Norepinephrine released by intestinal Paneth cells exacerbates ischemic AKI

Abstract

Small intestinal Paneth cells play a critical role in acute kidney injury (AKI) and remote organ dysfunction by synthesizing and releasing IL-17A. In addition, intestine-derived norepinephrine is a major mediator of hepatic injury and systemic inflammation in sepsis. We tested the hypothesis that small intestinal Paneth cells synthesize and release norepinephrine to exacerbate ischemic AKI. After ischemic AKI, we demonstrated larger increases in portal venous norepinephrine levels compared with plasma norepinephrine in mice, consistent with an intestinal source of norepinephrine release after renal ischemia and reperfusion. We demonstrated that murine small intestinal Paneth cells express tyrosine hydroxylase mRNA and protein, a critical rate-limiting enzyme for the synthesis of norepinephrine. We also demonstrated mRNA expression for tyrosine hydroxylase in human small intestinal Paneth cells. Moreover, freshly isolated small intestinal crypts expressed significantly higher norepinephrine levels after ischemic AKI compared with sham-operated mice. Suggesting a critical role of IL-17A in Paneth cell-mediated release of norepinephrine, recombinant IL-17A induced norepinephrine release in the small intestine of mice. Furthermore, mice deficient in Paneth cells (SOX9 villin Cre mice) have reduced plasma norepinephrine levels after ischemic AKI. Finally, supporting a critical role for norepinephrine in generating ischemic AKI, treatment with the selective α-adrenergic antagonists yohimbine and phentolamine protected against murine ischemic AKI with significantly reduced renal tubular necrosis, inflammation, and apoptosis and less hepatic dysfunction. Taken together, we identify Paneth cells as a critical source of norepinephrine release that may lead to intestinal and liver injury and systemic inflammation after AKI.

INTRODUCTION

Although acute kidney injury (AKI) is a frequent and serious clinical complication, especially during the perioperative period, no effective therapy or preventive measures exist (2, 5, 21). The morbidity and mortality from AKI are very high and have remained virtually unchanged for the past 70 yr (5, 6). This lack of progress in treating clinical AKI is in part due to a high incidence of extrarenal complications, including hepatic dysfunction, pulmonary dysfunction, intestinal barrier disruption, and systemic inflammatory response syndrome, which frequently lead to sepsis and multiorgan failure (7, 38, 57). Indeed, patients with isolated AKI have a much better prognosis than patients with AKI complicated by extrarenal organ dysfunction (11, 49). These extrarenal systemic complications, secondary to AKI, are the major causes of morbidity and mortality from AKI (7, 57). Therefore, understanding the mechanisms of AKI-induced remote organ dysfunction may eventually lead to effective therapy for clinical AKI.

Paneth cells located in the base of intestinal crypts provide important mucosal immunity against commensal bacterial pathogens by actively secreting antimicrobial peptides (e.g., α-defensins) as well as several proinflammatory mediators (IL-17A and lysozyme) (4, 35, 44). We have previously shown that small intestinal Paneth cells play a critical role in generating hepatic and intestinal injury after ischemic AKI by synthesizing and releasing IL-17A (44). Renal ischemia-reperfusion (IR) injury in mice causes Paneth cell degranulation and increases the secretion of Paneth cell-derived IL-17A. Paneth cell-derived IL-17A is a major contributor to extrarenal organ injury after AKI as genetic deletion or pharmacological depletion of Paneth cells not only provides significant protection against hepatic and intestinal injury and systemic induction of IL-17A after renal IR but also attenuates ischemic AKI (44).

The small intestine has been implicated as the source of systemic inflammation and infection that contributes significantly to multiorgan failure in patients who are critically ill (31, 54). In particular, intestine-derived norepinephrine has been shown to be a major mediator of hepatic injury and systemic inflammation in septic shock (31, 59). Studies have shown that gut-derived norepinephrine plays a critical role in hepatic dysfunction in sepsis via activation of α₂-adrenergic receptors (53–55). However, the exact cell type responsible for the synthesis and release of norepinephrine remains unclear. Since small intestinal Paneth cells play a critical role in hepatic and intestinal injury and inflammation after AKI by releasing IL-17A, we tested the hypothesis that small intestinal Paneth cells synthesize and release norepinephrine after ischemic AKI and that this induction of norepinephrine is driven by Paneth cell IL-17A.

METHODS

Mice and induction of ischemic AKI.

