Proximal tubular injury attenuates outer medullary hypoxic damage: studies in perfused rat kidneys

The straight segment (S3) of the proximal tubule is predominantly damaged during renal ischemia-reflow, whereas medullary thick ascending limbs (mTALs) are principally affected in other models of hypoxic acute tubular necrosis (ATN). Since the latter injury pattern largely depends on the extent of reabsorptive activity during hypoxic stress, we hypothesized that proximal tubular damage might attenuate downstream mTAL injury by means of diminished distal solute delivery for reabsorption. In isolated rat kidneys perfused for 90 min with oxygenated Krebs-Henseleit solution, mTAL necrosis developed in 75 +/- 3% of tubules in the mid-inner stripe of the outer medulla. By contrast, S3 segments in the outer stripe were minimally affected, with tubular fragmentation involving some 5 +/- 2% of tubules. In kidneys subjected in vivo to proximal tubular injury and subsequently used for isolated perfusion studies, the injury pattern was inverted: following 20 and 30 min ischemia and reflow for 24 h, S3 fragmentation rose to 18 +/- 16% and 72 +/- 13%, while mTAL damage was reduced to 33 +/- 10 and 24 +/- 8%, respectively. In kidneys subjected in vivo to D-serine S3 necrosis rose to 100%, while mTAL damage fell to 1 +/- 1% (p < 0.001). Substantial S3 tubular collapse (involving approximately 30% of tubules) and inner stripe interstitial hemorrhage were also noted, exclusively in kidneys subjected to ischemia-reflow. Proximal tubular necrosis alone or in combination with collapse inversely correlated with mTAL necrosis (R = -0.51 and -0.72, respectively, p < 0.003). This cogent inverse association might imply that disruption of the proximal nephron attenuates downstream mTAL necrosis by a reduction of distal tubular reabsorptive workload.     

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.     

Protective role of furosemide and saline in radiocontrast-induced acute renal failure in the rat

Acute renal failure (ARF) can be produced in rats by a combination of insults which augment transport activity and blunt regulatory mechanisms designed to maintain medullary oxygen sufficiency. This type of ARF is characterized by necrosis of medullary thick ascending limbs (mTALs). Uninephrectomized, salt-depleted rats injected with indomethacin (10 mg/kg) develop ARF following the administration of the radiocontrast agent, iothalamate. Furosemide (20 mg/kg, intravenous), administered immediately before the contrast material, attenuated the severity of ARF and reduced mTAL necrosis. Treatment with furosemide and/or normal saline prevented both the decline in renal function and mTAL injury. It is concluded that furosemide and normal saline may ameliorate the course of ARF if administered before radiocontrast.     

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.     

Early renal medullary hypoxic injury from radiocontrast and indomethacin.

We evaluated the acute changes in cortical and outer medullary oxygen tension and the alterations in renal function and morphology within the first 90 minutes after the administration of indomethacin and iothalamate to anesthetized Sprague-Dawley rats. Both agents were found to produce marked and protracted outer medullary hypoxia averaging 12 +/- 4 and 9 +/- 2 mm Hg, respectively (mean +/- SE). Given together to salt depleted uninephrectomized rats they produced an early hypoxic injury localized selectively in the outer medulla. This lesion progressed from 3 +/- 1% of medullary thick ascending limbs (mTALs) at 15 minutes to 22 +/- 7% at 24 hours. Condensed "dark" cells were observed at 15 minutes, probably representing a type of early injury. Residual red cell mass, quantified in the outer medullary vasculature of perfusion-fixed kidneys and presumably reflecting stasis, was substantially increased in iothalamate treated rats. Red cell mass in the interbundle zone correlated with mTAL necrosis. Taken together, these results show an early period of medullary hypoxia, accompanied by a selective injury to mTALs in the central interbundle zone with apparent stasis. These findings contrast sharply with the ischemia-reflow pattern of renal damage and emphasize the important role of medullary hypoxia in the genesis of acute renal failure in this model.     

Effect of glycine and hypertrophy on renal outer medullary hypoxic injury in ischemia reflow and contrast nephropathy.

