Organ cross talk is increasingly appreciated in human disease, and inflammatory mediators are shown to mediate distant organ injury in many disease models. Colitis and intestinal injury are known to be mediated by infiltrating immune cells and their secreted cytokines. However, its effect on other organs, such as the kidney, has never been studied. In the current study, we examined the effect of dextran sulfate sodium (DSS)-colitis on kidney injury and inflammation. In addition, we hypothesized that netrin-1 could modulate colon-kidney cross talk through regulation of inflammation and apoptosis. Consistent with our hypothesis, DSS-colitis induced acute kidney injury in mice. Epithelial-specific overexpression of netrin-1 suppressed both colitis and colitis-induced acute kidney injury, which was associated with reduced weight loss, neutrophil infiltration into colon mucosa, intestinal permeability, epithelial cell apoptosis, and cytokine and chemokine production in netrin-1 transgenic mice colon and kidney. To determine whether netrin-1-protective effects were mediated through suppression of IL-6, IL-6 knockout mice were treated with DSS and acute kidney injury was determined. IL-6 knockout was resistant to colitis and acute kidney injury. Moreover, administration of IL-6 to netrin-1 transgenic mice did not affect the netrin-1-protective effects on the colon and kidney, suggesting that netrin-1 may reduce both IL-6 production and its activity. The present study identifies previously unrecognized cross talk between the colon and kidney, and netrin-1 may limit distant organ injury by suppressing inflammatory mediators and apoptosis.
- acute kidney injury
- organ cross talk
chronic diseases of the gastrointestinal tract such as Crohn's disease (CD) and ulcerative colitis (UC) are a large burden on patients and society. Over a million sufferers are known to exist around the world (3). Disease progression is persistent, with the eventual outcome being surgical intervention for the majority of patients (40). Extraintestinal manifestations and complications are common in patients with inflammatory bowel disease (IBD) and can involve almost any organ or system. Renal or urinary complications occur in 4–23% of patients, often in those with severe, long-standing disease. The most common manifestations are kidney stones, enterovesical fistulas, and ureteral obstruction (32). Fistulas between the gastrointestinal tract and the urinary system are uncommon, occurring in 1–8%, being more common in patients with ileal or ileocecal disease than in those with colonic disease. Ureteral obstruction is not caused by stones in 50–73% of cases of CD and 50% of cases of UC (32). This noncalculus obstruction (NCO) is on the right in the great majority of patients. Renal tubular injury was also seen in many IBD patients (7). A recent study also showed the presence of an anti-brush border antibody in these IBD patients that cross-reacts with a kidney proximal tubular brush-border membrane antigen. There is also an antigenic relationship between the human kidney, colon, and the common antigen of enterobacteriaceal (35, 39). An antigenic relationship was also shown in an animal model of UC (9). In rats, the use of hyperosmolar enemas can induce nephritis through massive acidosis and electrolyte disturbances with hypocalcemia and hypernatremia. Inflammatory changes also occur in kidneys of mutant mice with IL-2 receptor γ chain deficiency (12). A description of tubulointerstitial disease unrelated to the consumption of nephrotoxic agents in such patients is not extensive. However, the presence of tubular injury in IBD patients is increasingly appreciated even before the start of nephrotoxic drugs such as aminosalicylate (7, 10, 16, 18). Moreover, it is now even recommended for the clinicians to look for the presence of tubulointerstitial disease and renal failure in IBD patients (16). Advances in understanding the pathogenic mechanism has given us hope that disease can be treated with recombinant proteins; however, this approach is not without serious complication and adverse events (3). Therefore, there is an urgent need for novel therapeutics, particularly to control the exaggerated inflammation within the intestine of patients with IBD.
Although the above-mentioned studies suggest the presence of an association between UC and kidney involvement, the nature of kidney involvement, role of inflammation, mechanism of kidney injury, and whether UC and kidney injury can be suppressed with anti-inflammatory therapy have never been examined.
Netrin-1 is a laminin-related guidance cue recently shown to have antiapoptotic and anti-inflammatory functions in many models of acute and chronic diseases (24, 26, 38, 43, 45, 48, 49). However, its role in colitis-induced acute kidney injury has never been examined. Here, we report for the first time that epithelial-specific overexpression of netrin-1 suppressed DSS-induced colitis, inflammation, and acute kidney injury. Our study suggests that organ cross talk in DSS-induced colitis can be effectively treated with the anti-inflammatory molecule netrin-1.
