Micropuncture studies of proximal tubule albumin concentrations in normal and nephrotic rats.

The concentration of serum albumin in proximal tubule fluid of normal rats and animals with aminonucleoside nephrosis was studied using renal micropuncture techniques. Albumin was quantitated by an ultramicrodisc electrophoresis method capable of measuring 3 chi 10(-11) g of albumin, in 10 nl volumes. With this sensitivity, only small samples of tubule fluid were required for analysis. Collection times could be kept short, therefore decreasing the opportunity for sample contamination with extraneous serum albumin. The measured mean concentration of albumin in proximal tubule fluid (1 mg/100 ml in females and 0.7 mg/100 ml in males) was somewhat lower than values reported by others, but even these values are apt to have been artifactually high as a result of animal preparation and trace contamination of samples during micropuncture. Rats injected with aminonucleoside of puromycin 4 days earlier, showed a significant increase in tubule-fluid albumin concentration coincident with a fall in serum albumin concentration and a 43-fold increase in urine albumin concentration. Tubular absorption of albumin was small relative to that of water. Although albumin filtration was significantly increased over that in normal animals, the glomerular basement membrane still served as a highly efficient barrier to albumin transfer.

Glomerular hemodynamics in established glycerol-induced acute renal failure in the rat.

The glomerular dynamic correlates of failed filtration were studied in volume replete rats with established glycerol-induced acute renal failure (ARF). Over one-half of all nephrons formed virtually no filtrate, while the single nephron glomerular filtration rate (SNGFR) of fluid-filled nephrons, measured at the glomerulotubular junction to preclude the possibility of covert tubular leakage, averaged one-sixth of control (P less than 0.001). Even that low mean value was elevated by a few nephrons with a near normal SNGFR. Renal failure thus reflected both total filtration failure in the majority of nephrons and massively reduced filtration in most of the remainder. Glomerular capillary pressure (Pg) averaged some 14 mmHg below control (P less than 0.001), whereas the arterial colloid osmotic and Bowman's space pressures were not significantly altered. Renocortical and whole kidney blood flow were also unchanged. Marked internephron functional heterogeneity precluded estimates of the ultrafiltration coefficient. However, the fall in SNGFR correlated well with the markedly depressed Pg and afferent net filtration pressure (delta PnetA, P less than 0.001), which in turn were caused by increased preglomerular resistance and a reciprocal fall in efferent arteriolar resistance. This complex change in intrarenal resistances was largely, if not entirely, responsible for failed filtration in this ARF model.

Micropuncture studies of the recovery phase of myohemoglobinuric acute renal failure in the rat.

Micropuncture studies of the recovery phase of glycerol-induced myohemoglobinuric acute renal failure were performed in rats whose blood urea nitrogen (BUN) had fallen at least 20% below its peak value. The glomerular filtration rate (GFR) of individual nephrons in a single kidney in the recovery period generally either was in the normal range or minimal. Each animal's BUN concentration at the time of the study was inversely related to the proportion of functioning surface nephrons, but did not correlate with individual nephron GFR values. Proximal tubule fractional water absorption was significantly depressed as manifested by both depressed inulin (TF/P) values and supernormal volumes of collections, a finding which, in the absence of a urea-induced osmotic diuresis, suggests impaired sodium transport by the damaged nephron. The mean proximal tubule hydrostatic pressure in recovery was normal and there was little variation in pressure among functioning nephrons. It is concluded that recovery from this model of acute renal failure reflects the progressive recruitment of increasing numbers of functioning nephrons. The recovery of individual nephron glomerular filtration, once begun, was rapid and complete. No evidence could be adduced that the gradual return of renal function towards normal reflects a slow release of tubular obstruction or repair of disrupted tubular epithelium. Rather, recovery appeared to be directly attributable to the return of an adequate effective glomerular filtration pressure. Significant limitation in proximal tubule water absorption persisted after individual nephron GFR had returned to normal or supernormal values in this model of experimental acute renal failure in the rat, a finding which readily accounts for the diuresis associated with the recovery phase of this syndrome.

Hemodynamic basis for human acute renal failure (vasomotor nephropathy).

Oliguric acute renal failure in man is characterized by intense outer cortical vasoconstriction and a marked increase in preglomerular resistance. The degree of preglomerlar resistance change needed to cause the expected 50 to 80 percent fall in blood flow far exceeds the level that would totally abolish filtration. By contrast, equal 3.0-fold increases in both pre- and postglomerular resistance provide this same degree of ischemia but leave filtration very well maintained. Such a scenario seems unlikely, however, since it would entail a mere 15 to 25 percent decrease in preglomerular resistance vessel caliber rather than the extreme attenuation observed. By contrast, there are reasons to believe that preglomerular constriction may be accompanied by postglomerular vascular relaxation. In sum, unless cortical ischemia reflects precisely matched increases in pre- and postglomerular resistances, filtration failure is inevitable in human vasomotor nephropathy.

Acute renal failure caused by nephrotoxins.

