Ahuva Shina

Hypoxia-inducible factor-2α-expressing interstitial fibroblasts are the only renal cells that express erythropoietin under hypoxia-inducible factor stabilization

The adaptation of erythropoietin production to oxygen supply is determined by the abundance of hypoxia-inducible factor (HIF), a regulation that is induced by a prolyl hydroxylase. To identify cells that express HIF subunits (HIF-1alpha and HIF-2alpha) and erythropoietin, we treated Sprague-Dawley rats with the prolyl hydroxylase inhibitor FG-4497 for 6 h to induce HIF-dependent erythropoietin transcription. The kidneys were analyzed for colocalization of erythropoietin mRNA with HIF-1alpha and/or HIF-2alpha protein along with cell-specific identification markers. FG-4497 treatment strongly induced erythropoietin mRNA exclusively in cortical interstitial fibroblasts. Accumulation of HIF-2alpha was observed in these fibroblasts and in endothelial and glomerular cells, whereas HIF-1alpha was induced only in tubular epithelia. A large proportion (over 90% in the juxtamedullary cortex) of erythropoietin-expressing cells coexpressed HIF-2alpha. No colocalization of erythropoietin and HIF-1alpha was found. Hence, we conclude that in the adult kidney, HIF-2alpha and erythropoietin mRNA colocalize only in cortical interstitial fibroblasts, which makes them the key cell type for renal erythropoietin synthesis as regulated by HIF-2alpha.

ApoSense: a novel technology for functional molecular imaging of cell death in models of acute renal tubular necrosis

PurposeAcute renal tubular necrosis (ATN), a common cause of acute renal failure, is a dynamic, rapidly evolving clinical condition associated with apoptotic and necrotic tubular cell death. Its early identification is critical, but current detection methods relying upon clinical assessment, such as kidney biopsy and functional assays, are insufficient. We have developed a family of small molecule compounds, ApoSense, that is capable, upon systemic administration, of selectively targeting and accumulating within apoptotic/necrotic cells and is suitable for attachment of different markers for clinical imaging. The purpose of this study was to test the applicability of these molecules as a diagnostic imaging agent for the detection of renal tubular cell injury following renal ischemia.MethodsUsing both fluorescent and radiolabeled derivatives of one of the ApoSense compounds, didansyl cystine, we evaluated cell death in three experimental, clinically relevant animal models of ATN: renal ischemia/reperfusion, radiocontrast-induced distal tubular necrosis, and cecal ligature and perforation-induced sepsis.ResultsApoSense showed high sensitivity and specificity in targeting injured renal tubular epithelial cells in vivo in all three models used. Uptake of ApoSense in the ischemic kidney was higher than in the non-ischemic one, and the specificity of ApoSense targeting was demonstrated by its localization to regions of apoptotic/necrotic cell death, detected morphologically and by TUNEL staining.ConclusionApoSense technology should have significant clinical utility for real-time, noninvasive detection of renal parenchymal damage of various types and evaluation of its distribution and magnitude; it may facilitate the assessment of efficacy of therapeutic interventions in a broad spectrum of disease states.

Activation of hypoxia-inducible factors ameliorates hypoxic distal tubular injury in the isolated perfused rat kidney