MATERIALS AND METHODS
The Institutional Animal Care and Use Committee of the Georgia Health Sciences University approved all of the protocols and procedures using animals (approval no. 2011-0348). Netrin-1 transgenic mice were characterized for transgene expression and phenotype (27, 45). Eight-week-old netrin-1 transgenic mice, which express chicken netrin-1 in proximal tubular epithelial cells under control of the fatty acid binding protein promoter, their wild-type littermates, and IL-6 knockout and C57BL/6 mice were used in DSS studies. IL-6 knockout mice and C57BL/6J mice were purchased from Jackson Laboratories. DSS (3.5%, MP Biomedicals) was added to the drinking water. The DSS was removed after 5–9 days, and the animals were fed with normal water. Animals were euthanized on day 2 after removal of DSS. Some transgenic animals were treated with IL-6 at a dose of 10 ng·animal−1·day−1 (intraperitoneally) starting on day 2 of DSS treatment and lasting until the end of the experiments.
Quantification of mRNA by real-time RT-PCR.
Real-time RT-PCR was performed in an Applied Biosystems 7700 Sequence Detection System (Foster City, CA). Total RNA (1.5 µg) was reverse transcribed in a reaction volume of 20 µl using an Omniscript RT kit and random primers. The product was diluted to a volume of 150 µl, and 6-µl aliquots were used as templates for amplification using the SYBR Green PCR amplification reagent (Qiagen) and gene-specific primers. The primer sets used were the following: mouse TNF-α (forward: GCATGATCCGCGACGTGGAA, reverse: AGATCCATGCCGTTG GCCAG); MCP-1 (forward: ATGCAGGTCCCTGTCATG, reverse: GCTTGAGGTGGTTGTGGA); ICAM-1 (forward: AGATCACATTCACGGTGCTG, reverse: CTTCAGAGGCAGGAAACAGG); netrin-1 (forward: AAGCCTATCACCCACCGGAAG, reverse: GCGCCACAGGAATCTTGATGC); osteopontin (forward: TCACCATTCGGATGAGTCTG-3', reverse: ACTTGTGGCTCTGATGTTCC); hemeoxygenase-1 (HO-1; forward: AGCATGCCCCAGGATTTG, reverse: AGCTCAATGTTGAGCAGGA); and transforming growth factor (TGF)-β1 (forward: TGACGTCACTGGAGTTGTACGG; reverse: GGTTCATGTCATGGATGGTGC). The amount of DNA was normalized to the β-actin signal amplified in a separate reaction (forward primer: AGAGGGAAATCGTGCGTGAC; reverse: CAATAGTGATGACCTGGCCGT).
Serum cytokine measurement.
Serum cytokines and chemokines were measured using an ELISA array kit from SA Biosciences and ELISA kit from eBiosciences.
Intestinal permeability in vivo.
Intestinal permeability was examined using a FITC-labeled dextran method as described previously (14). Wild-type mice or netrin-1 transgenic mice were exposed to 3.5% DSS for 5 days before oral gavage with FITC-dextran (4 kDa, 0.6 mg/g at 80 mg/ml; TdB Consultancy). Five hours after FITC-dextran administration, blood was collected by tail vein bleeding, and the serum concentration of FITC was quantified using a Biotech fluorescent plate reader.
TACS TdT in situ apoptosis detection.
To identify apoptotic cells, colon tissue sections were stained using a TACS TdT in situ Apoptosis Detection kit (R&D Systems) according to the manufacturer's instructions. Briefly, tissue sections were deparaffinized, hydrated, and washed with PBS. Sections were digested with proteinase K for 15 min at 24°C. Slides were then washed, and endogenous peroxidase activity was quenched with 3% H2O2 in methanol. Slides were washed and incubated with TdT labeling reaction mix at 37°C for 1 h and then with streptavidin-horseradish peroxidase. Color was developed using TACS blue label substrate solution. Slides were washed, counterstained, and mounted with Permount. Sections were photographed, and labeled cells were counted and quantified.
Renal function was assessed by measurements of serum creatinine (DZ072B, Diazyme Labs).
Histology and immunostaining.
