Isolated perfused rat kidney as a tool in the investigation of renal handling and effects of nonsteroidal antiinflammatory drugs.

An isolated perfused rat kidney (IPK) preparation is described in which renal perfusion flow, perfusion pressure, urinary flow, urinary pH, and glomerular filtration rate (GFR) are recorded continuously during the perfusion experiment. The usefulness of this IPK system in studying the renal handling and the effects of non-steroidal antiinflammatory drugs (NSAIDs) is shown using salicyluric acid (SU), salicylic acid (SA), and naproxen (NA). Excretion of SU involves glomerular filtration, active secretion, and passive reabsorption. The excretion rates of SA and NA were both much lower than their filtration rate, indicating extensive reabsorption. All three drugs accumulate in the IPK but at different levels. SU accumulates much more than either SA or NA. The effects on renal function were different for the three drugs studied. SU had no effect on kidney function. SA perfusate concentrations greater than 100 micrograms/mL caused diuresis and natriuresis, while SA concentrations less than 100 micrograms/mL did not influence kidney function. NA perfusate concentrations ranging from 0.16 to 25 micrograms/mL caused a decrease in urinary flow and sodium excretion. Very high NA concentrations (greater than 500 micrograms/mL) caused an increase in urinary flow and sodium excretion. We conclude that the IPK is a suitable preparation for characterizing and comparing renal handling and effects of NSAIDs.

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.

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.

The specificity of nephritogenic antibodies. V. Glomerular localization of anti-GP 330 and anti-GP 90 antibodies present in passive Heymann serum.

Administration in the rat of rabbit antibodies directed against rat tubular brushborder antigens (FXIA) leads to membranous glomerulopathy (HICN) associated with glomerular immune complexes (ICXS). These anti-FXIA antibodies (Abs) contain two major specificities, anti-GP 330 and anti-GP 90. The contribution of each specificity to the localization of anti-FXIA Abs was studied by direct immunofluorescence. Perfusion studies, under conditions which avoid ischaemia, confirmed that in this system glomerular localization of anti-FXIA Abs depends only on anti-GP 90 Abs, and the failure of anti-GP 330 Abs to localize after perfusion could not be attributed to ischaemia. In contrast, intravenous (i.v.) injection of anti-GP 330 Abs results in granular deposits of rabbit IgG, similar to those observed using anti-FXIA Abs. Injection i.v. of anti-GP 90 Abs results only in (faint) linear deposits. Using alternating and combined perfusion studies with these Abs, it is shown that anti-GP 90 Abs do not influence localization of anti-GP 330 Abs. In addition, systemic administration of anti-GP 90 Abs combined with perfusion of anti-GP 330 Abs does not facilitate localization of anti-GP 330 Abs. Although directly after i.v. injection of anti-FXIA Abs some anti-GP 90 Abs probably localize in the glomerular capillary wall, our results exclude a dominant or ancillary role for anti-GP 90 Abs in the formation of glomerular Icxs in HICN.

Urinary excretion rates of calcium and magnesium in normal and complicated pregnancies.

In this cross-sectional study calcium and magnesium metabolism was investigated in normal pregnancies (n = 34) and pregnancies complicated by either fetal growth retardation of hypertension with or without fetal growth retardation (SGA newborns) (n = 30). Special attention has been given to the renal excretion rates of calcium and magnesium and their relationship to creatinine and sodium clearances. No differences were noted in the third trimester of pregnancy between the normal and complicated pregnancies in calcium or magnesium metabolism except for an increased serum magnesium in the SGA group. Comparing the post-partum period to normal pregnancy the following results were observed: (i) serum ionic calcium levels showed no differences; (ii) urinary calcium excretion was increased as a result of increased calcium clearance. A striking feature was the fact that the fractional calcium clearance was not increased, in contrast to the increase in relative calcium clearance. The observed results can be explained by an increased GFR and a possible dissociation between the sodium and calcium handling in the cortical thick ascending Limb of Henle's Loop.

Effects of chlorothiazide, furosemide and PTH on Na+ and Ca2+ handling in isolated perfused kidneys of the spontaneously hypertensive rat.

The role of the kidney in a disturbed calcium metabolism in spontaneously hypertensive rats (SHR) was investigated. The hemodynamics of isolated perfused SHR kidneys were not basically altered compared to Wistar-Kyoto (WKY) control kidneys. Renal calcium handling by isolated perfused WKY and SHR kidneys at 12 weeks of age was not significantly different. In addition, the effects of chlorothiazide and furosemide on renal calcium handling were studied in isolated perfused kidneys from both rat strains. Both diuretics increased glomerular filtration rate, diuresis and excretion of sodium and calcium. However, both diuretics stimulated diuresis and calcium excretion significantly less in SHR than in WKY kidneys. The calciuric action of chlorothiazide was completely abolished by administration of human-parathyroidhormone (hPTH), while the natriuric effect was unchanged by hPTH. This observation suggests that a hypocalciuric action of chlorothiazide 'in vivo' is possibly mediated by PTH. Our study suggests that the kidney is not responsible for the disturbance in calcium metabolism in SHR. A surprising finding is that the SHR kidney was less responsive to the diuretics furosemide and chlorothiazide than the kidney of the WKY control.

Volumetrical microcomputer-based ultrafiltration monitor for hemodialysis.