C57BL/6 mice (20–25 g) were obtained from Harlan (Indianapolis, IN). Paneth cell-deficient mice (Sox9^f/f villin Cre) were generated as previously described (34). Sox9^f/f littermate mice were used as wild-type controls. After Columbia University Institutional Animal Care and Use Committee approval, male mice under pentobarbital anesthesia were subjected to sham surgery (sham) or 30-min renal ischemia, as previously described (17, 29). Sham animals underwent the same surgical procedures (anesthesia, laparotomy, bowel manipulations, and wound closure) without renal ischemia. For pain control, all mice received 0.5–1 mg/kg sc buprenorphine SR 24 h before surgery. Body temperature was maintained at ~37°C using a surgical heating pad during surgery and recovery from anesthesia. To test whether α-adrenergic receptor blockade attenuates ischemic AKI, some mice received 20 mg/kg phentolamine, 1 mg/kg yohimbine, or vehicle (saline) 30 min before the induction of renal ischemia. Kidney and small intestine tissues were collected 5 or 24 h after reperfusion. We also collected portal venous and systemic plasma for the measurement of creatinine and norepinephrine at 5 and 24 h after reperfusion. Some mice were injected with recombinant mouse IL-17A (300 and 1,000 ng/mouse iv) to determine whether IL-17A induces norepinephrine synthesis in the small intestine. To determine whether renal IR injury increases blood pressure (BP) in mice, we used a noninvasive tail-cuff system (CODA 2, Kent Scientific, Torrington, CT) to measure BP in sham mice and mice subjected to renal IR 24 h after surgery as previously described (24).

Assessment of renal and hepatic injury after renal IR.

Plasma creatinine and blood urea nitrogen (BUN) as markers of renal injury were measured by an enzymatic creatinine reagent kit according to the manufacturer’s instructions (ThermoFisher Scientific, Waltham, MA). This method of creatinine measurement largely eliminates the interferences from mouse plasma chromagens well known to the Jaffe method (50). We also measured the induction of kidney neutrophil gelatinase-associated lipocalin (NGAL) mRNA in mice subjected to sham surgery or renal IR injury. NGAL is an early and sensitive marker of renal tubular injury (32, 33). Plasma alanine aminotransferase (ALT) activity was measured using the Infinity ALT Assay Kit according to the manufacturer’s instructions (ThermoFisher Scientific).

Histological detection of kidney injury.

Twenty-four hours after renal IR injury, kidney hematoxylin and eosin-stained sections after renal IR surgery or sham surgery were blindly assessed using a grading scale of kidney necrotic IR injury to the proximal tubules (renal injury score, scale: 0–4) as previously described outlined by Jablonski et al. (23).

Laser capture microdissection of mouse and human small intestinal Paneth cells.

Laser capture microdissection (LCM) of individual mouse and human small intestinal Paneth cells was performed with the PixCell I LCM System (Arcturus Engineering, Mountain View, CA) as previously described (43). We obtained freshly discarded surgical small intestine specimens from four patients subjected to small bowel resection for pancreas cancer surgery [Columbia University Institutional Review Board determined these deidentified human tissue specimens to be “nonhuman subject research” under 45CFR46 (IRB-AAAM5869)]. Tissues were excised and embedded in OCT compound (Sakura, Torrance, CA), sectioned at a thickness of 10 μm, and mounted on 1.0 PEN Membrane Slides (Carl Zeiss, Thornwood, NY). Sections were then prepared for microdissection using a LCM staining kit (Ambion, Austin, TX) through a graded alcohol series (95%, 75%, and 50%) followed by cresyl violet staining. After sections had been destained via second graded alcohol series (50%, 75%, and 95%), they were dehydrated in 100% ethanol followed by xylene. LCM was performed on a Zeiss Axiovert 200M microscope equipped with PALM RoboSoftware (Carl Zeiss), and the total area of tissue collected per slide was tracked and recorded. RNA was isolated from the dissected tissue following the protocol provided by the RNAqueous-Micro Kit (Ambion) via column purification.

Isolation of mouse small intestinal crypts.

Intact small intestinal crypts were isolated with the distended intestinal sac method as previously described by Traber et al. (52). The small intestine from the duodenum to the ileum was removed and rinsed thoroughly with intestinal wash solution (0.15 M NaCl, 1 mM DTT, and 40 pg/mL PMSF) and then filled with buffer A (96 mM NaCl, 27 mM sodium citrate, 1.5 mM KCl, 8 mM KH₂PO₄, 5.6 mM Na₂HPO₄, and 40 pg/mL PMSF, pH 7.4). The ends were clamped with microclips, and the intestine was filled to a pressure of 50 cmH₂O. The filled intestine was submerged in oxygenated 0.15 M NaCl at 37°C for 40 min and then drained, and the solution was discarded. The intestine was then filled with buffer B (109 mM NaCl, 2.4 mM KCl, 1.5 mM KH₂PO₄, 4.3 mM Na₂HPO₄, 1.5 mM EDTA, 10 mM glucose, 5 mM glutamine, 0.5 mM DTT, and 40 pg/mL PMSF, pH 7.4) and then incubated at 37°C for another 20 min, and the intestinal contents were drained and collected. Cells from the 40- to 60-min fraction containing intact and isolated crypts were collected by pelleting at 100 g for 5 min at 4°C and washed once with PBS.

