Uwe Hoff

Inhibition of 20-HETE synthesis and action protects the kidney from ischemia/reperfusion injury.

20-Hydroxyeicosatetraenoic acid (20-HETE) production is increased in ischemic kidney tissue and may contribute to ischemia/reperfusion (I/R) injury by mediating vasoconstriction and inflammation. To test this hypothesis, uninephrectomized male Lewis rats were exposed to warm ischemia following pretreatment with either an inhibitor of 20-HETE synthesis (HET0016), an antagonist (20-hydroxyeicosa-6(Z),15(Z)-dienoic acid), an agonist (20-hydroxyeicosa-5(Z),14(Z)-dienoic acid), or vehicle via the renal artery and the kidneys were examined 2 days after reperfusion. Pretreatment with either the inhibitor or the antagonist attenuated I/R-induced renal dysfunction as shown by improved creatinine clearance and decreased plasma urea levels, compared to controls. The inhibitor and antagonist also markedly reduced tubular lesion scores, inflammatory cell infiltration, and tubular epithelial cell apoptosis. Administering the antagonist accelerated the recovery of medullary perfusion, as well as renal medullary and cortical re-oxygenation, during the early reperfusion phase. In contrast, the agonist did not improve renal injury and reversed the beneficial effect of the inhibitor. Thus, 20-HETE generation and its action mediated kidney injury due to I/R. Whether or not these effects are clinically important will need to be tested in appropriate human studies.

Estrogen Receptor-β Signals Left Ventricular Hypertrophy Sex Differences in Normotensive Deoxycorticosterone Acetate-Salt Mice

We found earlier that deoxycorticosterone acetate-salt treatment causes blood pressure–independent left ventricular hypertrophy, but only in male mice. To test the hypothesis that the estrogen receptor-&bgr; (ER&bgr;) protects the females from left ventricular hypertrophy, we treated male and female ER&bgr;-deficient (ER&bgr;−/−) mice and their male and female littermates (wild-type [WT]) with deoxycorticosterone acetate-salt and made them telemetrically normotensive with hydralazine. WT males had increased (+16%) heart weight/tibia length ratios compared with WT females (+7%) at 6 weeks. In ER&bgr;−/− mice, this situation was reversed. Female WT mice had the greatest heart weight/tibia length ratio increases of all of the groups (+23%), even greater than ER&bgr;−/− males (+10%). Echocardiography revealed concentric left ventricular hypertrophy in male WT mice, whereas ER&bgr;−/− females developed dilative left ventricular hypertrophy. The hypertrophic response in female ER&bgr;−/− mice was accompanied by the highest degree of collagen deposition, indicating maladaptive remodeling. ER&bgr;+/+ females showed robust protective p38 and extracellular signal–regulated kinase 1/2 signaling relationships compared with other groups. Calcineurin A&bgr; expression and its positive regulator myocyte-enriched calcineurin-interacting protein 1 were increased in deoxycorticosterone acetate-salt female ER&bgr;−/− mice, yet lower than in WT males. Endothelin increased murine cardiomyocyte hypertrophy in vitro, which could be blocked by estradiol and an ER&bgr; agonist. We conclude that a functional ER&bgr; is essential for inducing adaptive p38 and extracellular signal–regulated kinase signaling, while reducing maladaptive calcineurin signaling in normotensive deoxycorticosterone acetate female mice. Our findings address the possibility of sex-specific cardiovascular therapies.

High temporal resolution parametric MRI monitoring of the initial ischemia/reperfusion phase in experimental acute kidney injury.

Ischemia/reperfusion (I/R) injury, a consequence of kidney hypoperfusion or temporary interruption of blood flow is a common cause of acute kidney injury (AKI). There is an unmet need to better understand the mechanisms operative during the initial phase of ischemic AKI. Non-invasive in vivo parametric magnetic resonance imaging (MRI) may elucidate spatio-temporal pathophysiological changes in the kidney by monitoring the MR relaxation parameters T2* and T2, which are known to be sensitive to blood oxygenation. The aim of our study was to establish the technical feasibility of fast continuous T2*/T2 mapping throughout renal I/R. MRI was combined with a remotely controlled I/R model and a segmentation model based semi-automated quantitative analysis. This technique enabled the detailed assessment of in vivo changes in all kidney regions during ischemia and early reperfusion. Significant changes in T2* and T2 were observed shortly after induction of renal ischemia and during the initial reperfusion phase. Our study demonstrated for the first time that continuous and high temporal resolution parametric MRI is feasible for in-vivo monitoring and characterization of I/R induced AKI in rats. This technique may help in the identification of the timeline of key events responsible for development of renal damage in hypoperfusion-induced AKI.

