Simple HPLC-UV method for determination of iohexol, iothalamate, p-aminohippuric acid and n-acetyl-p-aminohippuric acid in human plasma and urine with ERPF, GFR and ERPF/GFR ratio determination using colorimetric analysis.

A simple high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of iohexol, iothalamate, p-aminohippuric acid (PAH) and n-acetyl-p-aminohippuric acid (n-acetyl-PAH) in human plasma and urine. A C(18) column at a flow rate of 1 ml/min with an aqueous mobile phase of trifluoroacetic acid (0.1% TFA in deionized water (pH 2.2), v/v) and methanol gradient was used for component separation. The plasma and urine assay demonstrated linearity from 10 to 50 microg/ml for iohexol and iothalamate, 5 to 40 microg/ml for PAH and 2.5 to 40 microg/ml for n-acetyl-PAH. The HPLC plasma and urine results obtained for PAH were used to calculate the subject kidney effective renal plasma flow (ERPF) and the iohexol results were used to calculate the subject kidney glomerular filtration rate (GFR). The HPLC results for PAH were then compared to an alternative colorimetric method for analyzing PAH to determine if subject metabolism (acetylation) of PAH affected the ERPF results obtained using the colorimetric method, the subsequent ERPF/GFR ratio and clinical impression of subject patient kidney function. The method was utilized in several different clinical studies evaluating the effect of kidney function from medications (phase IV evaluations) marketed for patients with cardiovascular disease.

Colorimetry of p-aminohippurate in the presence of sulfamethoxazole.

Colorimetric quantification of p-aminohippurate (PAH), which is helpful in assessment of renal function, is subject to interference from sulfa compounds such as sulfamethoxazole. We were unable to measure serum PAH in two patients receiving a trimethoprim-sulfamethoxazole combination because of interference by the latter with the colorimetric estimation. We found that isoamyl acetate removes sulfamethoxazole from the samples without influencing PAH. The extraction is simple, inexpensive, and reproducible.

Effect of digoxin on plasma clearance and anticoagulant effect of warfarin in rats.

The possible effects of digoxin on the elimination and anticoagulant action of warfarin were examined in rats. The pharmacokinetic parameters of warfarin after a single i.v. (1.2 mg/kg) or repeated oral coadministration with anticoagulant (0.6 mg/kg on day 1, thereafter 0.3 mg/kg) and digoxin (50 micrograms/kg) were not significantly different as compared with those in the group treated with warfarin alone. However, prothrombin complex activity (PCA) following coadministration with the diuretic was significantly and relatively rapidly recovered as compared with that in the warfarin group. The amounts of warfarin extracted by liver 2 and 6 h after a single i.v. dosing or 3 and 8 h after repeated oral dosing in the coadministered group were significantly decreased as compared with those in the group received warfarin alone. The renal function (renal plasma flow rate (RPF) and glomerular filtration rate) in the group coadministered with digoxin was significantly higher than that in the group receiving warfarin alone. On the other hand, the fraction of warfarin bound to BSA or rat plasma and the plasma water were little changed in the presence of digoxin. These results suggest that a pharmacological interaction, the decrease in the anticoagulant action, is induced between warfarin and digoxin coadministered.

Evidence for PAH extraction from superficial cortical efferent vessel plasma.

Consistent with its anatomical association with the proximal tubule we have previously shown that superficial cortical efferent vessel blood contains an admixture of early and late proximal tubular reabsorbate. Since tubular secretion of p-aminohippurate (PAH) occurs predominantly in the late proximal tubule, extraction of this compound should occur preferentially from efferent vessel blood. As a result, the midportion of the proximal convoluted tubule supplied by the more downstream peritubular capillaries would receive blood containing a disproportionately reduced concentration of PAH. To study this, proximal and distal tubular fluid and efferent vessel blood samples were collected from rats. The data confirm that preferential secretion of PAH occurs in the pars recta and demonstrate that PAH is extracted from efferent vessel plasma by the pars recta. This in turn preferentially reduces PAH concentration in early postglomerular blood before it reaches the peritubular capillary network. We speculate that PAH and similar substances secreted by the pars recta are short-circuited by rapid extraction from early postglomerular blood, reducing their delivery to the mid-proximal convoluted tubule. Such circumstances must be considered in any analysis of organic compound secretion by the in vivo proximal tubule.