Kidney tissue was fixed in buffered 10% formalin for 12 h and then embedded in paraffin wax. For assessment of injury, 5-µm sections were stained with periodic acid-Schiff (PAS) followed by hematoxylin. Tubular injury was assessed in PAS-stained sections using a semiquantitative scale (37) in which the percentage of cortical tubules showing epithelial cell necrosis, brush-border loss, cast formation, and apoptotic bodies in the cortex were assigned a score: 0 = normal; 1 = <10%; 2 = 10–25%; 3 = 26–75%; 4 = >75%. Sections were scored independently by two investigators who were blinded to the treatment of the animal. Ten fields of ×40 magnification were examined and averaged. The individual scoring of the slides was blinded to the genotype of the animal. To quantify leukocyte infiltration, sections were stained with a rat anti-mouse neutrophil antibody or anti-mouse macrophage antibody (1:200 dilution, Abcam, Cambridge, MA) followed by a goat anti-rat biotin conjugate. Color was developed after incubation with ABC reagent (Vector Labs). Stained sections were photographed and five ×40 fields of neutrophils were examined for quantification of leukocytes. To determine endogenous mouse netrin-1 and chicken netrin-1 (transgene) expression, sections were stained with a rabbit anti-mouse netrin-1 antibody (1:100 dilution, Calbiochem) or goat anti-chicken netrin-1 antibody (R&D Systems) followed by a goat anti-rabbit biotin or rabbit anti-goat biotin conjugate. Color was developed after incubation with ABC reagent (Vector Labs). Stained sections were photographed using an Olympus inverted microscope with a color CCD camera.
Samples of proximal colon were fixed in 10% buffered formalin and stained with hematoxylin and eosin. The histological examination was performed in a blinded fashion using a scoring system previously validated and described (4). Three independent parameters were measured: severity of inflammation (0–3: none, slight, moderate, severe); depth of injury (0–3: none, mucosal, mucosal and submucosal, transmural); crypt damage (0–4: none, basal damaged, basal damaged, only surface epithelium intact, entire crypt and epithelium lost); and percentage of the involved area (0–4: 0%, and 1–10, 10–25, 25–50, and 50–100%). All scores on the individual parameters together could result in a total score ranging from 0 to 14.
Kidney neutrophil sequestration was quantified using a fluorescence based MPO assay kit (K745–100, BioVision, Milpitas, CA). In short, animals were euthanized, and kidney were perfused with 5 ml of PBS through the right ventricle. The kidney was excised, homogenized in 4 volumes of Assay Buffer, and centrifuged (13,000 g for 10 min) to remove insoluble material. Fifty microliters of the sample was used for the assay. Enzyme activity was calculated from the standard curve. Enzyme activity was normalized initially for per milligram of protein, and then the fold-increase over control samples was calculated.
All assays were performed in duplicate or triplicate. The data are reported as means ± SE. Statistical significance was assessed by an unpaired, two-tailed Student's t-test for single comparisons or ANOVA for multiple comparisons.
Regulation of endogenous netrin-1 expression in the colon and kidney during colitis.
A previous study suggests that inflammation regulates netrin-1 expression that varied among different cells and tissues (1, 24, 31, 46). To examine the effects of inflammation on intestinal expression of endogenous netrin-1, we assessed netrin-1 mRNA levels by RT-PCR and protein levels by immunohistochemistry in mouse colon tissues (Fig. 1). Netrin-1 mRNA expression is significantly downregulated following DSS treatment in wild-type and netrin-1 transgenic animals. The downregulation of endogenous netrin-1 was much more pronounced in netrin-1 transgenic animals treated with DSS (Fig. 1B). Endogenous mouse netrin-1 protein is not detectable in the normal colon. However, after DSS treatment, netrin-1 protein expression is readily detectable in colonic epithelial cells despite the downregulation of mRNA following DSS (Fig. 1A). These observations indicate induction of netrin-1 expression during experimental colitis with significant expression by the epithelium.
Overexpression of chicken netrin-1 in transgenic mice.
Netrin-1 induction after DSS is low in colonic epithelial cells. To determine whether colonic epithelial cell-specific overexpression of netrin-1 can protect the colon against DSS-induced inflammation and injury, we used mice that overexpress chicken netrin-1 using the liver fatty acid binding protein (L-FABP) promoter. Overexpression was confirmed by RT-PCR and immunohistochemistry. Consistent with a previous report (25), transgene chicken netrin-1 mRNA expression was seen in the colon, small intestine, spleen, and kidney (Fig. 1C). A very high level of transgene expression was seen in colon epithelial cells (Fig. 1A). After DSS, chicken netrin-1 mRNA expression is increased even more.