Detection of kidney apoptosis.

TUNEL staining detected fragmented DNA 24 h after hepatic IR as previously described (39). Apoptotic TUNEL-positive cells were quantified in five to seven randomly chosen ×200 magnified microscope image fields in the liver and small intestine.

Detection of kidney neutrophil infiltration.

Kidney neutrophil infiltration after renal IR injury was detected with immunohistochemistry using rat anti-mouse Ly6G monoclonal antibody (ThermoFisher Scientific) (42, 45). Neutrophils were counted from five to seven randomly chosen ×200 magnified microscope image fields.

RT-PCR.

Conventional RT-PCR was performed to analyze the expression of tyrosine hydroxylase as previously described (17, 29). Tyrosine hydroxylase mRNA expression was normalized to GAPDH mRNA (Table 1). Renal inflammation after IR was assessed by measuring proinflammatory mRNA markers including IL-6, IL-8, ICAM-1, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein-2, and TNF-α by quantitative RT-PCR as described previously, with primers listed in Table 1 (40, 42). Primer design was based on published GenBank sequences. To confirm equal RNA loading, GAPDH mRNA expression was also measured.

Table 1.

Primers and annealing temperatures used in quantitative RT-PCRs to amplify mouse cDNAs based on published GenBank sequences

Primers	Sequence (Sense/Antisense)	Annealing Temperature, °C
Human TH
Sense	5′-GCTAAACCTGCTCTTCTCCC-3′	63
Antisense	5′-TCCAAGTCCAGGTCAGGGTC-3′
Mouse TH
Sense	5′-GCCGTCTCAGAGCAGGATAC-3′	66
Antisense	5′-ACCAGGGAACCTTGTCCTCT-3′
Mouse TNF-α
Sense	5′-TACTGAACTTCGGGGTGATTGGTCC-3′	65
Antisense	5′-CAGCCTTGTCCCTTGAAGAGAACC-3′
Mouse MCP-1
Sense	5′-ACCTGCTGCTACTCATTCAC-3′	60
Antisense	5′-TTGAGGTGGTTGTGGAAAAG-3′
Mouse MIP-2
Sense	5′-CCAAGGGTTGACTTCAAGAAC-3′	60
Antisense	5′-AGCGAGGCACATCAGGTACG-3′
Mouse KC
Sense	5′-CAATGAGCTGCGCTGTCAGTG-3′	60
Antisense	5′-CTTGGGGACACCTTTTAGCATC-3′
Mouse IL-6
Sense	5′-CCGGAGAGGAGACTTCACAG-3′	62
Antisense	5′-GGAAATTGGGGTAGGAAGGA-3′
Mouse ICAM-1
Sense	5′-TGTTTCCTGCCTCTGAAGC-3′	60
Antisense	5′-CTTCGTTTGTGATCCTCCG-3′
Mouse NGAL
Sense	5′-CACCACGGACTACAACCAGTTCGC-3′	66
Antisense	5′-TCAGTTGTCAATGCATTGGTCGGTG-3′
GAPDH
Sense	5′-ACCACAGTCCATGCCATCAC-3′	65
Antisense	5′-CACCACCCTGTTGCTGTAGCC-3′

TH, tyrosine hydroxylase; MCP, monocyte chemoattractive protein; MIP, macrophage inflammatory protein; KC, keratinocyte-derived cytokine; NGAL, neutrophil gelatinase-associated lipocalin.

ELISA for norepinephrine.

Five hours after the induction of AKI, norepinephrine levels of the plasma, jejunum, and isolated crypts were measured with mouse-specific ELISA kits according to the manufacturer’s instructions (Rocky Mountain Diagnostics, Colorado Springs, CO). Plasma samples were added with 1 mM EDTA and 4 mM sodium metabisulfite to prevent catecholamine degradation. Small intestine tissues and isolated crypts were homogenized in 0.01 N HCl in the presence of EDTA and sodium metabisulfite, and samples were processed for mouse-specific ELISA kits.

Statistical analysis.

Data were analyzed with a t test when means between two groups were compared or with one-way ANOVA (e.g., plasma creatinine or BUN) plus a Tukey post hoc multiple-comparison test to compare mean values across multiple treatment groups. Ordinal values of the renal injury score were analyzed by a Mann-Whitney nonparametric test. In all cases, P < 0.05 was taken to indicate significance. All data are expressed as means ± SE.

RESULTS

Small intestinal Paneth cells express tyrosine hydroxylase.

