The role for kidney TLR9 in ischemic acute kidney injury (AKI) remains unclear. In this study, we tested the hypothesis that renal proximal tubular TLR9 activation exacerbates ischemic AKI by promoting renal tubular epithelial apoptosis and inflammation. To test this hypothesis, we generated mice lacking TLR9 in renal proximal tubules (TLR9fl/fl PEPCK Cre mice). Contrasting previous studies in global TLR9 knockout mice, mice lacking renal proximal tubular TLR9 were protected against renal ischemia/reperfusion (IR) injury, with reduced renal tubular necrosis, inflammation (decreased proinflammatory cytokine synthesis and neutrophil infiltration), and apoptosis (decreased DNA fragmentation and caspase activation) when compared with wild-type (TLR9fl/fl) mice. Consistent with this, a selective TLR9 agonist oligonucleotide 1668 exacerbated renal IR injury in TLR9fl/fl mice but not in renal proximal tubular TLR9-null mice. Furthermore, in cultured human and mouse proximal tubule cells, TLR9-selective ligands induced NF-κB activation, proinflammatory cytokine mRNA synthesis, as well as caspase activation. We further confirm in the present study that global TLR9 deficiency had no impact on murine ischemic AKI. Taken together, our studies show that renal proximal tubular TLR9 activation exacerbates ischemic AKI by promoting renal tubular inflammation, apoptosis as well as necrosis, after IR via NF-κB and caspase activation. Our studies further suggest the complex nature of TLR9 activation, as renal tubular epithelial TLR9 promotes cell injury and death whereas TLR9 signaling in other cell types may promote cytoprotective effects.
Acute kidney injury (AKI) is a major clinical problem (1). Renal ischemia/reperfusion (IR) injury is a leading cause of AKI, and patients undergoing major surgical procedures (cardiac, vascular, or liver transplantation) have ∼50–80% chance of developing ischemic AKI (2, 3). Renal IR results in proximal tubular necrosis and apoptosis with rapid upregulation of proinflammatory cytokines and chemokines that causes influx of inflammatory leukocytes into the renal parenchyma (4–6). Unfortunately, despite nearly seven decades of research, there is no effective preventive measures or therapy for ischemic AKI (7, 8).
TLRs are pattern recognition receptors critical for regulating innate as well as adaptive immunity and play important roles in protecting against microbial invasion (9–11). Of 13 identified TLRs for mice and 11 for humans (9, 10), TLRs can be classified into cell surface TLRs (TLR1, 2, 4, and 6 that recognize bacterial or fungal products) and intracellular TLRs (TLR3, 7, 8, and 9 that recognize DNA and RNA products) (12). Numerous endogenous ligands can also activate TLRs, including histones, high-mobility group box 1, heat shock proteins, and mitochondrial DNA—all of these endogenous damage-associated pattern molecular patterns (DAMPs) are rapidly released after IR injury. Indeed, previous studies showed that cell surface TLR4 and TLR2 play important roles in liver and kidney IR injury (12–15).
TLR9 is a cytosolic receptor for unmethylated CpG DNA found in microbial DNA and DNA viruses (12, 16). Directly relevant to ischemic tissue injury, TLR9 also recognizes endogenous mitochondrial DNA products released from injured cells to trigger MyD88-dependent NF-κB–mediated gene transcription, leading to inflammation and apoptosis (16–19). Indeed, TLR9 activation plays a critical role in hepatic IR injury (16, 18). Furthermore, mitochondrial DNA released after trauma causes neutrophil extracellular trap formation (20). Recent studies suggest that neutrophil extracellular traps formed by DAMPs exacerbate sterile liver IR injury in mice (21).
However, the role for kidney TLR9 in ischemic AKI remains unclear. Previous studies suggest that TLR9 does not play a role in ischemic AKI, as mice deficient in TLR9 were not protected against ischemic AKI (22, 23). However, TLR9 has diverse effects depending on the cell types and organs studied (12). Indeed, TLR9 activation induces inflammation in hepatic IR and plays a role in septic AKI (17, 18, 24, 25). In contrast, TLR9 induces cytoprotective signaling in immune, cardiac, and neuronal cells (26–28). Additionally, global TLR9 deficiency may lead to compensatory changes in multiple nonrenal tissue and cell types (29). Taken together, global TLR9 deficiency may not allow for direct examination of TLR9 in ischemic AKI. Therefore, we generated mice with proximal specific deletion of TLR9 and tested the hypothesis that selective renal proximal tubular TLR9 activation exacerbates ischemic AKI by promoting renal tubular epithelial apoptosis and inflammation.