Glycine preserves tubular cell integrity under hypoxic and toxic conditions in vitro. It also ameliorates cisplatin nephrotoxicity in vivo. We studied the effect of glycine on tubular necrosis from ischemia reflow and on inner stripe injury in an animal model of radiocontrast nephropathy. In all experiments, glycine (75 mg/100 g/h) increased tubular damage in the inner stripe. In the model of radiocontrast nephropathy, the percentage of medullary thick ascending limb (mTAL) necrosis at 24 hours increased from 22% +/- 6% to 41% +/- 9% or 55% +/- 7% with glycine infusion of 75 or 135 minutes, respectively (mean +/- SE, P less than 0.05, analysis of variance [ANOVA]). Renal function was not significantly affected. In rat kidneys subjected to ischemia reflow, mTAL injury following glycine increased from 1% +/- 0% to 12% +/- 6% (P less than 0.05) and from 8% +/- 5% to 49% +/- 8% (P less than 0.01) 24 hours after 30 minutes and 45 minutes ischemia, respectively. Tubular injury in the inner stripe was maximal in the deep interbundle zone, typical of hypoxic, rather than reperfusion, injury. Prior uninephrectomy increased inner stripe damage, but protected the proximal tubules. Both uninephrectomy and glycine infusion were found to contribute to mTAL necrosis. The infusion of glycine for 1 hour in intact rats increased renal blood flow by 44% and tripled urine volume (P less than 0.01). A parallel increase in glomerular filtration rate GFR; by 22% over 90 minutes) fell short of statistical significance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endotoxin-induced renal failure. II. A role for tubular hypoxic damage

Endotoxin-induced hypotension and altered renal microcirculation could lead to tubular injury, particularly at the physiologically hypoxic outer medulla. We explored this hypothesis in isolated perfused kidneys and in vivo in rats subjected to endotoxemia. Rat kidneys were removed 15 min after endotoxin injection in vivo (from Escherichia coli 0127:B8, 1 mg/kg i.p.) and perfused with oxygenated medium supplemented with 20 amino acids and endotoxin. Glomerular filtration rate and filtration fraction markedly declined (0.4 +/- 0. 1 ml/min and 1.1 +/- 0.1, respectively) as compared with control kidneys (0.7 +/- 0.1 ml/min and 1.8 +/- 0.1, n = 8-12 per group; p < 0.05). Hypoxic injury to medullary thick ascending limbs in the innermost outer medulla increased (47 +/- 9% of tubules vs. 16 +/- 8% in controls, p < 0.05). When rats were preconditioned with an additional endotoxin injection 16 h earlier (a manipulation that markedly reduces cortical and medullary blood flow), glomerular filtration rate and filtration fraction further declined to 0.1 +/- 0.0 ml/min and 0.4 +/- 0.1, respectively (p < 0.01), and tubular sodium reabsorption fell to 81 +/- 12 vs 98 +/- 0% in controls (p < 0.05). Tubular damage, however, did not increase (20 +/- 7%), probably reflecting a decline in reabsorptive workload and oxygen requirement. In rats subjected to a single or two repeated daily doses of endotoxin (1 mg/kg i.p.) plasma creatinine comparably rose 41% on the average over 24 h, creatinine clearance fell by 27% (p < 0.0001), but tubular damage was absent. By contrast, in rats preconditioned with indomethacin and the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (10 mg/kg), the addition of endotoxin markedly augmented outer medullary hypoxic tubular damage both in S(3) segments (27 +/- 10 vs 1 +/- 1%) and in medullary thick ascending limbs (38 +/- 11 vs. 10 +/- 5%, n = 7-8; p < 0.05). It is concluded that under special conditions, such as altered medullary oxygen balance or defective nitric oxide or prostaglandin synthesis, endotoxin may predispose to hypoxic outer medullary tubular damage.     

