Amany Abdelkader

Renal oxygenation in acute renal ischemia-reperfusion injury

Tissue hypoxia has been demonstrated, in both the renal cortex and medulla, during the acute phase of reperfusion after ischemia induced by occlusion of the aorta upstream from the kidney. However, there are also recent clinical observations indicating relatively well preserved oxygenation in the nonfunctional transplanted kidney. To test whether severe acute kidney injury can occur in the absence of widespread renal tissue hypoxia, we measured cortical and inner medullary tissue Po2 as well as total renal O2 delivery (Do2) and O2 consumption (Vo2) during the first 2 h of reperfusion after 60 min of occlusion of the renal artery in anesthetized rats. To perform this experiment, we used a new method for measuring kidney Do2 and Vo2 that relies on implantation of fluorescence optodes in the femoral artery and renal vein. We were unable to detect reductions in renal cortical or inner medullary tissue Po2 during reperfusion after ischemia localized to the kidney. This is likely explained by the observation that Vo2 (-57%) was reduced by at least as much as Do2 (-45%), due to a large reduction in glomerular filtration (-94%). However, localized tissue hypoxia, as evidence by pimonidazole adduct immunohistochemistry, was detected in kidneys subjected to ischemia and reperfusion, particularly in, but not exclusive to, the outer medulla. Thus, cellular hypoxia, particularly in the outer medulla, may still be present during reperfusion even when reductions in tissue Po2 are not detected in the cortex or inner medulla.

Determinants of renal tissue hypoxia in a rat model of polycystic kidney disease

Renal tissue oxygen tension (PO2) and its determinants have not been quantified in polycystic kidney disease (PKD). Therefore, we measured kidney tissue PO2 in the Lewis rat model of PKD (LPK) and in Lewis control rats. We also determined the relative contributions of altered renal oxygen delivery and consumption to renal tissue hypoxia in LPK rats. PO2 of the superficial cortex of 11- to 13-wk-old LPK rats, measured by Clark electrode with the rat under anesthesia, was higher within the cysts (32.8 ± 4.0 mmHg) than the superficial cortical parenchyma (18.3 ± 3.5 mmHg). PO2 in the superficial cortical parenchyma of Lewis rats was 2.5-fold greater (46.0 ± 3.1 mmHg) than in LPK rats. At each depth below the cortical surface, tissue PO2 in LPK rats was approximately half that in Lewis rats. Renal blood flow was 60% less in LPK than in Lewis rats, and arterial hemoglobin concentration was 57% less, so renal oxygen delivery was 78% less. Renal venous PO2 was 38% less in LPK than Lewis rats. Sodium reabsorption was 98% less in LPK than Lewis rats, but renal oxygen consumption did not significantly differ between the two groups. Thus, in this model of PKD, kidney tissue is severely hypoxic, at least partly because of deficient renal oxygen delivery. Nevertheless, the observation of similar renal oxygen consumption, despite markedly less sodium reabsorption, in the kidneys of LPK compared with Lewis rats, indicates the presence of inappropriately high oxygen consumption in the polycystic kidney.

Chronic treatment with tempol does not significantly ameliorate renal tissue hypoxia or disease progression in a rodent model of polycystic kidney disease

In the present study, we tested whether polycystic kidney disease (PKD) is associated with renal tissue hypoxia and oxidative stress, which, in turn, contribute to the progression of cystic disease and hypertension. Lewis polycystic kidney (LPK) rats and Lewis control (Lewis) rats were treated with tempol (1 mmol/L in drinking water) from 3 to 13 weeks of age or remained untreated. The LPK rats developed polyuria, uraemia and proteinuria. At 13 weeks of age, LPK rats had greater mean arterial pressure (1.5‐fold), kidney weight (sixfold) and plasma creatinine (3.5‐fold) than Lewis rats. Kidneys from LPK rats were cystic and fibrotic. Renal hypoxia was evidenced by staining for pimonidazole adducts and hypoxia‐inducible factor (HIF)‐1α in cells lining renal cysts and upregulation of HIF‐1α and its downstream targets vascular endothelial growth factor (VEGF), glucose transporter‐1 (Glut‐1) and heme oxygenase 1 (HO‐1). However, total HO activity did not differ greatly between kidney tissue from LPK compared with Lewis rats. Renal oxidative and/or nitrosative stress was evidenced by ninefold greater immunofluorescence for 3‐nitrotyrosine in kidney tissue from LPK compared with Lewis rats and a > 10‐fold upregulation of mRNA for p47phox and gp91phox. Total renal superoxide dismutase (SOD) activity was sevenfold less and expression of SOD1 mRNA was 70% less in kidney tissue from LPK compared with Lewis rats. In LPK rats, tempol treatment reduced immunofluorescence for 3‐nitrotyrosine and HIF1A mRNA while upregulating VEGF and p47phox mRNA expression, but otherwise had little impact on disease progression, renal tissue hypoxia or hypertension. Our findings do not support the hypothesis that oxidative stress drives hypoxia and disease progression in PKD.

