Saturable accumulation and diuretic activity of hydrochlorothiazide in the isolated perfused rat kidney.

The role of tubular accumulation in renal disposition and diuretic efficacy of hydrochlorothiazide was studied in the isolated perfused rat kidney. Hydrochlorothiazide resulted in a dose-dependent increase in the fractional excretion of sodium, chloride and potassium, and in urinary flow and pH. Renal clearance of the drug was low as a result of a low extraction ratio and extensive nonionic back-diffusion. Hydrochlorothiazide was subject to saturable tubular secretion, following Michaelis-Menten kinetics. Parameters obtained after nonlinear regression analysis were a maximum tubular transport velocity of 42 +/- 6 micrograms/min, a Michaelis-Menten constant of secretion of 38 +/- 11 micrograms/ml and a fraction of excreted drug reabsorbed passively of 0.49 +/- 0.03. The thiazide diuretic accumulated extensively in kidney tissue due to active cellular uptake (maximum capacity of renal accumulation of 500 +/- 270 micrograms/g; affinity constant of renal accumulation of 28 +/- 16 micrograms/ml) and passive diffusion. Plots were constructed of the sodium excretion rate versus hydrochlorothiazide perfusate concentration or the renal excretion rate. The perfusate plot could be described by the sigmoid Emax model, while a simplification of the model had to be used for the response curve in urine because a maximum effect was not observed. The apparent maximum effect resulting from the perfusate concentration-response curve and the discrepancy with the renal excretion rate-response curve indicates that the diuretic effect of hydrochlorothiazide is restricted by saturable accumulation and secretion.

Renal handling and effects of S(+)-ibuprofen and R(-)-ibuprofen in the rat isolated perfused kidney.

1. The renal handling and effects of S(+)- and R(-)-ibuprofen have been studied in the isolated perfused kidney (IPK) of the rat. 2. Both ibuprofen enantiomers were extensively reabsorbed and accumulated in the kidney in a concentration-dependent manner. No pharmacokinetic differences were observed between the two enantiomers. 3. S(+)-ibuprofen concentrations ranging from 0.25 to 25 micrograms ml-1 (1.2 to 120 microM) caused a decrease in urinary flow, glomerular filtration rate (GFR) and electrolyte excretion. Urinary pH and excretion of glucose were not influenced. R(-)-ibuprofen concentrations ranging from 2.5 to 25 micrograms ml-1 (12 to 120 microM) also decreased urinary flow and electrolyte excretion. This decrease, however, was less than observed with S(+)-ibuprofen. GFR, urinary pH and glucose excretion were not affected by R(-)-ibuprofen. Prostaglandin E2 (PGE2) concentrations of 133 ng ml-1 reversed the effects on renal function of both enantiomers. 4. Very high S(+)- and R(-)-ibuprofen concentrations (greater than 400 micrograms ml-1) resulted in an increase in urinary flow and fractional excretion of sodium, chloride, potassium, glucose and calcium. 5. It is concluded that the pharmacokinetic behaviour of ibuprofen in the kidney is not stereoselective. Relatively high concentrations of both enantiomers increased the urinary flow and electrolyte excretion in a nonstereoselective manner. Lower concentrations of S(+)-ibuprofen decreased urinary flow and electrolyte excretion. The pharmacologically inactive R(-)-ibuprofen was also able to affect renal function in a similar way, but at different concentrations. These effects on renal function are probably caused by inhibition of PGE2 synthesis.

Renal handling and effects of salicylic acid in the isolated perfused rat kidney.