BACKGROUND Preconditional activation of HIF with specific prolyl-hydroxylase inhibitors (PHD-I) attenuates proximal tubular injury, induced by warm ischaemia/ reperfusion (Bernhardt, JASN, 2006). Distal tubular damage occurs in humans with acute kidney injury (AKI), in experimental contrast media-induced nephropathy (CIN), as well as in cell-free isolated perfused kidneys (IPKs). Since in the IPK distal tubular damage inversely correlates with HIF activation (Rosenberger, KI, 2005), we explored the potential of PHD-I to improve morpho-functional outcome in this model. METHODS Male SD rats were randomly given the synthetic PHD-inhibitor FG-4497 (FibroGen, 50 mg/kg IV) or its vehicle (CTR, n = 10 per group). Six hours later, the right kidney was perfused for 90 min with cell-free oxygenated medium and subsequently perfusion-fixed for morphologic assessment. The left kidney was used for HIF immunostaining. RESULTS As compared with CTR kidneys, at 6 h after FG-4497 HIF-alpha isoforms were markedly up-regulated in all renal zones: HIF-1alpha in tubules and in papillary interstitial cells (IC), HIF-2alpha in IC and vascular endothelial cells. FG-4497 treatment resulted in a higher perfusate flow rate (P < 0.04, ANOVA). Tubular injury to medullary thick ascending limbs (mTALs) was significantly attenuated in the treatment versus control group (38.9 +/- 7.4% versus 62.7 +/- 4.9% of mTALs in the mid-inner stripe (P < 0.02); 23.8 +/- 6.8% versus 45.6 +/- 7.4% in the innermost zone of the inner stripe (P < 0.05). CONCLUSIONS These findings illustrate that PHD-I preconditioning attenuates hypoxic distal tubular injury produced in the IPK in the same fashion in which it protects proximal tubules. mTAL conservation may be related to the stabilization of cellular HIF, as well as to preserved endothelial function and microcirculation.

Acute-on-chronic renal failure in the rat: functional compensation and hypoxia tolerance.

Background: We hypothesized that chronic renal parenchymal disease may predispose to acute renal failure (ARF), facilitating the induction of hypoxic medullary tubular injury. Methods: To induce chronic renal parenchymal injury, rats underwent sham operation (control) or bilateral 50-min clamping of the renal artery [ischemia-reperfusion (IR)]. One or 3 months later, both groups were subjected to an ARF protocol, consisting of radiocontrast and the inhibition of prostaglandin and nitric oxide synthesis. Renal function and morphology were determined 24 h later. Results: Chronic tubulointerstitial changes (fibrosis, atrophy and hypertrophy) in the IR group correlated with baseline tubular function, but glomerular function was preserved. Functional deterioration after the ARF protocol was only marginally more pronounced in the IR group, and the degree of medullary acute tubular necrosis (ATN) was unaffected by prior IR. The extent of both tubular necrosis and chronic tubulointerstitial changes independently predicted the acute decline in renal function. Immunostaining of IR kidneys disclosed critically low medullary pO2 (determined by pimonidazole adducts), regional hypoxic cell response (hypoxia-inducible factors) and upregulation of endothelin-B receptors. Conclusions: Compensatory changes result in normal plasma creatinine 1 and 3 months after IR, despite diminished tubular function. Preexisting renal disease only marginally predisposes to ARF, and the extent of ATN is not significantly enhanced. These findings illustrate the complex interaction between chronic and acute renal injury and dysfunction and parallel the difficulty of their assessment in the clinical practice. Adaptive cellular responses to chronic hypoxia in conjunction with parenchymal loss and decreased oxygen demand might alleviate acute hypoxic injury.

Endotoxin-Induced Renal Failure

The pathogenesis of sepsis-induced renal failure is multifactorial and only partially understood. In these studies we evaluated intrarenal microcirculatory changes during endotoxemia and the potential role of nitric oxide (NO) and endothelin in these changes. In anesthetized rats endotoxin infusion [lipopolysaccharide (LPS), Escherichia coli serotype 0127:B8; 10 mg/kg/h] resulted in hypotension and a transient enhancement of renal blood flow, with cortical vasodilation and a loss of outer medullary vasodilatory response to hypotension. The initial cortical vasodilation was abolished by the NO synthase inhibitor NG-nitro-L-arginine methyl ester, but not by indomethacin. Direct NO measurements disclosed a gradual rise in cortical NO, despite the waning vasodilatory effect, suggesting antagonizing vasoconstrictive stimuli. In rats pretreated by LPS (1 mg/kg i.p. 1 day earlier) the renal blood flow was reduced to 55% of that of controls. Moreover, the vasodilatory response to LPS infusion was converted into profound cortical and medullary vasoconstriction. In these preconditioned rats the endothelin receptor antagonist bosentan evoked a vasodilatory response and attenuated the vasoconstrictive reaction to LPS infusion. The infusion of another LPS (E. coli serotype 0111:B4) exerted predominant and protracted renal vasodilation without hypotension. In conclusion, different LPS exert diverse systemic and renal hemodynamic responses. The 0127:B8 serotype attenuates renal medullary vasodilation during hypotension, exerts transient cortical vasodilation, and following repeated exposure induces profound renal vasoconstriction. NO and endothelin participate in LPS-induced vascular responses that may predispose to hypoxic tubular damage.