We performed LCM to selectively capture small intestinal Paneth cells and measure tyrosine hydroxylase mRNA. Figure 1A shows representative micrographs of cresyl violet-stained Paneth cells in the mouse jejunum before and after selective capture and collection of Paneth cells for RT-PCR (×50 and ×400 magnified images shown, representative of 6 LCM experiments). Figure 1B shows representative images of cresyl violet-stained Paneth cells in the human small intestine before and after selective capture and collection of Paneth cells for RT-PCR (×200 shown). We obtained sufficient mRNA to perform RT-PCR for tyrosine hydroxylase and GAPDH in mouse (Fig. 1A, representative of 6 experiments) and human (n = 4; Fig. 1B) small intestinal Paneth cells. Small intestinal immunohistochemistry revealed that Paneth cells express tyrosine hydroxylase protein (Fig. 2, top, representative of 4 experiments). Myenteric plexus tyrosine hydroxylase served as a positive control for the staining. Isotype control antibodies for tyrosine hydroxylase showed no staining (Fig. 2, bottom).

Fig. 1.

Mouse and human small intestine express tyrosine hydroxylase mRNA. A: mouse small intestinal Paneth cells before and after laser capture microdissection (LCM; ×50 and ×400 magnifications shown; top). RT-PCR analyses of Paneth cells extracted from LCM showed tyrosine hydroxylase mRNA (representative of 6 experiments; bottom). The negative (Neg) control was a water blank. The mouse brain was used as a positive (Pos) control for RT-PCR. B: human small intestinal Paneth cells before and after LCM (×200 magnification shown; top). Discarded and deidentified human small intestine tissues from pancreas cancer surgery were used. RT-PCR analyses of tissues extracted from LCM showed tyrosine hydroxylase mRNA (representative of 4 experiments; bottom). GAPDH mRNA served as lane loading controls for both human and mouse small intestine LCM RT-PCR.

Fig. 2.

Mouse small intestines express tyrosine hydroxylase protein. Top: representative photomicrographs (×400, dark brown stain indicated by black arrows) of 5 experiments of immunohistochemistry for tyrosine hydroxylase in mouse small intestine tissue. Red arrows indicate tyrosine hydroxylase stain in the myenteric plexus. Bottom: lack of staining using isotype control antibody.

Small intestinal Paneth cells synthesize and release norepinephrine.

Consistent with tyrosine hydroxylase mRNA and protein expression, we demonstrated that small intestinal Paneth cells produce and release norepinephrine. Norepinephrine ELISA performed in freshly isolated small intestinal crypts showed increased norepinephrine 5 h after 30-min renal ischemia and reperfusion compared with intestinal crypts isolated from sham animals (n = 3–4; Fig. 3A). Further supporting Paneth cells as the source of small intestinal norepinephrine release, we showed that mice deficient in Paneth cells (Sox9^f/f villin Cre mice) had significantly reduced norepinephrine release in their plasma after renal IR (n = 4; Fig. 3B). Portal and peripheral blood norepinephrine increased significantly more after renal IR compared with sham mice (Fig. 3C). Consistent with the intestinal source for norepinephrine release, portal venous norepinephrine levels were significantly higher compared with the blood-sampled systemic circulation (n = 4–6; Fig. 3C). To determine whether the increase in systemic norepinephrine by renal IR affects BP, we measured BP 1 day after sham surgery or renal IR. We found that renal IR injury did not increase mean systemic BP (111.8 ± 4.3 mmHg, n = 4) compared with sham mice (127.7 ± 5 mmHg, n = 3). Finally, we demonstrated that IL-17A is the key stimulator for Paneth cell norepinephrine synthesis, as recombinant IL-17A injection increased jejunum norepinephrine levels in a dose-dependent fashion (n = 4; Fig. 3D).

Fig. 3.

Paneth cells synthesize and release norepinephrine. A: supernatant norepinephrine levels in freshly isolated jejunum crypts from mice subjected to sham operation or 30-min renal ischemia-reperfusion (IR) and 5-h reperfusion (n = 4–5). Norepinephrine levels were higher in isolated crypts after renal IR injury. *P < 0.05 vs. sham-operated mice. B: plasma norepinephrine levels from wild-type (SOX9^f/f) or Paneth cell-deficient (SOX9^f/f villin Cre) mice, demonstrating significantly lower plasma norepinephrine levels in Paneth cell-deficient mice subjected to renal IR injury (n = 4). *P < 0.05 vs. SOX9^f/f mice. C: portal venous and systemic plasma norepinephrine levels in mice subjected to sham surgery or to renal IR and 5-h reperfusion. Consistent with intestine being the source for circulating norepinephrine, portal venous norepinephrine levels were higher than systemic plasma norepinephrine (n = 4–5). *P < 0.05 vs. sham-operated mice; #P < 0.05 vs. systemic plasma. D: IL-17A induced small intestine norepinephrine synthesis. Mice were injected with recombinant IL-17A intravenously, and the jejunum was collected 5 h later for norepinephrine assay (n = 4). *P < 0.05 vs. vehicle-treated group. Error bars represent 1 SE.

α-Adrenergic receptor blockade protects against ischemic AKI.