Novel signalling mechanisms and targets in renal ischaemia and reperfusion injury.

Acute kidney injury (AKI) induced by ischaemia and reperfusion (I/R) injury is a common and severe clinical problem. Vascular dysfunction, immune system activation and tubular epithelial cell injury contribute to functional and structural deterioration. The search for novel therapeutic interventions for I/R‐induced AKI is a dynamic area of experimental research. Pharmacological targeting of injury mediators and corresponding intracellular signalling in endothelial cells, inflammatory cells and the injured tubular epithelium could provide new opportunities yet may also pose great translational challenge. Here, we focus on signalling mediators, their receptors and intracellular signalling pathways which bear potential to abrogate cellular processes involved in the pathogenesis of I/R‐induced AKI. Sphingosine 1 phosphate (S1P) and its respective receptors, cytochrome P450 (CYP450)‐dependent vasoactive eicosanoids, NF‐κB‐ and protein kinase‐C (PKC)‐related pathways are representatives of such ‘druggable’ pleiotropic targets. For example, pharmacological agents targeting S1P and PKC isoforms are already in clinical use for treatment for autoimmune diseases and were previously subject of clinical trials in kidney transplantation where I/R‐induced AKI occurs as a common complication. We summarize recent in vitro and in vivo experimental studies using pharmacological and genomic targeting and highlight some of the challenges to clinical application of these advances.

Linking non-invasive parametric MRI with invasive physiological measurements (MR-PHYSIOL): towards a hybrid and integrated approach for investigation of acute kidney injury in rats

Acute kidney injury of various origins shares a common link in the pathophysiological chain of events: imbalance between renal medullary oxygen delivery and oxygen demand. For in vivo assessment of kidney haemodynamics and oxygenation in animals, quantitative but invasive physiological methods are established. A very limited number of studies attempted to link these invasive methods with parametric Magnetic Resonance Imaging (MRI) of the kidney. Moreover, the validity of parametric MRI (pMRI) as a surrogate marker for renal tissue perfusion and renal oxygenation has not been systematically examined yet. For this reason, we set out to combine invasive techniques and non‐invasive MRI in an integrated hybrid setup (MR‐PHYSIOL) with the ultimate goal to calibrate, monitor and interpret parametric MR and physiological parameters by means of standardized interventions. Here we present a first report on the current status of this multi‐modality approach. For this purpose, we first highlight key characteristics of renal perfusion and oxygenation. Second, concepts for in vivo characterization of renal perfusion and oxygenation are surveyed together with the capabilities of MRI for probing blood oxygenation‐dependent tissue stages. Practical concerns evoked by the use of strong magnetic fields in MRI and interferences between MRI and invasive physiological probes are discussed. Technical solutions that balance the needs of in vivo physiological measurements together with the constraints dictated by small bore MR scanners are presented. An early implementation of the integrated MR‐PHYSIOL approach is demonstrated including brief interventions of hypoxia and hyperoxia.