Renal micropuncture studies have greatly changed our views on the pathophysiology of acute renal failure caused by nephrotoxins. Formerly, this type of renal insufficiency was attributed to a direct effect of the nephrotoxins on tubule epithelial permeability. According to that theory, glomerular filtration was not greatly diminished, the filtrate formed being absorbed almost quantitatively and nonselectively across damaged tubule epithelium. Studies in a wide variety of rat models have now shown glomerular filtration to be reduced to a level which will inevitably cause renal failure in and of itself. Passive backflow of filtrate across tubular epithelium is either of minor degree or nonexistent even in models where frank tubular necrosis has occurred. This failure of filtration cannot be attributed to tubular obstruction since proximal tubule pressure is distinctly subnormal in most models studied. Instead, filtration failure appears best attributed to intrarenal hemodynamic alterations. While certain facts tend to incriminate the renin-angiotensin system as the cause of the hemodynamic aberrations, others argue to the contrary. The issue is underactive investigation.

Does the ultrafiltration coefficient play a key role in regulating glomerular filtration in the rat.

Various experimental maneuvers, hormones, and pharmacological agents are reported to exert their effects on glomerular filtration largely or exclusively by changing the ultrafiltration coefficient (Kf) of the filtration barrier. In light of a number of uncertainties discussed in this review, however, it is by no means certain that the Kf actually does play this central role in the physiological regulation of glomerular function.

Renal and extrarenal considerations in high-dose mannitol therapy.

It is well recognized that the large doses of mannitol used in treating cerebral edema alter extracellular fluid (ECF) volume, osmolality, and composition to a degree which, under some circumstances, can lead to acute renal failure, cardiac decompensation, and other difficulties. It is less well appreciated that the patient's body habitus, age, total body water content relative to body weight, pretreatment plasma sodium concentration and plasma osmolality, and the presence of edema or ascites also can influence the degree of ECF change and the rate of mannitol excretion to a significant degree. Here, we show how these changes occur and the way in which their magnitude can be predicted prior to therapy so as to minimize the risk of an adverse result.

An analysis of glomerular dynamics in rat, dog, and man.

A network thermodynamic model was utilized to assess similarities and dissimilarities in the predicted response of human, rat, and dog glomeruli to change in the independent variables regulating glomerular filtration. The analysis in rat and dog employed basal values reported in the micropuncture literature. The analysis in man was based on a calculated total nephron vascular resistance (Rt) of 1.2 X 10(10) dyne sec cm-5 with a range of pre- (Ra) and postglomerular (Re) resistances and capillary hydraulic conductivities (Kf) that would provide a nephron blood flow (GBF) of approximately 550 nl/min and single nephron filtration rate (SNGFR) of approximately 65 nl/min. The maximal putative value for Ra/Re in man was approximately 1.1, a ratio demanding a Kf greater than 20 nl/min mm Hg to obtain the required SNGFR. Solitary changes in Ra and Re, glomerular capillary resistance, proximal tubule pressure, serum protein concentration, total vascular resistance, and Kf were induced and the resultant effect on SNGFR was examined in the three species. The relationship between changes in individual resistances, glomerular blood flow, glomerular filtration, and glomerular capillary pressure also was assessed. The patterns of response in man and dog, determined by the model, were remarkably similar and distinct from those of the rat in many regards. Except when the maximal possible Ra/Re ratio is assumed for man, filtration pressure equilibrium was not found; plasma flow dependence of SNGFR was not evident in rat, dog, or man. The differences in SNGFR control predicted for the rat, on the one hand, and dog and man on the other may have distinct physiologic significance.

Theoretical analysis of pathogenetic mechanisms in experimental acute renal failure.

A network thermodynamic model of glomerular dynamics has been employed to determine the degree of change in individual glomerular vascular resistances, hydraulic conductivity and proximal tubule pressure that, singly or in concert, could lower GFR to the degree expected in experimental acute renal failure (ARF). In both the rat and dog, the analysis shows that filtration failure is not achieved until preglomerular resistance (RA) is increased at least twofold or postglomerular resistance (RE) is decreased by 74% or more with all other determinants held at control values. Tubular obstruction alone will not provide failed filtration until tubule pressure is increased to 30 to 40 mm Hg in the rat and 44 mm Hg in the dog. A much smaller change in tubular pressure can contribute greatly to the development of filtration failure, however, when occurring in association with major change in individual vascular resistances. Glomerular capillary resistance must be increased to a value more than twice the normal sum of RA and RE (greater than fivefold in the dog), and glomerular capillary hydraulic conductivity lowered to below 5% of control, as isolated changes, before full filtration failure is approached. There is no reason to believe that most forms of ARF relate to only a single abnormality, however, and the effect of concomitant changes in individual resistances, hydraulic conductivity and proximal tubule pressure on glomerular filtration and blood flow is presented in the text and figures. A possible mechanism by which altered blood viscosity at the efferent arteriole may contribute to ARF is discussed and quantified. The degree of change in any determinant required to exert a given effect on filtration is independent of etiology, thus rendering the results of this analysis equally valid for any other pathological event which causes a significantly reduced GFR in the rat or dog.

