Hormonal regulation of postnatal development of renal tubular transport processes

Renal tubular transport of p-aminohippurate (PAH) is immature at birth. Repeated saturation of transport sites by treatment with various organic anions is without any influence on the postnatal development of kidney transport capacity. Hormonal regulation of postnatal maturation of PAH transport must therefore be taken into consideration. It was tried to stimulate immature PAH transport by treating rats of different ages with thyroid hormones, corticosteroids or testosterone, respectively. In rats with immature kidney function, renal PAH excretion can be stimulated by daily treatment with thyroid hormones. Experiments on renal cortical slices have shown that PAH excretion is preferentially stimulated by an increase of transport capacity. Whereas thyroid hormones stimulate the renal excretion of PAH both in young and in adult rats, dexamethasone treatment is more effective in rats with immature kidney function. Dexamethasone treatment is without any influence on PAH accumulation in renal cortical slices. Kidney weight and the protein content of kidney tissue was increased after dexamethasone treatment. Repeated testosterone administration did not stimulate the PAH transport in rats of different ages. The data have demonstrated the influence of thyroid hormones or of dexamethasone on renal tubular transport processes in rats with immature kidney function. Treatment with such hormones could be useful in the management of renal insufficiency in full-term and pre-term neonates with immature kidney function.     

Effect of stevioside on PAH transport by isolated perfused rabbit renal proximal tubule

Stevioside, a non-caloric sweetening agent, is used as a sugar substitute. An influence of stevioside on renal function has been suggested, but little is known about its effect on tubular function. Therefore, the present study was designed to explore the direct effect of stevioside on transepithelial transport of p-aminohippurate (PAH) in isolated S2 segments of rabbit proximal renal tubules using in vitro microperfusion. Addition of stevioside at a concentration of 0.45 mM to either the tubular lumen, bathing medium, or both at the same time had no effect on transepithelial transport of PAH. Similarly, a concentration of 0.70 mM (maximum solubility in the buffer) when present in the lumen, had no effect on PAH transport. However, this concentration in the bathing medium inhibited PAH transport significantly by about 25-35%. The inhibitory effect of stevioside was gradually abolished after it was removed from the bath. Addition of 0.70 mM stevioside to both lumen and bathing medium at the same time produced no added inhibitory effect. Stevioside at this concentration has no effect on Na+/K+-ATPase activity as well as cell ATP content. These findings suggest that stevioside, at a pharmacological concentration of 0.70 mM, inhibits transepithelial transport of PAH by interfering with the basolateral entry step, the rate-limiting step for transepithelial transport. The lack of effect of stevioside on transepithelial transport of PAH on the luminal side and its reversible inhibitory effect on the basolateral side indicate that stevioside does not permanently change PAH transport and should not harm renal tubular function at normal human intake levels.     

Effect of S-(2-chloroethyl)-DL-cysteine on the transport of p-aminohippurate ion in renal plasma membrane vesicles

The effects of S-(2-chloroethyl)-DL-cysteine (CEC) (a potent nephrotoxin) on the transport of p-aminohippurate ion (PAH) in renal plasma membrane vesicles isolated from rat renal cortex were studied in vitro. The uptake of PAH was significantly reduced in a dose-dependent manner in both the brush border membrane (BBM) and basolateral membrane (BLM) vesicles. These results demonstrate that CEC is capable of interfering with the accumulation of PAH (a model organic anion for renal tubular transport system) by both energy-independent and energy-dependent carrier-mediated transport processes. Probenecid, a typical inhibitor of the organic anion transport system, showed the highest inhibition of PAH uptake in both the membranes vesicles. These data indirectly suggest that transport by renal tubular cells may result in the accumulation of CEC in renal cellular organelles eventually in toxic concentrations. Thus, CEC showed both dose- and time-dependent inhibition of the activities of gamma-glutamyl transferase (a BBM marker enzyme) and Na+, K(+)-ATPase (a BLM marker enzyme), while no such inhibition was noticed with probenecid. Pretreatment with probenecid prevented the inhibition of the gamma-glutamyl transferase activity due to CEC in BBM, but failed to do so for the Na+,K(+)-ATPase activity in BLM vesicles. Thus, the data suggest that the inhibition of the activities of these membrane-specific enzymes by CEC could lead to the initial development of its nephrotoxicity.     

