Renal elimination of organic anions in rats with bilateral ureteral obstruction

Urinary tract obstruction is an important cause of acute renal failure. Several abnormalities in renal tubular function may occur in obstructive nephropathy. The tubular secretion of organic anions is an important function of the kidney that eliminates potentially toxic organic anions from the body, however, the mechanisms involved in organic anions renal elimination in rats with bilateral ureteral obstruction (BUO) have not been elucidated. In this study, it was evaluated the renal handling of p-aminohippurate (PAH) in adult male Wistar rats with BUO. A diminished renal clearance of PAH was observed in BUO rats as consequence of a diminution in the secreted load of this organic anion. The increase in the abundance of organic anions transporter 1 (OAT1) and the absence of modification in cortical renal blood flow, measured with fluorescence microspheres, do not explain the altered secretion of PAH. The diminished Na,K-ATPase activity in cortex from obstructed kidneys might condition OAT1 function. Additionally, it is also possible to conclude that in the presence of BUO, PAH clearance is not a good estimate of renal plasma flow.     

Evidence for a secretory component in the handling of unconjugated bilirubin by the isolated perfused rat kidney

Previous studies in rats have suggested that the urinary excretion of unconjugated bilirubin (UB) comprises only a small fraction of the pigment that reaches the tubular lumen by glomerular filtration and escapes from tubular cell reabsorption. However, additional data also indicated that UB interacts with renal peritubular cell membranes impairing the secretion of p-aminohippurate (PAH). In this study we examined the possibility of a secretory step which could also be involved in the renal excretory mechanism for UB. An isolated rat kidney preparation was used, and the uptake of UB by renal tissue, the UB appearance in the urine, and the secretion of PAH were analyzed throughout the perfusion. The results indicated that the UB urinary excretion rate changed independently of UB filtered load. The latter remained almost unchanged during the perfusion, whereas the excretion rate of UB and the UB-to-creatinine (Cr) clearance ratio increased significantly. Furthermore, a relationship between the uptake of UB by the kidney, the UB-to-Cr clearance ratio, and the decrease in PAH secretion rate, was proved. In addition, when probenecid was added to the perfusate solution the cumulative uptake of UB by the kidney and the rate of excretion of UB in the urine were diminished. We conclude that the mechanism of UB excretion by the kidney may be considered as the result of a process involving glomerular filtration plus tubular secretion followed by a back diffusion step from the lumen in a similar way to other endogenous compounds, thus explaining the virtual absence of UB from the normal urine.     

Renal response to intravenous phosphate infusion in the sheep.

Renal clearance experiments were performed on six Merino ewes in which plasma phosphate concentrations were increased by the intravenous infusion of isohydric sodium phosphate. As the phosphate load to the kidney increased, the renal tubular reabsorptive capacity became saturated and a definite tubular maximum for phosphate reabsorption (Tmp) was demonstrated. The Tmp was directly related to the glomerular filtration rate and had a mean value of 333-1+/-27-0 (s.e.m.) mumol/min or 416-6+/-13-5 mumol/100 ml glomerular filtrate. Calcium infused concurrently with phosphate in order to maintain plasma total calcium levels did not alter the Tmp. Ultrafilterability of calcium and phosphate in the plasma decreased with phosphate infusion and this was accentuated by an accompanying calcium infusion. The Tmp in sheep's kidney is higher than in non-ruminant animals and the implications of this are discussed.     

Abnormal tubular permeability in hypothermic perfused kidneys.

Isolated canine kidneys perfused with cryoprecipitated plasma at 15 degrees C exhibit unexpectedly low inulin clearance (CIn) and creatinine clearance (CCr) rates. CIn and CCr, as well as p-aminohoppurate (PAH) clearance, varied linearly with urine flow rate, whether the variations in urine flow were spontaneous or induced, either by elevating perfusion pressure or by adding mannitol to the perfusate. Retrograde intraureteral injection (RII) of an isotonic fluid containing dextran, inulin, and PAH, followed by a period of ureteral occlusion and subsequent serial recollection of the injected fluid, revealed that inulin and PAH, relative to dextran, were lost from distal tubular fluid. Similar experiments in anesthetized dogs indicated no loss of inulin or PAH from tubules of in situ kidneys. Renal venous perfusate, collected from isolated kidneys during the low pressure phase of the RII, contained the following percentages of the quantities injected intraluminally: dextran, 9.22%; inulin, 11.0%; and PAH, 22.0%. These data indicate that a low measured glomerular filtration rate in hypothermic perfused kidneys is partly due to diffusion of inulin or creatinine out of the tubular lumen.     

