Sang Won Park

Cytokines induce small intestine and liver injury after renal ischemia or nephrectomy

Patients with acute kidney injury (AKI) frequently suffer from extra-renal complications including hepatic dysfunction and systemic inflammation. We aimed to determine the mechanisms of AKI-induced hepatic dysfunction and systemic inflammation. Mice subjected to AKI (renal ischemia reperfusion (IR) or nephrectomy) rapidly developed acute hepatic dysfunction and suffered significantly worse hepatic IR injury. After AKI, rapid peri-portal hepatocyte necrosis, vacuolization, neutrophil infiltration and pro-inflammatory mRNA upregulation were observed suggesting an intestinal source of hepatic injury. Small intestine histology after AKI showed profound villous lacteal capillary endothelial apoptosis, disruption of vascular permeability and epithelial necrosis. After ischemic or non-ischemic AKI, plasma TNF-α, IL-17A and IL-6 increased significantly. Small intestine appears to be the source of IL-17A, as IL-17A levels were higher in the portal circulation and small intestine compared with the levels measured from the systemic circulation and liver. Wild-type mice treated with neutralizing antibodies against TNF-α, IL-17A or IL-6 or mice deficient in TNF-α, IL-17A, IL-17A receptor or IL-6 were protected against hepatic and small intestine injury because of ischemic or non-ischemic AKI. For the first time, we implicate the increased release of IL-17A from small intestine together with induction of TNF-α and IL-6 as a cause of small intestine and liver injury after ischemic or non-ischemic AKI. Modulation of the inflammatory response and cytokine release in the small intestine after AKI may have important therapeutic implications in reducing complications arising from AKI.

α2-Adrenergic agonists protect against radiocontrast-induced nephropathy in mice

Radiocontrast nephropathy (RCN) is a common clinical problem for which there is no effective therapy. Utilizing a murine model, we tested the hypothesis that alpha(2)-adrenergic receptor agonists (clonidine and dexmedetomidine) protect against RCN induced with iohexol (a nonionic low-osmolar radiocontrast). C57BL/6 mice were pretreated with saline, clonidine, or dexmedetomidine before induction of RCN. Some mice were pretreated with yohimbine (a selective alpha(2)-receptor antagonist) before saline, clonidine, or dexmedetomidine administration. alpha(2)-Agonist-treated mice had reduced plasma creatinine, renal tubular necrosis, renal apoptosis, and renal cortical proximal tubule vacuolization 24 h after iohexol injection. Yohimbine reversed the protective effects of clonidine and dexmedetomidine pretreatment. Injection of iohexol resulted in a rapid ( approximately 90 min) fall of renal outer medullary blood flow. Clonidine and dexmedetomidine pretreatment significantly attenuated this perfusion decrease without changing systemic blood pressure. To determine whether proximal tubular alpha(2)-adrenergic receptors mediate the cytoprotective effects, we treated cultured human proximal tubule (HK-2) cells and rat pulmonary microvascular endothelial cells with iohexol after vehicle, clonidine, or dexmedetomidine pretreatment. Iohexol caused a direct dose-dependent reduction of HK-2 and rat pulmonary microvascular endothelial cell viability, but alpha(2)-agonists failed to preserve the viability of both cell types. We conclude that alpha(2)-adrenergic receptor agonists protect mice against RCN by preserving outer medullary renal blood flow. As alpha(2)-agonists are widely utilized during the perioperative period, our findings may have significant clinical relevance to improving outcomes following radiocontrast exposure.

Kidney-specific reconstitution of the A1 adenosine receptor in A1 adenosine receptor knockout mice reduces renal ischemia-reperfusion injury.