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.     

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.     

Sodium-sensitive, probenecid-insensitive p-aminohippuric acid uptake in cultured renal proximal tubule cells of the rabbit.

In the intact kidney, renal proximal tubule cells accumulate p-aminohippurate (PAH) via a basolateral, probenecid- and sodium-sensitive transport system. Primary cultures of rabbit proximal tubule cells retain sodium-glucose co-transport in culture, but little is known about PAH transport in this system. Purified proximal tubule cells from a rabbit were grown in culture and assessed for PAH and alpha-methyl-D-glucoside uptake capacities as well as proximal tubule marker enzyme activities. Control PAH uptake on collagen-coated filters (20 +/- 3 pmol/mg protein.min; n = 8) was not significantly different from uptake in the presence of 1 mM probenecid (19 +/- 4 pmol/mg protein.min; n = 8). Uptake from the basal side of the cell was 3.9 +/- 0.7 times greater than that from the apical side. In multi-well plate studies, the uptake was significantly reduced by removing sodium from the medium and stimulated by coating the wells with collagen. Glutarate (10 mM) had no effect on the uptake of PAH. Other differentiated proximal tubule characteristics were retained in culture, including the ability to form domes and to transport glucose by a phlorizin-sensitive system. Phlorizin-sensitive 1 mM alpha-methyl-D-glucoside uptake was 134 +/- 42 pmol/mg protein.min (n = 7; P less than 0.02). The proximal tubule marker enzymes alkaline phosphatase and gamma-glutamyltranspeptidase, increased in activity in the cultures after confluence. It was concluded that whereas some differentiated properties were retained during primary culture of rabbit proximal tubule cells, the PAH transport system was selectively lost or modified from that present in the intact kidney.     

Acceleration of p-aminohippurate excretion in immature rats by dexamethasone treatment

In 5-, 10- and 15-day-old rats repeated administration of dexamethasone caused a dose-dependent increase of p-aminohippurate (PAH) excretion. Remarkably, dexamethasone has no influence on PAH excretion in adult rats. The effect of treatment in young rats cannot be explained by an increase in the glomerular filtration rate. In 10-day-old rats GFR shows a tendency to increase. In renal cortical slices from 5-day-old rats PAH transport is increased following dexamethasone treatment, whereas in 10- and 15-day-old rats an increase of kidney mass seems to be responsible for acceleration of renal excretion of PAH after administration of dexamethasone. In 5-day-old rats only the protein content is statistically significantly increased after dexamethasone treatment.     

Stimulation of renal tubular transport by ethacrynic acid in rats of different ages.

The transport system for organic acids in the kidney is not fully developed in the neonatal period. The effect of repeated administrations of ethacrynic acid on the renal excretion of p-aminohippurate (PAH) was studied in rats of different ages. Pretreatment with ethacrynic acid was followed by an increase in the renal excretion of PAH in 33-, 55-, 105- and 240-day-old rats but not in newborn rats. In 55-day-old rats the increase in renal excretion of PAH after pretreatment with ethacrynic acid was not associated with any consistent change of the glomerular filtration rate. It is concluded from these results that the stimulation of transport processes in the kidney by ethacrynic acid and some other drugs is linked with their affinity to tissue proteins.     

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.     

Identification of a novel voltage-driven organic anion transporter present at apical membrane of renal proximal tubule