The renal handling of salicylic acid (SA) and its effects on renal function were studied in the isolated perfused rat kidney (IPK). The renal handling of SA is dominated by reabsorption and only a small fraction of the filtered SA is excreted into the urine. Reabsorption is a passive process and is dependent on urinary pH. Because of the extensive reabsorption, no decrease in perfusate concentration can be observed in the course of the IPK experiment. SA accumulated slightly in the IPK and this accumulation is concentration dependent. Small amounts of SA were converted to salicyluric acid (SU), the glycine conjugate of SA. SA concentrations higher than 100 micrograms/ml caused an immediate increase in urinary flow and in fractional excretion of sodium, potassium, chloride and calcium. Fractional excretion of glucose increased gradually. Glomerular filtration rate, renal perfusion flow, renal pressure and fractional excretion of magnesium were not affected by SA. The effects were dependent on the SA concentration. Although SA is a classical non-steroidal antiinflammatory drug (NSAID), its influence on renal function appears to be different from other NSAIDs which are usually associated with a reduction in urinary flow and salt excretion.

Renal disposition and effects of naproxen and its l-enantiomer in the isolated perfused rat kidney.

Renal handling, metabolism and effects on kidney function of naproxen and its l-enantiomer were examined in the isolated perfused rat kidney (IPK). Urinary excretion rate of naproxen was much lower than the filtration rate, indicating extensive reabsorption. Naproxen is accumulated considerably in the IPK. This accumulation is concentration-dependent and is probably the result of active secretion of naproxen. Considerable amounts of desmethyl-naproxen were formed in the IPK. The kinetic behavior of the l-enantiomer of naproxen did not differ from naproxen. Addition of 37.5 to 3750 micrograms naproxen caused a decrease in urinary flow, glomerular filtration rate and fractional excretion of sodium, chloride, potassium, magnesium and calcium. The presence of prostaglandin E2 in the perfusate fully opposed the effects of naproxen on kidney function. Addition of 375 micrograms l-enantiomer of naproxen did not influence kidney function. Addition of very high doses (1 x 10(5) micrograms) of naproxen and its l-enantiomer to the IPK caused diuresis and increased the fractional excretion of sodium, chloride, potassium, glucose and calcium. We conclude that the pharmacokinetic behavior and the metabolism of naproxen in the IPK is probably not stereoselective; that relatively low doses of naproxen exert a specific, stereoselective effect on kidney function caused by inhibition of the prostaglandin E2 synthesis and that high doses of naproxen exert a nonstereoselective effect on kidney function.

Effects of potassium and calcium on magnesium and lithium handling in the isolated rat kidney.

Acute effects of potassium and calcium on renal lithium and magnesium handling were studied in the isolated rat kidney. Fractional and absolute lithium as well as magnesium transport varied with extracellular potassium in the range of 3.0-6.0 mM. The changes in lithium reabsorption, induced by extracellular potassium are linearly related to the variations simultaneously observed in magnesium reabsorption. Independently of extracellular potassium, lithium reabsorption was stimulated by low calcium comparable to magnesium reabsorption. Bumetanide (3.10(-6) M) lowered lithium and magnesium reabsorption. The results favor the concept that lithium can also be reabsorbed in the thick ascending limb of Henle by as much as 17% of the filtered load, under the conditions of the present experiments.

Renal handling of salicyluric acid in the isolated perfused rat kidney: evidence for accumulation in tubular cells.

The renal handling of salicyluric acid (SU) was studied over a broad concentration range (0-400 micrograms/ml) in the isolated perfused rat kidney (IPK). The accumulation of SU was determined by difference calculations between the SU dose given and the SU amount present in the perfusate and excreted into the urine. SU accumulates highly in the IPK and this accumulation is concentration-dependent. At low perfusate concentrations (5-20 micrograms/ml) there is a sharp increase in the accumulation (100-400 micrograms/g), whereas at higher concentrations (20-100 micrograms/ml) a small increase (400-500 micrograms/g) is seen. The largest part of the accumulation is probably caused by accumulation of SU in the tubular cells. This is a result of the active uptake of SU over the basolateral membrane followed by facilitated diffusion over the brush-border membrane. As a result of the saturation of the active SU uptake, accumulation reaches a maximal value. Excretion of SU in the IPK involves glomerular filtration, active secretion and reabsorption. Reabsorption is probably a passive process, dependent on the urinary flow and pH. The apparent Michaelis-Menten constant of the excretion is 18.7 +/- 1.8 micrograms/ml and the maximum transport capacity is 69.8 +/- 1.4 micrograms/min. The pharmacokinetic parameters of the excretion of SU were in good agreement with previously reported in vivo values.

