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.

Evidence for sustained renal hypoxia and transient hypoxia adaptation in experimental rhabdomyolysis-induced acute kidney injury.

BACKGROUND: Indirect evidence suggests that hypoxia contributes to the pathophysiology of rhabdomyolysis-induced acute kidney injury (AKI). However, the cellular location and kinetics of hypoxia, as well as potential hypoxia adaptation are unclear. METHODS: Rhabdomyolysis was induced in rats by IM glycerol (GLY) injection, which largely recapitulates the full clinical syndrome. Additional rats received IV myoglobin (MYO), in order to assess the contribution of MYO per se. We performed immunohistochemistry for hypoxia markers [pimonidazole (PIM) adducts and hypoxia-inducible factors (HIFs)] and the cell-protective HIF target gene heme oxygenase-1 (HO-1). Furthermore, we sought a potential negative feedback loop to terminate HIF activation, driven by HIF prolyl-hydroxylase-2 (PHD-2). RESULTS: In GLY, progressive tubular injury, mainly of proximal tubules (PT), developed over time, but its extent was heterogeneous. PIM, HIFalpha and HO-1 were all absent in controls, but strongly positive in GLY, with a specific spatio-temporal pattern. In PT, (a) PIM was detectable throughout the study with a maximum at 6 h, (b) HIF was activated only at 3 h and (c) HO-1 and PHD-2 appeared at 6 h and persisted at a lower level at 24 h. Apart from tubular cast formation, MYO did not cause overt tissue damage, but led to strong activation of HIFs, in a pattern similar to 3 h of GLY. CONCLUSIONS: Our data suggest that renal hypoxia occurs in rhabdomyolysis, and that MYO, at least partly, contributes to hypoxia generation. Since in the most affected tubules transcriptional hypoxia adaptation is transient and inhomogeneous, pharmacologic HIF enhancement holds the potential to improve outcome in rhabdomyolysis-induced AKI.

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 N(omega) 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.

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.

Effect of nicotine on the renal microcirculation in anesthetized rats: a potential for medullary hypoxic injury?

BACKGROUND: Cigarette smoking has been associated with accelerated renal dysfunction among patients with chronic renal disease. Conceivably, repeated parenchymal hypoxic injury, induced by nicotine-related vasomotor changes, might contribute to the progression of renal failure in smokers. METHODS: Renal blood flow and selective cortical and outer medullary blood flows were determined in anesthetized rats. Changes in total renal, cortical and medullary vascular resistance were calculated. Nicotine was repeatedly infused at rising doses (50-200 microg/kg) to intact (CTR) animals and to rats chronically administered with nicotine in their drinking water (NIC). In a complementary study, nicotine-treated and control rats were subjected to medullary hypoxic stress, induced by radiocontrast and indomethacin. RESULTS: Chronic nicotine exposure led to lower baseline renal blood flow and creatinine clearance. Nicotine infusion induced a transient dose-dependent rise in blood pressure, renal blood flow and cortical flow, with a corresponding decline in renal vascular resistance and cortical resistance in both experimental groups. However, while medullary flow increased in CTR by up to 16 +/- 6%, it remained unchanged or even somewhat declined in the NIC group. Calculated medullary resistance reciprocally declined in CTR while it rose in the NIC group (p < 0.001). In animals subjected to radiocontrast and indomethacin, nicotine intensified renal dysfunction, associated with focal medullary hypoxic damage. CONCLUSIONS: Chronic exposure to nicotine selectively compromises the outer medullary microcirculation, blunting a local vasodilatory response to acute nicotine administration. Repeated acute-on-chronic exposure to nicotine may predispose to hypoxic medullary injury.

Up-regulation of HIF in experimental acute renal failure: evidence for a protective transcriptional response to hypoxia.

BACKGROUND: Medullary hypoxia is believed to play an important role in the pathogenesis of acute renal failure (ARF). Hypoxia-inducible transcription factors (HIF) are recognized as master regulators of hypoxic adaptation, but little is known about their role in renal disease. METHODS: A multi-insult rat model of ARF combining the application of contrast medium with nitric oxide synthase (NOS) and cyclooxygenase (COX) inhibition was used to study chronology and distribution of the oxygen regulated HIF isoforms HIF-1alpha and HIF-2alpha in comparison with the hypoxia-marker pimonidazole between 10 minutes and 48 hours after injury induction. Treatment with furosemide was used to study HIF expression under conditions of ameliorated tissue injury. RESULTS: Contrast medium in combination with NOS and COX inhibition resulted in widespread induction of HIF in the outer and inner medulla that was initiated within 10 minutes, reached the highest levels at 2 hours and diminished 8 hours to 24 hours thereafter. HIF isoforms were expressed in a cell type-specific fashion: HIF-1alpha in tubular and HIF-2alpha in interstitial and endothelial cells. The degree of HIF-1alpha accumulation varied between nephron segments, being much stronger in collecting ducts than in medullary thick ascending limb of the loop of Henle (mTAL). Comparison with pimonidazole staining and the effect of furosemide indicated that HIF induction in mTAL is maximal with moderate hypoxia and declines with increasing severity of hypoxia. CONCLUSION: A complex pattern of HIF activation appears to play an important role in tissue preservation as a response to regional renal hypoxia. The limited capacity of mTAL cells for HIF activation may explain their susceptibility to injury.