A novel transport protein with the properties of voltage-driven organic anion transport was isolated from pig kidney cortex by expression cloning in Xenopus laevis oocytes. A cDNA library was constructed from size-fractionated poly(A)+ RNA and screened for p-aminohippurate (PAH) transport in high potassium medium. A 1856-base pair cDNA encoding a 467-amino acid peptide designated as OATV1 (voltage-driven organic anion transporter 1) was isolated. The predicted amino acid sequence of OATV1 exhibited 60-65% identity to those of human, rat, rabbit, and mouse sodium-dependent phosphate cotransporter type 1 (NPT1), although OATV1 did not transport phosphate. The homology of this transporter to known members of the organic anion transporter family (OAT family) was about 25-30%. OATV1-mediated PAH transport was affected by the changes in membrane potential. The transport was Na+-independent and enhanced at high concentrations of extracellular potassium and low concentrations of extracellular chloride. Under the voltage clamp condition, extracellularly applied PAH induced outward currents in oocytes expressing OATV1. The current showed steep voltage dependence, consistent with the voltage-driven transport of PAH by OATV1. The PAH transport was inhibited by various organic anions but not by organic cations, indicating the multispecific nature of OATV1 for anionic compounds. This transport protein is localized at the apical membrane of renal proximal tubule, consistent with the proposed localization of a voltage-driven organic anion transporter. Therefore, it is proposed that OATV1 plays an important role to excrete drugs, xenobiotics, and their metabolites driven by membrane voltage through the apical membrane of the tubular epithelial cells into the urine.     

Effect of piperonyl butoxide on organic anion and cation transport in rabbit kidneys

Piperonyl butoxide has been shown to reduce accumulation of cephaloridine in rabbit renal cortex; however, the mechanism responsible for this effect remains unclear. Cephaloridine is a zwitterion and its accumulation in renal cortex has been suggested to be regulated by both organic anion and cation transport systems. Thus, it was of interest to determine the effect of piperonyl butoxide on renal transport of p-aminohippurate (PAH, an organic anion) and tetraethylammonium (TEA, an organic cation). Although pretreatment with piperonyl butoxide markedly inhibited renal cortical uptake of cephaloridine, the same treatment had less inhibitory effect on either PAH or TEA uptake. Efflux of PAH from preloaded renal cortical slices was enhanced by pretreatment with piperonyl butoxide; however, TEA efflux was unaffected. Thus, piperonyl butoxide appears to have effects on renal membrane functions which result in differential effects on PAH, TEA, and cephaloridine transport.     

Epidermal growth factor (EGF) increases the renal amino acid transport capacity in amino acid loaded rats

Summary In anaesthetized adult female rats, the influence of epidermal growth factor (EGF) on renal amino acid handling was investigated in glutamine, arginine (both 50 mg/100 g b. wt. per hour), or alanine (90 mg/ 100 g b. wt. per hour) loaded animals. Continuous infusions of the three amino acids were followed by an increase in the fractional excretion (FE) of the administered amino acids as well as of the other endogenous amino acids. Under load conditions (alanine, arginine or glutamine), EGF pretreatment (8g/100g b. wt. subcutaneously for 8 days, twice daily 8 a.m. and 4 p.m.) was followed by a stimulation of renal amino acid reabsorption. The increase in the fractional excretion of the administered amino acids was significantly lower than in non-EGF-treated rats. These changes in amino acid transport were connected with a significant reduction of GFR after EGF pretreatment (0.96 ± 0.10 vs. 0.62 ± 0.07 ml/min X 100 g b. wt.) and a distinct increase in sodium excretion (2.98 ± 0.55 vs. 4.97 ± 0.71val/100 g b. wt. X 20 min). After loading with p-aminohippurate (PAH; 200mg/100g b. wt.), PAH excretion in EGF rats was increased by about 20%, whereas urinary protein excretion was lower in EGF pretreated rats (control: 0.45 ± 0.04 vs. EGF: 0.18 ± 0.03 mg/ 100 g b. wt. X 20 min). The PAH load reduced amino acid reabsorption as a sign of overloading of renal tubular transport capacity, but in EGF pretreated animals the amino acid excretion was only slightly increased under these conditions. Furthermore, EGF pretreatment depressed normal kidney weight gain significantly (874 ± 18 vs. 775 ± 32mg/100g b. wt.). EGF can improve the renal tubular transport capacity, but, compared to well-known stimulators of renal transport like dexamethasone or tri-iodothyronine, its effect is only of a moderate degree.     