Renal medullary Na-K-ATPase and hypoxic injury in perfused rat kidneys

We wished to see if chronic alterations in Na-K-ATPase activity in the medullary thick ascending limb would modify the susceptibility of its cells to the hypoxic injury produced by perfusion of the isolated kidney. Rats were fed a diet high (64%) or low (8%) in protein for three weeks. Renal medullary Na-K-ATPase was 75 +/- 12 U/mg protein/hr (mean +/- SE) in the high protein group and 44 +/- 3 in rats given low protein. After 90 minutes of perfusion, the kidneys of rats fed a high protein diet showed almost all mTAL cells near the inner medulla with severe damage (93 +/- 4.8%), whereas the same zone in perfused kidneys of rats on a low protein diet showed only 47 +/- 7.7% injury. In a similar fashion, damage to mTAL cells seen in perfused kidneys was greatly augmented by compensatory renal hypertrophy produced by removal of the contralateral kidney two weeks earlier, and by a diet high in potassium given for two weeks, procedures which also increased the activity of medullary Na-K-ATPase. The results suggest that the level of transport work of medullary cells mediated by Na-K-ATPase is a determinant of the vulnerability of mTAL cells to hypoxic injury.     

Determinants of intrarenal oxygenation: factors in acute renal failure.

Oxygen tension within the renal parenchyma is influenced by two factors: metabolic demand and oxygen supply. There are three regions within the kidney in which there is an anatomical basis for limited oxygen availability. The first is the inner stripe where oxygen diffusion between arterial and venous vasa recta reduces PO2. The other two are the outer stripe and medullary rays which are fed by O2-poor blood from venous vasa recta. The balance between oxygen demand and supply is most critical in the inner stripe where the PO2 is most influenced by transport activity. In contrast, altering transport activities in the outer stripe will not change the prevalence of hypoxic S3 injury but will alter its type (i.e., cell fragmentation related to high GFR and increased workload versus cell edema related to low GFR and minimal workload). The effect of transport activity on medullary ray PO2 has not been well defined. Using sensitive oxygen microelectrodes, cortical PO2 (52 +/- 2 mm Hg) in the rat was found to be higher than medullary PO2 (21 +/- 2 mm Hg, p less than 0.001). How are these observations reflected in current models of acute renal failure? The ischemia-reflow model affects proximal tubules with a predilection for S3 (located within the outer stripe of medulla) after short-term ischemia. With hyperfiltration (induced by glycine or renal hypertrophy) and the pursuant increase in transport related O2 demand, hypoxic mTAL inner stripe injury becomes prominent. Renal parenchymal hypertrophy exaggerates injury in the contrast nephropathy model, in which mTAL inner stripe injury is a predominant feature and medullary PO2 is very low.(ABSTRACT TRUNCATED AT 250 WORDS)     

L-arginine reduces tubular cell injury in acute post-ischaemic renal failure.

BACKGROUND: The pathophysiology of renal ischaemia, resulting in tubular cell injury and leading to acute renal failure (ARF), remains unclear. An ever-increasing number of investigations focus on a possible role of nitric oxide (NO) in regulating circulation during ARF. In this context, we investigated the influence of chronic stimulation or inhibition of NO synthesis, or both, on haemodynamic parameters, histology and plasma renin activity (PRA) after ischaemia-reperfusion injury of rat kidneys. METHODS: Experiments were performed on adult, male Wistar rats. Before induction of ARF, a group of animals was treated with a NO synthesis inhibitor (L-NAME) and another group was treated with a precursor of NO synthesis (L-arginine). The animals received those substances for 4 weeks. Control groups received the same amount of tap water for 4 or 8 weeks and were divided into groups with ARF (4 weeks--ARF group and 8 weeks ARF group) and a sham-operated group. Another group of rats was treated first with L-NAME and then with L-arginine in their drinking water, for 4 weeks for each of these two substances. All parameters were evaluated 24 h after the induction of ischaemic ARF or the sham operation. RESULTS: Our results show that such long-term stimulation of NO release by L-arginine improved renal haemodynamics in the ischaemic form of ARF. Renal blood flow (RBF) increased by 96% in the L-arginine-treated rats with ARF compared with the group with ARF alone. Inhibition of NO synthesis worsens renal haemodynamics after ARF. However, this aggravation can be reversed by L-arginine. The rate of water reabsorption was reduced in all groups with ARF, but this reduction was least in the group treated with L-arginine. The rate of Na+ reabsorption was reduced in all groups 24 h after renal ischaemia, but a significant decrease was observed after the inhibition of NO synthesis. Histological examination of the kidney specimens showed that morphological changes were least in the rats treated with L-arginine, when compared with all other groups with ARF. Nevertheless, the lesions were most prominent in the L-NAME+ARF group. In this group, the areas of corticomedullar necrosis were more widespread in comparison with other groups, especially the L-arginine group where only swelling of the proximal tubular cells was observed. Treatment with L-NAME was not accompanied by any significant alteration in the plasma concentration of angiotensin I (ANG I), while in the group treated with L-arginine ANG I had a tendency to decrease. CONCLUSIONS: Acute post-ischaemic renal failure may be alleviated by administering the NO substrate (L-arginine). NO acts cytoprotectively on tubular epithelial cells in ischaemia--reperfusion injury of rat kidney. Evidence of this comes from both histopathological findings and increased tubular water and sodium reabsorption. However, inhibition of NO synthesis (provoked by L-NAME) worsens renal haemodynamics and aggravates morphological changes after ARF. These aggravations can, however, be reversed by L-arginine.     

