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Effect of dithiothreitol on mercuric chloride- and uranyl nitrate-induced acute renal failure in the rat. The current study was undertaken to examine the effects of dithiothreitol (DTT), a sulfhydryl reducing agent and heavy metal chelator, on the course of heavy metal-induced acute renal failure in the rat. Groups of rats inmetabolic cages received uranyl nitrate (UN) alone, UN plus DTT, mercuric chloride (HgCl2) alone, and HgCl2 plus DTT. UN injected alone produced azotemia, decreased creatinine clearance,and rising fractional sodium excretion over the 48hr of study. These effects of UN on renal function were not observed when DTT was administered 30min after UN injection. Qualitativelysimilar results were obtained with HgCl2-induced acute renal failure. Groups of rats were killed at 6hr after UN alone, UN plus DTT, HgCl2 alone, or HgCl2 plus DTT; and determinations ofplasma renin activity (PRA) and renin activities of the superficial and deep juxtaglomerular apparatus (JGA) were performed. PRA's and JGA renins were increased in animals receiving either UN or HgCl2 alone, but not in the rats receiving both DTT and UN or HgCl2. The effect of DTT on distribution of 203Hg was alsoexamined. Treatment with DTT did not alter the renal accumulation of 203Hg, suggesting that this agent does not act by limiting renal exposure to the heavy metals. Thus, DTT ameliorates the course of heavy metal-induced ARF, and this effect is associated with prevention of heavy metal-induced alterations in sodium excretion and renin-angiotensin system activity.Effet du dithiothreitol sur l'insuffisance rénale aiguë déterminée chez le rat par le nitrate d'uranyl et l'HgCl2. Ce travail a été entrepris pour étudier les effets du dithiothreitol (DTT), agent réducteur des groupements sulfhydryl et chélateur des métaux lourds, sur l'évolution de l'insuffisance rénale aiguë déterminée chez le rat par les métaux lourds. Des groupes de rats placés en cages métaboliques ont reçu soit seulement du nitrate d'uranyl (UN), soit UN + DTT, soit HgCl2 seul, soit HgCl2 + DTT. UN seul produit une hyperazotémie, une diminution de la clearance de la créatinine et une augmentation de l'excrétion fractionnelle de sodium pendant 48hr de l'étude. Les effets de UN sur la fonction rénale ne sont pas observés si DTT est administré 30min après UN. Qualitativement les mêmès résultats sont obtenus dans l'insuffisance rénale aiguë induite par HgCl2. Des groupes de rats ont été sacrifiés è la 6ehr après UN seul, UN + DTT, HgCl2 seul ou HgCl2 + DTT et l'on a dosé l'activité rénine plasmatique (PRA) et les activités rénine des appareils juxta-glomérulaires (JGA) superficiels et profonds. Les activités n'étaient augmentées que chez les rats qui avaient reçu soit UN soit HgCl2 seuls mais par chez les rats traités par DTT et UN ou HgCl2. L'effet de DTT sur la distribution de203Hg a été étudié. Le traitement par DTT ne modifie pas l'accumulation rénale de203Hg, ce qui suggère que cet agent n'agit pas par la limitation de l'exposition du rein aux métaux lourds. Ainsi DTT améliore l'évolution de l'ARF due au métal lourd et cet effet est associé è l'empêchement des modifications, induites par le métal lourd, de l'excrétion de sodium et de l'activité du système rénine-angiotensine.

The tubuloglomerular feedback (TGF) response was studied in control rats and after either hypotensive hemorrhage or aortic clamping (AC). TGF was assessed both by differences in proximally and distally determined single nephron glomerular filtration rate (SNGFR) and by proximally determined SNGFR responses to orthograde microperfusion at 0 or 36 nl/min. Hypotensive hemorrhage was induced by the removal of blood equivalent to 0.5-1% of body weight. In control rats, proximal SNGFR was 29.74 +/- SE 0.87 nl/min and distal SNGFR was 28.64 +/- 0.82 nl/min, values not significantly different from each other. After moderate hemorrhagic hypotension (MH: BP = 86 +/- 1 mm Hg) or AC (BP = 70 +/- 4 mm Hg), both proximal and distal SNGFR decreased, with no significant differences between the values in either group. After severe hemorrhagic hypotension (SH; BP = 70 +/- 1 mm Hg), proximal SNGFR was 25.23 +/- 2.07 nl/min and distal SNGFR was 19.69 +/- 1.50 nl/min, values significantly different from each other and consistent with an enhanced feedback response. Using orthograde microperfusion, a significant reduction in SNGFR at a perfusion rate of 36 nl/min was observed under all circumstances. However, with SH hypotension the percent change in SNGFR at the two perfusion rates was significantly increased to 35.0 +/- 5.5%, compared to 21.6 +/- 6.6% in controls. In contrast, AC with reduction in renal perfusion pressure to a degree comparable to SH hypotension did not augment the relative decrease in SNGFR, the percentage change being 22.2 +/- 7.2%. Neither was TGF enhanced after MH hypotension when similar volumes of blood were removed but a similar decrease in BP was not obtained. These results suggest that some factor related to severe systemic hypotension enhanced the TGF response.