The fibrinolytic system attenuates vascular tone: effects of tissue plasminogen activator (tPA) and aminocaproic acid on renal microcirculation.

1. The renal medulla is a major source of plasminogen activators (PA), recently shown to induce vasodilation in vitro. Treatment with PA inhibitors has been associated with renal dysfunction, suggesting compromised renal microvasculature. We investigated the impact of the PA inhibitor epsilon amino-caproic acid (EACA) upon vascular tone in vitro, and studied the effect of both tPA and EACA upon intrarenal hemodynamics in vivo. 2. In vitro experiments were carried out in isolated aortic rings and with cultured vascular smooth muscle cells. Studies of renal microcirculation and morphology were conducted in anesthetized Sprague-Dawley rats. 3. In isolated aortic rings, EACA (but not the other inhibitors of the fibrinolytic system PAI-1 or alpha-2 antiplasmin) reduced the half-maximal effective concentration of phenylephrine (PE) required to induce contraction (from 32 nm in control solution to 2 and 0.1 nm at EACA concentrations of 1 and 10 microm, respectively). Using reteplase (retavase) in the same model, we also provide evidence that the vasoactivity of tPA is in part kringle-dependent. In cultured vascular smooth muscle cells, Ca(2+) internalization following PE was enhanced by EACA, and retarded by tPA. 4. In anesthetized rats, EACA (150 mg x kg(-1)) did not affect systemic blood pressure, total renal or cortical blood flow. However, the outer medullary blood flow declined 12+/-2% below the baseline (P<0.03). By contrast, tPA (2 mg x kg(-1)), transiently increased outer medullary blood flow by 8+/-5% (P<0.02). Fibrin microthrombi were not found within the renal microvasculature in EACA-treated animals. 5. In conclusion, both fibrinolytic and antifibrinolytic agents modulate medullary renal blood flow with reciprocal effects of vasodilation (PA) and vasoconstriction (EACA). In vitro studies suggest that these hemodynamic responses are related to direct modulation of the vascular tone.

Effect of poly(ADP-ribose) polymerase inhibition on outer medullary hypoxic damage.

Poly(ADP-ribose) polymerase (PARP) activation after free-radical-induced DNA damage depletes cellular energy stores and participates in ischemia-reflow injury. We studied the potential protective effect of the water-soluble PARP inhibitor 3-aminobenzamide (3-AB) in a rat model of acute renal failure (ARF) from combined administration of radiocontrast, indomethacin and N(omega)-nitro-L-arginine methyl ester. Kidney function at 24 h was better preserved in rats treated with 3-AB as compared to control animals. However, the extent of tubular hypoxic damage was not significantly mitigated. It is concluded that PARP inhibition may attenuate renal dysfunction in this model of ARF with medullary hypoxic tubular injury even while the extent of tubular necrosis is not significantly altered. Further studies of this dyssynchrony of structure and function may provide important insights into the sequence of events that promotes renal failure after medullary injury.

N-acetylcysteine ameliorates renal microcirculation: studies in rats.

BACKGROUND: N-acetylcysteine (NAC) administration has been shown to ameliorate experimental acute renal failure induced by ischemia-reflow, and was found to prevent radiocontrast nephropathy in high-risk patients. While the protective effect of NAC has been primarily attributed to scavenging oxygen free radicals, improving renal microcirculation also may play a role in the prevention of acute renal failure. METHODS: This study was designed to explore the effect of NAC on renal microcirculation. Blood pressure, total renal blood flow and selective regional cortical and outer medullary blood flow were continuously monitored in anesthetized Sprague Dawley rats with ultrasonic and laser-Doppler probes during the infusion of NAC (60 mg/kg). RESULTS: In control intact rats blood pressure and renal microcirculation were unaffected by NAC. By contrast, following renal vasoconstriction induced by the radiocontrast agent iothalamate meglumine, NAC decreased total, cortical and medullary vascular resistance by 7 to 10% (P < 0.05). NAC also reduced renal vascular resistance by 16% when given during angiotensin II infusion (P < 0.05). Altered renal microcirculation, induced by the cyclooxygenase inhibitor indomethacin, by the nitric oxide synthase-inhibitor, Nomeganitro-l-arginine (L-NAME), or with their combination was partially restored by NAC. Nevertheless, NAC administration failed to attenuate renal function and morphology in a rat model of acute renal failure with selective outer medullary hypoxic injury, induced by indomethacin, L-NAME and iothalamate. CONCLUSIONS: NAC ameliorates renal vasoconstriction, an effect that seems to be mediated by mechanisms other than prostaglandins and nitric oxide. The potential renoprotective outcome of NAC and the role of its vasodilating effect on the pre-constricted renal vasculature should be evaluated further.