Accumulation of p-aminohippuric acid and cyclopenthiazide in renal cortex slices from rats of various ages and their dependence on the energy supply]

The accumulation of p-aminohippuric acid (PAH) and cyclopenthiazide, two drugs of acidic character and extremely different physico-chemical properties, was determined in renal cortical slices of rats aged 5, 15, 33, 55, 105, and 240 days. PAH is accumulated in the cortical slices of 5- and 15-day-old animals to a lesser extent than in those of adult rats. Cyclopenthiazide is accumulated in much higher amounts than PAH in all age groups. The age dependence of cyclopenthiazide accumulation is not so pronounced as with PAH accumulation. There is no accumulation of PAH in the cortical slices of all age groups through active tubular transport, when the energy supply is inhibited by means of 2,4-dinitrophenol (DNP) or nitrogen 2 times bubbling (i.e. anaerobic incubation). By subsequent addition of DNP to the incubation medium or subsequent N2 atmosphere bubbling, an already existing PAH accumulation may be completely nullified. Contrary to PAH accumulation, cyclopenthiazide accumulation can be neither prevented nor abolished by inhibiting the energy supply.     

Stimulation of renal excretion of p-aminohippuric acid by repeated administration of drugs in young and adult rats]

The velocity of renal excretion of p-aminohippuric acid (PAH) could be raised by repeated administration of phenol red, probenecid and penicillin, which are actively transported by the acid carrier into the urine like PAH. These drugs produced an effect in fairly lower doses than PAH. The renal excretion of PAH can be accelerated by repeated pretreatment with the lipid-soluble drugs sulfaclomide, sulfamethoxypyridazine and cyclopenthiazide. It could be demonstrated that the stimulation of renal excretion of PAH by repeated administration of the investigated compounds is less pronounced in young rats than in adult animals.     

Extent and duration of drug-induced stimulation of renal excretion of p-aminohippuric acid]

Repeated pretreatment with p-aminohippuric acid (PAH), probenecide, cyclopenthiazide, and phenobarbital stimulates the renal excretion of PAH. For an interval of at least 6 hrs following i.p. application pretreated rats excrete more PAH excretion than the controls. All the drugs studied were found to stimulate renal PAH excretion within a period of 3 hrs by 50-60% of the control value. The required duration of pretreatment varies with the substance used. With cyclopenthiazide, the excretion of PAH is demonstrable for 2-3 weeks. Phenobarbital has a brief stimulating action. Correlations of a binding of drugs to structures of their tubular cell and the length of the stimulating action are discussed.     

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.     

Influence of neomycin pretreatment on stimulated and maturative renal transport of p-aminohippurate (PAH)

Renal tubular transport of organic anions is immature at birth and can be stimulated in adult rats by repeated administration of xenobiotics. There is some evidence of an increased synthesis of carrier proteins in renal tubular cells following stimulation as well as during postnatal development of renal tubular transport processes. The effect of pretreatment with an inhibitor of protein biosynthesis (neomycin) on stimulated and maturative transport of p-aminohippurate (PAH) was measured. Neomycin has a dose dependent long acting and reversible effect on stimulated PAH transport and on postnatal maturation of PAH excretion, and increased protein biosynthesis is the likely common basic phenomenon of both processes.     

Determination of the glomerular filtration rate (GFR): methodological problems, age-dependence, consequences of various surgical interventions, and the influence of different drugs and toxic substances

Determinations of renal clearance of fluorescein isothiocyanate (FITC)-inulin were used for assessing the glomerular filtration rate (GFR) in rats and to characterize factors influencing the glomerular filtration capacity. In anesthetized rats, GFR develops after birth up to day 30. Thereafter, GFR remains relatively constant for up to 3 months of age and drops continuously until the 8th month. GFR can be determined in utero, already one day before birth, however, only at a very low level. It increases significantly on the first day of life. Even at this time the effect of furosemide on GFR can be proven. After reduction of renal mass, GFR is decreased in dependence on the extent of kidney tissue removal. However, within 2 days after unilateral nephrectomy (NX) or one week after 5/6 NX, GFR reaches values about 3/4 of the controls with two intact kidneys. Furthermore, the compensation of GFR after renal ischemia reaches 80% of baseline values after one week. On the other hand, GFR is enhanced after bile duct ligation as a model of hepato-renal failure. It has been shown in previous experiments that pretreatment with hormones can stimulate renal tubular transport processes. Pretreatment with dexamethasone or triiodothyronine after 5/6 NX improves glomerular filtration capacity whereas in animals with ligated bile ducts dexamethasone seems to prevent the increase in GFR. After subchronic treatment with epidermal growth factor (EGF) GFR is significantly reduced. A continuous infusion of amino acids does not change GFR in the controls but enhances the filtration capacity in EGF-treated rats. But immediately after bolus injection of amino acids GFR also increases significantly in the controls. Diuretics such as furosemide, most nephrotoxic agents (cyclosporine A [CsA], heavy metals) and imidazole reduce the GFR significantly. Diltiazem reported to act nephroprotectively in CsA nephrotoxicity in human beings was without beneficial effect in rats. This could be due to species differences in GFR because the rat is one of the species with the highest glomerular filtration capacity.     