Phorbol esters inhibit phosphate uptake in opossum kidney cells: a model of proximal renal tubular cells

The effects of phorbol esters and diacylglycerol on phosphate uptake in opossum kidney (OK) cells were investigated to assess the possible role of Ca2+-activated, phospholipid dependent protein kinase (protein kinase C) on renal phosphate handling. OK cells are widely used as a model of proximal renal tubular cells and are reported to possess a Na+-dependent phosphate transport system. Phorbol-12,13-dibutyrate (PDBu) inhibited phosphate uptake. This inhibitory effect was synergistically enhanced with A23187. 4 beta-phorbol 12,13-didecanoate inhibited phosphate uptake, while 4 alpha-phorbol 12,13-didecanoate did not. 1-oleoyl-2-acetyl-glycerol (OAG), a synthetic diacylglycerol, also exhibited an inhibitory effect on phosphate uptake. These data suggest the possible involvement of protein kinase C in proximal renal tubular phosphate transport.     

Renal tubular transport of delta-aminolevulinic acid in rat

delta-Aminolevulinic acid (ALA) interferes with cell membrane and metabolic functions in a variety of tissues. To determine if ALA interacts with renal tubular transport functions, we examined concentrative transport of this heme precursor in rat kidneys. ALA was accumulated against a concentration gradient in rat renal cortical slices. Section freeze-dry autoradiography demonstrated selective accumulation in cells of proximal tubules. Concentrative uptake of ALA was inhibited by KCN, probenecid and p-aminohippurate (PAH). ALA inhibited slice uptake of PAH but failed to block slice accumulation of galactose, cycloleucine, lysine, glycine, proline, or alpha-aminoisobutyric acid and did not alter O2 utilization. Massive intraperitoneal injection of ALA did not increase 24 hr fractional excretion of amino acids in vivo. Concentrative transport of ALA in proximal tubules does not lead to generalized renal tubular transport defects but ALA appears to share the organic acid secretory system in rat kidney.     

p-Aminohippurate transport in basal-lateral membrane vesicles from rabbit renal cortex: stimulation by pH and sodium gradients

p-Aminohippuric acid (PAH) uptake was studied in basal-lateral membrane vesicles prepared from rabbit renal cortex. An outwardly directed hydroxyl gradient (pHo = 6.0, pHi = 7.6) stimulated PAH uptake slightly over that when the internal and external pH values were equal at 7.6. A 100 mM sodium gluconate gradient directed into the basal-lateral membrane vesicles increased PAH uptake about 2-fold over that when N-methyl-D-glucamine or potassium gluconate gradients were present. When hydroxyl and sodium gradients were simultaneously imposed (pHo = 6.0, pHi = 7.6 and 100 mM sodium gluconate extravesicularly) PAH uptake was stimulated greater than with the pH or Na+ gradient alone. In fact, an 'overshoot' was observed. Countertransport experiments showed that either intravesicular PAH or intravesicular PAH and Na+ could stimulate 3H-PAH uptake. Probenecid, an inhibitor of organic anion transport, inhibited both the hydroxyl-stimulated and Na+ gradient-stimulated PAH uptake but the greatest inhibition by probenecid was seen when the hydroxyl and sodium gradients were both present. Thus, it is proposed that the driving force for PAH accumulation across the basal-lateral membrane of the proximal tubule is a transport system which moves Na+ and PAH into the cell for an hydroxyl ion leaving the cell, i.e. a sodium-dependent anion-anion exchange system.     

Renal elimination of p-aminohippurate (PAH) in response to three days of biliary obstruction in the rat. The role of OAT1 and OAT3

Pharmacokinetic studies of the drugs administered to subjects with mechanical cholestasis are scarce. The purpose of the present study was to examine the effects of bile duct ligation of 3 days (peak of elevation of serum bile acids and bilirubin) on the systemic and renal PAH clearance and on the expression of cortical renal OAT1 and OAT3 in a rat model. PAH is the prototypical substrate of the renal organic anion transport system. Male Wistar rats underwent a bile duct ligation (BDL rats). Pair-fed sham-operated rats served as controls. BDL rats displayed a significantly lower systemic PAH clearance. Renal studies revealed a reduction in the renal clearance and in the excreted and secreted load of PAH in BDL rats. The OAT1 protein expression in kidney homogenates was not modified, but it decreased in the basolateral membranes from BDL rats. In contrast, OAT3 abundance in both kidney cortex homogenates and in basolateral membranes increased by 3 days after the ligation. Immunocytochemical studies (light microscopic and confocal immunofluorescence microscopic analyses) confirmed the changes in the renal expression of these transport proteins. The present study demonstrates the key role of OAT1 expression in the impaired elimination of PAH after 3 days of obstructive cholestasis.     