Diabetes and radiocontrast media increase endothelin converting enzyme-1 in the kidney

Plasma endothelin-1 levels rise in diabetes and after exposure to contrast media suggesting a role in progressive diabetic and acute radiocontrast nephropathies. Here we studied individual and combined effects of streptozotocin-induced diabetes and contrast media on renal endothelin converting enzyme-1 levels in the rat. In vivo, medullary (but not cortical) endothelin converting enzyme protein gradually increased 4 to 5-fold following the induction of diabetes or after the administration of contrast media but rose 15-fold when diabetic rats were given contrast media. Changes in mRNA expression paralleled those of the protein. Immunohistochemistry confirmed that increased tubular and endothelial cell endothelin converting enzyme-1 were most pronounced in the medulla. In vitro, endothelin-1 levels increased 3-fold following incubation of endothelial cells with media high in glucose or with contrast and 4-fold with their combination. Endothelin converting enzyme-1 protein and mRNA expression changed in a similar pattern while prepro endothelin-1 mRNA increased with each insult but not in an additive way. Our study shows that diabetes and contrast media up-regulate renal medullary endothelin converting enzyme-1 expression and synthesis.

A Role for Erythropoietin in the Attenuation of Radiocontrast-Induced Acute Renal Failure in Rats

Background. Radiocontrast-induced nephropathy (CIN) remains an important iatrogenic cause of acute renal failure in high-risk patients, despite the development of safer contrast media, the improvement of hydration protocols, and the introduction of additional preventive strategies. Erythropoietin (EPO) pretreatment may confer protection against acute renal failure through the induction of stress response genes. Methods. The effect of EPO has been evaluated in a rat model of CIN, induced by iothalamate, following the inhibition of nitric oxide- and prostaglandin-synthesis with indomethacin and Nω nitro-L-arginine methyl ester (L-NAME). Twenty-two male Sprague-Dawley rats were subjected to saline (CTR) or EPO injections (3000 U/kg and 600 U/kg, 24 and 2 h before the induction of CIN, respectively). Results. The decline in creatinine clearance in CTR animals from 0.38 ± 0.03 to 0.28 ± 0.03 mL/min/100 g (p < 0.005), was prevented by EPO pretreatment (from 0.34 ± 0.02 to 0.32 ± 0.03 mL/min/100 g, NS). The extent of medullary thick ascending limb- and S3-tubular damage in the outer medulla, however, was comparable in the two experimental groups. Conclusions. EPO pretreatment prevents renal dysfunction in a rat model of CIN. Further experimental and clinical studies are required to confirm these preliminary conclusions regarding a potential protective potency of EPO against CIN.

Effect of Radiocontrast Agents on Intrarenal Nitric Oxide (NO) and NO Synthase Activity