Materials and Methods
Generation of mice with renal proximal tubule cell-specific TLR9 deficiency
We bred mice with floxed TLR9 gene (TLR9fl/fl mice on a C57BL/6 [B6] background) (30) with mice that express Cre-recombinase selectively in proximal tubular epithelia (Cre recombinase under the control of the phosphoenolpyruvate carboxykinase promoter or PEPCK Cre, generated by Dr. V. Haase, Vanderbilt University) (31). PEPCK Cre mice were obtained through the laboratory of Dr. H. Eltzschig (University of Colorado School of Medicine) after they were backcrossed with a B6 strain for several generations. This approach allowed us to generate sibling mice with proximal tubule-specific deletion of TLR9 (TLR9fl/fl PEPCK Cre mice) or wild-type (TLR9fl/fl) mice. At least four generations of sibling breeding in our laboratory resulted in mice used in this study. Tail PCR with PEPCK Cre and TLR9 loxP-specific primers (Table I) confirmed the genotypes of proximal tubule cell-specific TLR9-null mice and the control wild-type mice (TLR9fl/fl) generated from breeding.
Confirmation of renal proximal tubular TLR9 deletion in TLR9fl/fl PEPCK Cre mice
Paraffin-embedded kidney tissues were cut at 5 μm, deparaffinized, and rehydrated in graded ethanol series. Endogenous peroxidase was inhibited using 3% hydrogen peroxide in methanol for 30 min, and then the sections were autoclaved in sodium citrate buffer (10 mM, pH 6) at 120°C for 10 min. The sections were incubated with 3% BSA in PBS for 60 min and then incubated overnight at 4°C with rabbit monoclonal anti-TLR9 Ab (1:100; 1% BSA in PBS; Abcam, Cambridge, MA). After washing three times with PBS buffer, the kidney sections were incubated with Alexa Fluor 594–conjugated goat anti-rabbit secondary Ab (1:100; 1% BSA in PBS; Abcam). To stain the brush border of proximal tubules, kidney sections were also stained using Phaseolus vulgaris leucoagglutinin lectin conjugated to a green fluorescent dye according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA) as described previously (32). P. vulgaris leucoagglutinin lectin binds avidly to the brush border of proximal tubules (33).
We also confirmed selective deletion of renal proximal tubular TLR9 in TLR9fl/fl PEPCK Cre mice by measuring TLR9 mRNA in isolated proximal tubule cells (34) as well as bone marrow cells, small intestine, and spleen with RT-PCR as described previously (35, 36). Primer design was based on published GenBank sequences. To control for RNA loading, GAPDH mRNA expression was also measured.
Renal IR injury in mice
After Columbia University Institutional Animal Care and Use Committee approval, 8- to 10-wk-old male TLR9 proximal tubule-deficient (TLR9fl/fl PEPCK Cre) mice or control wild-type (TLR9fl/fl) mice weighing 20–25 g were anesthetized with pentobarbital i.p. (50 mg/kg body weight or to effect; Sigma-Aldrich, St. Louis, MO). Mice were then subjected to right nephrectomy and 30-min left renal ischemia as described previously (37, 38). A separate cohort of TLR9fl/fl mice or renal tubular TLR9-null mice were pretreated with a selective TLR9 agonist (1 mg/kg oligonucleotide [ODN]-1668 or control ODN; InvivoGen, San Diego, CA) and subjected to 20-min renal IR injury. Some mice were treated with vehicle for ODN (saline) before 20-min renal ischemia. To determine the effects of global TLR9 deletion in ischemic AKI, we also subjected TLR9 knockout (TLR9KO) mice (C57BL/6JTlr9M7Btlr/Mmjax) and their wild-type controls (B6 mice) to 30-min renal IR injury (The Jackson Laboratory, Bar Harbor, ME). For pain management, all mice received 0.5–1 mg/kg s.c. buprenorphine SR prior to surgery. Sham-operated animals underwent anesthesia followed by laparotomy, right nephrectomy, bowel manipulations, and wound closure without renal ischemia. Body temperature of all mice was sustained at ∼37°C using a surgical heating pad during surgery as well as during recovery from anesthesia.