Gentamicin nephrotoxicity. II. Physiological, biochemical and morphological effects of prolonged administration to rats.

The purpose of this investigation was to determine the morphological, physiological and biochemical effects of gentamicin upon the rat kidney following prolonged administration of the antibiotic. Sprague-Dawley and Fischer 344 strain rats were given 3, 10, 20 or 40 mg gentamicin per kg body weight per day for 28 days. Morphologic alterations were evaluated by light and electron microscopy. Functional parameters included glomerular filtration rate, PAH secretion, renal plasma flow, sodium reabsorption, potassium excretion, urine volume and protein, and serum urea nitrogen. Oxidative metabolism of mitochondrial fractions from renal cortical homogenates was evaluated by oxygen uptake and P:O ratios. The results indicate focal proximal tubular injury, decreased tubular maximum secretion of PAH, and altered oxidative metabolism at the higher dose levels of gentamicin. Neither elevations of serum urea nitrogen nor alterations in glomerular filtration rate, renal plasma flow, or sodium or potassium excretion were observed. Thus, it appears that high dose levels (40 mg per kg per day) alter the structure and function of some proximal tubular segments when administered over prolonged periods. The alterations appear reversible. Although nephro-toxicity is identified under these conditions in rats, extrapolation to human patients usually receiving much lower doses must be guarded.     

Renal Excretion of Digoxin in Swine and Goats

In experiments on swine and goats the renal excretion of digoxin was examined, and it was found that the renal clearance of non-protein-bound digoxin in swine was lower than creatinine clearance which expresses filtration clearance. Correlation analysis showed that the renal clearance of digoxin in swine was not significantly influenced by the concentration of non-protein-bound digoxin in plasma and the pH of the urine, while there was a significant positive correlation between the clearance and the urine flow rate (Table 4). On the other hand, the renal clearance of digoxin in goats was significantly influenced by the concentration of non-proteinbound digoxin in plasma and by urine pH (Table 4). From these results it is concluded that glomerular filtration and back-diffusion are involved in the renal handling of digoxin in both swine and goats. In addition active tubular secretion is also involved in the renal excretion of digoxin in goats.     

Antidiuretic action of prolactin in the rat with diabetes insipidus.

Rats with hereditary hypothalamic diabetes insipidus, devoid of endogenous ADH, exhibited a prompt antidiuresis when injected subcutaneously or intraarterially with ovine prolactin. The antidiuresis was accompanied by a decrease in free water clearance and an increase in urine osmolality without a change in osmolal clearance or creatinine excretion. Measurement of PAH and insulin clearances indicated that prolactin had no effect on renal plasma flow or glomerular filtration rate. Prolactin injection caused a transient decrease in urinary sodium excretion, but proximal tubular sodium reabsorption, estimated by lissamine green transit time, was unaffected. The antidiuretic effect of prolactin could not be attributed to ADH contamination of the ovine prolactin preparation. Kidney cyclic AMP content was increased significantly 5 min after injection of prolactin. Thus, prolactin has an antidiuretic effect similar to that which occurs as a result of ADH action on the kidney and does not require either the release or the presence of ADH in order to cause the antidiuresis. Further, the impaired water excretion cannot be attributed to an increase in proximal tubular sodium reabsorption or to alteration of renal hemodynamics. It is suggested that prolactin has a direct ADH-like action on the kidney resulting in antidiuresis.     

p-aminohippuric acid transport at renal apical membrane mediated by human inorganic phosphate transporter NPT1