Sex-Specific mTOR Signaling Determines Sexual Dimorphism in Myocardial Adaptation in Normotensive DOCA-Salt Model

The deoxycorticosterone acetate (DOCA)-salt mouse model exhibits adverse cardiac remodeling in male mice and cardiac protection in female mice, even when blood pressure is normalized. We hypothesized that intact mammalian target of rapamycin (mTOR) signaling is necessary for cardiac protection in females. We first tested sex differences and intracellular signaling after mTOR targeting with rapamycin in wild-type mice. Radio-telemetric blood pressure was maintained at normal for 6 weeks. Rapamycin significantly reduced left ventricular hypertrophy, preserved ejection fraction, inhibited fibrosis, and maintained capillary structure in male mice. Decreased mTORC1 and increased mTORC2 activity were detected in rapamycin-treated male mice compared with vehicle controls. In contrast, female mice developed dilative left ventricular hypertrophy, cardiac fibrosis, and capillary loss similar to DOCA-salt females lacking the estrogen receptor &bgr; (ER&bgr;−/−) that we described earlier. Because rapamycin downregulated ER&bgr; in female mice, we next studied ER&bgr;−/− normotensive DOCA-salt females. Vehicle-treated wild-type females maintained their high constitutive mTORC1 and mTORC2 in response to DOCA-salt. In contrast to males, both mTORCs were decreased by rapamycin, in particular mTORC2 by 60%. ER&bgr;−/− DOCA-salt females showed similar mTORC1 and mTORC2 response patterns. We suggest that ER&bgr;-dependent regulation involves sex-specific use of mTOR signaling branches. Maintenance of both mTORC1 and mTORC2 signaling seems to be essential for adaptive cardiac remodeling in females and supports a rationale for sex-specific therapeutic strategies in left ventricular hypertrophy.

Protein kinase C inhibition ameliorates posttransplantation preservation injury in rat renal transplants

Background Prolonged cold preservation frequently causes delayed renal graft function resulting from tubular epithelial injury. Inhibition of signal transduction downstream from protein kinase C (PKC) may reduce renal ischemia-reperfusion injury and confer renal graft protection. We therefore evaluated the effect of sotrastaurin, a small-molecule inhibitor of Ca2+-dependent and Ca2+-independent PKC isoforms, in comparison with mycophenolic acid (MPA) on rat renal transplants with prolonged cold preservation. Methods Donor kidneys from male Lewis rats were cold stored in University of Wisconsin solution for 24 hr before syngeneic grafting. Recipients received sotrastaurin (30 mg/kg twice daily), MPA (20 mg/kg/day), or vehicle through gavage starting 1 hr after surgery. Renal function was evaluated by serum creatinine and histology on day 2 (acute injury) and day 7 (repair phase) after transplantation. Postreperfusion inflammation was determined by real-time polymerase chain reaction of proinflammatory genes and histology. Signaling mechanisms were studied by Western blotting and immunohistochemistry. Results Sotrastaurin enhanced immediate transplant function, attenuated epithelial injury, and accelerated renal function recovery compared with MPA. Despite the stronger anti-inflammatory capacity of MPA, only sotrastaurin treatment achieved significant cellular protection with persisting reduced apoptosis of tubular epithelial cells. Decreased phosphorylation of extracellular signal–regulated protein kinase and p66Shc adaptor protein, both involved in cellular stress and apoptosis, were likely the responsible mechanism of action. Conclusions The PKC inhibitor sotrastaurin effectively ameliorated ischemia-reperfusion organ damage and promoted cytoprotection in a clinically relevant model of extended renal cold preservation followed by transplantation. Pharmacologic targeting of PKC may be beneficial for recipients receiving renal transplants at risk for delayed graft function.

Cytoprotective Actions of FTY720 Modulate Severe Preservation Reperfusion Injury in Rat Renal Transplants.