In vitro and in vivo analysis of para-aminohippuric acid (PAH) transport: effect of indomethacin.

Effect of the prostaglandin synthesis inhibitor, indomethacin (indo) were studied on renal PAH transport (a) in renal cortical slices in vitro and (b) in anaesthetized dogs with various levels of plasma PAH concentrations (b1:PPAH less than 3 mg/dl and b2:PPAH V greater than 20 mg/dl] in vivo. It was found that: (a) indomethacin in concentrations of 0.4 and 4.0 micrograms/ml failed to influence PAH accumulation (S/M) and K or Na contents in renal cortical slices, whereas in a concentration of 40 micrograms/ml it potently decreased PAH accumulation (S/Mcontrol: 4.62 +/- 0.76, S/Mindo: 2.38 +/- 0.41; p less than 0.001). The oxygen uptake by tissue slices remained unaltered in the presence of indomethacin. (b1) Indomethacin in a dose of 4 mg/kg produced a pronounced antidiuresis and antinatriuresis with no change in GFR. PAH secretion was slightly reduced (TPAH control: 3.97 +/- 1.21; TPAH indo: 3.21 +/- 1.55 mg/min/100 g; p less than 0.01). (b2) Indomethacin appreciably decreased the secretory maximum of tubules (TmPAH control: 10.0 +/- 4.39; TmPAH indo: 4.95 +/-3.83 mg/min/100 g; p less than 0.001). Based on these results it is concluded that indomethacin, even when applied in the usual concentration, might partially block certain enzymes in the kidney.

Renal venous portal contribution to PAH and uric acid clearance in the chicken.

The contributions of the renal venous portal and the renal arterial circulations to the renal clearance of p-aminohippuric acid (PAH) and uric acid were determined in the unanesthetized chicken by the simultaneous use of the urinary clearance technique and the Sperber preparation. The [3H]PAH apparent tubular excretion fraction (ATEF) and the clearance of inulin (CIn) were used as indicators of the renal portal and renal arterial contributions, respectively, in a planar equation for total PAH clearance. This equation accurately reflects the renal clearance of PAH as well as the renal venous portal and renal arterial contributions to the total renal clearance of PAH. The equation suggests that under normal conditions approximately 50% of the renal clearance of PAH or uric acid comes from the renal venous portal circulation, with the remaining 50% coming from the arterial circulation. An inverse hyperbolic relationship between the filtration fraction (CIn/CPAH) and the [3H]PAH ATEF was found, demonstrating that the filtration fraction in the chicken decreases from a value of 18 to approximately 6% as the renal portal plasma flow increases from zero to a maximum value. Our equation for total clearance was also used with results of experiments on inhibition of uric acid excretory transport by L- and D-dopa and by probenecid to locate the probable sites of action of those inhibitors.

Effect of quinidine on the renal handling of digoxin.

The effect of left renal artery infusion of quinidine, 37.5 microgram (base)/kg/min on the renal clearance of ultrafilterable digoxin was studied in eight dogs given 0.06 mg/kg digoxin 24 hr previously. Digoxin was measured by 125I RIA, whose specificity was confirmed by HPLC. Within 15 min of the start of the infusion, the digoxin clearance decreased on the left 7.3 +/- 5.8 ml/min and on the right 6.1 +/- 6.4 ml/min, p < 0.02 and 0.05, respectively, compared to control. Quinidine infusion did not alter Cin. CDIG returned toward prequinidine infusion values after the infusion was stopped. We conclude that quinidine has a prompt and direct effect on the renal clearance of digoxin.

Individual renal clearances determined at renal vein catheterization.