Organic anions are secreted into urine via organic anion transporters across the renal basolateral and apical membranes. However, no apical membrane transporter for organic anions such as p-aminohippuric acid (PAH) has yet been identified. In the present study, we showed that human NPT1, which is present in renal apical membrane, mediates the transport of PAH. The K(m) value for PAH uptake was 2.66 mM and the uptake was chloride ion sensitive. These results are compatible with those reported for the classical organic anion transport system at the renal apical membrane. PAH transport was inhibited by various anionic compounds. Human NPT1 also accepted uric acid, benzylpenicillin, faropenem, and estradiol-17beta-glucuronide as substrates. Considering its chloride ion sensitivity, Npt1 is expected to function for secretion of PAH from renal proximal tubular cells. This is the first molecular demonstration of an organic anion transport function for PAH at the renal apical membrane.     

Renal excretion of chlorphenol red and related organic acids in the intact flounder Pseudopleuronectes americanus

Renal clearance experiments were performed on unanesthetized winter flounder from which bladder urine was collected continuously and caudal vein blood was sampled periodically; renal tissue was also obtained terminally for comparison of test organic acid content in vivo and after incubation in vitro. Urine flow rates and inulin U/P (urine to plasma concentration) ratios were relatively constant for a given fish and averaged 1.0 ml/hr X kg fish and 2.6, respectively. In contrast, U/P ratios for all three test acids cycled from minima of near 100 to maxima of over 1000 roughly every 24 hr when plasma concentrations of unbound acid were below 1 micron; correction of plasma protein binding was required in the case of chlorphenol red, but not PAH or Diodrast. Both in vivo and in vitro the organic acid content of renal tissue was intermediate between plasma and urine concentrations. These results demonstrate that kidneys of intact flounder exhibit the remarkable concentrative capacity for exogenous organic acids previously observed with isolated tubules and suggest that the tubular urine concentration is established in two steps by cell transport first at the peritubular and second at the luminal membrane. The anterior kidney position and the magnitude of maximal PAH and Diodrast clearances, about 1000 ml/hr X kg flounder, are consistent with most of the cardiac output returning to the heart through the renal portal circulation; a regulatory shunt bypassing the peritubular capillaries is proposed to explain cycling of organic acid clearances to minimal values.     

Exogenous fluorescent tracer agents based on pegylated pyrazine dyes for real-time point-of-care measurement of glomerular filtration rate

Novel pyrazine carboxamides bearing hydrophilic poly(ethylene glycol) (PEG) moieties were designed, synthesized, and evaluated for use as fluorescent glomerular filtration rate (GFR) tracer agents. Among these, compounds 4d and 5c that contain about 48 ethylene oxide units in the PEG chain exhibited the most favorable physicochemical and renal clearance properties. In vitro studies show that these two compounds have low plasma protein binding, a necessary condition for renal excretion. In vivo animal model results show that 4d and 5c have a higher urine recovery of the injected dose than iothalamate (a commonly considered gold standard GFR agent). Pharmacokinetic studies show that these two compounds exhibit a plasma clearance equivalent to iothalamate, but with a faster (i.e. lower) terminal half-life than iothalamate (possibly from restricted distribution into the extracellular space due to large molecular size and hydrodynamic volume). Furthermore, the plasma clearance of 4d and 5c remained unchanged upon blockage of the tubular secretion pathway with probenecid, a necessary condition for establishment of clearance via glomerular filtration exclusively. Finally, noninvasive real-time monitoring of this class of compounds was demonstrated by pharmacokinetic clearance of 5c by optical measurements in rat model, which correlates strongly with plasma concentration of the tracer. Hence, 4d and 5c are promising candidates for translation to the clinic as exogenous fluorescent tracer agents in real-time point-of-care monitoring of GFR.     

The effect of renal arterial infusion of albumin and dextran on tubular fluid reabsorption in the dog.

