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Selective sphingosine 1-phosphate 1 receptor activation reduces ischemia-reperfusion injury in mouse kidney. Am J Physiol Renal Physiol 290: F1516 -F1524, 2006. First published January 10, 2006 doi:10.1152/ajprenal.00311.2005.-The mechanisms involved in renal ischemia-reperfusion injury (IRI) are complex and appear to involve the early participation of bone marrow-derived cells. T lymphocytes participate in the pathogenesis of IRI. Sphingosine 1-phosphate (S1P) induces peripheral T cell depletion. Therefore, we hypothesized that S1P 1 receptor activation protects kidney from IRI. FTY-720, a non-receptor-selective sphingosine analog, was given intraperitoneally to C57BL/6 mice, and animals were subjected to ischemia for 32 min followed by reperfusion for 24 h. Plasma creatinine, blood count, myeloperoxidase (MPO) activity, and renal histology were determined. IRI led to a marked increase in plasma creatinine, MPO activity, leukocyte infiltration, and vascular permeability. FTY-720 significantly decreased plasma creatinine in a doseresponse manner with a maximal reduction of ϳ73 and ϳ69% with doses of 240 and 48 g/kg, respectively. MPO, leukocyte infiltration, vascular permeability, and peripheral blood lymphocyte counts were markedly decreased with FTY-720 treatment. The protective effect of FTY-720 was reversed with VPC-44116, a selective S1P 1 receptor antagonist. Furthermore, SEW-2871, a selective S1P 1 agonist, significantly decreased plasma creatinine in a dose-response manner with a maximal reduction of ϳ70% with a dose of 10 mg/kg. Analysis of kidneys by light microscopy revealed minimal histological signs of ischemic injury with FTY-720 or SEW-2871 treatment compared with the vehicle group. Using RT-PCR, we found a time-dependent increase in the S1P 1 mRNA expression following IRI that begins after 2 h with the maximum expression at ϳ4 h. We conclude that the protective effect of FTY-720 is due primarily to activation of S1P1 receptors. The mechanism of protection is not known but may be related to peripheral lymphocyte depletion or direct effects on kidney cells expressing S1P1 receptor.FTY-720; inflammation; lymphocyte; acute renal failure NOVEL THERAPEUTIC INTERVENTIONS for preventing or attenuating renal tissue injury following ischemia-reperfusion (IR) remain a focus of significant interest. Although the exact pathogenic mechanisms remain poorly defined, accumulating evidence supports the potential role of inflammation and involvement of the immune system. Previous studies have mainly focused on the participation of neutrophils as mediators of IR injury (IRI) (17, 32); however, recent reports show that lymphocytes contribute to injury (8,40). The observation that injury is ameliorated by depletion of T cells and reconstituted with adoptive transfer of CD4 ϩ cells is evidence that the tissue injury following IR is dependent on CD4 ϩ cells (8), although the mechanism is not fully understood. Renal damage secondary to IRI is frequent in organ transplantation and adversely affects allograft survival. An...

During renal ischemia-reperfusion, local and distant tissue injury is caused by an influx of neutrophils into the affected tissues. Here we measured the kinetics of margination and transmigration of neutrophils in vivo in the kidney and lungs following renal ischemia-reperfusion. After bilateral renal injury, kidney neutrophil content increased threefold at 24 h. The neutrophils were found primarily in the interstitium and to a lesser degree marginated to the vascular endothelium. These interstitial neutrophils had significantly lower levels of intracellular IFN-γ, IL-4, IL-6, and IL-10 a tendency for decreased amounts of IL-4 and TNF-α compared to the marginated neutrophils. Localization of the neutrophils to the kidney interstitium was confirmed by high resolution microscopy and these sites of transmigration were directly associated with areas of increased vascular permeability. Activation of the adenosine 2A receptor significantly decreased both kidney neutrophil transmigration by about half and vascular permeability by about a third. After unilateral renal ischemia-reperfusion, the unclipped kidney and lungs did not accumulate interstitial neutrophils or have increased vascular permeability despite a marked increase of neutrophil margination in the lungs. Our findings suggest there is a sequential recruitment and transmigration of neutrophils from the vasculature into the kidney interstitium at the site of tissue injury following renal ischemia-reperfusion.

Monocyte/macrophage recruitment correlates strongly with the progression of diabetic nephropathy. Tumor necrosis factor-alpha (TNF-α) is produced by monocytes/macrophages but the direct role of TNF-α and/or macrophage-derived TNF-α in the progression of diabetic nephropathy remains unclear. Here we tested whether inhibition of TNF-α confers kidney protection in diabetic nephropathy via a macrophage-derived TNF-α dependent pathway. Compared to vehicle-treated mice, blockade of TNF-α with a murine anti-TNF-α antibody conferred kidney protection in Ins2Akita mice as indicated by reductions in albuminuria, plasma creatinine, histopathologic changes, kidney macrophage recruitment and plasma inflammatory cytokine levels at 18 weeks of age. To assess the direct role of macrophage-derived TNF-α in diabetic nephropathy, we generated macrophage specific TNF-α deficient mice (CD11bCre/TNF-αFlox/Flox). Conditional ablation of TNF-α in macrophages significantly reduced albuminuria, the increase in plasma creatinine and BUN, histopathologic changes and kidney macrophage recruitment compared to diabetic TNF-αFlox/Flox control mice after 12 weeks of streptozotocin-induced diabetes. Thus, production of TNF-α by macrophages plays a major role in diabetic renal injury. Hence, blocking TNF-α could be a novel therapeutic approach for treatment of diabetic nephropathy.

