The pathogenesis of renal ischemia/reperfusion (I/R) injury involves activating several signal transduction cascade systems in endothelial cells. Sphingosine 1-phospate (S1P) maintains endothelial cell integrity and inhibits lymphocyte egress via the specific S1P 1 receptor, and may play a role in reducing ischemic renal injury. We examined the protective effects of a newly identified S1P 1 -selective agonist, SEW2871, on mouse renal I/R injury. Kidneys were harvested 1-4 days after I/R injury for histopathology, immunofluorescence studies, and quantitative real-time reverse transcriptasepolymerase chain reaction analyses to assess the change in gene expression profiles of inflammation-associated cytokines and adhesion molecules. SEW2871 improved renal function with a 40% reduction in plasma creatinine levels (Po0.01) and a significant reduction in tubular necrosis scores (I/R only: 4.370.2 vs I/R þ SEW2871: 2.570.4, Po0.05) 24 h after ischemia. These changes were accompanied by 69% reduction in circulating lymphocytes, and 77 and 66% reduction in infiltrating neutrophils and macrophages in renal outer medulla, respectively (all Po0.01). The mRNA abundance of tumor necrotic factor-a (TNF-a), P-selectin, E-selectin, and intercellular adhesion molecule-1 (ICAM-1) was markedly increased by I/R injury (3.5-, 4.1-, 3.5-, and 4.8-folds, respectively, all Po0.05 vs sham). SEW2871 treatment partially reversed the upregulation of TNF-a, P-selectin, and ICAM-1 (47, 59, 54%, respectively, vs I/R control: 100%, all Po0.05). The reduction in protein expression of TNF-a, P-selectin, and ICAM-1 was further confirmed with immunofluorescence studies. These results suggest that SEW2871 ameliorates renal I/R injury by inhibiting lymphocyte egress and reducing pro-inflammatory molecules. This new class of renoprotective agent shows promise as a novel approach in preventing/treating ischemic acute renal failure.

T cells are thought to be involved in the pathogenesis of renal ischemia-reperfusion injury (IRI); however, earlier studies have not found significant T-cell numbers in the kidney following injury. In this study we test the hypothesis that T cells transiently infiltrate the kidney following reperfusion and leave behind T-cell-derived cytokines such as interferons and interleukins, thus triggering an inflammatory reaction. An early rise of infiltrating T cells was coupled with a decrease in both circulating lymphocytes and CD4+ cells of periarterial lymphocyte aggregates. The renal expression of several chemokines was rapidly and markedly increased by ischemia-reperfusion (IR). Sphingosine-1-phosphate type 1 receptor agonists have been shown to protect kidneys from injury. One of these agonists given before IR significantly reduced histologically assessed renal injury, circulating lymphocyte numbers, and renal T-cell infiltration. This pretreatment did not, however, affect the increase in T-cell chemokines but caused an increase in CD4+ cells in the renal lymphatic system. We conclude that T-cell infiltration is an early event after IRI and is mediated by several chemokines. Sphingosine-1-phosphate receptor agonists reduce renal injury and T-cell infiltration in spite of chemokine generation by inhibiting T-cell mobilization from both renal and extra-renal lymphoid tissue.

Regulatory T cells (Tregs) are key components of the peripheral tolerance system and have become an immunotherapeutic agent for treating inflammatory processes. This therapeutic option, however, is hampered by problems arising from isolating and expanding desirable Tregs. Here we used an alternative approach with a pharmacologic agent to stimulate Tregs to achieve immunosuppressive effects. Pretreatment of mice with the naturally occurring sphingosine N, N-dimethylsphingosine (DMS) was found to increases both tissue infiltrating T effectors (Teff, CD4+Foxp3−) and Tregs (CD4+Foxp3+), in the early phase of bilateral renal ischemia/reperfusion injury. DMS itself had no effects on renal function or histopathology, but rapidly and transiently increased both Teff and Tregs and increased the expression of chemokines CXCL9, CCL5, and CXCL10 in non-ischemic kidneys (sham operation). This renoprotection was abolished by administration of the Treg suppressing agents, anti-CTLA-4 or anti-CD25 monoclonal antibodies, suggesting that Tregs play a key role in DMS-induced renoprotection. Thus, Tregs recruited to the kidney by DMS, ameliorate acute kidney injury and provide a new approach to control inflammatory diseases.

Thiazide diuretics are specific inhibitors of the Na-Cl cotransporter in the distal convoluted tubule (DCT). In addition to producing diuresis and natriuresis, they have a hypocalciuric effect. Recently, two apical calcium channels have been identified, transient receptor potential vanilloid 5 (TRPV5) and TRPV6; both are expressed in the DCT. We studied the effects of thiazides on mouse renal calcium handling and renal gene expression of TRPV5 and TRPV6, as well as calbindin-D(28k) and calbindin-D(9k), both of which are calcium transport facilitators located in the DCT. Upregulation of renal TRPV5 was found 4 h after intraperitoneal injection of chlorothiazide (CTZ) at both 25 and 50 mg/kg, but not at 100 mg/kg. Chronic treatment with CTZ at 25 mg/kg twice daily for 3 days, with or without salt supplementation of 0.8% NaCl and 0.1% KCl in the drinking water, caused hypocalciuria, but the gene expression patterns were different. Without salt supplementation, mice developed volume contraction and there were no changes in gene expression. When volume contraction was prevented by salt supplementation, there was a significant increase in gene expression of TRPV5, calbindin-D(28k), and calbindin-D(9k). Salt supplementation alone also induced significant upregulation of TRPV5, TRPV6, and both calbindins. The upregulation of TRPV5 by CTZ and salt supplementation and salt alone was further confirmed with immunofluorescent staining studies. Our studies suggest that thiazides induce hypocalciuria through different mechanisms depending on volume status. With volume contraction, increased calcium reabsorption in the proximal tubule plays the major role. Without volume contraction, hypocalciuria is probably achieved through increased calcium reabsorption in the DCT by the activation of a transcellular calcium transport system and upregulation of apical calcium channel TRPV5, calbindin-D(28k), and calbindin-D(9k).

IQGAP1 is a multifunctional junction molecule that is involved in cell migration, proliferation, differentiation, cell polarity, and cell-cell adhesion. It is highly expressed in the kidney and has recently been identified in the glomerular basement membrane as a nephrin-associated protein. However, the distribution of IQGAP1 in renal tubular epithelial cells is unknown. We performed confocal microscopic studies to localize IQGAP1 in each nephron segment using dual immunofluorescence staining with various antibodies against segment-specific markers. We found that IQGAP1 was strongly expressed in the distal convoluted tubule (DCT), collecting duct, and macula densa and moderately in the thick ascending limb and proximal tubule. In the DCT, the IQGAP1-F-actin complex forms a comb-like structure with multiple parallel spikes sitting on the basal membrane. In the macula densa cells, IQGAP1 is strongly expressed in the apical membrane, whereas in type A intercalated cells, IQGAP1 is expressed in the basolateral membrane, where it colocalizes with anion exchanger 1, and in principal cells, it is diffusely expressed. In conclusion, we showed the expression and subcellular localization of IQGAP1 in various nephron segments. The site-specific expression pattern of this potent modulator of multiple biological pathways in the renal tubules suggests that IQGAP1 may have multiple important roles in various renal functions.