To test whether α-adrenergic receptor activation exacerbates ischemic AKI, some mice were pretreated with phentolamine (a nonselective α-adrenergic receptor antagonist, 20 mg/kg) or yohimbine (a selective α₂-adrenergic receptor antagonist, 1 mg/kg) 30 min before ischemic AKI. Figure 4 shows that vehicle-treated mice subjected to renal IR had significantly higher plasma BUN and creatinine as well as kidney NGAL mRNA (n = 4–7) compared with sham mice. Both phentolamine-treated mice and yohimbine-treated mice showed significantly attenuated renal injury after ischemic AKI compared with vehicle-treated mice (n = 4–7). These findings suggest that Paneth cell synthesis and release of norepinephrine exacerbates ischemic AKI via α₂-adrenergic receptor activation.

Fig. 4.

α-Adrenergic receptor antagonists protect against ischemic acute kidney injury. Plasma blood urea nitrogen (BUN; n = 4–7) and creatinine (n = 4–7) as well as kidney neutrophil gelatinase-associated lipocalin (NGAL) mRNA (n = 4–7) were measured in mice subjected to sham surgery or renal ischemia and reperfusion (RIR) after vehicle treatment or after treatment with phentolamine or yohimbine. Plasma BUN and creatinine levels from mice subjected to sham surgery or to renal ischemia were measured at 5 or 24 h after reperfusion. P < 0.05 vs. vehicle-treated mice subjected to sham surgery; #P < 0.05 vs. vehicle-treated mice subjected to RIR. Error bars represent 1 SE.

Figure 5A shows representative kidney hematoxylin- and eosin-stained images of vehicle-treated mice subjected to sham surgery or 30-min renal IR and 24-h reperfusion (magnification: ×200, n = 6). Mice subjected to renal IR showed severe tubular necrosis and proteinaceous casts as well as increased tubular dilatation and congestion compared with sham mice. In contrast, phentolamine-treated or yohimbine-treated mice subjected to renal IR had decreased renal tubular necrosis, congestion, and cast formation compared with vehicle-treated mice subjected to renal IR. Kidneys from phentolamine-treated or yohimbine-treated mice had significantly reduced renal tubular injury score compared with vehicle-treated mice after IR (Fig. 5B).

Fig. 5.

α-Adrenergic receptor antagonists reduce renal tubular necrosis after ischemic acute kidney injury. A: representative hematoxylin and eosin (H&E)-stained images (from 5 experiments) of kidneys from vehicle-treated mice subjected to sham surgery or 30-min renal ischemia and 24-h reperfusion (magnification: ×200). Some mice were pretreated with phentolamine or with yohimbine before renal ischemia-reperfusion (RIR) injury. B: renal injury scores assessing the degree of renal tubular necrosis are also shown (scale: 0–4) 24 h after RIR (n = 6). *P < 0.05 vs. vehicle-treated mice subjected to sham surgery; #P < 0.05 vs. vehicle-treated mice subjected to RIR. Error bars represent 1 SE. For statistical analysis, a Mann-Whitney nonparametric test was used to detect significant changes.

α-Adrenergic receptor blockade attenuates neutrophil infiltration after ischemic AKI.

Figure 6A shows representative immunohistochemistry images and Fig. 6B shows counts of infiltrating kidney neutrophils (n = 6) in the kidneys of vehicle-treated mice subjected to sham surgery or 30-min renal IR and 24-h reperfusion (magnification: ×200, n = 6). Kidney neutrophil infiltration was significantly higher in mice subjected to renal IR injury. Consistent with the pathological role for α-adrenergic receptors in ischemic AKI, phentolamine-treated or yohimbine-treated mice had a significantly reduced number of neutrophils infiltrating after renal IR injury.

Fig. 6.

α-Adrenergic receptor antagonists decrease kidney neutrophil infiltration after ischemic acute kidney injury. Representative images of immunohistochemistry for neutrophils (dark brown) and counts of infiltrating kidney neutrophils (n = 6) in the kidneys of vehicle-treated mice subjected to sham surgery or 30-min renal ischemia and 24-h reperfusion (magnification ×200) are shown. Some mice were pretreated with phentolamine or with yohimbine before renal ischemia-reperfusion (RIR) injury. *P < 0.05 vs. vehicle-treated mice subjected to sham surgery; #P < 0.05 vs. vehicle-treated mice subjected to RIR Error bars represent 1 SE. PMN, polymorphonuclear neutrophils.

α-Adrenergic receptor blockade attenuates proinflammatory chemokine and cytokine induction after ischemic AKI.

Figure 7 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 vehicle-treated mice compared with sham mice. Consistent with the anti-inflammatory and renal protective role of α-receptor antagonism via attenuated induction of neutrophil and macrophage attracting chemokines, we showed that macrophage inflammatory protein-2, keratinocyte-derived cytokine, and MCP-1 expression was significantly attenuated in phentolamine-treated or yohimbine-treated mice subjected to renal IR. Moreover, IL-6, as well as ICAM-1 induction, was attenuated in phentolamine-treated or yohimbine-treated mice subjected to ischemic AKI.

