone marrow replicated the extent of IRI seen in wild-type mice (Fig. 7⇓A). As an alternative approach we depleted renal myeloid cells by intraperitoneal injection of clodronate, which consistently reduces the number of myeloid APCs shortly after injection (25). This entirely prevented the aggravation of acute renal failure after bilateral IR (45 min) in Sigirr-deficient mice (Fig. 7⇓B). We therefore conclude SIGIRR suppresses renal IRI by specifically inhibiting the postischemic activation of intrarenal myeloid cells.

FIGURE 7.
FIGURE 7.

IRI studies with Sigirr chimeric mice. Sigirr+/+ mice were lethally irradiated and reconstituted with bone marrows from either Sigirr+/+ or Sigirr−/− donors. Eight weeks after bone marrow transplantation all mice underwent IRI. Postischemic kidneys were obtained after 24 h. A, Left panel, Semiquantitative morphometry of tubular injury is shown as means ± SEM from five to six mice of each group. Right panel, Neutrophil immunostaining was performed on renal sections and neutrophil counts from 10 high-power fields from each section are shown as means ± SEM from five to six mice of each group. N.d.m not done. B, Sigirr+/+ or Sigirr−/− mice underwent bilateral IR for 45 min after having received an intraperitoneal injection of clodronate which specifically depletes macrophages and dendritic cells. Data represent serum creatinine levels of individual mice 24 h after bilateral IR.

Discussion

SIGIRR is a known inhibitor of LPS signaling and modulates carcinogenesis after toxic injury of the intestinal epithelial barrier (17, 19, 22). The results of our present study now first identify the constitutive expression of SIGIRR as a novel mechanism that prevents postischemic acute renal failure by suppressing the early activation of resident myeloid cells.

During the last decade the concept has evolved that postischemic acute renal failure represents a state of sterile inflammation and that the recruitment of immune cells, that is, mostly neutrophils and macrophages, rather aggravate tissue injury than promoting the healing phase (1). For example, leukocyte recruitment to the tubulointerstitial compartment of the kidney is initiated by proinflammatory CC and CXC chemokines, which specifically regulate the molecular events that determine the spatial and temporal recruitment patterns of leukocyte subsets (31). Local production of CXCL2/MIP2 determines the early influx of neutrophils and the production of CCL2/MCP-1 determines the subsequent recruitment of macrophages in renal IRI (32, 33). Our data demonstrate that SIGIRR, constitutively expressed in the murine kidney, is required to inhibit the early production of CXCL2/MIP2 and CCL2/MCP-1, which prevents an excessive influx of neutrophils and macrophages to the postischemic kidney. Because in Sigirr-deficient mice such excessive interstitial leukocyte infiltrates were associated with increased immunity-related tubular necrosis, SIGIRR represents another endogenous inhibitor of inappropriate immune activation that prevents unnecessary postischemic tissue damage. This novel function of SIGIRR is similar to that of Tamm-Horsfall protein or that of endogenous immunoregulatory lipid mediators like lipoxin A4, protectin D1, and resolvins that all limit renal IRI (34, 35, 36).

The way SIGIRR suppresses IRI is determined by its structural characteristics and its cell type-specific function. SIGIRR is a single transmembrane receptor that can regulate IRI by modulating the activation of cells that express SIGIRR. In the kidney, tubular epithelial cells constitutively express SIGIRR mRNA and protein, but distinct glycosylation patterns inhibit SIGIRR’s immunoregulatory function (23). Hence, the SIGIRR genotype does not affect tubular epithelial cell activation by endotoxin (23) and, as shown here, during IR. In contrast, SIGIRR potently suppresses endotoxin-induced activation of CD11b-positive myeloid cells isolated from healthy murine kidneys (23). In addition to endotoxin, hypoxia drives dendritic cell activation via hypoxia-inducible factor 1-α signaling, especially when the cells are coactivated via TLR4 (37). Here we show that lack of SIGIRR massively enhances IR-induced activation of renal myeloid cells under similar culture conditions. The results from our in vitro studies are consistent with our previous finding that SIGIRR specifically modulates the activation of dendritic cells, but not of intrinsic renal cells (23). Additionally, lack of SIGIRR in myeloid cells was sufficient to replicate the phenotype of renal IRI in Sigirr-deficient mice. Additionally, depletion of renal myeloid cells with clodronate entirely prevented the aggravation of acute renal failure in SIGIRR-deficient mice. The physiological intrarenal network of APCs has only recently been phenotypically characterized (38, 39), but little is yet known about their functional roles in the initiation and progression of kidney diseases. Renal IR studies with Tlr2- and Tlr4-chimeric mice demonstrated that TLR2 and TLR4 signaling in parenchymal cells and not resident immune cells predominantly contributes to renal IRI (11, 12). Our data now first show that SIGIRR suppresses innate immune signaling in intrarenal immune cells during IR, which is a novel mechanism demonstrating how intrarenal immune cells limit inappropriate kidney injury after IR. However, it remains unclear whether this effect can be attributed to a single cytokine or chemokine that is attracting a specific “nephrotoxic” immune cell type. However, published data suggest that renal IRI is mediated rather by multiple proinflammatory cytokine mediators and cell types. As another mechanism, SIGIRR may control the role of IL-18 on intrarenal immune cells, which was recently shown to contribute to innate immunity in renal IRI (40). We conclude that resident myeloid cells have a major role in regulating IR-related danger signaling. Future studies will have to evaluate whether gene polymorphisms in genes like SIGIRR affect the risk for IRI. Conceptually, interventions that reduce the activation of resident immune cells might offer novel strategies to prevent or to limit IRI.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by grants from the Deutsche Forschungsgemeinschaft (AN372/9-1 and GRK 1202) and by the European Union Integrated Projects INNOCHEM (FP6-518167) to H.J.A. and MUGEN to C.G. and A.M. Parts of this project were prepared as a doctoral thesis at the Faculty of Medicine, University of Munich, by A.A.-F.

  • ↵2 M.L. and A.A-F. contributed equally to the results of this study.

  • ↵3 Address correspondence and reprint requests to Dr. Hans-Joachim Anders, Medizinische Poliklinik, Universität München, Pettenkoferstrasse 8a, 80336 München, Germany. E-mail address: hjanders{at}med.uni-muenchen.de

  • ↵4 Abbreviations used in this paper: IR, ischemia-reperfusion; IRI, ischemia-reperfusion injury; PAS, periodic acid-Schiff; SIGIRR, single Ig IL-1-related receptor; TIR, Toll-IL-1 receptor.

  • Received January 26, 2009.
  • Accepted July 15, 2009.

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