Colon epithelial-specific overexpression of netrin-1 suppresses disease activity in a model of experimental colitis.
A recent study indicates that mice with epithelial cell-specific overexpression of netrin-1 experience a blunted inflammatory response in models of acute and chronic inflammation (27, 36), thus prompting our investigation of these mice in experimental colitis and its impact on kidney function. We observed that netrin-1 transgenic mice experienced blunted disease severity compared with wild-type controls as measured by weight loss, colon and spleen weight, and histological damage (Figs. 2 and 3, respectively). Most importantly, netrin-1 overexpression suppresses DSS-induced intestinal permeability, suggesting that netrin-1 protects the intestinal barrier (Fig. 2D). Thus we conclude that netrin-1 overexpression in colon epithelial cells is protective against the development of DSS-colitis.
Netrin-1 overexpression suppresses inflammation of the colon during colitis.
Recent studies had demonstrated that netrin-1 can inhibit neutrophil and monocyte infiltration into tissues such as lung and kidney in acute inflammation (24, 43). Inappropriate neutrophil accumulation within the lamina properia (LP) is a key feature of early and active UC (4, 6, 19). DSS-colitis is characterized by rapid neutrophil influx into the LP, closely modeling human disease. Additionally, expression levels of the proinflammatory cytokines TNF-α, IL-1β, IP-10, IL-6, MCP-1, and proinflammatory enzyme cyclooxygenase (COX)-2 (Fig. 4) were significantly enhanced in wild-type mice following DSS treatment, confirming the exaggerated inflammatory response observed in these mice. These changes were minimal in netrin-1 transgenic mice. Immunostaining of colon sections showed that treatment with DSS leads to an increase in neutrophil infiltration in the LP of wild-type mice compared with netrin-1 transgenic animals (Fig. 5A).
Netrin-1 overexpression suppresses apoptosis in colon epithelial cells during colitis.
Colon epithelial cell apoptosis is known to occur during DSS-colitis (34, 42). Since netrin-1 has the ability to suppresses apoptosis in colon epithelial cells (25), we determined the effect of netrin-1 overexpression on DSS-induced colon epithelial cell apoptosis. As shown Fig. 5B, DSS treatment significantly increased apoptosis in wild-type mice, which was significantly suppressed in netrin-1 transgenic colon, suggesting that netrin-1 also regulates colon epithelial cell apoptosis.
Experimental colitis induces acute kidney injury.
Human studies indicate that involvement of nonintestinal organs in UC. Therefore, we determined whether DSS-colitis causes acute kidney injury. DSS treatment of wild-type mice caused acute kidney injury as seen by increased serum creatinine (Fig. 6).
Tubular epithelial cell-specific overexpression of netrin-1 suppressed colitis-induced acute kidney injury and inflammation.
In addition to overexpression of netrin-1 in colon epithelial cells, the L-FABP promoter is also found to be active in kidney proximal tubular epithelial cells (Fig. 7). DSS treatment did not alter the expression of the transgene in the kidney. Previous observations demonstrate that netrin-1 suppresses kidney injury and inhibits neutrophil and monocyte recruitment in acute inflammation (36, 43). As shown in Fig. 6, netrin-1 overexpression was protective of the kidney from DSS-induced acute kidney injury. Inappropriate neutrophil accumulation within the kidney interstitium is a key feature of early-phase acute kidney injury (21, 43). Immunostaining of kidney sections showed that treatment of DSS led to an increase in neutrophil infiltration in the kidney interstitium of wild-type mice compared with netrin-1 transgenic animals (Fig. 8), which was associated with increased histological injury in wild-type compared with netrin-1 transgenic animals (Fig. 8). Additionally, expression levels of the proinflammatory cytokines IP-10, IL-6, and MCP-1 and proinflammatory enzyme COX-2 (Fig. 9) were significantly enhanced in wild-type mouse kidneys following DSS treatment, confirming the exaggerated inflammatory response observed in these mice. These changes were minimal in netrin-1 transgenic mouse kidneys.
Netrin-1 overexpression confers resistance to DSS+IL-6-induced acute kidney injury.
