Marina Goldfarb

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.

Tissue oxygenation modifies nitric oxide bioavailability.

Objective: Because changes in blood oxygenation acutely alter vascular tone, we explored a possible modulation of nitric oxide‐induced vasodilation (nitrovasodilation) by oxygen.

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.

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 µg/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.

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 Nω-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.

Renal Effects of Nabumetone, a COX-2 Antagonist: Impairment of Function in Isolated Perfused Rat Kidneys Contrasts with Preserved Renal Function in vivo

The constitutive cyclooxygenase (COX)-1 enzyme has been considered the physiologically important isoform for prostaglandin synthesis in the normal kidney. It has, therefore, been suggested that selective inhibitors of the ‘inducible’ isoform (COX-2) may be free from renal adverse effects. We studied the renal effects of the predominantly COX-2 antagonist nabumetone in isolated perfused kidneys. As compared with controls, kidneys removed after in vivo administration of oral nabumetone (15 mg/kg) disclosed altered renal function with reduced glomerular filtration rate, filtration fraction, and urine volume and enhanced hypoxic outer medullary tubular damage. By contrast, renal function and morphology were not affected in vivo by nabumetone or its active metabolite 6-methoxy-2-naphthylacetic acid. The latter agent (10–20 mg/kg i.v.) did not significantly alter renal microcirculation, as opposed to a selective substantial reduction in medullary blood flow noted with the nonselective COX inhibitor indomethacin (5 mg/kg i.v.). In a rat model of acute renal failure, induced by concomitant administration of radiocontrast, nitric oxide synthase, and COX inhibitors, the decline in kidney function and the extent of hypoxic medullary damage with oral nabumetone (80 mg/kg) were comparable to a control group, and significantly less than those induced by indomethacin. In rats subjected to daily oral nabumetone for 3 consecutive weeks, renal function and morphology were preserved as well. Both nabumetone and 6-methoxy-2-naphthylacetic acid reduced renal parenchymal prostaglandin E2 to the same extent as indomethacin. It is concluded that while nabumetone adversely affects renal function and may intensify hypoxic medullary damage ex vivo, rat kidneys are not affected by this agent in vivo, both in acute and chronic studies. COX selectivity may not explain the renal safety of nabumetone.

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

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. 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. In isolated aortic rings, EACA (but not the other inhibitors of the fibrinolytic system PAI‐1 or α‐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 μM, 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, Ca2+ internalization following PE was enhanced by EACA, and retarded by tPA. In anesthetized rats, EACA (150 mg 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 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. 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.

Transient urethral obstruction predisposes to ascending pyelonephritis and tubulo-interstitial disease: studies in rats

Abstract Chronic tubulo-interstitial disease, an important cause of end-stage renal disease, often results from the combined effects of a disturbed urinary outflow tract and urinary tract infection. Acute unilateral ureteral obstruction in rats rapidly induces foci of medullary necrosis, confined to the region of the papilla and fornices. This injury may provide a nidus for bacterial invasion and may invoke reactive and regenerative changes, ultimately leading to chronic pyelonephritis and tubulo-interstitial nephropathy. To explore this possibility, adult rats underwent renal morphological evaluation 2–7 days following transient 24-h unilateral ureteral obstruction. In some experiments the bladder was inoculated with bacteria (108–109 cfu/ml Escherichia coli in 0.5 ml) after release of ureteral obstruction, with subsequent cultures obtained from the pelvis of both kidneys and from the urinary bladder. Morphologic evaluation of perfusion-fixed kidneys, 2–7 days after the release of 24-h ureteral obstruction disclosed papillary necrosis, urothelial proliferation, marked inner-stripe interstitial expansion, and fibrosis and proximal tubular (S3) dilatation. The lateral (perihilar region) was predominantly affected, with lesions spreading from the fornices. There was some progression of interstitial fibrosis during the postobstructive time course or following more prolonged ureteral obstruction. By contrast, infection hardly contributed to the tubulointerstitial changes. In rats subjected to infection, cultures were positive in all 15 postobstructive kidneys, as opposed to five contralateral kidneys (P < 0.0001). Viable counts from the postobstructive kidney were also higher than those from the contralateral side (79,000 ± 12,000 vs 2900 ± 1600 cfu/ml, mean ± SEM, P < 0.0001), and were comparable to those obtained from the bladder (77,000 ± 13,000 cfu/ml). We conclude that transient ureteral obstruction predisposes to ascending pyelonephritis and to tubulointerstitial disease. This vulnerability may relate to altered urodynamics and medullary tissue destruction.