Genetic deletion of the adenosine A1 receptor (A1AR) increased renal injury following ischemia-reperfusion injury suggesting that receptor activation is protective in vivo. Here we tested this hypothesis by expressing the human-A(1)AR in A(1)AR knockout mice. Renal ischemia-reperfusion was induced in knockout mice 2 days after intrarenal injection of saline or a lentivirus encoding enhanced green fluorescent protein (EGFP) or EGFP-human-A(1)AR. We found that the latter procedure induced a robust expression of the reporter protein in the kidneys of knockout mice. Mice with kidney-specific human-A(1)AR reconstitution had significantly lower plasma creatinine, tubular necrosis, apoptosis, and tubular inflammation as evidenced by decreased leukocyte infiltration, pro-inflammatory cytokine, and intercellular adhesion molecule-1 expression in the kidney following injury compared to mice injected with saline or the control lentivirus. Additionally, there were marked disruptions of the proximal tubule epithelial filamentous (F)-actin cytoskeleton in both sets of control mice upon renal injury, whereas the reconstituted mice had better preservation of the renal tubule actin cytoskeleton, which co-localized with the human-A(1)ARs. Consistent with reduced renal injury, there was a significant increase in heat shock protein-27 expression, also co-localizing with the preserved F-actin cytoskeleton. Our findings suggest that selective expression of cytoprotective A(1)ARs in the kidney can attenuate renal injury.

Paneth cell-derived interleukin-17A causes multiorgan dysfunction after hepatic ischemia and reperfusion injury.

Hepatic ischemia and reperfusion (IR) injury is a major clinical problem that leads to frequent extrahepatic complications including intestinal dysfunction and acute kidney injury (AKI). In this study we aimed to determine the mechanisms of hepatic IR‐induced extrahepatic organ dysfunction. Mice subjected to 60 minutes of hepatic IR not only developed severe hepatic injury but also developed significant AKI and small intestinal injury. Hepatic IR induced small intestinal Paneth cell degranulation and increased interleukin‐17A (IL‐17A) levels in portal vein plasma and small intestine. We also detected increased levels of IL‐17A messenger RNA (mRNA) and protein in Paneth cells after hepatic IR with laser capture dissection. IL‐17A‐neutralizing antibody treatment or genetic deletion of either IL‐17A or IL‐17A receptors significantly protected against hepatic IR‐induced acute liver, kidney, and intestinal injury. Leukocyte IL‐17A does not contribute to organ injury, as infusion of wildtype splenocytes failed to exacerbate liver and kidney injury in IL‐17A‐deficient mice after hepatic IR. Depletion of Paneth cell numbers by pharmacological (with dithizone) or genetic intervention (SOX9 flox/flox Villin cre+/− mice) significantly attenuated intestinal, hepatic, and renal injury following liver IR. Finally, depletion of Paneth cell numbers significantly decreased small intestinal IL‐17A release and plasma IL‐17A levels after liver IR. Conclusion: Taken together, the results show that Paneth cell‐derived IL‐17A plays a critical role in hepatic IR injury and extrahepatic organ dysfunction. Modulation of Paneth cell dysregulation may have therapeutic implications by reducing systemic complications arising from hepatic IR. (HEPATOLOGY 2011;)

Inhibition of Sphingosine 1-Phosphate Receptor 2 Protects against Renal Ischemia-Reperfusion Injury

Activation of the sphingosine 1-phosphate receptor 1 (S1P(1)R) protects against renal ischemia-reperfusion (IR) injury and inflammation, but the role of other members of this receptor family in modulating renal IR injury is unknown. We found that a selective S1P(2)R antagonist protected against renal IR injury in a dose-dependent manner. Consistent with this observation, both S1P(2)R-deficient mice and wild-type mice treated with S1P(2)R small interfering RNA had reduced renal injury after IR. In contrast, a selective S1P(2)R agonist exacerbated renal IR injury. The S1P(2)R antagonist increased sphingosine kinase-1 (SK1) expression via Rho kinase signaling in renal proximal tubules; the S1P(2)R agonist decreased SK1. S1P(2)R antagonism failed to protect the kidneys of SK1-deficient mice or wild-type mice pretreated with an SK1 inhibitor or an S1P(1)R antagonist, suggesting that the renoprotection conferred by S1P(2)R antagonism results from pathways involving activation of S1P(1)R by SK1. In cultured human proximal tubule (HK-2) cells, the S1P(2)R antagonist selectively upregulated SK1 and attenuated both H(2)O(2)-induced necrosis and TNF-α/cycloheximide-induced apoptosis; the S1P(2)R agonist had the opposite effects. In addition, increased nuclear hypoxia inducible factor-1α was critical in mediating the renoprotective effects of S1P(2)R inhibition. Finally, induction of SK1 and S1P(2)R in response to renal IR and S1P(2)R antagonism occurred selectively in renal proximal tubule cells but not in renal endothelial cells. Taken together, these data suggest that S1P(2)R may be a therapeutic target to attenuate the effects of renal IR injury.