Kidney TLR9 protein expression after sham surgery and renal IR injury
Paraffin-embedded kidney sections were stained rabbit monoclonal anti-TLR9 Ab as described above. The sections were subsequently incubated with anti-rabbit secondary Ab (1:100, 2% goat serum in PBS; Vector Laboratories) for 1 h, washed, and incubated with avidin-biotin complex (ABC kit; Vector Laboratories). The sections were developed with 3,3′-diaminobenzidine. Negative controls were performed on serial sections by omitting primary Ab and by using nonspecific isotype control primary Ab. Kidney TLR9 expression was quantified from three to five randomly chosen ×200 microscope image fields surrounding the corticomedullary junction as described by Ruifrok et al. (39).
Measurement of renal injury after IR injury
Twenty-four hours after renal IR injury, we measured plasma blood urea nitrogen (BUN) and creatinine using an enzymatic creatinine reagent kit (Thermo Fisher Scientific). This creatinine measurement method limits the interferences from mouse plasma chromagens known to occur in the Jaffe method. We also performed quantitative RT-PCR for kidney neutrophil gelatinase-associated lipocalin (NGAL) mRNA from mice subjected to sham surgery or to renal IR injury. NGAL is an early and sensitive marker of renal tubular injury (40).
Histological detection of kidney injury
Twenty-four hours after renal IR injury, kidney H&E sections after renal IR surgery or sham surgery were blindly assessed using a grading scale of kidney necrotic IR injury to the proximal tubules (0–4, renal injury score) as outlined by Khalid et al. (41). In brief, the following scale was applied: 0, no damage; 1, loss of brush border in <25% of tubular cells; 2, loss of brush border in >25% of tubular cells; 3, necrosis up to 60% of tubular cells; and 4, necrosis in >60% of tubular cells.
Detection of kidney tubular apoptosis
We used two independent assays to assess kidney apoptosis after IR, that is, with TUNEL staining as well as detection of caspase 3 and caspase 8 fragmentation by immunoblotting. Twenty-four hours after sham surgery or renal IR injury, TUNEL staining detected fragmented DNA as described (42) using a commercially available kit (Roche, Indianapolis, IN). Apoptotic TUNEL+ cells were quantified in five to seven randomly chosen ×100 microscope images fields in the corticomedullary junction, and results were expressed as apoptotic cells counted per ×200 field. Kidney caspase 3 and caspase 8 immunoblotting were performed as described previously (43). Primary Abs for cleaved mouse caspase 3 and caspase 8 were from Cell Signaling Technology (Danvers, MA).
Detection of kidney neutrophil infiltration
Kidney neutrophil infiltration after IR injury was detected with immunohistochemistry using rat anti-mouse Ly6B mAb (AbD Serotec, Raleigh, NC) as described (36, 44). Primary IgG2a Ab (MCA1212; AbD Serotec) was used as a negative isotype control. Quantification of kidney infiltrating neutrophils was performed using five to seven randomly chosen ×200 microscope image fields (corticomedullary junction for kidney neutrophils), and results were expressed as neutrophils counted per ×200 field.
Quantitative RT-PCR for proinflammatory cytokine and chemokine mRNA expression
Renal inflammation after IR was also assessed by measuring proinflammatory mRNA markers, including IL-6, IL-8, ICAM-1, MCP-1, MIP-2, and TNF-α quantitative RT-PCR, as described previously with primers listed in Table II (35, 36). Primer design was based on published GenBank sequences. To confirm equal RNA loading, GAPDH mRNA expression was also measured.
Proximal tubule cell culture and TLR9 agonist treatment
Immortalized human proximal tubular cells (HK-2; American Type Culture Collection, Manassas, VA) were grown as described (45, 46). Mouse kidney proximal tubules were isolated and grown using Percoll density gradient separation as described previously (34). Confluent cells were treated with control ODNs or with selective 1–5 μM TLR9 ligands [ODN-2006 (human specific), ODN-BW006 (human and mouse specific) or ODN-1668 (mouse specific)] for 3 d as described (47). Proximal tubule cells were subjected to quantitative RT-PCR for proinflammatory genes, with primers listed in Table II. We also performed caspase 3 and caspase 8 immunoblotting in HK-2 cells (43). Finally, HK-2 cell nuclear and cytosolic fractions were prepared and subjected to p65 and the phospho-p65 subunit of NF-κB with Abs from Santa Cruz Biotechnology (Santa Cruz, CA) as described (48).