Fig. 7.

α-Adrenergic receptor antagonists attenuate kidney proinflammatory chemokine/cytokine induction and neutrophil infiltration after ischemic acute kidney injury. With quantitative RT-PCR, we measured the expression of proinflammatory cytokine and chemokine mRNAs in the kidney [keratinocyte-derived cytokine (KC), monocyte chemoattractive protein (MCP)-1, macrophage inflammatory protein (MIP)-2, TNF-α, IL-6, and ICAM-1] 24 h after sham surgery or 30-min renal ischemia. Some mice were pretreated with phentolamine or yohimbine before renal ischemia-reperfusion (RIR) injury. *P < 0.05 vs. vehicle-treated mice subjected to sham surgery; #P < 0.05 vs. vehicle-treated mice subjected to RIR. Error bars represent 1 SE.

α-Adrenergic receptor blockade attenuates kidney apoptosis after ischemic AKI.

Figure 8A shows representative TUNEL staining images indicative of renal apoptosis and Fig. 8B shows counts of TUNEL-positive kidney cells (n = 5) mice subjected to sham surgery or 30-min renal IR and 24-h reperfusion (magnification: ×200). Many TUNEL (fragmented DNA)-positive cells were detected, suggestive of renal tubular apoptosis, in the kidneys from vehicle-treated mice subjected to renal IR injury. TUNEL-positive kidney cell counts were significantly reduced in phentolamine-treated or yohimbine-treated mice subjected to renal IR injury.

Fig. 8.

α-Adrenergic receptor antagonists attenuate kidney apoptosis after ischemic acute kidney injury. A: representative images of TUNEL staining indicative of renal tubular apoptosis. B: counts of TUNEL-positive kidney cells (n = 5) in the kidneys of vehicle-treated mice subjected to sham surgery or to 30-min renal ischemia and 24-h reperfusion (magnification: ×200). Some mice were pretreated with phentolamine or yohimbine before renal ischemia-reperfusion (RIR) injury. *P < 0.05 vs. vehicle-treated mice subjected to sham surgery; #P < 0.05 vs. vehicle-treated mice subjected to RIR. Error bars represent 1 SE.

α-Adrenergic receptor blockade attenuates hepatic injury after ischemic AKI.

We measured plasma ALT levels as a marker of hepatocyte injury in mice. Sham mice had similar plasma ALT levels after vehicle, phentolamine, or yohimbine treatment (Fig. 9). However, vehicle-treated mice developed mild but significant hepatic injury 24 h after ischemic AKI injury, with significantly higher plasma ALT levels compared with sham mice. In contrast, phentolamine-treated or yohimbine-treated mice were protected against hepatic injury after renal IR.

Fig. 9.

α-Adrenergic receptor antagonists reduce hepatic injury after ischemic acute kidney injury (AKI). Plasma alanine aminotransferase (ALT) levels detected increased hepatic injury after renal renal ischemia-reperfusion (RIR) in mice subjected to ischemic AKI (n = 6). Some mice were pretreated with phentolamine or yohimbine before RIR injury. *P < 0.05 vs. vehicle-treated mice subjected to sham surgery; #P < 0.05 vs. vehicle-treated mice subjected to RIR. Error bars represent 1 SE.

DISCUSSION

AKI is a major cause of mortality and morbidity during the perioperative period (7, 57). Unfortunately, interventions to attenuate this morbidity and mortality are lacking and only kidney-supportive measures (e.g., hemodialysis and hemodynamic/electrolyte support) currently are in use. A better understanding of the mechanisms of multiple organ injury caused by AKI would lead to improved therapeutic approaches for patients suffering from AKI. In the present study, we demonstrated that small intestinal Paneth cells express tyrosine hydroxylase to synthesize and release norepinephrine. This release of norepinephrine increased after renal IR injury, and IL-17A directly drives the synthesis of norepinephrine in small intestinal Paneth cells. Finally, we showed that α₂-adrenergic receptor blockade protects against ischemic AKI as well as AKI-induced hepatic dysfunction, consistent with the hypothesis that norepinephrine released from small intestinal Paneth cells exacerbates renal IR injury.