Selective renal overexpression of human heat shock protein 27 reduces renal ischemia-reperfusion injury in mice

We have previously shown that exogenous and endogenous A(1) adenosine receptor (A(1)AR) activation protected against renal ischemia-reperfusion (IR) injury in mice by induction and phosphorylation of heat shock protein 27 (HSP27). With global overexpression of HSP27 in mice, however, there was a paradoxical increase in systemic inflammation with increased renal injury after an ischemic insult due to increased NK1.1 cytotoxicity. In this study, we hypothesized that selective renal expression of HSP27 in mice would improve renal function and reduce injury after IR. Mice were subjected to renal IR injury 2 days after intrarenal injection of saline or a lentiviral construct encoding enhanced green fluorescent protein (EGFP) or human HSP27 coexpressing EGFP (EGFP-huHSP27). Mice with kidney-specific reconstitution of huHSP27 had significantly lower plasma creatinine, renal necrosis, apoptosis, and inflammation as demonstrated by decreased proinflammatory cytokine mRNA induction and neutrophil infiltration. In addition, there was better preservation of the proximal tubule epithelial filamentous (F)-actin cytoskeleton in the huHSP27-reconstituted groups than in the control groups. Furthermore, huHSP27 overexpression led to increased colocalization with F-actin in renal proximal tubules. Taken together, these findings have important clinical implications, as they imply that kidney-specific expression of HSP27 through lentiviral delivery is a viable therapeutic option in attenuating the effects of renal IR.

Sphingosine kinase 1 protects against renal ischemia-reperfusion injury in mice by sphingosine-1-phosphate1 receptor activation.

The roles of sphingosine kinases SK1 and SK2 in ischemia-reperfusion injury have not been fully elucidated since studies have found beneficial effects of SK1 while others showed no role in this injury. To help resolve this, we used SK1 or SK2 knockout mice and confirmed that renal ischemia-reperfusion injury induced SK1, but not SK2, in the kidneys. Furthermore, knockout or pharmacological inhibition of SK1 increased injury after renal ischemia-reperfusion injury. In contrast, lack of SK2 conferred renal protection following injury. In addition, we used lentiviral gene delivery to selectively express enhanced green fluorescent protein (EGFP) or human SK1 coexpressed with EGFP (EGFP-huSK1) in the kidney. Mice with kidney-specific overexpression of EGFP-huSK1 had significantly improved renal function with lower plasma creatinine, renal necrosis, apoptosis, and inflammation. Moreover, EGFP-huSK1 overexpression in cultured human proximal tubule (HK-2) cells protected against peroxide-induced necrosis. Selective overexpression of EGFP-huSK1 led to increased HSP27 mRNA and protein expression in vivo and in vitro. Functional protection as well as induction of HSP27 with EGFP-huSK1 overexpression in vivo was blocked with sphingosine-1-phosphate-1 receptor(1) (S1P(1)) antagonism. Thus, our findings suggest that SK1 is renoprotective by S1P(1) activation and perhaps HSP27 induction. Kidney-specific expression of SK1 through lentiviral delivery may be a viable therapeutic option to attenuate renal ischemia-reperfusion injury.

Sphinganine-1-phosphate protects kidney and liver after hepatic ischemia and reperfusion in mice through S1P1 receptor activation

Liver failure due to ischemia and reperfusion (IR) and subsequent acute kidney injury are significant clinical problems. We showed previously that liver IR selectively reduced plasma sphinganine-1-phosphate levels without affecting sphingosine-1-phosphate (S1P) levels. Furthermore, exogenous sphinganine-1-phosphate protected against both liver and kidney injury induced by liver IR. In this study, we elucidated the signaling mechanisms of sphinganine-1-phosphate-mediated renal and hepatic protection. A selective S1P1 receptor antagonist blocked the hepatic and renal protective effects of sphinganine-1-phosphate, whereas a selective S1P2 or S1P3 receptor antagonist was without effect. Moreover, a selective S1P1 receptor agonist, SEW-2871, provided similar degree of liver and kidney protection compared with sphinganine-1-phosphate. Furthermore, in vivo gene knockdown of S1P1 receptors with small interfering RNA abolished the hepatic and renal protective effects of sphinganine-1-phosphate. In contrast to sphinganine-1-phosphate, S1Ps hepatic protection was enhanced with an S1P3 receptor antagonist. Inhibition of extracellular signal-regulated kinase, Akt or pertussis toxin-sensitive G-proteins blocked sphinganine-1-phosphate-mediated liver and kidney protection in vivo. Taken together, our results show that sphinganine-1-phosphate provided renal and hepatic protection after liver IR injury in mice through selective activation of S1P1 receptors and pertussis toxin-sensitive G-proteins with subsequent activation of ERK and Akt.