Data were analyzed with a Student t test, a one-way ANOVA plus a Tukey post hoc multiple comparison test, or a Mann–Whitney nonparametric U test to analyze renal injury scores. All data are expressed throughout the text as means ± SEM.
Genotyping and confirmation of renal proximal tubular TLR9 deletion
Tail PCR confirmed the genotypes of proximal tubule-specific TLR9-null mice (TLR9fl/fl PEPCK Cre) and the control wild-type mice (TLR9fl/fl) generated (Fig. 1A). PEPCK Cre primers generated a 466-bp fragment. TLR9 flox primers amplified a 507-bp fragment for the TLR9 floxed allele and a 433-bp fragment for the TLR9 wild-type allele.
We simultaneously stained kidney sections for TLR9 immunofluorescence (red) as well as Phaseolus vulgaris leucoagglutinin lectin to identify the brush border of proximal tubules (green) (Fig. 1B, representative of four experiments). Merged images demonstrate that cytosolic TLR9 staining was visible in lectin-positive proximal tubule cells as well as lectin-negative renal cells in TLR9fl/fl mice (left panel). In contrast, TLR9 staining was nearly absent in lectin-positive proximal tubule cells but was visible in lectin-negative renal cells in TLR9fl/fl PEPCK Cre mice (right panel).
To further confirm renal proximal tubule cell-specific deletion of TLR9 in TLR9fl/fl PEPCK Cre mice, we measured the TLR9 mRNA expression in isolated proximal tubules (Fig. 1C). TLR9 mRNA expression in renal proximal tubules from TLR9fl/fl PEPCK Cre mice were decreased by >95% compared with TLR9fl/fl mice. However, TLR9 mRNA expressions in isolated bone marrow cells, intestine, and spleen were equivalent between TLR9fl/fl PEPCK Cre mice and TLR9fl/fl mice (Fig. 1D), again demonstrating renal proximal tubule-specific deletion of TLR9 in TLR9fl/fl PEPCK Cre mice.
Kidney IR injury induces renal tubular TLR9 expression
Fig. 2 shows a representative TLR9 staining in TLR9fl/fl mice subjected to sham operation or to 30-min renal IR injury. Faint cytosolic TLR9 staining is visible in renal tubules from sham-operated mice. Kidneys from mice subjected to renal IR injury show increased cytosolic TLR9 staining compared with sham-operated mice (×200, representative of six immunohistochemistry experiments).
Renal proximal tubular TLR9 plays a critical role in ischemic AKI
Plasma creatinine values were similar between TLR9fl/fl mice and renal proximal tubular TLR9-null mice subjected to sham operation (Fig. 3A). TLR9fl/fl mice subjected to renal IR had significantly increased plasma BUN and creatinine as well as kidney NGAL mRNA (n = 6–8) compared with sham-operated mice (n = 4–5). We show in the present study that mice lacking renal proximal tubular TLR9 were protected against ischemic AKI compared with TLR9fl/fl mice as demonstrated by reduced plasma BUN and creatinine as well as kidney NGAL mRNA expression (n = 6–8). In contrast, global TLR9KO mice are not protected against renal IR injury and had similar increases in plasma BUN and creatinine and kidney NGAL mRNA expression compared with B6 mice (n = 5–7) (Fig. 3B).
Reduced renal tubular necrosis in proximal tubular TLR9-deficient mice after ischemic AKI
Fig. 4A shows representative H&E images of renal proximal tubular TLR9-null mice and TLR9fl/fl mice subjected to sham surgery or 30-min renal IR and 24-h reperfusion (original magnification ×200; n = 6). TLR9fl/fl mice subjected to renal IR showed severe tubular necrosis and proteinaceous casts as well as increased tubular dilatation and congestion. Renal proximal tubular TLR9-null mice had decreased renal tubular necrosis, congestion, and cast formation compared with TLR9fl/fl mice subjected to renal IR. Kidneys from renal proximal tubular TLR9-null mice had slightly but significantly reduced renal tubular injury score compared with control TLR9fl/fl mice after IR (Fig. 4B). Unlike reduced necrosis observed in renal proximal tubule TLR9-null mice, global TLR9KO mice and control B6 wild-type mice showed similarly severe renal tubular necrotic injury after IR (Fig. 4C, 4D).