Albumin or Dextran solutions of varying concentration were infused into the renal artery of hydropenic dogs. Their effect on urine flow, sodium excretion, creatinine and PAH clearance, single nephron GFR, fractional and absolute fluid reabsorption in the proximal convolution, reabsorptive t1/2, and hydrostatic pressures in the proximal tubules and adjacent capillaries was compared with a similar infusion of isotonic saline solution. Six, 9, 12, 18 and 25% albumin and 6% Dextran solution did not significantly change the measured parameters. Infusion of 9 and 12% Dextran solution elicited a decrease in water and sodium excretion as well as absolute and fractional proximal tubular fluid reabsorption to a 5% level of significance. Infusion of 18% Dextran was accompanied by a marked decrease in total and proximal reabsorption combined with a decline of GFR, PAH clearance, and hydrostatic pressures in tubules and peritubular capillaries. The results do not support the hypothesis of a direct action of oncotic pressure on tubular fluid reabsorption; the above described effects of Dextran seem to be accounted for by its other "pharmacological" effect.     

Renal effects of SK&F 82526 in anesthetized rats

Dopamine affects renal hemodynamics, renal tubular functions, and the secretion of renin. We have studied the renal effects of SK&F 82526 (an agonist which is selective for the DA1 subclass of dopamine receptors) in anesthetized rats. Infused intravenously at 0.005 mumol/min/kg, this drug increased renal plasma flow and the clearances of PAH and insulin, effects which are consistent with decreased renovascular resistance. Concomitantly, urine flow and K excretion increased, and Na excretion tended to increase. All these effects of SK&F 82526 were antagonized by intravenous metoclopramide (1 mumol/min/kg). Despite its diuretic effect and despite its lack of effect on arterial blood pressure, SK&F 82526 increased arterial plasma renin concentration, suggesting a stimulatory effect on renin secretory rate. Taken together, our results demonstrate that the renal effects of SK&F 82526 mimic those of dopamine.     

Compensation of renal drug excretion after unilateral nephrectomy

Twenty hours after unilateral nephrectomy (uNX) the PAH excretion of uninephrectomized rats reaches about 80% of the controls. Immediately after removal of one kidney the parenchyma loss can be compensated by an intensification of glomerular filtration. Thereafter the active tubular secretion capacity raises. 24 h after uNX, a significant increase of renal mass could be measured. The specific PAH accumulation capacity per 1 g renal cortical tissue increases significantly 96 h after uNX if the animals had been pretreated with cyclopenthiazide before the operation. Administration of azauracil or fluoruracil or neomycin causes a dose-dependent reduction of PAH elimination in sham operated as well as in uNX-rats. The effect of stimulation by cyclopenthiazide, also occurring after uNX could be reduced significantly by the inhibitors. The relative extent of compensation (80 +/- 10%) was not influenced by the inhibitors of protein synthesis. The compensation after uNX and the stimulation of renal tubular function are mediated by different mechanisms.     

Dose-dependent elimination of 5-fluoro-2'-deoxyuridine in the monkey

The kinetics of 5-fluoro-2'-deoxyuridine (FdUrd) and 5-fluorouracil (FUra) disposition after bolus intravenous injection were determined in anesthetized rhesus and cynomolgus monkeys. FdUrd disappearance from plasma was an apparent triexponential process with average half-lives of 0.5, 2, and 8 min; FUra disappearance was biphasic with average half-lives of 2 and 13 min. After FdUrd injection, FUra reached peak plasma concentrations of 15-30% of the initial FdUrd concentrations within 3 min, and then disappeared more slowly than FdUrd. Total FdUrd clearance fell from 105 to 73 to 56 ml/kg/min as the dose increased from 10 to 20 to 40 mg/kg. Metabolic clearance was about 85% of total clearance and fell similarly with increasing dosage. Total and metabolic FUra clearances were about 30% of FdUrd values at an equimolar dose. Renal FdUrd clearance exceeded glomerular filtration rate and was decreased by probenecid, indicating tubular secretion; renal FUra clearance was close to glomerular filtration rate. There was no apparent correlation between dose and renal clearance or volume of distribution. It was concluded that FdUrd, like FUra, is eliminated primarily by a dose-dependent process. The metabolic basis of the dose-dependent kinetics remains to be determined.     

Long-term follow-up of the tubular secretion of creatinine in renal graft recipients.

