Pentoxifylline in the isolated perfused rat kidney

The effects of pentoxifylline, a new methylxanthine with marked hemorrheologic properties, were studied following brief renal artery occlusion in the isolated rat kidney model perfused with cell-free Krebs-Henseleit buffer. Anuria was observed in 3 of 6 control kidneys within 5 min after reperfusion; urine flow was maintained in all rat kidneys perfused with pentoxifylline (2500 ng/ml). Glomerular filtration rate was significantly greater in kidneys administered pentoxifylline compared with controls following 40 min of postocclusion reperfusion (460 +/- 100 vs. 100 +/- 110 microliters/min/gKW; P less than 0.01). Pretreatment of kidneys with indomethacin, a nonspecific cyclooxygenase inhibitor, blocked the protective effects of pentoxifylline in this setting. These data suggest that the addition of pentoxifylline may prevent hypoxia-related changes in renal function of transplanted kidneys. Stimulation of renal prostaglandin synthesis, as well as an interaction at the level of the adenosine receptors, were most likely responsible for the observed beneficial effects of pentoxifylline.     

Effects of cyclosporine on the isolated perfused rat kidney

Although cyclosporine (CsA) has been shown to cause decreased renal function in humans, the mechanisms important in cyclosporine nephrotoxicity are not well understood. Investigations of cyclosporine nephrotoxicity in animal models have been complicated by systemic toxic effects not seen in humans. In the present study, the direct renal effects of cyclosporine were investigated in the isolated perfused rat kidney (IPRK) model. Cyclosporine delivered by nontoxic liposomes had no effect on IPRK resistance, perfusate flow, inulin clearance, or fractional reabsorption of sodium, despite marked tissue accumulation of CsA (55.1 +/- 7.2 micrograms/g kidney tissue). In contrast, a 63% decrease in inulin clearance was observed following the administration of intravenous cyclosporine (0.1 ml). However, similar changes in IPRK function were seen after the administration of 0.1 ml of the intravenous cyclosporine vehicle, cremophor, suggesting that the alterations in function were secondary to the vehicle. All together, these findings suggest that cyclosporine nephrotoxicity may be secondary to renal innervation, toxic metabolites, or other systemic effects of cyclosporine not present in the IPRK.     

Effects of suprofen on the isolated perfused rat kidney.

Although suprofen has been associated with the development of acute renal failure in greater than 100 subjects, the mechanism of damage remains unclear. The direct nephrotoxic effects of a single dose of 15 mg of suprofen were compared in the recirculating isolated rat kidney perfused with cell-free buffer with or without the addition of 5 mg/dL of uric acid. There were no significant differences in renal sodium excretion, oxygen consumption, or urinary flow rates in kidneys perfused with suprofen compared with the drug-free control groups. In contrast, a significant decline in glomerular filtration rate was found after the introduction of suprofen to the kidney perfused with uric acid; no changes were found with suprofen in the absence of uric acid. A significant decrease in the baseline excretion rate of uric acid was found in rats given suprofen, compared with drug-free controls. However, the fractional excretion of uric acid was unchanged between the groups over the experimental period. In summary, suprofen causes acute declines in renal function, most likely by directly altering the intrarenal distribution of uric acid.     

Hypoxic injury in the proximal tubule of the isolated perfused rat kidney

The effect of hypoxia on the morphology of the proximal tubule was examined in isolated rat kidneys perfused with Krebs-albumin medium, gassed with 95% N2/5% CO2 for 45, 90 and 190 min. The major findings were heterogeneity of types of cell injury and definable topographical zones of protection from damage. The characteristic injury in S1 and S2 was that of mitochondrial swelling and brush border alterations that progressed to complete disorganization of this zone. S3 manifested two distinct lesions, one characterized by cell swelling and the other by tubular epithelial fragmentation. Protection from these injuries was seen in tubules located in periarterial zones, apparently due to gradients of oxygenation. The same types of injury but without zones of protection were seen after perfusion with cyanide (10 mM) in oxygenated medium. A "reflow" period of oxygenated perfusion did not alter the fundamental character of these lesions but affected tubules showed progression of damage while there was preservation of tubules in periarterial areas. Thus the responses of the proximal tubule to hypoxic injury depends both on segment type (S1, S2 versus S3) and on tubular location in relation to O2 availability. Additional factors appear to determine the type of response in S3 tubules.     