Since epithelial-specific netrin-1 overexpression suppresses IL-6 expression in the colon and kidney as well as its concentration in plasma, we hypothesized that netrin-1 suppresses DSS-induced acute kidney injury through suppression of IL-6 and its activity. To determine this directly, netrin-1 transgenic mice were fed DSS and received IL-6 or vehicle injection. Colitis was confirmed by measuring body weight loss and histological analysis, and acute kidney injury was determined by measuring serum creatinine. As shown Fig. 10, wild-type mice treated with DSS showed significantly increased body weight loss, histological damage (Fig. 10H), and serum creatinine level (Fig. 10B), which was suppressed in netrin-1 transgenic mice. Moreover, administration of IL-6 to netrin-1 transgenic mice does not induce renal dysfunction, suggesting that netrin-1 not only suppresses IL-6 production but also suppresses IL-6 activity. It is interesting to note that even extended administration of DSS and IL-6 (11 days) did not cause acute kidney injury.
IL-6 knockout mice are resistant to DSS-induced acute kidney injury.
To confirm further that IL-6 mediates colitis-induced acute kidney injury, wild-type and IL-6 knockout mice were fed DSS for 7 days, and the mice were euthanized on day 9. As shown in Fig. 10C, wild-type mice lost body weight more than the IL-6 knockout mice. In addition, wild-type mice developed acute kidney injury, which was significantly reduced in IL-6 knockout mice, suggesting that IL-6 mediates part of the DSS-induced acute kidney injury. Consistent with functional protection, IL-6 knockout mice showed much better preservation of colon morphology (Fig. 10J) than wild-type (Fig. 10H) mice treated with DSS.
CD and UC are serious relapsing disorders of the gastrointestinal tract that have devastating consequences in more than a million sufferers and society (3). Disease progression is relentless, with the eventual outcome being surgical intervention for up to 70% of patients (40). Clinical and epidemiological evidence suggest that IBD also affects other organs, including the kidney. However, IBD-induced acute kidney injury has not been examined, and the mechanism is unclear. Inflammation is a major player in IBD and IBD-associated disease processes in other organs. Therefore, effective control of inflammation may be a good and effective way of treating IBD. However, currently used therapeutic approaches are ineffective, and there is an urgent need for novel therapeutics, particularly to control the exaggerated inflammation within the intestine of patients with IBD. Recent studies have implicated the neuronal guidance molecule netrin-1 in regulating tissue inflammatory responses (1, 24, 26, 38, 43, 46). However, it was not known whether netrin-1 suppresses IBD-induced inflammation and acute kidney injury or whether epithelial cell-specific overexpression of netrin-1 in the colon and kidney is sufficient to suppress disease severity and inflammation of colon and kidney. Interestingly, our studies revealed that mice that overexpress netrin-1 in the colon and kidney proximal tubular epithelial cells showed protection against DSS-induced colitis. DSS colitis in wild-type mice was associated with robust neutrophil and mononuclear cell infiltration into the colonic LP and a significant increase in tissue cytokine expression, which was completely suppressed in netrin-1 transgenic mice. In addition, netrin-1 overexpression was associated with attenuated weight loss, improved tissue histology, and diminished colonic inflammation.
For the first time, we also show that DSS-colitis-induced acute kidney injury, which was associated with increased infiltration of neutrophils and cytokine and chemokine expression. Netrin-1 overexpression in proximal tubular epithelial cells was also suppressed acute kidney injury, inflammatory cytokine expression, and neutrophil infiltration. Together, these studies demonstrate for the first time that epithelial cell-specific netrin-1 overexpression is sufficient to suppress intestinal inflammation as occurs in the context of IBD and IBD-induced kidney inflammation and acute kidney injury. The use of anti-inflammatory agents such as aminosalicylate is a major cause of renal failure in patients with IBD. Amyloidosis and, very rarely, glomerulopathies (IgA nephropathy and membranous glomerulopathy) may also accompany IBD. However, several clinical studies have documented the presence of tubulointerstitial disease unrelated to the consumption of nephrotoxic agents in IBD patients. For example, Fraser et al. (7) had shown that a minimal degree of renal tubular dysfunction (manifested as tubular proteinuria) exists even before the introduction of nephrotoxic drugs (1). Urinary N-acetyl-β-d-glucosaminidase and α-1-microglobulin at diagnosis were increased in 10 (48%) and 11 (52%) patients, respectively. Another published case report (16) described a patient with UC who developed new onset and unprovoked renal failure with a high levels of serum creatinine (the maximum was 4.6 mg/dl). A renal biopsy demonstrated no amyloidosis on Congo red staining. However, intense interstitial mononuclear infiltration with occasional eosinophils, fibrosis, and tubular atrophy was detected. This particular patient did not receive any nephrotoxic agent for at least 1 yr, suggesting that kidney disorder might be part of a common immunological dysregulation that had begun with juvenile rheumatoid arthritis and ended in UC and tubulointerstitialitis.