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.     

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.     

Succinate dehydrogenase activity in rat kidney after repeated administrations of p-aminohippurate or sodium chloride

During PAH excretion and 18 h after repeated PAH administrations to rats, renal cortical SDH activity was unchanged in comparison with untreated controls. On the other hand, 18 h after repeated administrations of 0.9% or 1.8% NaCl solution, SDH activity was decreased by about 20% in kidney cortex. In outer medulla SDH activity was decreased 18 h after all pretreatments. The decline of SDH activity was observed whenever an increased urinary Na excretion occurred, except that PAH was repeatedly administered. The probability is discussed that substrate-induced stimulation of the carrier system for weak organic acids is accompanied by an increase of mitochondrial activity in kidney cortex.     

Kidney extract reversibly inhibits compensatory cell proliferation after unilateral nephrectomy

Unilateral nephrectomy (uNX) in mice is followed by a transitory increase in cell proliferation in the remaining kidney. To examine whether this response could be related to a negative feedback control of kidney epithelial cell renewal, water extracts were made of kidney homogenate. Five mg freeze-dried extract was injected 18 h post-operatively, and the animals were sacrificed at intervals during the following 54 h. The mitotic rate and the incorporation of tritiated thymidine (3H-TdR) into DNA were measured in the remaining kidney. The results show that the kidney extract reduces both the mitotic rate and the incorporation of 3H-TdR into DNA. In the tubular epithelium in the kidney, the strongest inhibitory effect was found by injecting the extract at 18 or 39 h postoperatively.     

Inhibitors of poly (ADP-ribose) synthetase reduce renal ischemia-reperfusion injury in the anesthetized rat in vivo.

The activation of poly (ADP-ribose) synthetase (PARS) subsequent to DNA damage caused by reactive oxygen or nitrogen species has been implicated in several pathophysiological conditions, including ischemia-reperfusion injury and shock. The aim of this study was to investigate whether PARS inhibitors could provide protection against renal ischemia-reperfusion injury in the rat in vivo. Male Wistar rats were subjected to 45 min bilateral clamping of the renal pedicles, followed by 6 h reperfusion (control animals). Animals were administered the PARS inhibitors 3-aminobenzamide, 1, 5-dihydroxyisoquinoline, or nicotinamide during the reperfusion period. Ischemia, followed by reperfusion, produced significant increases in plasma concentrations of urea, creatinine, and fractional excretion of Na(+) (FE(Na)) and produced a significant reduction in glomerular filtration rate (GFR). However, administration of the PARS inhibitors significantly reduced urea and creatinine concentrations, suggesting improved renal function. The PARS inhibitors also significantly increased GFR and reduced FE(Na), suggesting the recovery of both glomerular and tubular function, respectively, with a more pronounced recovery of tubular function. In kidneys from control animals, histological examination revealed severe renal damage and immunohistochemical localization demonstrated PARS activation in the proximal tubule. Both renal damage and PARS activation were attenuated by administration of PARS inhibitors during reperfusion. Therefore, we propose that PARS activation contributes to renal reperfusion injury and that PARS inhibitors may be beneficial in renal disorders associated with oxidative stress-mediated injury.     

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.     

Early changes in renal function following chemically induced nephropathy

The functional changes in the rat kidney 24 h after administration of 2-bromoethanamine hydrobromide (BEA) have been extensively described. There is, however, little information regarding earlier alterations. The present study was designed to measure early changes in renal function in order to clarify further pathomechanisms of the BEA-induced lesion. Experiments were performed in two groups of Wistar rats with different infusion rates during the first 3 h following injection of 100 mg/kg BW BEA compared to sham-injected rats. Analysis included measuring urine flow, osmolality, urea, sodium and potassium as well as inulin and para-aminohippuric acid clearance. Our studies show a tubular as well as a glomerular involvement in BEA-induced nephropathy. A significantly higher urine flow occurred already in the first 30 min following injection of BEA. Urine osmolality began to decrease after 90 min, Na excretion was elevated at 3 h, K excretion was not significantly different from the control group, urea excretion was increased after 30 min. Contrary to other studies we found a continuously decreasing glomerular filtration rate and PAH clearance during the first 3 h. Our results suggest an early effect of BEA on tubular function (increasing sodium excretion), papillary concentration capacity (increasing urine flow combined with decreasing osmolality) and glomerular function (decreasing glomerular filtration rate).     