Uranyl nitrate-induced acute renal failure in the rat: Micropuncture and renal hemodynamic studies. Rats were studied after uranyl nitrate administration to define the mechanism(s) responsible for acute renal failure (ARF). Early (six hours) in the course of ARF, proportional decreases occurred in renal blood flow rate (RBF) and whole kidney and single nephron glomerular filtration rates (GFR), to 64%, 67% and 69% of control values, respectively. The stability of single nephron GFR at puncture sites more distal to the glomerulus, appearance of i.v. administered lissamine green in distal tubules and relatively normal proximal intratubular hydrostatic pressure are not consistent with a dominant role for transtubular "leak" of glomerular filtrate or tubular obstruction in the diminution of GFR at six hours. At 48 hr, further proportioned decrements in RBF and GFR, to 29% and 26% of control values, respectively, were observed. Proximal single nephron GFR values could only be obtained at earlier puncture sites suggesting that micropuncture studies late in this model of ARF were limited by uranyl nitrate-induced anatomic alterations. Analyses of 133 Xe washout, 85 Kr autoradiographs and silicone rubber renal vascular casts demonstrated progressive, patchy renal cortical ischemia. The data suggest that altered renal hemodynamics were responsible for diminished renal function after uranyl nitrate administration in the rat although "leak", especially at 48 hr, was not conclusively excluded. Insuffisance renale aigue par le nitrate d'urane: Etude hemodynamique et par microponctions. Des rats ont ete etudies apres administration de nitrate d'urane afin de preciser le ou les mecanismes de l'insuffisance renale aigue (ARF). Au cours de cette ARF il apparait precocement (six heures) une diminution proportionnelle du debit sanguin renal total (RBF), de la filtration glomerulaire globale et des filtrations glomerulaires individuelles respectivement a 64%, 67% et 69% des valeurs controles. La stabilite de la filtration glomerulaire individuelle a distance du glomerule, l'apparition du vert de lissamine dans les tubes distaux et la pression hydrostatique intratubulaire proximale proche de la normale ne sont pas en faveur d'un role important d'une "fuite" transtubulaire du filtrat glomerulaire ou d'une destruction tubulaire dans la diminution de GFR a la 6e heure. Apres 48 heures, une diminution proportionnelle encore accrue de RBF et GFR est observee, respectivement, a 29% et 26% des valeurs controles. Les debits de filtration individuels des nephrons ne peuvent etre mesures qu'en des sites proximaux proches des glomerules, ce qui suggere que les etudes tardives par microponctions de ce modele d'ARF sont limitees par les alterations anatomiques liees au nitrate d'urane. L'analyse du lavage au 133 Xe, des autoradiographies avec 85 Kr et des moulages vasculaires par le silicone montrent une ischemie corticale progressive et irreguliere. Ces resultats suggerent que des modifications hemodynamiques sont responsables de la diminution de la fonction renale apres nitrate d'urane chez le rat, quoique la "fuite", particulierement a la 48e heure, ne soit pas definitivement exclue.

The proximal renal tubules have a marked affinity for gentamicin; they also are the major site of nephrotoxicity caused by this drug. The uptake of radiolabeled gentamicin in separated, viable renal tubules prepared by enzymatic digestion of rabbit kidneys was studied. The preparations showed rapid initial uptake of gentamicin followed by continued slower uptake. Accumulation was not affected by pH, but was significantly inhibited by ouabain, dinitrophenol, anoxia, and hypothermia in the absence of evident cellular damage. At gentamicin concentrations of greater than 50 microgram/ml in the medium, there was competition for drug uptake. Gentamicin efflux in tubules that were taken from a medium containing antibiotic and placed into antibiotic-free fluid was slow and incomplete. From these data it appears that gentamicin uptake by separated renal tubules occurs by a process that requires metabolic energy; thereafter, the drug resides in a poorly exchangeable cellular pool.