OBJECTIVETo determine 1) whether renal arginase activity or expression is increased in diabetes and 2) whether arginase plays a role in development of diabetic nephropathy (DN).RESEARCH DESIGN AND METHODSThe impact of arginase activity and expression on renal damage was evaluated in spontaneously diabetic Ins2Akita mice and in streptozotocin (STZ)-induced diabetic Dilute Brown Agouti (DBA) and arginase-2–deficient mice (Arg2−/−).RESULTSPharmacological blockade or genetic deficiency of arginase-2 conferred kidney protection in Ins2Akita mice or STZ-induced diabetic renal injury. Blocking arginases using S-(2-boronoethyl)-l-cysteine for 9 weeks in Ins2Akita mice or 6 weeks in STZ-induced diabetic DBA mice significantly attenuated albuminuria, the increase in blood urea nitrogen, histopathological changes, and kidney macrophage recruitment compared with vehicle-treated Ins2Akita mice. Furthermore, kidney arginase-2 expression increased in Ins2Akita mice compared with control. In contrast, arginase-1 expression was undetectable in kidneys under normal or diabetes conditions. Arg2−/− mice mimicked arginase blockade by reducing albuminuria after 6 and 18 weeks of STZ-induced diabetes. In wild-type mice, kidney arginase activity increased significantly after 6 and 18 weeks of STZ-induced diabetes but remained very low in STZ-diabetic Arg2−/− mice. The increase in kidney arginase activity was associated with a reduction in renal medullary blood flow in wild-type mice after 6 weeks of STZ-induced diabetes, an effect significantly attenuated in diabetic Arg2−/− mice.CONCLUSIONSThese findings indicate that arginase-2 plays a major role in induction of diabetic renal injury and that blocking arginase-2 activity or expression could be a novel therapeutic approach for treatment of DN.

Monocyte/macrophage recruitment correlates strongly with the progression of renal impairment in diabetic nephropathy (DN), yet their direct role is not clear. We hypothesized that macrophages contribute to direct podocyte injury and/or an abnormal podocyte niche leading to DN. Experiments were conducted in CD11b-DTR mice treated with diphtheria toxin (DT) to deplete macrophages after streptozotocin-induced diabetes. Additional experiments were conducted in bone marrow chimeric (CD11b-DTR→ C57BL6/J) mice. Diabetes was associated with an increase in the M1-to-M2 ratio by 6 wk after the induction of diabetes. Macrophage depletion in diabetic CD11b-DTR mice significantly attenuated albuminuria, kidney macrophage recruitment, and glomerular histological changes and preserved kidney nephrin and podocin expression compared with diabetic CD11b-DTR mice treated with mutant DT. These data were confirmed in chimeric mice indicating a direct role of bone marrow-derived macrophages in DN. In vitro, podocytes grown in high-glucose media significantly increased macrophage migration compared with podocytes grown in normal glucose media. In addition, classically activated M1 macrophages, but not M2 macrophages, induced podocyte permeability. These findings provide evidence showing that macrophages directly contribute to kidney injury in DN, perhaps by altering podocyte integrity through the proinflammatory M1 subset of macrophages. Attenuating the deleterious effects of macrophages on podocytes could provide a new therapeutic approach to the treatment of DN.

We found an early rise in renal TNF-alpha levels after induction of diabetes with STZ, which precedes the rise in UAE by about 2 weeks. These findings suggest a possible contribution of TNF-alpha in the complicated pathogenic process resulting in microalbuminuria in diabetes.

We previously demonstrated the anti-inflammatory effects and renal tissue protection in response to adenosine A(2A)-receptor (A(2A)R) activation in acute renal injury. We sought to extend these studies and determine the efficacy of A(2A)R agonists in a chronic model of renal injury. We hypothesized that A(2A) agonists mediate renal tissue protection in diabetic nephropathy by reducing glomerular inflammation. Diabetes was induced with single intravenous injection of streptozotocin in Sprague-Dawley rats (50 mg/kg). Increases in urinary albumin excretion (UAE) and plasma creatinine at week 6 in the diabetes group (26- and 6-fold over control, respectively) were markedly reduced by continuous subcutaneous administration of ATL146e (10 ng x kg(-1) x min(-1)), a selective A(2A) agonist. The increase in UAE in the diabetes group was associated with a significant reduction in the expression of slit diaphragm-associated molecules compared with control (nephrin; P < 0.05 and podocin; P < 0.005) that was reversed by ATL146e treatment. Diabetes led to an increase in urinary excretion of monocyte chemoattractant protein-1 (705% of control), TNF-alpha (1,586% of control), IFN-gamma (298% of control), kidney fibronectin mRNA (457% of control), and glomerular infiltration of macrophages (764% of control), effects significantly reduced by ATL146e treatment. Mesangial expansion and basement membrane thickness were reduced with ATL146e. To further confirm the selectivity of ATL146e, we used wild-type (WT) or A(2A)knockout (A(2A)-KO) mice. Four weeks after diabetes, UAE increased significantly in both WT and A(2A)-KO diabetic mice (3.0- and 3.3-fold over control). A(2A) agonist treatment blocked the increase in UAE in WT diabetic mice (P < 0.001), whereas it had no effect on the A(2A)-KO diabetic mice. These results demonstrate that chronic A(2A)R activation in diabetic rats 1) ameliorates histological and functional changes in kidneys induced by diabetes and 2) causes reduced inflammation associated with diabetic nephropathy.