Handling of human pepsinogens by the isolated rat kidney: evidence for a high glomerular sieving coefficient.

Pepsinogen A (PGA) and pepsinogen C (PGC) are low-molecular-weight proteins synthesized by the gastric mucosa. Data in man suggest that both pepsinogens are almost freely filtered through the glomerular basement membrane despite a molecular weight of about 43,000 dalton and a strongly negative charge. This promoted us to investigate the glomerular sieving of PGA and PGC in the isolated rat kidney model by measuring their fractional excretions before and after inhibition of tubular function with sodium iodoacetate. During the control episode fractional excretion of PGA was 40 +/- 0.04% and of PGC 42 +/- 0.04% (mean +/- SEM from 9 experiments). After complete inhibition of tubular function a large increase in fractional excretion was found for both pepsinogens: 87 +/- 0.04% for PGA and 95 +/- 0.09% for PGC. It is concluded that tubular secretion does not contribute to the high fractional excretion of pepsinogens and that both PGA and PGC are almost freely filtered through the glomerular basement membrane.

Influence of renal capsule on kidney function in hypertension.

Proximal tubular hydrostatic pressure (PT) and subcapsular pressure i.e. renal interstitial pressure (PS) were measured in isolated perfused kidneys of normotensive Wistar Kyoto rats (WKY) and of spontaneously hypertensive rats (SHR). At a renal perfusion pressure of 90 mmHg PT and PS were significantly lower in kidneys of SHR rats, while the fractional potassium and magnesium excretion rates were elevated. Superimposed venous occlusion of 15 mmHg increased PT as well as PS in both strains of rats but to the same level, while the increase in fractional electrolyte excretion (sodium, potassium, chloride, calcium and magnesium) was again much more pronounced in kidneys of the SHR rats. Decapsulation of the kidneys of the WKY strain lowered the PT values to that observed in SHR rats. Venous pressure elevation under these conditions yielded the same electrolyte excretion pattern in both types of kidneys. At comparable elevated PT and PS values in kidneys of the SHR rats the electrolyte excretion rats were much higher during venous occlusion than at increased perfusion pressure. The same experimental conditions in kidneys of WKY rats resulted in different values of PT and PS, whereas the electrolyte excretion rates were the same. The results strongly suggest that: 1) the absolute values of the intrarenal pressures are not correlated with electrolyte excretion pattern; 2) venous pressure elevation has a profound effect on electrolyte excretion rate only in SHR kidneys and in the decapsulated kidney of the WKY rat; 3) the capsular compliance in kidneys of SHR rats is higher.

Glomerular filtration in the isolated perfused kidney. I. Sieving of macromolecules.

The permeability of the glomerular capillary wall to neutral macromolecules was studied in isolated perfused rat kidneys. Pluronic F108 (BASF, Wyandotte, MI, USA), a polyoxyethylene-polyoxypropylene block copolymer of mol weight approximately 14,000, was used as plasma expander. Pore theory was applied to the fractional clearances of Pluronic F108 and dextran (mol weight 19,400) molecules measured both as a function of glomerular filtration rate. Using the pore model of Verniory et al. [30] the effective pore radius (60.9 A) and the ratio of total pore area and pore length (4.0 cm/nephron) were estimated, and a hydraulic permeability coefficient KF (0.036 nl/s . mm Hg) was calculated. There was no significant difference between the fractional clearance of Pluronic F108 obtained with different Pluronic F108 concentrations over the range 15-35 g/l, hence with largely differing osmotic pressures. It was concluded that the sieving properties of the glomerular membrane of the isolated perfused rat kidney are not detectably different from those in the intact rat, at least in the case of uncharged macromolecules.