Renal proximal tubular TLR9 regulates proinflammatory chemokine and cytokine induction after ischemic AKI
Fig. 5A shows fold increases in proinflammatory mRNAs normalized to GAPDH for each indicated mRNA (n = 4–6). Ischemic AKI increased all of the proinflammatory genes measured in TLR9fl/fl mice. Consistent with the critical role of renal proximal tubular TLR9 for the induction of neutrophil- and macrophage-attracting chemokines, we show that MIP-2 and MCP-1 expression was significantly attenuated in renal proximal tubular TLR9-null mice. Moreover, IL-6 as well as ICAM-1 induction was attenuated by renal proximal tubular TLR9 deficiency. In contrast, we determined that global TLR9KO mice had a similar degree of induction in keratinocyte chemoattractant (KC), TNF-α, MIP-2, and MCP-1 after ischemic AKI when compared TLR9-competent mice (Fig. 5B). Interestingly, global TLR9KO mice had significantly reduced kidney IL-6 mRNA expression but had higher kidney ICAM-1 mRNA expression. These data again suggest fundamental differences in renal inflammatory response between renal proximal tubular TLR9-null mice and global TLR9KO mice.
Renal proximal tubule TLR9-deficient mice have reduced kidney neutrophil infiltration after ischemic AKI
Fig. 6A shows representative immunohistochemistry images and Fig. 6B shows counts of infiltrating kidney neutrophils (n = 6) in the kidneys of TLR9fl/fl mice and TLR9fl/fl PEPCK Cre subjected to sham surgery or renal IR (original magnification ×200). Kidney neutrophil infiltration was significantly higher in the TLR9fl/fl wild-type mice compared with TLR9fl/fl PEPCK Cre mice after renal IR. In contrast, kidney neutrophil infiltration was similar between B6 mice and global TLR9KO mice after ischemic AKI (Fig. 6C, 6D).
Reduced kidney apoptosis in renal proximal tubule TLR9-deficient mice after ischemic AKI
Fig. 7A shows representative TUNEL staining images indicative of renal apoptosis, and Fig. 7B shows counts of TUNEL+ kidney cells (n = 5) from TLR9fl/fl mice and TLR9fl/fl PEPCK Cre subjected to sham surgery or to renal IR (original magnification ×200). Many TUNEL+ (fragmented DNA) cells were detected, suggestive of renal tubular apoptosis in the kidneys from TLR9fl/fl mice subjected to renal IR injury. TUNEL+ kidney cell counts were significantly reduced in proximal tubular TLR9-null mice compared with TLR9fl/fl mice. We also detected caspase 3 and caspase 8 activation by measuring cleaved caspase 3 and caspase 8 in the kidney lysates from sham-operated mice and mice subjected to renal IR injury (Fig. 7C). Caspase 3 and caspase 8 cleavage was increased in TLR9fl/fl mice subjected to renal IR that were significantly reduced in proximal tubular TLR9-null mice. In contrast, TUNEL+ kidney cells were equivalent between B6 mice and global TLR9KO mice after ischemic AKI (Fig. 7D, 7E).
TLR9 activation exacerbates ischemic AKI and increases renal tubular necrosis, inflammation, and apoptosis
There were no differences of plasma creatinine, kidney NGAL mRNA, as well as renal tubular necrosis between vehicle (saline)-treated mice and control ODN-treated mice (Fig. 8A, 8B). TLR9fl/fl mice subjected to 20-min renal IR after ODN-1668 treatment had significantly increased plasma creatinine as well as kidney NGAL mRNA compared with control ODN-treated mice (n = 5–6, Fig. 8A). In contrast, not only mice lacking renal proximal tubular TLR9 had reduced renal injury after 20-min renal IR, ODN-1668 treatment failed to exacerbate ischemic AKI in these mice (n = 5–6). Moreover, selective TLR9 activation with ODN-1668 increased renal tubular necrosis (Fig. 8B), neutrophil infiltration (Fig. 8C, 8E), proinflammatory cytokine mRNA induction (Fig. 8G), and TUNEL+ cells (Fig. 8D, 8F) in TLR9fl/fl mice compared with control ODN-treated TLR9fl/fl mice. However, ODN-1668 failed to increases these markers of renal injury and inflammation in renal proximal tubular TLR9-null mice. Furthermore, mice lacking renal proximal tubular TLR9 had reduced renal injury scores, neutrophil infiltration, proinflammatory cytokine induction, and TUNEL staining compared with TLR9fl/fl mice after 20-min renal IR injury.
|Primers .||Sequence (Sense/Antisense) .||Annealing Temperature (°C) .|
|Primers .||Sequence (Sense/Antisense) .||Annealing Temperature (°C) .|
Annealing temperatures used for each primer are also provided.
|Primers .||Sequence (Sense/Antisense) .||Annealing Temperature (°C) .|
|Primers .||Sequence (Sense/Antisense) .||Annealing Temperature (°C) .|
Annealing temperatures used for each primer are also provided.