Renal para-aminohippurate (PAH) clearances were predicted in 16 kidneys of eight hypertensive patients with renal artery stenosis, pyelonephritis, or obstructive nephropathy, without individual ureteral catheterization. Predictions of left or right kidney clearance (CL or CR) were based on roentgenographic renal frontal areas (A), on total PAH clearances (CT), and on individual PAH extractions (E) measured at renal vein catheterization according to the formula (formula: see text). When these patients underwent ureteral catheterization for diagnostic reasons, individual PAH clearances were measured and ranged from 22 to 286 cm3/min. After correction for differences in total PAH clearance on the two occasions, predicted and individually measured values corresponded closely along a line of identity. The 95% confidence limit (+/- 2 SEM) for predictions of individual PAH clearance was approximately +/- 38 cm3/min and for percet of total PAH clearance distributed to left or right kidney, +/- 6%. Individual renal PAH clearances can therefore be predicted at renal vein catheterization with acceptable error. Thus, the substantially invasive procedure of ureteral catheterization is not required to ascertain left and right kidney PAH clearance in patients already at risk from renal disease.

The sodium-retaining effect of renal nerve activity in the cat: role of angiotensin formation.

1. The effect of low-frequency stimulation of the renal nerves on renal function and renin release has been investigated. The experiments were performed in unilaterally nephrectomized, anaesthetized cats in which the nerves to the remaining kidney were sectioned. 2. When stimulation frequency was adjusted to reduce renal blood flow by approximately 15% for 15 min, glomerular filtration rate was hardly affected. The ratio sodium clearance/glomerular filtration rate was significantly reduced and plasma renin activity was significantly increased. 3. When the renal nerves were similarly stimulated in the presence of the beta-adrenergic receptor blocking agent, propranolol, the glomerular filtration rate was significantly reduced and the rise in plasma renin activity was significantly inhibited. The reduction of sodium clearance/glomerular filtration rate was as great as in the control animals. 4. The results are consistent with the view that the maintenance of glomerular filtration rate, during renal nerve stimulation which reduced renal blood flow, may be mediated by the local generation of angiotensin. The results also suggest that angiotensin does not play an important role in the sodium retention associated with increased renal nerve activity.

Effects of azotemia on renal extraction and clearance of PAH and TEA.

Dogs were made azotemic by bilateral ureteral-venous anastomosis. Subsequently, the left ureter, a systemic artery, and the left renal vein were cannulated. Left renal blood flow (RBF) was measured by an electromagnetic flowmeter. Left renal clearances and extractions of [14C]tetraethylammonium ([14C]TEA) and p-aminohippuric acid (PAH) were determined at five 40-min intervals after administraiton of loading doses and institution of a maintenance infusion containing PAH and [14C]TEA. The renal extraction of TEA was independent of blood urea nitrogen (BUN), whereas the extraction of PAH correlated inversely with BUN. Renal plasma flows calculated from urinary clearances and renal extractions agreed with renal plasma flows calculated from flowmeter data. The extraction of TEA remained constant over the 200-min experimental period, whereas the extraction of PAH increases progressively as a function of time. It is probable that those organic acids which accumulate in azotemia, and interfered with renal tubular secretion of PAH, were themselves eliminated after opening the ureteral-venous anastomosis. Thus, TEA is superior to PAH for evaluation of the renal circulation in azotemia.

Effects of anesthetic agents on autoregulation of renal hemodynamics in the rat and dog.

Controversy has existed over apparent dissimilarities in the autoregulatory capacities of the rat and dog. A protocol was designed to evaluate both the effects of the anesthetic agents. Nembutal (used most commonly in dogs) and Inactin (most frequently employed in rats) and the species peculiarities of these two mammals on autoregulation of renal blood flow (RBF) and glomerular filtration rate (GFR). With Nembutal autoregulation of RBF was present in both experimental animals. Inactin impaired RBF autoregulation similarly in both species. With impaired RBF autoregulation similarly in both species. With either anesthetic GFR was autoregulated well in both rat and dog. Comparison of the two species revealed a greater RBF per gram kidney weight and a higher renal perfusion pressure (RPP) at which autoregulation of both hemodynamic parameters was lost in the rat. It is concluded from these studies that 1) the frequent use of Inactin in the rat in large part accounts for the observed lack of autoregulation of RBF in this animal and 2) renal hemodynamic responses are qualitatively similar in rat and dog when the same anesthetic agents are used.