Effect of glycine on medullary thick ascending limb injury in perfused kidneys

The addition of 2 mM glycine to the recirculating perfusate of isolated perfused rat kidneys almost completely prevented the severe morphological injury to tubular cells lining the medullary thick ascending limb (mTAL) that normally develops in this preparation. Glycine was similarly effective in reducing mTAL injury associated with hypoxic perfusion, indomethacin and amphotericin. Fractional reabsorption of sodium was increased with glycine, without any change in perfusate flow to the whole kidney and without consistent improvement in GFR. L-alanine demonstrated a similar though less pronounced cytoprotective action, but glutamine, cysteine, glutamate, cysteine plus glutamate, 1-serine and 4-aminoisobutyric acid all had little or no effect in preventing severe mTAL injury. The protective effect of glycine was unimpaired by the arginine analogue NG-monomethyl-l-arginine (L-NMMA), suggesting that the endothelial-derived relaxing factor, NO, was not involved. The action of glycine was not reduced by the addition of a substrate (benzoate) or a product (hippurate) of the glycine N-acyltransferase reaction. Glycine did not depress the respiration of dispersed mTALs prepared from rat kidneys. The cytoprotective effect of glycine in the mTAL of perfused kidneys, shared with l-alanine, appears to be relatively specific for these amino acids and probably unrelated to a diminution in cell work.     

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.     

Cyclosporine nephropathy: morphometric analysis of the medullary thick ascending limb.

The effects of cyclosporine A (CsA) (12.5 mg/kg/d) on the medullary thick ascending limb (mTAL) were studied in five experimental groups: vehicle-treated control (C), salt depletion (SD), cyclosporine (CsA), and the combination of both salt depletion and cyclosporine for 3 (CsA-SD:S) and 8 (CsA-SD:L) weeks. Evaluation was performed on 1-micron plastic horizontal sections. mTALs were classified as either atrophic or nonatrophic by assessing mitochondrial density. The mean cross-sectional area of atrophic mTALs was found to be significantly smaller than the mean of nonatrophic mTALs in all treatment groups. The percentage of atrophic tubules was found to be significantly increased in both CsA-SD groups as compared with the other three treatment groups (P less than 0.01). Regression analysis indicated a rectangular hyperbolic relationship between the percentage of atrophic tubules and mean nonatrophic tubule cross-sectional area (P less than 0.0001). Thus, low levels of injury are associated with a rapid increase in cross-sectional tubular area (hypertrophy), and this response plateaued with increasing degrees of injury. Terminal plasma creatinine correlated with nonatrophic tubular cross-section area (r = 0.52, P less than 0.003). These studies indicate that CsA induces mTAL atrophy, which is more extensive with salt depletion. With limited injury, hypertrophy develops. However, the hypertrophic response cannot be sustained with increasing degrees of injury. The phenomenon of mTAL atrophy and hypertrophy is particularly important, since hypertrophy itself is a known risk factor for mTAL 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.     