Glomerular filtration in the isolated perfused kidney. II. Glomerular hemodynamics.

Glomerular hemodynamics were studied of isolated perfused kidneys of 12-wk-old normotensive (NR) and spontaneously hypertensive (SHR) rats, using Pluronic F108 (BASF, Wyandotte, MI, USA) as a plasma expander. Glomerular filtration rate (GFR), proximal tubular hydrostatic pressure (PT) and glomerular capillary hydrostatic pressure (PGC) were approximately linearly related with renal perfusion pressure. PGC measured directly by micropuncture was comparable to PGC calculated from other parameters of glomerular dynamics using pore theory. We conclude that GFR in isolated kidneys perfused with Pluronic F108 is lower than in vivo, mainly as a result of an increase in PT. This rise in tubular pressure is due to an increased urine flow rate and an elevated tubular fluid viscosity. The difference in glomerular dynamics between NR and SHR kidneys is the result of an increased preglomerular vascular resistance in SHR, possibly due to an adaptive hypertrophic reaction to a sustained hypertension.

Inhibition of calcium release of diazoxide studied in the isolated rat kidney.

Isolated kidneys of normotensive Wistar rats (NWR) and of genetically hypertensive Kyoto rats (SHR) were perfused. Contraction of intrarenal vascular smooth muscle cells were induced by barium ions (0.1 to 1.0 mmol) as well as by high potassium concentrations (40 mmol). It was shown that barium stimulates intracellular calcium stores to release activator calcium. Repeated stimulation with barium under calcium-free perfusion conditions resulted in depletion of these stores. In kidneys of SHR rats the tension development in response to barium was stronger as compared with NWR rats and the time needed to deplete the stores was prolonged. This suggests that smooth muscle cells of SHR rats contain more sequestered calcium. Diazoxide inhibited the barium contracture rapidly, reversibly and dose dependently. The inhibition was non-competitive. Percentual inhibition was equal in NWR and in SHR rat kidneys. Moreover, diazoxide did not block the calcium influx across the sarcolemma membrane. The magnitude of the potassium contractures depended entirely upon the extracellular calcium concentration. In the range of 0.1 to 1.0 mmol calcium, tension development varied linearly with log calcium concentration. Diazoxide also inhibited the potassium contractures, provided normally filled calcium stores were available. The combined results strongly suggest that diazoxide prevents intracellular calcium stores to release calcium and, furthermore, that this action is the basic mechanism of the vasodilating activity of diazoxide in smooth muscle cells of rat intrarenal vessels.

The contribution of a chloride shunt to the transmucosal potential of the rabbit submaxillary duct.

Chloride movement across the wall of the rabbit submaxillary duct has been studied. It was shown that the chloride diffusion from blood to luminal side was determined primarily by the existing transmucosal potential difference. From the fact that the ouabain-poisoned duct showed symmetrical behavior with respect to the chloride diffusion potentials in both directions and the fact that the measured chloride flux behaved as predicted according to the Goldman equation, it was suggested that a single barrier, rather than a series membrane system, determined the chloride movement. The permeability coefficients for chloride, in the order of 5.5 x 10(-5) cm sce-1 are much larger than would be expected for cell membranes. These findings in combination with the observations that mannitol permeability is higher during chloride perfusion than during sulfate perfusion and the observed electron-microscopic changes favor the concept of the existence of an extracellular route in chloride diffusion. An equivalent electrical circuit is given in order to evaluate the contribution of the chloride shunt more quantitatively. Calculations showed that the ductal resistivity during sulfate perfusion has a value in the order of 434 omega cm2, while during chloride perfusion this value is lowered to 48 omega cm2, indicating that the ductal wall can change from a tight to a leaky epithelium. The implications of these findings are discussed.