Role of prostaglandins in the pathogenesis of acute renal failure.

It is suggested that impaired glomerular infiltration in acute renal failure may be due, if only in part, to the derangement of a normal local feedback mechanism involving prostaglandins. According to this hypothesis, prostaglandins made in the medulla normally enter the loop of Henle and thus are present in distal tubule fluid reaching the macula densa. In the absence of direct vascular channels connecting the renal medulla with the outer cortex, this may be the only route by which medullary prostaglandins can reach the cortex without being destroyed by dehydrogenases. Under most circumstances in which renal perfusion and glomerular filtration fall, flow through Henle's loop continues, albeit at a reduced rate. This slowed flow through the loop and augmented prostaglandin release combine to produce a high concentration of prostaglandin in fluid leaving the medulla which, acting on the macula densa, counteracts the vasoconstrictor stimulus. If, however, filtration stops completely because of overwhelming vasoconstriction, flow along the loop also stops ans prostaglandins no longer reach the macula densa. Thus, the prostaglandin feedback loop would be opened, constrictor mechanism remain unchecked, and filtration failure perpetuated. It is proposed that such a self-perpetuating mechanism might operate in acute renal failure.

Microcomputer simulation as an aid in analyzing data and teaching the principles of glomerular dynamics.

This report describes a simple program, written in BASIC language, that closely emulates a previously published network thermodynamic model of glomerular dynamics. While the latter requires the SPICE 2 simulation program and a mainframe computer for its execution, the present program operates on any IBM-compatible microcomputer. It has equal utility as an aid in the interpretation of laboratory studies of glomerular dynamics and as a tool for teaching the intricacies of the control of glomerular function. The program is available in 'user friendly' format that obviates the need for any expertise in the use of computers.

Simultaneous quantitation of twenty-two amino acids in nanolitre samples of biological fluid.

The technique of thin-layer chromatography and 14C-dansylation has been used for simultaneous measurements of 22 individual amino acids, taurine, serotonin, and gamma-aminobutyric acid in nanolitre samples of glomerular and renal tubule fluid. Standard curves of each amino acid mixture containing all the others showed excellent linearity at amounts ranging from 5.7.10(-13) to 1.45.10(-11) mole (regression coefficients all greater than 0.95). With careful standardization of dansylation conditions, the technique permits highly reproducible measurement of less than 1 pmole of an amino acid.

Micropuncture studies of the transport of individual amino acids by the Necturus proximal tubule.

Absorption of amino acids by the necturus proximal tubules was measured under free-flow conditions The coexistence of proximal tubular amino acid influx was determined by infusing saline into tubular lumens by the stopped-flow microperfusion technique. Under free-flow conditions, fractional absorption of individual amino acids ranged from 0.30 +/- 0.18 (glutamic acid) to 0.96 +/- 0.02 (proline), with 14 of 19 values greater than 0.75. The transport avidity for a given amino acid bore no relationship to its molecular weight, transport class, or plasma concentration. The values obtained for tubular fluid/plasma (TF/P) were very comparable to those reported for the rat. In stopped-flow microperfusion experiments, samples of isotonic saline residing in tubule lumens for 20 min were found to contain all the amino acids present in plasma (filtrate). The concentrations of all except the acidic anionic pair, glutamic acid and aspartic acid, were remarkably similar to those obtained by collection of end proximal samples in free-flow studies. The very high concentrations of the acidic amino acids may reflect their passive distribution across the luminal cell membrane, active absorption having been impaired by the absence of some substance normally present in glomerular filtrate.

Internephron heterogeneity of filtration fraction and disparity between protein- and hematocrit-derived values.

The variability of single nephron filtration fraction (SNFF) and the relationship between filtration fractions determined simultaneously from 125I albumin (SNFFALB) and hematocrit (SNFFHCT) values were assessed by multiple samplings, 5 to 8 per kidney, in normal, hydropenic, Munich-Wistar rats. Single nephron filtration rate (SNGFR) measurements served as an independent marker of internephron heterogeneity. Whether derived as SNFFALB or SNFFHCT, a wide degree of internephron variability in SNFF, comparable to that found for SNGFR (P greater than 0.2) was observed in all kidneys. Hematocrit-derived SNFF values, however, were some 80% higher than those obtained with 125I albumin (SNFFHCT 0.447 +/- SEM 0.016 versus SNFFALB 0.258 +/- 0.009, P less than 0.001). The disparity in results obtained by the two methods disappears if the efferent arteriolar hematocrit (Hctg) is assigned a value 17 +/- SEM 1.4% higher than the corresponding femoral arterial hematocrit and might well be a reflection of the perfusion of superficial glomeruli with erythrocyte rich blood produced by the "plasma-skimming" phenomenon. The data suggest that multiple estimates of SNGFR and SNFF in a given rat kidney are needed to provide representative mean values for glomerular dynamic studies.