The clinical relevance of our studies was also supported by two other reports describing overt renal failure due to IBD. In a study of 43 UC patients, 23% of cases had pathological enzymuria (a marker of early renal tubular injury) that almost normalized with anti-inflammatory therapies (18). Furthermore, a strong correlation between disease activity and tubular proteinuria has also been found in IBD patients (10). These findings may support our observation that organ cross talk exists between the colon and kidney, and tubulointerstitial disorder may be a natural part of IBD.
Regulation of endogenous netrin-1 expression in endothelial cells and epithelial cells of different organs may involve different mechanisms. In vitro and in vivo studies had demonstrated that colonic epithelial cell netrin-1 is regulated at the transcription level during colitis through hypoxia-inducible factor (HIF)-1- and NF-κB-dependent mechanisms (31, 38). However, inflammation was shown to downregulate netrin-1 expression in endothelial cells in lung and other tissues (24, 46). Moreover, during acute kidney injury, epithelial cell expression of netrin-1 mRNA is downregulated but, at the same time, protein expression is upregulated through increased translation of netrin-1 mRNA (13). Our current study suggests that very little netrin-1 is expressed under basal conditions in both the colon and kidney. DSS administration downregulated endogenous netrin-1 expression in both wild-type and netrin-1 transgenic animals. Previous studies had demonstrated that DSS induced netrin-1 expression, but the induction is modest at most (<1.5-fold) (1). The reason for the downregulation of endogenous netrin-1 mRNA in our study is not clear. However, it is consistent with studies in an ischemia-reperfusion injury model in the kidney, hypoxia-reoxygenation in kidney epithelial cells (13), and data from endothelial and lung tissues (24). However, netrin-1 protein was easily detectable after DSS treatment in the intestinal epithelium. This correlates with enhanced netrin-1 expression observed in the intestinal epithelia of patients with CD and UC (31) and the view that netrin-1 may be regulated at the level of mRNA translation (13). Unlike endogenous netrin-1 mRNA with regulatory elements, the chicken netrin-1 transgene does not have 5′- and 3′-regulatory elements. Our studies show that transgene chicken netrin-1 mRNA and protein are highly expressed, and the expression increased several-fold after DSS treatment. Transgenic expression was driven by the L-FABP promoter (partial/modified), which is known to be induced after epithelial injury (5, 30). Therefore, that transgene expression increases over baseline is consistent with L-FABP promoter regulation.
Supporting our observations, an anti-inflammatory effect of netrin-1 has been observed in models of acute inflammation such as kidney ischemia-reperfusion injury, hypoxia-induced inflammation, acute lung injury, and peritonitis (8, 24, 26, 43, 46). Netrin-1 has a number of receptors that are known to mediate its protective function, including deleted in colorectal cancer (DCC), uncoordinated receptor 5 (mouse UNC5A-D; human UNC5H1–4), and the A2B adenosine receptor (ADORA2B/Adora2b). We did not determine the receptor subtype that mediates the protective effects of netrin-1. However, our previous studies demonstrated that UNC5B mediates the netrin-1 anti-inflammatory effect in kidney ischemia-reperfusion injury. Consistent with our results, netrin-1 mediated suppression of inflammation, and migration of leukocytes was also mediated by UNC5B in vitro (24). In contrast, Aherne et al. (1) demonstrated that Adora2b mediates the anti-inflammatory effects of netrin-1. It is not clear whether the tissue difference could account for this difference.