Stimulation of renal phosphate secretion in the stenohaline freshwater teleost: Carassius auratus gibelio Bloch

1. Renal excretion of phosphate in the Prussian carp was modulated by tubular reabsorption and tubular secretion. 2. Under the conditions of an i.v. phosphate-load during which the plasma concentration of phosphate was doubled, 65% of the phosphate load was excreted by the kidney, mainly by tubular secretion. 3. The renal clearance of PAH markedly exceeded the clearance of polyfructosan which could be used for the measurement of the GFR. 4. A transport maximum for tubular PAH secretion was reached at plasma concentrations of about 250 mumol/l.     

The effect of norethandrolone on organic ion transport by rat renal cortical slices.

Norethandrolone (NE) and other androgenic steroids have been shown to be renotropic in various species and have also been reported to have salutary effects in patients with diminished renal function. Renal cortical slices prepared from rats pretreated with NE showed an increased capability to concentrate p-aminohippuric acid (PAH). Pretreatment with NE failed to stimulate the transport of the organic base tetraethylammonium and the organic acid benzylpenicillin. Stimulation of PAH transport was observed after eight daily subcutaneous injections of NE. No stimulation was observed with shorter pretreatment intervals. When NE was given subcutaneously for 14 days at doses of 2.6 or 20 mg kg-1 day-1, significant stimulation of PAH transport was seen at all three dose levels but no dose-effect relationship was apparent. Stimulation of PAH transport was seen in female rats as well as castrated and intact males. In addition to its general anabolic properties, NE induces the synthesis of hepatic microsomal drug-metabolizing enzymes. For comparative purposes, therefore, the effect of pregnenolone-16 alpha-carbonitrile (PCN) was also investigated. This agent is a potent inducer of drug metabolism but is neither anabolic nor renotropic. When rats were pretreated with an inducing dose of PCN (75 mg kg-1 day-1 for 3 days), there was no significant stimulation of PAH transport. It would seem, then, that the stimulatory effect of NE on PAH transport is more closely associated with its generalized anabolic effect than with its ability to induce hepatic microsomal enzymes.     

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.     

Free amino acids mimic the anabolic but not the proliferative effect of albumin in OK proximal tubular cells

When plasma proteins leak from circulation into the renal tubular lumen in the proteinuric renal diseases, nephrotoxicity of filtered albumin (and/or molecules bound to it) may be important in the subsequent development of tubulo-interstitial damage which contributes to the progression of the disease. When cultured opossum kidney (OK) proximal tubular cells were exposed to bovine serum albumin for 3 days in vitro, increased cell division ([3H]-thymidine incorporation) and cellular hypertrophy (increased protein/DNA ratio) were observed. Both effects were halved if defatted albumin was used. A trivial explanation for the growth responses is that free fatty acids carried on the albumin, and amino acids generated by intracellular degradation of the albumin, are exerting a non-specific growth effect as metabolic fuels which are oxidized to generate ATP. However, the water-soluble free fatty acid octanoate (1 mmol l(-1)) had no significant effect on protein/DNA ratio and a very variable stimulatory effect on [3H]-thymidine incorporation, whereas an essential amino acid mixture or 1 mmol/l(-1) l-Ala or l-Phe only increased the protein/DNA ratio. Furthermore no carnitine was added to the culture medium. This absence would have impaired mitochondrial transport (and hence oxidation) of long-chain free fatty acids derived from the albumin. l-Phe is also a poor substrate for mitochondrial oxidation in kidney. It is therefore concluded that the growth effects of albumin in OK proximal tubular cells are specific effects of the albumin protein and of the free fatty acids and amino acids derived from it, and not a non-specific effect on metabolic fuel supply.