Background/Aims: Contrast media (CM) induce a biphasic renal hemodynamic response, with late prominent cortical vasoconstriction and marked outer medullary vasodilation. The objective of the study was to explore a possible role for altered nitric oxide (NO) production or bioavailability in these hemodynamic responses. Methods: We explored the impact of CM (sodium iothalamate) upon rat renal NO synthase (NOS) activity (citrulline recovery) and NO (using a NO electrode). Results: The cortical NOS activity following CM was 11.5 ± 1.0 versus 13.8 ± 1.1 nmol/gww/min (gww = gram wet weight) in controls (p = 0.16, NS). In rats pretreated with the nonselective endothelin antagonist bosentan, CM reduced the cortical NOS activity to 8.5 ± 1.2 nmol/gww/min (p < 0.005 vs. controls). Cortial NO readings declined over 30 min following CM by 13 ± 8% (p < 0.05, Anova), in parallel with the decline in cortical blood flow. The outer medullary NOS activity was not affected by CM (5.2 ± 1.5 vs. 5.5 ± 1.3) nmol/gww/min in controls) or bosentan. Nevertheless, the outer medullary NO reading increased by 36 ± 23% (p < 0.05), with a concomitant increase in regional blood flow. Conclusion: In the cortex, CM might reduce the NOS activity (an effect blunted by endothelin release). This may potentiate the effect of endothelin to induce regional vasoconstriction. In the outer medulla, the vasodilatory response to CM does not seem to be mediated by enhanced NOS activity and might reflect increased local NO bioavailability as the result of regional hypoxia.

Acute Kidney Injury in the Diabetic Rat: Studies in the Isolated Perfused and Intact Kidney

Background/Aim: Diabetes leads to chronic renal hypoxia and cellular hypoxia response (mediated by hypoxia-inducible factors) and predisposes to acute kidney injury. We studied the impact of acute and chronic hypoxic stress on the development of acute kidney injury in the diabetic rat kidney. Methods: Control (CTR) and streptozotocin (STZ)-diabetic rats were studied following acute medullary hypoxic stress, induced by combinations of radiocontrast and inhibitors of cyclooxygenase and NO synthase. In addition, STZ and CTR kidneys were compared following ex vivo perfusion with oxygenated cell-free medium. Results: The extents of medullary acute tubular injury and renal dysfunction were largely comparable in CTR and STZ-diabetic kidneys in vivo. By contrast, functional deterioration and outer medullary injury were markedly enhanced in STZ kidneys perfused ex vivo. A peculiar collecting duct injury pattern, with cell swelling and detachment, noted in intact STZ kidneys, prominently intensified following isolated perfusion. Conclusions: The diabetic kidney is remarkably resistant to acute hypoxic injury in vivo, possibly due to chronic hypoxia adaptation. Thus, though diabetes predisposes to acute kidney injury in various clinical settings, reduced kidney function does not necessarily imply a greater extent of true tubular damage. The collecting duct injury pattern is an as yet unrecognized feature of early experimental diabetes.

Endothelin-converting enzyme is a plausible target gene for hypoxia-inducible factor

Renal endothelin-converting enzyme (ECE)-1 is induced in experimental diabetes and following radiocontrast administration, conditions characterized by renal hypoxia, hypoxia-inducible factor (HIF) stabilization, and enhanced endothelin synthesis. Here we tested whether ECE-1 might be a HIF-target gene in vitro and in vivo. ECE-1 transcription and expression increased in cultured vascular endothelial and proximal tubular cell lines, subject to hypoxia, to mimosine or cobalt chloride. These interventions are known to stabilize HIF signaling by inhibition of HIF-prolyl hydroxylases. In rats, HIF-prolyl-hydroxylase inhibition by mimosine or FG-4497 increased HIF-1α immunostaining in renal tubules, principally in distal nephron segments. This was associated with markedly enhanced ECE-1 protein expression, predominantly in the renal medulla. A progressive and dramatic increase in ECE-1 immunostaining over time, in parallel with enhanced HIF expression, was also noted in conditional von Hippel-Lindau knockout mice. Since HIF and STAT3 are cross-stimulated, we triggered HIF expression by STAT3 activation in mice, transfected by or injected with a chimeric IL-6/IL-6-receptor protein, and found a similar pattern of enhanced ECE-1 expression. Chromatin immunoprecipitation sequence (ChIP-seq) and PCR analysis in hypoxic endothelial cells identified HIF binding at the ECE-1 promoter and intron regions. Thus, our findings suggest that ECE-1 may be a novel HIF-target gene.