Julia L. Troy

The Dynamics of Glomerular Ultrafiltration in the Rat

Using a unique strain of Wistar rats endowed with glomeruli situated directly on the renal cortical surface, we measured glomerular capillary pressures using servo-nulling micropipette transducer techniques. Pressures in 12 glomerular capillaries from 7 rats averaged 60 cm H(2)O, or approximately 50% of mean systemic arterial values. Wave form characteristics for these glomerular capillaries were found to be remarkably similar to those of the central aorta. From similarly direct estimates of hydrostatic pressures in proximal tubules, and colloid osmotic pressures in systemic and efferent arteriolar plasmas, the net driving force for ultrafiltration was calculated. The average value of 14 cm H(2)O is lower by some two-thirds than the majority of estimates reported previously based on indirect techniques. Single nephron GFR (glomerular filtration rate) was also measured in these rats, thereby permitting calculation of the glomerular capillary ultrafiltration coefficient. The average value of 0.044 nl sec(-1) cm H(2)O(-1) glomerulus(-1) is at least fourfold greater than previous estimates derived from indirect observations.

Permselectivity of the glomerular capillary wall to macromolecules. II. Experimental studies in rats using neutral dextran

To determine the permselectivity characteristics of the glomerular capillary wall, known molecular size fractions of [3H]dextran, prepared by gel chromatography, were infused into normally hydrated Wistar rats, thus permitting simultaneous measurement of Bowmans space/plasma water (BS/P) and urine/plasma water (U/P) concentration ratios, along with glomerular pressures and flows. Since (BS/P)inulin = 1.01 +/- 0.01 SE(n = 34, radius = approximately 14 A) and since (BS/P)dextran/(BS/P)inulin equaled (U/P)dextran/(U/P)inulin for dextrans ranging in molecular radius from 21 to 35 A, these findings validate that dextrans are neither secreted nor reabsorbed. For dextran radii of 20, 24, 28, 32, 36, 40, and 44 A, (U/P)dextran/(U/P)inulin averaged 0.99, 0.92, 0.69, 0.42, 0.19, 0.06, and 0.01, respectively. In accord with theoretical predictions that these fractional dextran clearances should vary appreciably with changes in glomerular transcapillary pressures and flows, an increase in glomerular plasma flow rate, induced in these same rats by plasma volume expansion, resulted in a highly significant lowering of fractional clearance of all but the smallest and largest dextrans studied. These findings emphasize that fractional solute clearances alone are inadequate to describe the permselective properties of the glomerular capillary wall unless glomerular pressures and flows are also known. This sensitivity of fractional dextran clearance to changes in plasma flow indicates that dextrans are transported across the capillary not only by bulk flow but also to an important extent by diffusion.

Role for intrarenal mechanisms in the impaired salt excretion of experimental nephrotic syndrome.

A unilateral model of puromycin aminonucleoside (PAN)-induced albuminuria was produced in Munich-Wistar rats to examine the mechanisms responsible for renal salt retention. 2 wk after selective perfusion of left kidneys with PAN (n = 8 rats) or isotonic saline (control, n = 7 rats), increases in albumin excretion and decreases in sodium excretion were demonstrated in PAN-perfused but not in nonperfused kidneys of PAN-treated rats although systemic plasma protein concentration remained at control level. Total kidney glomerular filtration rate (GFR) and superficial single nephron (SN) GFR were also reduced selectively in PAN-perfused kidneys, on average by approximately 30%, due primarily to a marked decline in the glomerular capillary ultrafiltration coefficient (Kf), which was also confined to PAN-perfused kidneys. Values for absolute proximal reabsorption (APR) were also selectively depressed in PAN-perfused kidneys, in keeping with a similarly selective decline in peritubular capillary oncotic pressure measured in these kidneys, the latter also a consequence of the fall in Kf. In a separate group of seven PAN-treated rats, however, no differences were detected between PAN-perfused and nonperfused kidneys in the absolute amount of sodium reaching the early (0.77 +/- 0.09 neq/min vs. 0.74 +/- 0.08, P greater than 0.40) and late portions of superficial distal tubules (0.31 +/- 0.02) neq/min vs. 0.32 +/- 0.05, P greater than 0.50), despite the lesser filtered load of sodium in PAN-perfused kidneys. Suppressed sodium reabsorption in both proximal convoluted tubules and short loops of Henle of PAN-perfused kidneys contributed to this equalization of sodium delivery rates to the late distal tubule, as did comparable reabsorption along distal convolutions. In two additional groups of PAN-treated rats, infusion of saralasin (0.3 mg/kg per h, i.v.) led to substantial increases in total kidney GFR and SNGFR in PAN-perfused but not in nonperfused kidneys. Despite these increases in total and SNGFR, urinary sodium excretion by PAN-perfused kidneys remained at a level far below that for nonperfused kidneys, again indicating that the antinatriuresis characterizing the PAN-perfused kidney is due to alterations in sodium handling by the tubules rather than changes in GFR. These results therefore indicate (a) that reductions in Kf and depressed sodium reabsorption by proximal tubules and Henles loop segments in this model are brought about by intrarenal rather than circulating or systemic factors, and (b) assuming that superficial nephrons are representative of the entire nephron population, renal salt retention in this model is due primarily to intrarenal factor(s) acting beyond the distal convolution.