Mechanism of sodium chloride reabsorption in the ascending thin limb of Henle's loop

Summary The ascending thin limb of Henle's loop has been one of the most mysterious nephron segments because of the unique characteristics of its NaCl and water transport systems. The ascending thin limb is essentially impermeable to water under all circumstances. The majority of luminal sodium ions (Na⁺) are reabsorbed across the shallow tight junction between the ascending thin limb cells, as the apical membrane of the ascending thin limb is impermeable to Na⁺. Intracellular Na⁺ activity is maintained at a low level by a ouabain-sensitive Na⁺/K⁺-ATPase. Intracellular pH is maintained by an amiloridesensitive sodium ion-hydrogen ion (Na⁺/H⁺) antiporter, which depends on calmodulin. Intracellular calcium ion (Ca²⁺) activity is maintained at a low level by a calmodulin-sensitive Ca²⁺ pump and a dihydropyridine-sensitive Ca²⁺ channel. In the ascending thin limb, Cl⁻ is reabsorbed across the Cl⁻ channels in both the luminal and basolateral membranes. This channel is sensitive to various anion transport inhibitors. Chloride ion transport in the ascending thin limb is also sensitive to intra- and extracellular pH. Physiologic regulation of the Cl⁻ channel by the vasopressin V2 receptor has been identified. Our studies have elucidated the precise mechanism of NaCl transport in the ascending thin limb, and suggest that this countercurrent exchange system in the ascending thin limb is not effected by any energy-dependent process, but occurs as passive simple diffusion of Na⁺ via tight junctions as a result of facilitated transport of Cl⁻ across the cell membranes. This review was presented at the 39th Annual Meeting of the Japanese Society of Nephrology and received an Oshima Award for Young Investigators     

Mechanisms of ciclosporin A-induced vasoconstriction in the isolated perfused rat kidney.

Hypertension is a well-known side effect of ciclosporin A (CsA). In the present study the mechanisms of vasoconstriction in renal vessels were examined in the isolated perfused rat kidney. Kidneys were perfused with constant flow at a temperature of 37 degrees C with Tyrode's solution equilibrated with 95% O2/5% CO2. CsA was dissolved in ethanol. 500 and 2000 ng/ml increased resistance of renal vessels by 0.97 +/- 0.55 x 10(5) and 2.29 +/- 1.33 x 10(5) dyn s cm-5, respectively (mean values +/- SD, n = 12). The vasoconstriction developed gradually over 4 min. The vasopressor effect of CsA was not changed by saralasin (10(-6) M), nifedipine (10(-6) M) and ketanserin (10(-6) M), but was completely blocked by phentolamine and prazosin (each 10(-6) M). CsA-induced vasoconstriction was not prevented by perfusion with Ca(2+)-free solution containing 2 mmol EGTA. Similarly, pretreatment with reserpine to deplete sympathetic nerve endings from catecholamines did not affect CsA-induced vasoconstriction. The findings suggest that CsA-induced vasoconstriction is mediated by stimulation of alpha 1-receptors. Ca2+ influx does not play a role for CsA-induced vasoconstriction. Prolonged perfusion of rat kidneys with the vehicle cremophor EL elicits an irreversible increase in perfusion pressure.     

Renal accumulation of 99mTc-DMSA in the artificially perfused isolated rat kidney

In order to investigate aspects of the renal handling of 99mTc-DMSA, 68 isolated rat kidneys were artificially perfused. The experimental groups were: Group 1 (no. = 32)-oxygenated filtering kidneys; Group 2 (no. = 29)-oxygenated non-filtering kidneys; Group 3 (no. = 7)-anaerobic non-filtering kidneys. We conclude that the 99mTc-DMSA complex is strongly bound to albumin, is not filtered and is removed from perfusion fluid through the renal peritubular capillary route and that this occurs by an active process which depends upon aerobic metabolism. This process has a high capacity and is not inhibited by probenecid.     

Evaluation of citrate flushing solution using the isolated perfused rat kidney

The isolated rat kidney perfused at 37 C with dialyzed bovine serum albumin (6.5 g/100 ml) in Krebs-Henseleit buffer was used to examine why a hypertonic citrate flush permits rapid recovery of renal function after storage. The composition of the original hyperosmolar citrate solution was varied so that the roles of osmolality, magnesium, and citrate could be evaluated. All kidneys were flushed with the test solutions and stored for 24 hr in the test solutions at 0 C. The citrate flushing solution requires both the citrate anion and magnesium for efficacy. Hyperosmolality does not enhance its action, an isosmolar solution is more effective. Citrate can be replaced by a nonmetabolizable analogue, tricarballylate, if the solution is suitably buffered. The mechanism of action of citrate is still uncertain, it does not seem primarily to act as a metabolic fuel or inhibitor.     