Augmented expression and secretion of adipose-derived pigment epithelium-derived factor does not alter local angiogenesis or contribute to the development of systemic metabolic derangements

Impaired coupling of adipose tissue expansion and vascularization is proposed to lead to adipocyte hypoxia and inflammation, which in turn contributes to systemic metabolic derangements. Pigment epithelium-derived factor (PEDF) is a powerful antiangiogenic factor that is secreted by adipocytes, elevated in obesity, and implicated in the development of insulin resistance. We explored the angiogenic and metabolic role of adipose-derived PEDF through in vivo studies of mice with overexpression of PEDF in adipocytes (PEDF-aP2). PEDF expression in white adipocytes and PEDF secretion from adipose tissue was increased in transgenic mice, but circulating levels of PEDF were not increased. Overexpression of PEDF did not alter vascularization, the partial pressure of O2, cellular hypoxia, or gene expression of inflammatory markers in adipose tissue. Energy expenditure and metabolic substrate utilization, body mass, and adiposity were not altered in PEDF-aP2 mice. Whole body glycemic control was normal as assessed by glucose and insulin tolerance tests, and adipocyte-specific glucose uptake was unaffected by PEDF overexpression. Adipocyte lipolysis was increased in PEDF-aP2 mice and associated with increased adipose triglyceride lipase and decreased perilipin 1 expression. Experiments conducted in mice rendered obese by high-fat feeding showed no differences between PEDF-aP2 and wild-type mice for body mass, adiposity, whole body energy expenditure, glucose tolerance, or adipose tissue oxygenation. Together, these data indicate that adipocyte-generated PEDF enhances lipolysis but question the role of PEDF as a major antiangiogenic or proinflammatory mediator in adipose tissue in vivo.

Renal cellular hypoxia in adenine-induced chronic kidney disease.

We determined whether adenine‐induced chronic kidney disease (CKD) in rats is associated with renal tissue hypoxia. Adenine (100 mg) or its vehicle was administered to male Sprague‐Dawley rats, daily by oral gavage, over a 15‐day period. Renal function was assessed before, and 7 and 14 days after, adenine treatment commenced, by collection of a 24‐hour urine sample and a blood sample from the tail vein. On day 15, arterial pressure was measured in conscious rats via the tail artery. Renal tissue hypoxia was then assessed by pimonidazole adduct immunohistochemistry and fibrosis was assessed by staining tissue with picrosirius red and Massons trichrome. CKD was evident within 7 days of commencing adenine treatment, as demonstrated by increased urinary albumin to creatinine ratio (30 ± 12‐fold). By day 14 of adenine treatment plasma creatinine concentration was more than 7‐fold greater, and plasma urea more than 5‐fold greater, than their baseline levels. On day 15, adenine‐treated rats had slightly elevated mean arterial pressure (8 mmHg), anaemia and renomegaly. Kidneys of adenine‐treated rats were characterised by the presence of tubular casts, dilated tubules, expansion of the interstitial space, accumulation of collagen, and tubulointerstitial hypoxia. Pimonidazole staining (hypoxia) co‐localised with fibrosis and was present in both patent and occluded tubules. We conclude that renal tissue hypoxia develops rapidly in adenine‐induced CKD. This model, therefore, should prove useful for examination of the temporal and spatial relationships between tubulointerstitial hypoxia and the development of CKD, and thus the testing of the ‘chronic hypoxia hypothesis’.

Bladder urine oxygen tension for assessing renal medullary oxygenation in rabbits: experimental and modeling studies