TLR9 agonists induce renal proximal tubular NF-κB activation, caspase cleavage, and proinflammatory mRNA induction
Fig. 9A shows representative immunoblotting for the nuclear p65 NF-κB subunit and band intensity quantifications normalized to PARP-1 in HK-2 cells (n = 4). TLR9 activation with ODN-2006 increased nuclear translocation of p65 NF-κB subunit in HK-2 cells. Moreover, TLR9 activation with another TLR9 ligand (ODN-BW006) in HK-2 cells also increased the phosphorylated p65 NF-κB subunit in the cytosol as shown by the representative immunoblotting images for cytosolic p65 subunit (top) and band intensity quantifications normalized to p65 subunit or β-actin (n = 4, bottom) (Fig. 9B). Fig. 9C shows a representative immunoblotting experiment for cleaved caspase 3 and caspase 8 (top) and band intensity quantifications normalized to β-actin (n = 4, bottom) demonstrating increased TLR9 ligand–mediated cleavage of caspase 3 and caspase 8 in HK-2 cells.
Quantitative RT-PCR revealed that a selective TLR9 ligand ODN-BW006 treatment significantly induced the expression of IL-8, TNF-α, and ICAM-1 mRNAs measured without changing the MIP-2 expression (Fig. 9D, n = 4) in HK-2 cells. Additionally, we show that TLR9 agonist ODN-1668 treatment significantly induced the expression of proinflammatory mRNAs (IL-8, TNF-α, ICAM-1, MCP-1, and MIP-2) in renal proximal tubules from TLR9fl/fl mice but not in renal proximal tubules isolated from renal proximal tubule TLR9-null mice (Fig. 9E, n = 4).
TLR9 activation mediates IR injury in several organs, including the heart and liver, but plays no role in intestinal IR injury (16, 18, 49). In cardiac and hepatic IR injury, TLR9 activation by endogenous DAMPs plays a critical role in cardiomyocyte and hepatocyte death (18, 21, 30, 50). In particular, mitochondrial DNA has a high unmethylated CpG motif that can strongly stimulate TLR9 (30, 51). Previous studies have shown that circulating and locally released mitochondrial DNA can activate TLR9 to promote liver injury after trauma or IR (21, 30).
However, the role for TLR9 in ischemic AKI remains unclear. Suggesting a detrimental role of TLR9 in the kidney, mitochondrial DNA-induced TLR9 activation appears to play a critical role in septic AKI, as TLR9 signaling inhibition or genetic TLR9 deletion attenuates CLP-induced sepsis in mice (17, 24). Moreover, siRNA-mediated TLR9 deletion also protects against CLP sepsis (25). Additionally, TLR9 activation by endogenous DNA induces kidney podocyte apoptosis (51). These findings in various kidney pathologies suggest that kidney TLR9 may play a role in ischemic AKI. However, two previous studies using global TLR9-null mice failed to show a detrimental role for TLR9 in ischemic AKI (22, 23).
We demonstrate in the present study that renal proximal tubule TLR9 plays a critical role in ischemic AKI, as renal tubular TLR9-null mice had significantly reduced renal tubular necrosis and apoptosis after IR. Necrotic renal cells after IR release mitochondrial DNA that can target intracellular renal tubular TLR9 (24). In particular, we demonstrate that TLR9 signaling regulates renal proximal tubular apoptosis after kidney IR injury. Mice deficient in renal tubular TLR9 had reduced caspase 3 and caspase 8 activation after renal IR compared with control TLR9fl/fl mice. Moreover, the TLR9-selective ligand induced proximal tubule apoptosis in culture. Our data are consistent with the hypothesis that renal IR injury causes renal proximal tubular TLR9 activation to induce kidney apoptosis via caspase 3/8 activation.