Potassium transport in the isolated proximal convoluted tubule (PCT) of the rabbit was studied to determine the importance of concentration-dependent passive processes in potassium reabsorption. Net potassium flux was measured with an initial perfusate potassium concentration of 4 mM and bath potassium concentration of 2, 4, or 6 mM. When bath concentration was 6 mM, there was net potassium secretion in both superficial (SF) and juxtamedullary (JM) PCT. When bath concentration was 2 mM, there was net reabsorption in both groups of tubules. The apparent permeability coefficients were found to be significantly higher in JMPCT (2.96 +/- 0.37 pmol X mm-1 X min-1 X mM-1) than in SFPCT (1.94 +/- 0.34 pmol X mm-1 X min-1 X mM-1). We also attempted to uncover an active potassium reabsorption process by inhibiting water flux, but not other transport processes, by the imposition of a transtubular osmotic gradient. When the perfusate was made 30 mosmol/kg H2O hypertonic to the bath, there was net fluid entry of 0.38 +/- 0.09 nl/min, accompanied by a significant decline in net potassium flux. The collected fluid-to-perfusate potassium concentration ratio was not significantly less than zero, thereby not providing evidence for active potassium reabsorption. These studies suggest that a primary mode of potassium reabsorption in this segment is due to its movement along a transepithelial gradient established by fluid reabsorption. We have been unable to identify an active component of potassium reabsorption.

To determine the sites of tubular phosphate reabsorption in the nephron, microinjection studies were undertaken, utilizing isotonic electrolyte solutions, containing either 1.4 or 8.0 mM phosphate and radioactive PO4-33P and inulin-3H, in rats made mildly diuretic by infusion of mannitol. The injected sites were localized by the technique of latex dissection. The relation between proximal tubular length and per cent 33P recovery for injections of 1.4 mM phosphate (physiological amounts) suggest that relatively little reabsorption of phosphate occurs in the distal 30% of the proximal tubule compared with the proximal portion of the tubule. The corresponding recoveries for proximal tubular microinjections of 8.0 mM phosphate fall along a smooth curve tending to plateau with essentially complete 33P recovery (> 95%) beyond 50% of the tubule. Absolute reabsorption of injected phosphate for both concentrations (i.e., absolute efflux per unit tubular length in the proximal tubule) was independent of phosphate delivery, since the relationship between reabsorption and site of injection was no different for the two concentrations. Distal convoluted tubular microinjections for both phosphate concentrations showed complete recovery of 33P from all injection sites. The data indicate that: (a) no phosphate reabsorption occurs in the distal convoluted tubule or in the collecting duct, (b) phosphate efflux per unit tubular length is greater in the first one-third of the proximal tubule than in the remaining two-thirds, and (c) in the last two-thirds of the proximal tubule, absolute phosphate reabsorption is relatively small and might be limited by factors other than the amount or concentration of injected phosphate.

The measurement of electrolyte concentration in nanoliter quantities of fluid is a fundamental step in the utilization of micropuncture and isolated tubule microperfusion techniques for studying ion transport. A number of methodologies have been developed for these analyses, including helium glow photometry [1], electron probe microanalysis [2], and atomic absorption spectrophotometry [3]. Because of the significant cost of the equipment for performing atomic absorption spectrophotometry or electron probe microanalysis, and the lack of commercial availability of helium glow photometers, we decided to utilize ion-specific electrodes for the measurement of sodium and potassium in samples from isolated tubule microperfusion experiments. The application of ion-specific micro-electrodes to the measurement of intracellular ion activity and macroanalysis is well established [4], but the technique has not been widely applied to the analysis of electrolyte concentration in nanoliter droplets, although Garvin has recently developed a flow-through ion-selective electrode apparatus for measurement of potassium concentrations [5]. Since aqueous droplets must be analyzed under oil to prevent evaporation, one problem we had to overcome was the deterioration in the sensitivity of liquid ion exchanger microelectrodes on contact with oil. Other investigators have utilized ion-selective microelectrodes to study intracellular ion activities or ion activities in tubular fluid in situ, circumstances in which the tissue is bathed in an aqueous electrolyte solution rather than mineral oil [6–8]. In the case where measurements have been made in aqueous droplets under oil, an ion-selective glass rather than a liquid ion exchanger microelectrode was used [9]. With liquid ion exchangers, we found that the sensitivity of the electrode decreased markedly on contact with mineral oil, probably due to the lipophilicity of the ionophore. Although it is possible to utilize very small tips and holders with sidearms to express contaminated ionophore (S. Agulian, personal communication), we found this technique cumbersome and have adapted the methods described by Vogel et al [10], which utilizes a desilanization step to allow a droplet of aqueous solution to protect the ionophore from oil contamination. We have applied electrodes constructed using this technique to the measurement of electrolyte concentrations in aqueous nanoliter droplets under oil. The equipment for this analysis is relatively inexpensive, available in many microperfusion laboratories, and can be applied to a variety of ions, and therefore the technique may be valuable to a wide spectrum of investigators.