Glutathione in the isolated perfused rabbit kidney

Perfusion of the isolated rabbit kidney at normothermia leads to a marked depletion in the concentration of reduced glutathione in the kidney tissue. After 90 min of perfusion at 36 ± 1°C, the concentration of GSH found in the renal cortex is more than 80% lower than the levels found in fresh tissue. Comparable perfusion under hypothermic conditions reduced the amount of glutathione depletion considerably; however, after 90 min of perfusion at 15 ± 1°C, renal cortex GSH levels were still nearly 50% lower than the levels found in fresh tissue. It was found that the addition of GSH to the perfusate completely alleviated this glutathione-depletion phenomenon.     

Regulation of kallikrein and renin release by the isolated perfused rat kidney

Rat kidneys perfused in vitro released kallikrein in urine, and renin and kallikrein in the perfusate. The kallikrein was characterized by its kininogenase activity and released bradykinin from bovine and dog substrates. Inactive trypsin activatable kallikrein was present in both perfusate and urine. Kallikrein secretion in urine was influenced by changes in perfusion pressure (PP). Raising the PP strikingly increased urinary kallikrein and lowering PP reduced it. Urinary water and electrolyte output were augmented to the same extent by furosemide and mannitol administration as by raising the PP, but neither drug affected kallikrein. Isoproterenol stimulated the release of renin but not kallikrein. Stopping the oxygen supply to the perfusate suppressed kallikrein secretion in urine and renin release in the perfusate. The kidneys released ten times less kallikrein in the perfusate than in urine, and perfusate kallikrein was not influenced by changes in PP. It is concluded that in this model, changes in PP and/or renal blood flow and/or oxygen supply regulate kallikrein secretion in urine, but that this secretion is unaffected by changes in urinary output. We also conclude that kallikrein release in urine and renin release in perfusate are regulated simultaneously by renal hemodynamic changes but are not affected concomitant by beta-adrenergic stimulation or changes in distal urine composition.     

Different effects of dopamine and dopexamine on the isolated perfused rat kidney

We have investigated the effect of dopamine and dopexamine on the isolated perfused rat kidney. After an equilibration period of 20 min and two control periods of 10 min, dopexamine 1.0, 2.5 or 4.0 micrograms kg-1 min-1 or dopamine 2.0 micrograms kg-1 min-1 were perfused for a further 40 min in random order. Renal blood flow, urine volume, glomerular filtration rate, absolute sodium excretion and fractional sodium reabsorption of the isolated perfused kidney were measured every 10 min during the experiment. Dopamine increased significantly urine production from mean 61.54 (SEM 4.7) to 117.2 (9.7) microliters min-1 g-1 and absolute sodium excretion from 0.4 (0.1) to 1.2 (0.1) microgramsmol min-1 g-1, and decreased significantly fractional sodium reabsorption from 97.3 (0.5) to 90.7 (0.7)%. Renal blood flow and glomerular filtration rate were not altered. In contrast, dopexamine had no effect on the isolated kidney. These data suggest that the diuretic and natriuretic effects of dopexamine in humans may not result from a direct action on the kidney.      

Direct effects of endotoxin on the function of the isolated perfused rat kidney

When the endotoxin-lipopolysaccharide (LPS) derived from the cell wall of gram-negative bacteria is given to the rat in vivo, there are prompt, marked decreases in glomerular filtration rate (GFR), renal blood flow (RBF) and % Na+ reabsorption (%T-Na+). However, it has not been determined whether the endotoxin itself has a direct effect on these renal functions. To test whether endotoxin has a direct renal effect, isolated rat kidneys (N = 8) were perfused for 160 minutes with a Krebs-Ringer-HCO3- solution containing substrate-free albumin (40 g/liter), glucose (5 mM) and L(+) lactate (7.5 mM). After control observations (20 to 80 min) were made, purified LPS from E. coli was added (N = 4) to the perfusate to achieve [endotoxin] of 0.01 micrograms/ml (80 to 120 min) and 0.1 micrograms/ml (120 to 160 min). Endotoxin had no effect on GFR, Na+ reabsorption or tissue K+ content when compared to timed-control perfusions (N = 4). There was a small (approximately 10%) but significant decrease in mean perfusion flow rate (PFR) at the highest [endotoxin] when compared to the low [endotoxin]p but no change in GFR occurred. When the same LPS was given to four rats in vivo at a dose which achieved an [endotoxin] of approximately 0.08 micrograms/ml plasma, there were prompt decreases in GFR and %T-Na+ and an increase in body temperature when compared with timed-controls; there also was a large loss of K+ from the kidney tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of parathyroid hormone in isolated perfused rat kidney and liver combined