Renal proximal tubule TLR9-null mice had significantly attenuated kidney IL-6, MCP-1, MIP-2, and ICAM-1 synthesis expression after ischemic AKI when compared with TLR9fl/fl mice (Fig. 5). Consistent with reduced proinflammatory cytokines and chemokines, renal proximal tubule TLR9-null mice had reduced neutrophil infiltration after IR. Moreover, TLR9-selective ligand induced proinflammatory cytokines and chemokines in cultured human and mouse renal proximal tubule cells. Finally, the TLR9-specific agonist ligand only induced inflammatory cytokines in TLR9fl/fl mice but not in TLR9-null renal proximal tubules. Taken together, our studies suggest that renal proximal tubules are the major source of proinflammatory cytokines and chemokines and that renal tubular TLR9 is a key regulator for several of these critical inflammatory mediators.
Neutrophils infiltrating the kidney after renal IR are a major contributor to additional renal injury after reperfusion and are able to recruit other inflammatory leukocytes, including NK cells, monocytes, and macrophages (52–54). Reduced kidney neutrophil infiltration most likely attenuated additional cytokine generation in renal proximal tubule TLR9-null mice that may have contributed to improved renal function after ischemic AKI. Consistent with this, renal proximal tubule cell TLR9-null mice also had significantly reduced MCP-1 expression in the kidney after IR. MCP-1 (CCL2) is the major chemokine that promotes macrophage infiltration and migration after IR (55, 56).
Although our findings suggest that renal IR-induced TLR9 activation directly promotes inflammation and apoptosis in renal proximal tubular cells and exacerbates ischemic AKI, it is interesting that mice globally deficient in TLR9 were not protected against ischemic AKI as shown previously by other investigators (22, 23). These discrepancies in renal injury between tissue-specific TLR9-null mice and global TLR9KO mice point out major differences between these mice and suggest caution in interpreting studies with global genetic deletions studies. These differences also suggest divergent effects of TLR9 activation depending on the cell and tissue types studied. Although renal proximal tubular TLR9 activation causes kidney injury by inducing apoptosis and inflammation after IR, other cell types in the kidney may benefit from TLR9 signaling. Indeed, a renal-protective role for TLR9 in a cisplatin-induced AKI model has been described, as TLR9-deficient mice had exacerbated renal injury with higher BUN, tubular injury, and inflammatory cytokine induction (57). In cisplatin-induced AKI as well as in a murine lupus model, TLR9 may promote tissue protection by promoting accumulation of beneficial regulatory T cells (57, 58). Directly relevant to our studies, global TLR9 deficiency leads to enhanced renal inflammation in several murine models of lupus (59, 60). Therefore, it is possible that global TLR9KO mice were not protected against ischemic AKI, as they may lack the tissue-protective regulatory T cells modulated by TLR9 signaling. Furthermore, not all TLR9 signaling results in exacerbation of IR injury, as TLR9 activation protects against cerebral as well as cardiac IR injury via activation of PI3K/Akt signaling (26, 27). Finally, TLR9 activation may mediate cardiac and neuronal protection by modulating energy metabolism (61). Therefore, our study as well as previous studies suggest that kidney TLR9 signaling results in complex and perhaps divergent effects depending on the cells targeted. Additionally, our findings also suggest that these variations in TLR9 signaling suggest caution in interpreting studies with global TLR9 gene deletion.
In summary, we demonstrate in this study that renal proximal tubular TLR9 plays a critical role in murine ischemic AKI by exacerbating necrosis, apoptosis, and inflammation after IR. Our study suggests caution when interpreting data from global gene deletion studies, as there were critical differences between proximal tubule TLR9-deficient mice and global TLR9-deficient mice subjected to ischemic AKI. Fig. 10 shows a schematic of proposed mechanisms for renal proximal tubular TLR9-mediated exacerbation of ischemic AKI. After renal IR, damaged renal cells release endogenous TLR9 activators (presumably mitochondrial DNA products) leading to NF-κB–mediated induction of proinflammatory chemokines and cytokines as well as caspase 3/8–mediated induction of renal tubular apoptosis.
We thank Dr. Volker Haas (Vanderbilt University, Nashville, TN) for providing the PEPCK Cre mice.
This work was supported by the Department of Anesthesiology, Columbia University and in part by National Institutes of Health Grants DK-109544 and DK-115694.
Abbreviations used in this article:
acute kidney injury
blood urea nitrogen
damage-associated molecular pattern
neutrophil gelatinase-associated lipocalin
The authors have no financial conflicts of interest.