Sphinganine-1-phosphate attenuates both hepatic and renal injury induced by hepatic ischemia and reperfusion in mice

Hepatic ischemia/reperfusion (I/R) injury is a major complication after liver transplantation, major hepatic resection, or prolonged portal vein occlusion. Furthermore, acute kidney injury is frequent after hepatic I/R and greatly increases postoperative complications. Sphinganine-1-phosphate is a sphingolipid with uncharacterized physiological effects. We serendipitously determined that plasma levels of sphinganine-1-phosphate fell significantly after liver I/R in mice. In this study, we hypothesized that repletion of plasma sphinganine-1-phosphate would protect against liver and kidney injuries after liver I/R. C57BL/6 mice were subjected to 60 min of partial hepatic I/R and treated with either vehicle or with sphinganine-1-phosphate (given immediately before and 2 h after reperfusion). Vehicle-treated mice subjected to liver I/R developed acute liver and kidney injuries with elevated plasma alanine aminotransferase and creatinine 5 and 24 h after liver I/R. However, liver and kidney injuries were significantly attenuated with sphinganine-1-phosphate treatment. Sphinganine-1-phosphate markedly inhibited liver and kidney necrosis and apoptosis 24 h after liver I/R. Moreover, sphinganine-1-phosphate attenuated neutrophil infiltration, reduced plasma IL-6 and TNF-&agr; upregulation, and preserved liver and kidney vascular integrity while reducing liver and kidney F-actin degradation after liver I/R. Finally, sphinganine-1-phosphate-mediated hepatic and renal protection was blocked by VPC23019, an antagonist for sphingosine-1-phosphate type 1 receptor. Therefore, sphinganine-1-phosphate improves acute liver and kidney injuries after hepatic I/R via sphingosine-1-phosphate type 1 receptor-mediated inhibition of necrosis and apoptosis and by improving vascular integrity. Harnessing the mechanisms of cytoprotection with sphinganine-1-phosphate activation may lead to new therapies for perioperative hepatic I/R injury and subsequent remote organ injury.

Mice that overexpress human heat shock protein 27 have increased renal injury following ischemia reperfusion

We previously showed that activation of the A1 adenosine receptor protected the kidney against ischemia-reperfusion injury by induction and phosphorylation of heat shock protein 27 (HSP27). Here, we used mice that overexpress human HSP27 (huHSP27) to determine if kidneys from these mice were protected against injury. Proximal tubule cells cultured from the transgenic mice had increased resistance to peroxide-induced necrosis compared to cells from wild-type mice. However, after renal ischemic injury, HSP27 transgenic mice had decreased renal function compared to wild-type mice, along with increased renal expression of mRNAs of pro-inflammatory cytokines (TNF-alpha, ICAM-1, MCP-1) and increased plasma and kidney keratinocyte-derived cytokine. Following ischemic injury, neutrophils infiltrated the kidneys earlier in the transgenic mice. Flow cytometric analysis of lymphocyte subsets showed that those isolated from the kidneys of transgenic mice had increased CD3(+), CD4(+), CD8(+), and NK1.1(+) cells 3 h after injury. When splenocytes or NK1.1(+) cells were isolated from transgenic mice and adoptively transferred into wild-type mice there was increased renal injury. Further, depletion of lymphocytes by splenectomy or neutralization of NK1.1(+) cells resulted in improved renal function in the transgenic mice following reperfusion. Our study shows that induction of HSP27 in renal tubular cells protects against necrosis in vitro, but its systemic increase counteracts this protection by exacerbating renal and systemic inflammation in vivo.