Influence of endogenous prostaglandins on mTAL injury

We altered renal prostaglandin production by isolated rat kidneys in several ways to see if this would influence the susceptibility of cells lining the medullary thick ascending limb to injury. Rats were fed a diet containing either safflower oil (high in linoleic acid) or fish oil (low in arachidonate precursors) as a source of fat. After 90 min of perfusion, the kidneys of rats fed safflower oil showed only 32.7 +/- 6.7% of medullary thick ascending limb cells near the inner medulla with severe damage, whereas the same zone in perfused kidneys of rats fed fish oil showed 96.6 +/- 1.3% severely damaged cells (P less than 0.01). The protection afforded by safflower oil was accompanied by a doubling of urinary excretion of PGE2 and 6-keto-PGF1 alpha, and was eliminated by indomethacin, which suppressed prostaglandin synthesis. Perfusion with bradykinin also greatly increased prostaglandin excretion and reduced severe medullary thick ascending limb damage in the deepest zone of the outer medulla from 51.3 +/- 6.6% in controls to 28.5 +/- 5.9% (P less than 0.02). The protection provided by bradykinin was also completely reversed by indomethacin. The results suggest that endogenous prostaglandins serve a protective function against hypoxic injury for cells of the medullary thick ascending limb.     

Effects of nitric oxide on gentamicin toxicity in isolated perfused rat kidneys

BACKGROUND: There have been many studies in recent years concerning the role of nitric oxide (NO) in acute renal failure (ARF). In this study, the effects of the inhibition or the induction of NO synthase (NOS) on gentamicin-induced ARF was investigated in isolated perfused rat kidneys. METHODS: Kidneys from male Sprague-Dawley rats were perfused in situ for 90 min. Perfusion was conducted in the presence of inulin (60 mg/dL in perfusion buffer) as a glomerular filtration rate (GFR) marker. Six groups (total: 42 rats) were studied: group 1, controls with no treatment; group 2, L-arginine (2 mM in perfusate); group 3, L-nitro-arginine-methyl ester (L-NAME, 0.1 mM in perfusate); group 4, gentamicin (GM, 0.5 mg/mL in perfusate); group 5, GM + L-arginine (same dose as groups 2 and 4) and; group 6, GM + L-NAME (same dose as groups 3 and 4). Cell injury was assessed by measuring N-acetyl-beta-D-glucosaminidase (NAG), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity in urine. RESULTS: L-arginine prevented, whereas L-NAME enhanced, GM-induced enzyme release and GFR reduction. Histological studies showed that GM-treated kidneys had clear signs of tubular damage and this damage was increased by simultaneous L-NAME and GM administration. CONCLUSION: This study suggests that NO formation could prevent the GM-induced nephrotoxicity in this ARF model.     

Resistance of mTAL Na+-dependent transporters and collecting duct aquaporins to dehydration in 7-month-old rats

BACKGROUND: Aging is associated with a defect in urinary concentration in both human and experimental animals. The purpose of these studies was to examine the urinary concentrating ability, the expression of kidney water channels [aquaporins (AQP1 to AQP3)], and medullary thick ascending limb (mTAL) Na+-dependent transporters in old but not senescent versus young animals in response to water deprivation. METHODS: Two-month-old and 7-month-old rats were placed in metabolic cages and deprived of water for 72 hours. Kidney tissues were isolated and examined for the expression of AQP1 to AQP3 and mTAL, peptide-derived polyclonal antibody specific to kidney apical Na+-K+-2 Cl- cotransporter (BSC1), Na+/H+ exchanger isoform 3 (NHE3), and Na+ pump using semiquantitative immunoblotting and Northern hybridization. RESULTS: After 72 hours of water deprivation, urine osmolality increased from 1269 to 3830 mOsm/kg H2O in 2-month-old rats, but only from 1027 to 2588 mOsm/kg H2O in 7-month-old rats. In response to water deprivation, AQP2 and AQP3 expression increased significantly in the cortex and medulla of 2-month-old rats but remained unchanged in the medulla or slightly increase in the cortex of 7-month-old animals. AQP1 expression was not altered by dehydration in both groups. The protein abundance of mTAL BSC1, NHE3, and Na+ pump increased significantly in young but remained unchanged in 7-month-old rats subjected to water deprivation. CONCLUSION: Age-related decrease in urinary concentrating ability is an early event, developed before the onset of senescence. This defect results from reduced responsiveness of cortical AQP2 and AQP3 and a blunted response of medullary AQP2 and mTAL BSC1, NHE3, and Na+ pump to dehydration in aging kidneys.