The effects on juxtaglomerular apparatus (JGA) renin activity of maneuvers known to acutely alter renin secretory rates were studied in rats. Five groups of rats were studied: group I: control; group II: 1% volume expansion; group III: 10% volume expansion; group IV: hypotensive hemorrhage; and group V: aortic clamping. Superficial (S) JGA renin activity was decreased in group II and further decreased in group III. Deep (D) JGA renin activity did not change. After hypotensive hemorrhage both S- and D-JGA renin activity increased. Similar results were seen after aortic clamping, although the increase in D-JGA renin activity was significantly less. These results indicate that S- and D-JGA renin activity may be under the control of different stimuli and may vary independently. They also support the concept that acute changes in renin secretion may be related to changes in renin synthesis.

Intact and acutely parathyroidectomy (TPTX) dogs were studied during hydropenia, volume expansion (VE), volume expansion plus etharcyrnic acid (EA), and volume expansion plus acetazolamide (AZ), In intact dogs, VE produced marked increases in both Na + and phosphate (Pi) excretion; in TPTX dogs, Na + excretion increased but phosphaturia was minimal. Addition of EA increased Na + but not Pi excretion in both groups. Discontinuing EA and substituting AZ in intact dogs produced a marked increase in Pi excretion compared to both VE and VE + EA. In TPTX dogs, AZ failed to increased Pi excretion compared to VE alone. The results suggest that increased distal Pi absorption in acutely TPTX dogs is not associated with NaCL reabsorption in the thick ascending loop to Henle or maly occur at an alternative nephron site. Furthermore, the increased, the distal Pi reabsorptive capacity revealed by TPTX can overcome the increase distal Pi delivery produced by the superimposition of AZ on VE.

Previous investigations have suggested that there is a functional heterogeneity along the length of the proximal convoluted tubule. This study was designed to confirm and extend these suggestions by examining the intrinsic absorption of fluid and the effect of parathyroid hormone (PTH) on net fluid absorption in isolated, anatomically defined segments of rabbit superficial proximal tubules. The EPCT (early proximal convoluted tubule), LPCT (late proximal convoluted tubule), and PR (pars recta) segments were studied under controlled conditions by the isolated perfused tubule technique. In 23 EPCT, base-line fluid absorption was 1.38 +/- 0.04 (SE), a rate significantly higher than those of 11 LPCT (0.62 +/- 0.02; P less than 0.001) and 12 PR (0.52 +/- 0.03 nl mm-1 min-1, P less than 0.001) segments. In 10 EPCT, mean control fluid absorption was 1.31 +/- 0.04 nl mm-1 min-1; addition of PTH resulted in a decrease to 0.95 +/- 0.05 nl mm-1 min-1 (P less than 0.001); and, after removal of PTH, fluid absorption increased (P less than 0.001). Parathyroid hormone had no effect on either seven LPCT segments or six PR segments. These results demonstrate differences in intrinsic capacity to absorb fluid by anatomically defined segments of the rabbit proximal tubule. This functional heterogeneity is further supported by the observed differential response to PTH by the various anatomic segments of the proximal tubule.