The differences in glomerular filtration rate (GFR) based on creatinine clearance (Ccr) or obtained by the more exact methods are caused mainly by tubular creatinine secretion. In this study, we monitored creatinine clearance (Ccr), GFR on the basis of polyfructosan renal clearance (C(PF)) and parameters characterizing tubular creatinine secretion (Ccr/C(PF), Ccr - C(PF), Tcr/C(PF) x 100) in 12 individuals with renal grafts (Group A), 12 kidney graft donors for related transplantation (Group B), and in 27 individuals undergoing nephrectomy for a pathological process in one kidney (Group C). In the monitored groups, C(PF) and Ccr values were within the limits consistent with the normal function of a single kidney in a healthy individual. The values characterizing tubular creatinine secretion in Group A did not differ significantly from those obtained in Groups B and C. However, the parameters showed a wide range in all groups. In seven individuals with a renal graft, all the above functional parameters were monitored at three-month intervals for a period of 24 months. Significant differences in the time courses of Ccr and C(PF) due to marked intra-individual fluctuations were found in tubular creatinine secretion. The findings suggest that the rate of tubular creatinine secretion in the renal graft does not differ significantly from that in individuals with a single native (normally functioning) kidney. However, there are large inter-individual differences. The large intra-individual fluctuations in tubular creatinine secretion in the kidney graft result in significant differences in the time courses of Ccr and C(PF) and a possibility of erroneous evaluation of graft function if based exclusively on Ccr.     

Dietary protein-induced changes in excretory function: a general animal design feature

Mammals are ureotelic and respond to an increased protein intake with an increase in glomerular filtration rate and renal plasma flow. Birds and terrestrial insects are uricotelic and following a high protein intake increase tubular urate secretion by the kidney (birds) or Malpighian tubule (insects). Ureogenic fish given NH₄Cl increase gill and renal clearance of urea and gill clearance of ammonia. Renal mass increases in mammals, birds and reptiles given a high protein intake. Thus, animals in general respond to an increase in protein intake with a change in excretory function such as to increase the clearance of the major nitrogenous end-products of protein metabolism. The components of this general animal excretory response include; a redistribution of regional perfusion with increased renal and gill blood flow, increased GFR and gill ammonia clearance, increased renal tubular urate clearance, changes in urea transport protein abundance and/or function and renal hypertrophy. Animal groups differ as to which components are accentuated. Amino acid catabolism with generation of ammonia appears to be a necessary prerequisite for this excretory response to occur. A hypothesis is put forward that ammonia itself is a regulatory molecule and an important signal communicating between amino acid catabolism following an increase in protein intake and the sequence of events leading to a change in excretory function.     

Effects of penicillin pretreatment on renal tubular para-aminohippurate transport in the immature rat.

The effect of pretreatment with penicillin on para-aminohippurate (PAH) transport by the kidney of the immature rat was evaluated in vivo. After 3 days of penicillin administration, renal clearances of inulin (CIN), PAH (CPAH), and the renal tubular transport maximum (Tm) for PAH were measured in rats as young as 17 days of age. The CPAH in 19- to 21-day-old rats pretreated with procaine penicillin was 54% greater than that of their littermate controls. Similarly, CPAH of rats that received sodium penicillin was 31% greater than control. CIN was not increased after penicillin pretreatment. Pretreatment of rats older than 24 days did not change CIN or CPAH. The Tm for PAH of 17-day-old rats pretreated with sodium penicillin was 51% greater than that of control rats. It was concluded that pretreatment with penicillin enhances the renal secretion of organic anions by the immature rat.     