We previously demonstrated that intestinal Paneth cells play a critical role in generating remote hepatic and intestinal injury after ischemic AKI by synthesizing and releasing the proinflammatory cytokine IL-17A (44). Indeed, we demonstrated that ischemic AKI led to profound hepatic and small intestinal injury and inflammation that was mediated in part by small intestinal Paneth cell synthesis and release of IL-17A. The present study adds to our previous finding that Paneth cells synthesize and release norepinephrine as another proinflammatory mediator and that this is driven by Paneth cell-derived IL-17A. We demonstrated here that small intestinal Paneth cells express tyrosine hydroxylase mRNA and protein. Tyrosine hydroxylase is the rate-limiting enzyme for the synthesis of norepinephrine and epinephrine (30, 36, 59). Consistent with these findings, we demonstrated norepinephrine in small intestinal crypts that is driven by IL-17A, as recombinant IL-17A dose dependently increased crypt norepinephrine levels. Plasma norepinephrine increased significantly to ~2 ng/mL after renal IR (Fig. 3C) compared with sham mice (~1 ng/mL). However, this small increase in plasma norepinephrine does not lead to significant changes in systemic BP in mice after renal IR. Our study does have limitations. Although our experiments with isolated crypts and LCM-captured intestine tissue and experiments with Paneth cell-deficient mice strongly suggest small intestinal Paneth cells as the source of tyrosine hydroxylase and norepinephrine production, we cannot rule out nerve-derived tyrosine hydroxylase as a contributor of small intestinal norepinephrine induction after ischemic AKI.

Previous studies have implicated gut-derived norepinephrine as a key mediator of hepatic inflammation via activation of hepatic Kupffer cell α₂-adrenergic receptors (56, 59). Mesenteric organs (especially the intestines) contribute significantly to total body norepinephrine production (19). Studies have suggested that the intestine produces about half of the total norepinephrine formed in the body (1, 10). In contrast, the contribution of brain norepinephrine to total body norepinephrine is only between 3% and 9% (1). Furthermore, gut-derived norepinephrine has been implicated in generating hepatic injury and systemic inflammation in sepsis (26, 56, 59).

Intestine-derived norepinephrine activates Kupffer cell α₂-adrenergic receptors as well as peripheral macrophage α₂-adrenergic receptors to increase TNF-α release (26, 31). Consistent with these findings, catecholamines directly cause inflammation in sepsis and multiple organ failure via activation of leukocyte α₂-adrenergic receptors (53). In septic rats, α₂-adrenergic receptors induce in Kupffer cells to promote inflammatory response and organ injury (31, 56). In addition, α₁-adrenergic receptors increase LPS-mediated induction of proinflammatory cytokines in human macrophages (15). Therefore, both α₁- and α₂-adrenergic receptors are implicated in the proinflammatory effects of increased circulating catecholamines. Plasma catecholamines appear to play a critical role in ischemic AKI, as we have previously shown that renalase, an enzyme that degrades catecholamines, released by renal proximal tubules serves to protect against ischemic AKI by regulating plasma catecholamine levels (28). Our study now suggests that small intestinal Paneth cells tyrosine hydroxylase synthesize and release norepinephrine after ischemic AKI to generate renal as well as systemic inflammation. Consistent with these findings, a previous study by Zhou et al. (59) demonstrated increased intestinal hydroxylase expression in sepsis. However, their study did not implicate small intestinal Paneth cells as the cell type responsible for tyrosine hydroxylase expression.

Supporting a pathogenic role of α₂-adrenergic receptors against ischemic AKI, we show that blockade of α₂-adrenergic receptors provided significant renal protection in mice. This finding is not new, as other investigators also have demonstrated the renal protective effects of α₂-adrenergic antagonist against ischemic AKI (47, 48). Shimokawa et al. demonstrated renal protective effects of α₂-adrenergic antagonist yohimbine and JP-1302 in a rat model of ischemic AKI (47, 48). However, their studies did not elucidate the mechanisms for these renal protective effects of α₂-adrenergic receptor antagonist. We show here that intestinal Paneth cells synthesize and release norepinephrine that gets delivered to the liver as well as to the systemic circulation.

Studies in mice have demonstrated that phentolamine (a nonspecific α-adrenergic receptor antagonist) and yohimbine (an α2-adrenergic receptor antagonist) produce divergent effects on BP and heart rate. Phentolamine causes slight but insignificant decreases in BP and no changes in heart rate in mice (37, 46). In contrast, α₂-adrenergic receptor blockade produced by yohimbine increases both heart rate and BP in mice at 1 mg/kg ip (58). Since both of these α-adrenergic receptor antagonists protected against ischemic AKI in mice (presumably by blocking α-receptor activation by increased Paneth cell norepinephrine release), it is unlikely that changes in systemic hemodynamics contributed to improved renal function after these drug treatments.

Taken together, our current and previous studies support the hypothesis that intestinal Paneth cell-derived norepinephrine exacerbates ischemic AKI and systemic inflammation as well as remote liver and intestine injury via α₂-adrenergic receptor activation. Although we did not identify the cell type responsible for α₂-adrenergic receptor-mediated exacerbation of ischemic AKI, we speculate that hepatic Kupffer cells as well as peripheral macrophages are likely to be responsible based on previous studies demonstrating that these cells release proinflammatory mediators with α₂-adrenergic receptor activation (26, 31).