It has been previously demonstrated that single neophron filtration rate, whole kidney glomerular filtration rate and total renal blood flow decreased by 30-35% 6 h after uranyl nitrate induced acute renal failure in the rat. In order to evaluate a role of the renin-angiotensin system in the initiating phase (0-6 h) of this model of acute renal failure determinations of plasma renin activity, superficial (S) and deep (D) juxtaglomerular apparatus (JGA) renin activity and distal nephron [Na+] were obtained. Plasma renin activity increased from the control value of 1.5 +/- 0.3 (S.E.M.) to 2.9 +/- 0.4 ng/ml/h (P less than 0.005) at 6 h. Mean renin activity in S- and D-JGA's of control rats was 6.99 +/- 0.41 and 2.67 +/- 0.21 ng/JGA/h, respectively. After uranyl nitrate, renin activity in S-JGA's increased to 13.62 +/- 0.80 ng/JGA/h (P less than 0.001) at 2 h and remained elevated, 12.56 +/- 0.90 and 12.75 +/- 0.87 ng/JGA/h at 4 and 6 h. D-JGA renin activity increased (P less than 0.05) to 7.04 +/- 0.53, 6.23 +/- 0.31 and 3.44 +/- 0.33 ng/JGA/h at 2, 4 and 6 h after uranyl nitrate. Distal tubule [Na+], 27 samples in 6 rats, increased from a mean control value of 53.7 +/- 1.2 mEq/l to 116.9 +/- 2.5 mEq/l, 24 samples in 6 rats (P less than 0.001). Prompt increases in JGA renin activity were observed in the initiating phase of acute renal failure, suggesting a role for the renin-angiotensin system in the pathophysiology of this nephrotoxic model. The association of increased JGA renin activity and increased distal [Na+] is consistent with a role for the tubuloglomerular feedback mechanism in the initiating phase of uranyl nitrate induced acute renal failure in the rat.

A comparison of the efficacy of prostacyclin and herapin as anticoagulants during hemodialysis in uremic dogs was evaluated. Prostacyclin was infused continuously into the arterial limb of the dialyzer (100 ng/kg/min) while heparin was administered as a 5000 unit bolus at the beginning of dialysis. Prostacyclin and heparin were alternated and a total of 4 dialyses/dog were performed. Parameters followed to assess the efficiency of dialyses included: dialysance (ml/min) of urea, creatinine, ultrafiltration rate (ml/hr), residual volume and platelet count (% of baseline). A lesser degree of hemodialysis associated thrombocytopenia, and a higher ultrafiltration rate were observed with prostacylcin. These studies demonstrate that prostacyclin can be used alone as an anticoagulant in a uremic dog model, and in contrast to heparin it maintains the platelet count and improves the ultrafiltration rate during hemodialysis.

The intrarenal gradient of renin activity was determined in rats by using superficial (S) and deep (D) cortical juxtaglomerular apparatuses (JGA's), identified and microdissected after silicone-rubber compound injection. Angiotensin generated from single JGA's using partially purified sheep renin substrate was quantified by rat bioassay. When, in rats on a normal NaCl diet, silicone-rubber was injected into a carotid artery, alone or with abdominal aorta catheterization, S:D renin activity ratios were 1.18+/-0.08 (SEM) and 1.21+/-0.12, respectively. The S:D renin activity ratios obtained when silicone-rubber was injected into the abdominal aorta (2.52+/-0.09) or a chronic carotid artery catheter (3.44+/-0.40) were significantly higher (P0.001). The lower S:D renin activity ratios after carotid artery manipulation were due to significantly higher D-JGA renin activities. This increased D-JGA renin activity and the lack of a renin gradient appear to be related to acute carotid artery manipulation. Alterations in JGA renin activity were examined relative to NaCl intake. 2 wk after high-NaCl diet the absolute net renin activity decreased (P0.001) more in S (5.84+/-0.11 ng AI.JGA(-1).h(-1)) than D (1.73+/-0.06 ng AI.JGA(-1).h(-1)) JGA's, and the intrarenal renin gradient was lost (S:D-JGA renin activity, 1.00+/-0.07), as compared to the regular NaCl diet. 2 wk of a low-NaCl diet resulted in a greater (P0.01) increase in S (14.28+/-1.47 ng AI.JGA(-1).h(-1)) than D (9.62+/-1.19 ng AI.JGA(-1).h(-1)) JGA renin activity and a renin gradient (S:D-JGA renin activity) of 1.75+/-0.12. These results demonstrate that NaCl intake clearly influences total JGA renin content and may also affect the relative intrarenal distribution of renin activity.