Metabolism of synthetic, intact, human parathyroid hormone (PTH) 10(-9) M was studied in a new experimental model using the isolated perfused rat kidney and liver combined. The combined organs cleared intact PTH significantly faster than the single kidneys (P less than 0.02) or livers (P less than 0.02), but not faster than the sum of the clearances in the single organs. The kidneys cleared intact PTH without accumulation of NH2-terminal, mid-molecule or COOH-terminal iPTH, and high-performance liquid chromatography (HPLC) studies did not reveal any PTH fragments. The livers cleared mid-molecule and COOH-terminal iPTH significantly slower (P less than 0.002) than intact PTH, and HPLC demonstrated generation of mid-molecule and COOH-terminal PTH fragments. The combined organs accumulated significantly less mid-molecule (P less than 0.001) and COOH-terminal (P less than 0.03) iPTH than the single livers, and HPLC demonstrated mid-molecule peaks that were smaller but not qualitatively different. In conclusion, the predominance of COOH-terminal PTH fragments in plasma may be maintained by differential clearance mainly in the liver, excessive accumulation being prevented by filtration in the kidneys.     

Regional membrane specialization in the thin limbs of Henle's loops as seen by freeze-fracture electron microscopy

Rat thin limbs of Henle were studied by freeze-fracture electron microscopy. Thin limb segments in both short- and long-looped nephrons were identified by previously developed ultrastructural criteria, continuity with known thick segments, and architectural relationships in the outer medulla. Intramembrane particle (IMP) density and the number of intramembrane fibrils comprising the zonula occludens were determined for each morphologically identifiable thin limb segment. The IMP density on the protoplasmic faces of both the luminal and abluminal membranes of the upper portion of the descending thin limb (DTL) of the long-looped nephron is quantitatively greater than in the short-looped thin limb, lower portion of the long-looped DTL, and in the ascending thin limb. The zonulae occludens in the long-looped upper DTL consists of a single fibril; the long-looped lower DTL contains 3.13 +/- 0.14 fibrils; the ascending thin limb contains 1.31 +/- 0.09 fibrils; and the short-looped DTL contains 3.75 +/- 0.19 fibrils. These studies further support the contention that there is anatomic heterogeneity among the thin limb segments. Because direct physiologic studies in the thin limbs are incomplete and conflicting, the need for correlative physiologic studies on anatomically characterized structures is indicated.     

DNA fragmentation reduced by antioxidants following ischaemia-reperfusion in the isolated perfused rat kidney

SUMMARY: The role of oxygen-derived free radicals (OFR) in modifying structure and function after ischaemia-reperfusion (IR) injury was studied in isolated perfused rat kidneys (IPRK). Control kidneys were studied after 20 min of ischaemia followed by 15 or 60 min of reperfusion. the xanthine oxidase inhibitor allopurinol and the hydroxyl radical scavenger dimethylthiourea (DMTU) were used to prevent OFR-related damage. Morphological injury was assessed in cortex, inner and outer medulla and compared with indices of global renal function (inulin clearance, fractional sodium excretion and renal vascular resistance). Apoptosis was assessed using both morphological criteria and in situ end-labelling (ISEL) to identify DNA fragmentation. Tubular damage, as evidenced by cellular blebbing, tubular cast formation, epithelial necrosis, and occasional apoptosis, was greatest in the straight proximal tubule and thick ascending limb (TAL) in the outer zone of the outer medulla. Pretreatment with allopurinol or DMTU did not significantly improve renal function. However, structural damage and luminal debris were diminished in allopurinol- and DMTU-treated kidneys. These changes may lead to functional improvement after more prolonged reperfusion. In situ end-labelling was more frequent in distal tubular epithelial cells after IR than either morphological evidence of apoptosis or necrosis. Decreased ISEL was observed after pretreatment with both allopurinol and DMTU. the data demonstrate that OFR produce DNA damage after IR, increasing ISEL. This probably represents reversible DNA damage rather than incipient apoptosis. Thus, antioxidants reduce or prevent DNA and cellular injury after IR and may reduce functional impairment after prolonged reperfusion.