Intestinal epithelial cell apoptosis, along with alterations in epithelial tight junction formation, is central to the mucosal disruption observed in patients with IBD (41, 42). Similarly, epithelial cell apoptosis and tight junction breakdown are key features of DSS-colitis, while inhibition of apoptosis in this model is barrier protective (44). Therefore, it is conceivable that the antiapoptotic effects of netrin-1 signaling may be central to its beneficial effects during experimental colitis. Transgenic overexpression of netrin-1 in the colon suppressed epithelial cell apoptosis, which leads to colon polyps and malignant transformation (25). Consistent with this observation, we see a large reduction in DSS-induced epithelial cell apoptosis in the colon. Moreover, our in vivo studies indicate that epithelial cell-specific overexpression of netrin-1 suppressed intestinal permeability in contrast to previous in vitro studies where recombinant netrin-1 addition to the culture did not alter DSS-induced epithelial permeability (1). In vitro studies do not recapitulate the complex in vivo cell-cell interaction. Moreover, administration of recombinant netrin-1 intraperitoneally or intravenously will unlikely reach such a highly local concentration in the epithelium compared with epithelial-specific overexpression. It is interesting to note that HIF-1α induction in epithelial cells confers protection against DSS-colitis (14). Netrin-1 is proangiogenic and induced during hypoxia. However, it is not clear as to whether netrin-1 mediates its protective function on the epithelium through induction of HIF-1α in our study.
Organ cross talk is shown to play an important role in human disease (15, 47). In a mouse model of intestinal ischemia-reperfusion, inflammatory changes were detected in the brain, kidney, and other organs (11, 29, 33). Acute kidney injury is also known to cause inflammatory changes in the lung, heart, and brain (23). IL-6 was shown to be a mediator of distant organ injury in organ cross talk in these studies (2, 17). Interestingly, DSS induced a large increase in both intestinal expression as well as serum concentration of IL-6, suggesting that netrin-1 may suppress DSS-induced acute kidney injury through suppression of IL-6. Consistent with this idea, IL-6 knockout mice are resistant to colitis-induced acute kidney injury. In addition, administration of IL-6 to netrin-1 transgenic mice did not exacerbate acute kidney injury. Our data suggest that netrin-1 suppresses both IL-6 production as well as activity.
Neutrophil infiltration is a prominent feature of both colitis and acute kidney injury (1, 19, 21, 43, 46). Depletion or blocking of neutrophil chemokine receptors (CXCR2) is protective against acute kidney injury and colitis (6, 20–22). Consistent with the previous report, we see a large increase in infiltration of neutrophils in the kidney and the colon, which is suppressed by netrin-1. The mechanism as to how epithelial netrin-1 suppresses neutrophil infiltration is not clear. Epithelial cells are known to produce inflammatory cytokines and chemokines that are known to mediate leukocyte infiltration. Interestingly, we see a large reduction in colon, kidney, and serum cytokine and chemokine levels in transgenic animals compared with wild-type littermates. Therefore, netrin-1 may suppress neutrophil infiltration indirectly through suppression of chemokine production.
In the present findings, overexpression of netrin-1 is protective in experimental colitis. Interestingly, previous studies have shown that administration of recombinant netrin-1 in DSS-colitis, where its expression is induced, is also therapeutically effective (1). Similar to our studies, it was shown that increasing the local concentration of HIF-1α transcription factor during experimental colitis is of therapeutic benefit (14). However, this is the first study showing that colitis-induced inflammation and acute kidney injury can also be suppressed with increased expression of netrin-1. Therefore, enhancing in vivo protective responses for therapeutic intervention will have beneficial effects on disease outcomes.
In summary, our studies demonstrate, for the first time, that DSS-colitis induced acute kidney injury and inflammation. Netrin-1 overexpression in epithelial cells protects the colon and kidney by suppressing inflammation and permeability. Part of the protective effects of netrin-1 was mediated through suppression of IL-6 production and activity. Either increasing netrin-1 expression or administering recombinant netrin-1 may be useful in treatment of intestine-kidney cross talk and kidney leukocyte accumulation as occurs in colitis.
This work was supported by an R01 National Institute of Diabetes and Digestive and Kidney Diseases Grant (7R01DK083379-02) to G. Ramesh.
No conflicts of interest, financial or otherwise, are declared by the authors.
Author contributions: P.R., C.J., and M.S. performed experiments; P.R., M.S., and G.R. analyzed data; P.R., M.S., and G.R. interpreted results of experiments; P.R. and G.R. prepared figures; P.R. and G.R. drafted manuscript; P.R., C.J., M.S., and G.R. approved final version of manuscript; C.J., M.S., and G.R. edited and revised manuscript; G.R. provided conception and design of research.
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