Oxygen tension (Po2) of urine in the bladder could be used to monitor risk of acute kidney injury if it varies with medullary Po2 Therefore, we examined this relationship and characterized oxygen diffusion across walls of the ureter and bladder in anesthetized rabbits. A computational model was then developed to predict medullary Po2 from bladder urine Po2 Both intravenous infusion of [Phe(2),Ile(3),Orn(8)]-vasopressin and infusion of N(G)-nitro-l-arginine reduced urinary Po2 and medullary Po2 (8-17%), yet had opposite effects on renal blood flow and urine flow. Changes in bladder urine Po2 during these stimuli correlated strongly with changes in medullary Po2 (within-rabbit r(2) = 0.87-0.90). Differences in the Po2 of saline infused into the ureter close to the kidney could be detected in the bladder, although this was diminished at lesser ureteric flow. Diffusion of oxygen across the wall of the bladder was very slow, so it was not considered in the computational model. The model predicts Po2 in the pelvic ureter (presumed to reflect medullary Po2) from known values of bladder urine Po2, urine flow, and arterial Po2 Simulations suggest that, across a physiological range of urine flow in anesthetized rabbits (0.1-0.5 ml/min for a single kidney), a change in bladder urine Po2 explains 10-50% of the change in pelvic urine/medullary Po2 Thus, it is possible to infer changes in medullary Po2 from changes in urinary Po2, so urinary Po2 may have utility as a real-time biomarker of risk of acute kidney injury.

751 TREATMENT WITH THE ANTIOXIDANT TEMPOL DOES NOT SIGNIFICANTLY AMELIORATE HYPERTENSION OR PROGRESSION OF RENAL DISEASE IN A RODENT MODEL OF POLYCYSTIC KIDNEY DISEASE

Objectives: We have tested the hypothesis that renal tissue hypoxia and oxidative stress exist in polycystic kidney disease (PKD) and contribute to development of hypertension and renal disease progression. Methods: Lewis polycystic kidney rats (LPK) and Lewis controls were treated with tempol (1 mM in water) from 3-13 weeks of age, or remained untreated. At 13-weeks, arterial pressure was measured in conscious animals, and then renal tissue collected post-mortem for assessment of markers of tissue hypoxia and oxidative stress. Results: Relative to Lewis, LPK had significantly greater arterial-pressure (1.5-fold), kidney weight (6-fold) and plasma creatinine (3.5-fold). Renal tissue hypoxia was evidenced by staining for pimonidazole-adducts and hypoxia-inducible factor-1&agr; (HIF-1&agr;) in renal cysts, and up-regulation of mRNA for HIF-1&agr; and its downstream target glucose-transporter-1 & heme-oxygenase (HO)-1. Total HO activity did not differ in LPK compared to Lewis. Renal oxidative/nitrosative stress was evidenced by 9-fold greater immunofluorescence for 3-nitrotyrosine in LPK, and a 10-fold upregulation of mRNA for p47phox & gp91phox. Total renal superoxide-dismutase (SOD) activity was 7-fold less in LPK compared to control, consistent with reduced levels of SOD-1 mRNA. In LPK, tempol treatment reduced 3-nitrotyrosine labelling and HIF-1&agr; mRNA expression, while up-regulating P47phox mRNA, but otherwise had little impact on arterial-pressure, progression of kidney disease or renal hypoxia. Conclusion: Our findings support the hypothesis that renal oxidative stress and hypoxia exist in association PKD, but do not provide evidence for a role of oxidative stress in progression of hypertension or kidney disease in this model of PKD.

286 TELEMETRY-BASED OXYGEN SENSOR TO CONTINUOUSLY MONITOR KIDNEY OXYGENATION IN CONSCIOUS RATS; A NEW PLATFORM FOR EXPLORING DEVELOPMENT OF RENAL DISEASE

Objectives: Disturbed kidney oxygenation, i.e. renal hypoxia, may contribute to initiation and progression of both chronic kidney disease and acute kidney injury. A critical barrier to investigate this is the lack of available technology to chronically measure kidney tissue oxygenation. We have developed a novel solution for chronic measurement of oxygen concentration in the kidney. Methods: Using a telemetry-based carbon paste oxygen electrode we continuously recorded oxygen concentration in the inner medulla for 6 weeks in rats, unhindered by anaesthesia or restraint. The implantable potentiostat allows for potentially lifetime monitoring through the use of inductive powering of the implanted telemeter (Telemetry Research Ltd, Auckland, New Zealand). Results: Oxygen concentration values were stable over time and comparable between animals. Within an animal the level of tissue oxygen slightly increased over time from 7 days after implantation until 3 weeks. When the average of day 5, 6 and 7 is considered as baseline, oxygen concentration increased by 6-66% with a coefficient of variation of 22%. We observed a reproducible response to repeatedly applied changes in inspired oxygen. Hypoxia (10% oxygen) decreased medullary oxygen concentration by 48 ± 7% while hyperoxia (100% oxygen) increased it by 76 ± 5%. Three weeks after implantation, there was little fibrosis around the implanted electrodes. Conclusions: These findings illustrate the possibility of monitoring renal tissue oxygen concentration in conscious animals. This technology provides a platform for the investigation of the role of tissue hypoxia in development of kidney disease.