Fractional reabsorption of calcium,magnesium and phosphate in the kidneys of freshwater North American eels (Anguilla rostrata LeSueur) following removal of the corpuscles of Stannius

Renal function was observed in freshwater North American eels (Anguilla rostrata LeSueur) 2 weeks after the removal of the corpuscles of Stannius. There was a positive linear correlation between glomerular filtration rates and urine flow rates in both sham-operated and stanniectomized eels but there was no difference in slope or elevation between the two groups nor did urine flow rates ever exceed glomerular filtration rates. Osmolar clearance and free-water clearance were unchanged following stanniectomy. Plasma Ca²⁺ and K⁺ concentrations increased and plasma Mg²⁺, phosphate, Na⁺ and Cl^- concentrations decreased following stanniectomy. Plasma ultrafilterable Ca increased and ultrafilterable Mg decreased after stanniectomy but neither changed relative to its total plasma concentration. Stanniectomy was followed by a decreased renal tubular reabsorption of Mg²⁺ relative to the amount filtered (C _Mg/C _In); the same applies to C _Na/C _In. Even though the filtered load of Ca increased in conjuction with the predictable hypercalcemia, there was no change in the fraction of filtered Ca reabsorbed. Net tubular secretion of phosphate was observed in both sham-operated and stanniectomized cels together with a slight increase in C _phos/C _In following stanniectomy. Some or all of these changes in plasma electrolytes and/or the modified renal transport of Na⁺, Mg²⁺ and possibly phosphate may be caused by the changes in cardiovascular function that were recently shown to follow stanniectomy.Abbreviations CSX Stanniectomized/stanniectomy - ER endoplasmatic reticulum - FW fresh water - GFR glomerular filtration rate - Pi inorganic phosphate - RAS renin-angiotensin system - SHM sham-operated - SW sea water - UFR urine flow rate - U/P ratio urine/plasma ratio     

Developmental expression of renal organic anion transporters in rat kidney and its effect on renal secretion of phenolsulfonphthalein

Organic anion transporters (OAT1 and OAT3) and multidrug resistance-associated proteins (MRP2 and MRP4) play important roles in anionic drug secretion in renal proximal tubules. Changes in the expression of such transporters are considered to affect the tubular secretion of anionic drugs. The purpose of this study was to elucidate the developmental changes in the expression of OAT1, OAT3, MRP2, and MRP4 and their effects on the tubular secretion of drugs. The mRNA level of each transporter was measured by real-time PCR, and the protein expression was evaluated by Western blotting and immunohistochemical analysis. In addition, the tubular secretion of phenolsulfonphthalein (PSP) in infant (postnatal day 14) and adult rats was estimated based on in vivo clearance study. The protein expression of organic anion transporters were very low at postnatal day 0 and gradually increased with age. In postnatal day 14 rats, the expression of OAT1 and OAT3 seemed to be at almost mature levels, while MRP2 and MRP4 seemed to be at immature levels. Immunohistochemical analysis in the kidney of postnatal day 0 rats revealed OATs on the basolateral membrane and MRPs on the brush-border membrane. At postnatal day 0, the distribution of these transporters was restricted to the inner cortical region, while after postnatal day 14, it was identical to that in adult kidney. An in vivo clearance study revealed that the tubular secretion of PSP was significantly lower in postnatal day 14 rats than adult rats. These results indicate that age-dependent changes in organic anion transporter expression affect the tubular secretion of anionic drugs in pediatric patients.     

Plasma levels and renal handling of endogenous amino acids in snakes: a comparative study

Plasma levels of 22 endogenous amino acids were measured by ion-exchange chromatography in four species of snakes: Thamnophis sirtalis, T. radix, Aipysurus laevis, and Python molurus. Despite considerable interspecific variation in the amino acid composition, all species showed relatively high plasma concentrations of histidine, a feature apparently unique to reptiles. The renal handling of these amino acids was studied by renal clearance methods. As in other vertebrates, net tubular absorption of filtered amino acids predominated. However, net tubular secretion of taurine, cysteic acid and/or phosphoserine and beta-alanine was observed, with taurine being the predominant amino acid secreted. The percentage reabsorption of the total amino acids filtered by the snake kidneys ranged from 79 to 95%. Evidence for the postrenal absorption of amino acids in these reptiles is presented. In species that normally undergo hibernation (Thamnophis spp.), the ability of the kidney to reabsorb amino acids was depressed by cold acclimation. Cold acclimation significantly decreased plasma levels of all amino acids except taurine, whose concentration increased. The increase in plasma taurine level may have resulted from cellular osmoregulation. Under these conditions, renal excretion of taurine increased concomitantly with the increase in plasma taurine concentration.