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.     

Allicin attenuates calcium oxalate crystal deposition in the rat kidney by regulating gap junction function

Renal calculus is a global common urological disease that is closely related to crystal adhesion and renal tubular epithelial cell impairment. Gap junctions (GJs) and their components (connexins and Cxs) are involved in various pathophysiology processes, but their roles in renal calculi progression are not well defined. Our previous RNA microarray analysis suggests that GJs are one of the key predicted pathways involved in the renal calcium oxalate (CaOx) crystal rat model. In the current study, we found that the Cx43 and Cx32 expression and the GJ function decreased significantly after stimulation with CaOx or sodium oxalate (NaOx) in NRK-52E, MDCK, and HK-2 cells, and Cx43 expression also decreased in renal tissues in renal CaOx crystal model rats. Inhibition of Cx43 in NRK-52E cells by small interference RNA significantly increased the CD44 and androgen receptor expression, and the adhesion between CaOx crystals and cells, which were consistent with the function of GJ inhibitors. On the other hand, after GJ function and Cx43 expression were increased by allicin, diallyl disulfide, or diallyl trisulfide, the impairment of NRK-52E cells by NaOx or other GJ inhibitors and the adhesion between CaOx crystals and renal cells decreased significantly. Furthermore, allicin also increased Cx43 expression and inhibited crystal deposition in rat kidneys. Taken together, our results provide a basis that GJs and Cx43 may participate in renal CaOx stone progression and that allicin, together with its analogues, could be potential drugs for renal calculus precaution.     

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.     

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.     

Connective tissue growth factor regulates the key events in tubular epithelial to myofibroblast transition in vitro

Connective tissue growth factor (CTGF) has been reported to play an important role in mediating the profibrotic effects of transforming growth factor-beta (TGF-beta) in various renal diseases. To elucidate the role of CTGF in renal tubular epithelial-myofibroblast transdifferentiation, we examined the expression of alpha-smooth muscle actin (alpha-SMA), vimentin, tenascin-C, and collagen IV expression upon the stimulation of CTGF in cultured human proximal tubular epithelial cell line (HKC), and further investigated the effects of endogenous CTGF blockade on the transdifferentiation process induced by TGF-beta. It is revealed that upon the stimulation of recombinant human CTGF (rhCTGF, 2.5 or 5.0 microg/L), the expression of alpha-SMA and tenascin-C mRNA increased significantly (p<0.01), while collagen IV gene expression decreased significantly (p<0.01), all in a dose-dependent manner. The percentage of alpha-SMA-positive cells was significantly larger in the rhCTGF-stimulated groups than that in negative control (38.9%, 65.5% vs. 2.4%, respectively, p<0.01) as confirmed by flow cytometry. Both cytoplasmic and secretory tenascin-C expression was upregulated by the stimulation of rhCTGF (p<0.01). Under this condition, collagen IV secreted into the culture media was lowered markedly (p<0.01). On RT-PCR analysis, TGF-beta1 upregulated CTGF gene expression, preceding that of alpha-SMA. The alpha-SMA mRNA expression induced by TGF-beta1 was significantly inhibited by CTGF antisense oligodeoxynucleotide (ODN) transfection (p<0.01). With prolonged incubation time, CTGF antisense ODN also inhibited intracellular alpha-SMA protein synthesis, as demonstrated by indirect immuno-fluorescence. So it is concluded that CTGF could promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblasts in vitro, both directly and as a downstream mediator of TGF-beta, and CTGF blockade would be a possible therapeutic target against tubulointerstitial fibrosis.     

Renal transport of organic acids and bases in genetically obese mice.

Accumulation of p-aminohippurate (PAH) and N-methylnicotinamide (NMN) by renal cortical slices was used to estimate transport capacity for organic anions and cations, respectively. In a previous study, renal organic anion transport appeared to be selectively depressed in animals rendered obese by high fat feeding. However, the effects of obesity could not be discretely separated from the effects of the 30% fat diet used to produce the obesity. Genetically obese hyperglycemic mice provided a model to determine the effect of obesity on renal transport systems without the complication due to diet. Accumulation of both PAH and NMN was depressed in renal cortical slices from genetically obese mice. Addition of plasma from thin or obese animals increased PAH accumulation by slices from thin animals. Accumulation by slices from obese animals was unaffected by addition of plasma. Oxygen consumption with acetate in the medium was less in kidneys from obese mice than kidneys from thin mice. Thus, in addition to inhibition of transport capacity, renal cortex of genetically obese mice has a biochemical defect that prevents response to stimulators. It is concluded that several renal functions are depressed in the genetically obese hyperglycemic mouse. Whether the depressed function results from the obesity or is concomitant with the gene for obesity is as yet undertermined.