Background. Fingolimod (FTY720) is a potent agonist of sphingosine 1 phosphate receptors and thereby interferes with lymphocyte trafficking. We previously showed that FTY720 protects from mild preservation reperfusion injury induced by 4 hr of cold ischemia. The purpose of this study was to explore the role of FTY720 in ischemic injury and regeneration using a clinically relevant rat renal transplant model with 24 hr of cold ischemia. Methods. Donor kidneys were cold stored in the University of Wisconsin solution for 24 hr before transplantation into bilaterally nephrectomized syngeneic recipients (n=6 per group), which received 0.5 mg/kg/d FTY720 or vehicle through oral gavage. Grafts were harvested 2 or 7 days posttransplantation. Renal tissue was examined histologically, stained for apoptosis, proliferation, inflammatory cell infiltrates, and studied for transforming growth factor-β, and tumor necrosis factor-α expression. Rat proximal tubular cells were incubated with 0.1 to 30 μmol/L of phosphorylated FTY720 to test for in vitro cytopathic effects. Results. FTY720 induced peripheral lymphopenia and significantly reduced intragraft CD3+ and ED1+ infiltrates. Acute tubular damage scores and graft function were not influenced by FTY720. Tubular apoptosis was significantly reduced, whereas the number of proliferating cell nuclear antigen-positive tubular cells were markedly increased. FTY720 attenuated renal tumor necrosis factor-α and transforming growth factor-β expression. In vitro, pharmacologic concentrations up to 1 μmol/L of phosphorylated FTY720 did not affect tubular cell viability. Conclusion. FTY720 confers tubular epithelial protection in the presence of severe preservation reperfusion injury. Beneficial effects may in part be due to reduction in cell-mediated immune mechanisms. Furthermore, FTY720 could be helpful in patients with delayed graft function.

Renal Ischemia/Reperfusion Injury in Soluble Epoxide Hydrolase-Deficient Mice

Aim 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP)-dependent eicosanoids that play opposite roles in the regulation of vascular tone, inflammation, and apoptosis. 20-HETE aggravates, whereas EETs ameliorate ischemia/reperfusion (I/R)-induced organ damage. EETs are rapidly metabolized to dihydroxyeicosatrienoic acids (DHETs) by the soluble epoxide hydrolase (sEH). We hypothesized that sEH gene (EPHX2) deletion would increase endogenous EET levels and thereby protect against I/R-induced acute kidney injury (AKI). Methods Kidney damage was evaluated in male wildtype (WT) and sEH-knockout (KO)-mice that underwent 22-min renal ischemia followed by two days of reperfusion. CYP-eicosanoids were analyzed by liquid chromatography tandem mass spectrometry. Results Contrary to our initial hypothesis, renal function declined more severely in sEH-KO mice as indicated by higher serum creatinine and urea levels. The sEH-KO-mice also featured stronger tubular lesion scores, tubular apoptosis, and inflammatory cell infiltration. Plasma and renal EET/DHET-ratios were higher in sEH-KO than WT mice, thus confirming the expected metabolic consequences of sEH deficiency. However, CYP-eicosanoid profiling also revealed that renal, but not plasma and hepatic, 20-HETE levels were significantly increased in sEH-KO compared to WT mice. In line with this finding, renal expression of Cyp4a12a, the murine 20-HETE-generating CYP-enzyme, was up-regulated both at the mRNA and protein level, and Cyp4a12a immunostaining was more intense in the renal arterioles of sEH-KO compared with WT mice. Conclusion These results indicate that the potential beneficial effects of reducing EET degradation were obliterated by a thus far unknown mechanism leading to kidney-specific up-regulation of 20-HETE formation in sEH-KO-mice.

Decreased Transplant Arteriosclerosis in Endothelial Nitric Oxide Synthase-Deficient Mice

Background. Occlusive vascular changes, characterized by the formation of a neointima with lumen obstruction, are key histologic findings of allograft arteriosclerosis. Vascular integrity of the graft is critically dependent on nitric oxide (NO), synthesized by NO synthases (NOS), of which three isoforms have been located in the arterial wall: endothelial NOS (eNOS), inducible NOS, and neuronal NOS (nNOS). We have studied the role of NOS in a murine model of aortic allograft rejection. Methods. The descending thoracic aorta of donor mice (BALB/c mice) was transplanted into two groups of recipients: (a) C57BL/6J and (b) C57BL/6J mice homozygous (−/−) for a knockout of the eNOS gene (eNOS−/−). Results. After 4 weeks, pronounced neointima formation, upregulated expression of adhesion molecules, and increased infiltration by inflammatory cells were demonstrated in wild-type recipient mice, whereas eNOS−/− recipient mice were protected from neointima development by a significantly increased synthesis of NO, as shown by increased formation of cGMP; this was mainly explained by upregulation of inducible NOS and nNOS. Conclusions. Upregulation of inducible NOS and nNOS isoforms may be beneficial in preventing allograft arteriosclerosis in the early posttransplant period.