The natriuretic effect of p-aminohippurate in man.

The effects of p-aminohippurate (PAH) on electrolyte clearances were measured in 8 human volunteers. At plasma PAH levels used to measure renal plasm flow, the only significant increase was in sodium clearance, while with PAH levels used to measure maximum tubular secretion, the clearances of sodium, potassium and phosphorus were all significantly increased. The antriuresis combined with a depression of free water clearance and the absence of a chloruresis led to the conclusion that PAH- changed the anion composition of the filtrate delivered to the ascending limb, thus interfering with coupled Na+ reabsorption. This finding must be taken into account when electrolyte excretion is being measured during PAH infusion. A similar mechanism may operate in patients with chronic uremia whose endogenous hippurate level is increased.

Comparison of hilar and capsular renal lymph.

The sites of origin of renal lymph were studied by analysis of simultaneously collected samples of lymph and plasma. The samples included renal hilar (HL) and capsular lymph (CL), thoracic duct lymph (TD), renal venous (RVP) and arterial plasma (AP), and were analyzed for Na+, Cl-, K+, urea, glucose, inulin, and PAH concentrations. The glucose concentrations of HL (76 mg/100 ml, SE +/- 1.9), and CL (92 mg/100 ml, SE +/- 2.5) were significantly different (P less than 0.01) from that of RVP (86 mg/100 ml, SE +/- 2.2). Concentrations of both inulin and PAH in CL and HL fell between the AP and RVP values. The concentration of inulin in CL (CL/RVP = 1.27) exceeded that in HL (HL/RVP = 1.10), but no such difference was detected for PAH. The Na+ (152 meq/liter) and Cl- (129 meq/liter) concentrations in HL were significantly (P less than 0.01) higher than those in CL (Na+ = 148 meq/liter; Cl- = 120 meq/liter), in TD (Na+ = 146 meq/liter; Cl- = 121 meq/liter), and in RVP (Na+ = 144 meq/liter; Cl- = 114 meq/liter. These differences remained highly significant when the electrolyte concentrations were converted to milliequivalents per kilogram H2O. In contrast, no significant differences were detected between the concentrations of urea in the various fluids sampled. It is concluded that CL and HL do not drain a perfectly homogeneous intrarenal pool, and that they do not equilibrate with RVP or AP within the kidney. The results also indicate that CL and HL derive a small component from tubular reabsorbate; that for CL stemming from the cortex, and that for HL from the deeper cortex and outer medulla.

Autoregulation of renal blood flow in dogs at normal body temperature and at 27 degrees C.

1. Dogs cooled to 27 degrees C were compared with control dogs maintained at 38 degrees C. The mean arterial blood pressure, renal blood flow and glomerular filtration rate were lower in the hypothermic animals. 2. The relation between mean arterial blood pressure and renal blood flow was investigated. Auto-regulation of renal blood flow occurred in the kidneys of normothermic and hypothermic animals. Thus the reduction in renal blood flow during hypothermia is not due simply to the fall in mean arterial blood pressure. 3. Similarities between recordings of renal blood flow obtained at 38 degrees C and 27 degrees C suggest that its autoregulation occurs by the same mechanism at the two temperatures. 4. Autoregulation of renal blood flow occurred in hypothermic kidneys in the presence of a cold-induced vasoconstriction. The observed responses to cold and to alterations in mean arterial blood pressure may take place in different areas of the renal vasculature.

[Determination of glomerular and tubular clearance in children without urine collection]. / Détermination de la clearance glomérulaire et tubulaire de l'enfant sans recueil d'urines

The determination of glomerular and tubular clearance in a child was carried out by using a method excluding urine collection, with a continuous intravenous infusion of polyfructosan and para-amino-hippuric acid. A loading injection followed by a continuous perfusion provides a constant plasmatic level 150 minutes after the beginning of the study.