Clinically, a very small rise in plasma creatinine translates to a big rise in mortality and morbidity during the perioperative period (2, 27). For example, plasma creatinine increases of <0.3 mg/dL after surgery was associated with twofold to fivefold increases in the risk of death. This large increase in morbidity and mortality even in patients with mild AKI is, in part, due to the frequent development of extrarenal complications (11, 14, 57). Moreover, the prognosis after development of AKI is closely related to the severity of extrarenal complications that arise (20, 49). Indeed, hepatic dysfunction is very frequent in patients with perioperative AKI and frequently leads to other complications including gut dysfunction, lung failure, and sepsis (11, 57). We have previously shown that mice developed rapid hepatic injury after renal IR, which is characterized by periportal hepatocyte vacuolization, necrosis, and inflammation (44). We again show here that mice subjected to renal IR developed significant hepatic injury evidenced by increased plasma ALT and α₂-adrenergic receptor blockade significantly attenuated the hepatic dysfunction after ischemic AKI.

Although no clinical studies have been performed to test the protective role for α-adrenergic receptor blockade or neutralization against ischemic AKI, α-adrenergic receptor blockade appears to benefit patients with chronic renal disease through improved BP, better preservation of renal function, and improved metabolic markers (13). Furthermore, patients with AKI after cardiac surgery had significant decreases in plasma renalase (a catecholamine-metabolizing enzyme) 24 h after cardiopulmonary bypass, and the degree of decreases in plasma renalase was correlated with peak creatinine and changes in creatinine and serum NGAL levels (22). Therefore, a reduction of plasma norepinephrine concentration or blockade of α-adrenergic receptors may provide novel therapeutic approaches to ischemic AKI and AKI-induced remote organ dysfunction.

We and others have demonstrated that adenosine signaling protects against ischemic and inflammation-induced injury in multiple organs (12, 18, 25, 40). Hypoxia-inducible factor (HIF) signaling is also a master regulator of cellular and tissue protection in multiple organs, including the kidney, lung, heart, and liver (3, 8, 9, 16). The A_2B adenosine receptor, in particular, dampens inflammation in multiple organs including the kidney, liver, heart, and intestine (8, 9). Interestingly, HIF is a transcriptional mediator of A_2B adenosine receptor-mediated organ protection (9). A previous study (10) has shown that kidney renalase expression is increased after ischemic preconditioning and mediated by HIF signaling. Furthermore, HIF-1α is critical in protecting against toxin-induced hepatic injury, as HIF-1α deletion exacerbates hepatic injury and increases IL-17A synthesis (51). The multiorgan protective effects of A_2B adenosine receptor blockade and HIF signaling are analogous to our findings with α-adrenergic receptor blockade-mediated protection against ischemic AKI and hepatic dysfunction. It remains to be determined in future studies whether modulation of A_2B adenosine receptor and HIF signaling regulates Paneth cell-mediated synthesis of IL-17A and norepinephrine after ischemic AKI.

In summary, we show, in the present study, that small intestinal Paneth cells synthesize and release norepinephrine to exacerbate ischemic AKI. This release of norepinephrine after renal IR injury is driven by IL-17A and α-adrenergic receptor blockade protects against ischemic AKI, consistent with the hypothesis that norepinephrine release from small intestinal Paneth cells exacerbates renal IR injury. We hypothesize that norepinephrine synthesized and released by Paneth cells activates α₂-adrenergic receptors presumably located in intestinal macrophages and/or hepatic Kupffer cells to promote inflammation and tissue injury after renal IR injury (Fig. 10). Targeting Paneth cell release of norepinephrine may lead to effective therapy for ischemic AKI and remote organ dysfunction.

Fig. 10.

Proposed mechanisms of Paneth cell norepinephrine synthesis after ischemic acute kidney injury (AKI). We show, in the present study, that small intestinal Paneth cells synthesize and release norepinephrine (NE) to exacerbate ischemic AKI. This release of NE after renal ischemia-reperfusion injury is driven by IL-17A, and α₂-adrenergic receptor blockade protects against ischemic AKI, consistent with the hypothesis that NE release from small intestinal Paneth cells exacerbates renal ischemia-reperfusion injury. We hypothesize that NE synthesized and released by Paneth cells activates α₂-adrenergic receptors presumably located in intestinal macrophages and/or hepatic Kupffer cells to promote inflammation and tissue injury after renal ischemia-reperfusion injury.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-109544 and DK-115694 (to H. T. Lee).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.J.H. and H.T.L. conceived and designed research; S.J.H., M.K., and V.D.D. performed experiments; S.J.H., M.K., V.D.D., and H.T.L. analyzed data; S.J.H., M.K., and H.T.L. interpreted results of experiments; S.J.H., M.K., and H.T.L. prepared figures; S.J.H. and H.T.L. drafted manuscript; S.J.H. and H.T.L. edited and revised manuscript; S.J.H., M.K., V.D.D., and H.T.L. approved final version of manuscript.