Uniform and controlled ultrafiltration during hemodialysis can decrease dialysis side effects. One of the prerequisites for this treatment is accurate measurement of ultrafiltration. The best method currently available for ultrafiltration measurement is based on the volumetrically measured change of dialysate flow over the dialyzer. We have developed an ultrafiltration monitor (UFM) for hemodialysis using two micro-oval flowmeters that measure the flow rate of the dialysate entering and leaving the dialyzer. Correction for intrinsic error was achieved with an Acorn microprocessor, in a calibration run without ultrafiltration and constant, equal flow through both flow transducers. The accuracy of the UFM in vitro was 99.4% of the mean total ultrafiltration, with a correlation coefficient of 0.998 (n = 8) with actual ultrafiltration. In vivo, in which UFM was compared with bed scale body weight monitoring, a correlation coefficient of 0.97 (n = 82) was obtained, and an accuracy of 90% of total ultrafiltration as measured by the change in body weight. Therefore, this UFM provides a cheap and reliable method for ultrafiltration measurement.

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.

Intracellular activities of chloride, potassium and sodium ions in rabbit corneal epithelium.

The mechanism of ion transport in the epithelium of rabbit cornea was studied by determining the intracellular ion activity of Cl-, Na+ and K+ under various conditions. Ionic activities were measured by means of microelectrodes containing liquid ion-exchangers selective for Cl-, Na+ or K+. The Cl- activity in basal cells of the epithelium in Na+ containing bathing solutions amounts to 28 +/- 2 mM (n = 11). This value is 1.9-times greater than expected on the basis of passive distribution across the tear side membrane. This finding suggests the existence of a Cl- accumulating process. Replacement of Na+ in the aqueous bathing solution by choline or tetraethylammonium results in a reversible decrease in Cl- activity to 22 +/- 1 mM (n = 11, P less than 0.025). The ratio of observed and predicted Cl- activity decreased significantly from 1.9 to 1.4 (P less than 0.05). The decrease in Cl- activity due to Na+ replacement was rather slow. In contrast, after readmittance of Na+ to the aqueous bathing solution, Cl- activity rose to a stable level within 30 min. These results indicate involvement of Na+ in Cl- accumulation into the basal cells of the epithelium. The K+ and Na+ activities of the basal cells of rabbit corneal epithelium in control bathing solutions were 75 +/- 4 mM (n = 13) and 24 +/- 3 mM (n = 12), respectively. The results can be summarized in the following model for Cl- transport across corneal epithelium. Cl- is accumulated in the basal cells across the aqueous side membrane, energized by a favourable Na+ gradient. Cl- will subsequently leak out across the tear side membranes. Na+ is extruded again across the aqueous side membrane of the epithelium by the (Na+ + K+)-ATPase.

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.

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.

Natriuresis and renal Na-K-ATPase activity in kidneys of normotensive and spontaneously hypertensive rats.

The isolated perfused kidneys of spontaneously hypertensive (SHR) rats aged 10-12 weeks (early hypertensive phase) exhibited pressure natriuresis curves already shifted to the right, as compared with kidneys of the normotensive Wistar rats and Wistar-Kyoto rats. Sodium excretion in relation to tubular sodium load was higher in kidneys of SHR rats as compared with those of the normotensive controls. Renal Na-K-ATPase activity was significantly lowered only in the cortex during chronic hypertension but not in the outer medulla. In both parts of the kidney no differences could be detected in the apparent Km value for sodium stimulation or the apparent activation energies. Mg-ATPase activity was lowered in SHR rats during chronic hypertension. Protein content was significantly decreased in the outer medulla of SHR rats in the early hypertensive phase as well as during chronic hypertension. The results suggest that the functional changes observed in kidneys of young SHR rats are not due to a genetically determined defect in renal Na-K-ATPase.

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.

Absence of effect of diazoxide on sodium and water handling in the isolated perfused rat kidney.

The effect of the thiazide derivative diazoxide on water and sodium handling of the isolated perfused rat kidney was studied. In kidneys of normotensive rats no influence on diuresis, glomerular filtration rate or sodium and potassium excretion rates could be detected whereas hydrochlorothiazide showed the anticipated effects. The diuretic effect of the latter compound was not inhibited nor potentiated by pretreatment with diazoxide. In kidneys of hypertensive rats, studied at different ages: 10, 20, 30, and 45 weeks respectively, no acute effects of diazoxide on tubular function were found either. Diazoxide and hydrochlorothiazide both resulted into a small drop of renal perfusion pressure indicating that renal vascular resistance was lowered. The results suggest that the water and sodium retention frequently observed in patients treated with diazoxide are not caused by a direct effect of the drug on tubular function but are mediated by its peripheral and/or intrarenal vascular effects.

Instantaneous measurement of glomerular filtration rate in the isolated perfused rat kidney.

In perfusion studies on the isolated cellfree perfused rat kidney, the glomerular filtration rate (GFR) can be controlled if the GFR can be measured instantaneously. This paper reports on a GFR meter which opens this possibility. Vitamin B12 is used as a marker for the GFR. A bubble flow meter determines urine flow rates ranging from 10 up to 200 microliter-min-1. A fiberoptic colorimeter measures vitamin B12 concentrations in the urine up to 400 mg-l-1. The flow meter and the colorimeter are described in detail. The reliability of vitamin B12 as a marker for the GFR is demonstrated and, moreover, the identity of GFR values obtained with the GFR meter and those from B12 spectrophotometry is demonstrated.