Renal and systemic hemodynamic effects of synthetic atrial natriuretic peptide in the anesthetized rat.

To characterize the hemodynamic events responsible for alterations in renal function during administration of atrial natriuretic peptide, we studied the systemic, renal, and glomerular circulatory effects of intravenous rANP[126–149], administered as a 4 μg/kg prime and 0.5 μg/kg per minute continuous infusion in anesthetized, euvolemic rats. With this protocol, a small decline in mean systemic arterial blood pressure occurred in the context of markedly enhanced urinary sodium excretion, hemoconcentration, and reduced left ventricular end-diastolic pressure and +dP/dt. However, despite a significant decrement in renal vascular resistance, total peripheral resistance remained constant, thereby denoting a preferential renal vasodilatory effect of this peptide in vivo. Whole kidney and single nephron GFR increased by approximately 20%, while effective renal and glomerular plasma flow rates remained stable, resulting in a substantial rise in filtration fraction. Of all the parameters potentially capable of augmenting single nephron GFR, only glomerular capillary hydraulic pressure increased significantly and therefore accounted entirely for the hyperfiltration observed during ANP infusion. This rise in glomerular capillary pressure, in turn, resulted from afferent arteriolar vasodilatation and concurrent efferent arteriolar vasoconstriction, findings that proved independent of both endogenous angiotensin II activity and ANP-induced reductions in renal perfusion pressure. These renal hemodynamic effects are unique when compared with actions of previously studied renal vasodilatory agents.

Dynamics of Glomerular Ultrafiltration in the Rat. IV. DETERMINATION OF THE ULTRAFILTRATION COEFFICIENT

Pressures and flow rates were measured in accessible surface glomeruli of mutant Wistar rats under conditions deliberately designed to prevent achievement of filtration pressure equilibrium, that is, the equalization of transcapillary hydrostatic and oncotic pressures by the efferent end of the glomerulus as typically observed in the normal hydropenic rat. Disequilibrium was obtained at elevated levels of glomerular plasma flow (GPF) brought about by acute expansion of plasma volume with a volume of rat plasma equal to 5% of body weight. Glomerular hydrostatic and oncotic pressures measured at high GPF were used to calculate the ultrafiltration coefficient, K(f), the product of effective hydraulic permeability and surface area. GPF was then either lowered (by aortic constriction) or raised (by carotid occlusion) in order to examine the dependence of K(f) on GPF. The value of K(f) per glomerulus, 0.08 nl/(s.mm Hg), was found not to vary over an approximately twofold range of GPF. This finding, taken together with data from previous studies from this laboratory, leads us to conclude that plasma-flow dependence of glomerular filtration rate (GFR) results primarily from flow-induced changes in mean ultrafiltration pressure, rather than large changes in K(f).

Postglomerular vascular protein concentration: evidence for a causal role in governing fluid reabsorption and glomerulotubular balance by the renal proximal tubule

We tested the relationship between postglomerular microvascular protein concentration and rates of sodium and water transfer by rat proximal tubules. Using recently described microperfusion techniques, efferent arterioles and branch peritubular capillaries of normal hydropenic rats were perfused with colloid-free Ringers solution, and isoncotic (9.0-10.0 g/100 ml) and hyperoncotic (15 g/100 ml) albumin-Ringers solutions. Reabsorption in adjacent proximal tubules was studied using free-flow techniques, with initial collections obtained during normal blood perfusion, recollections during experimental microperfusion, and in some tubules, repeat recollections after microperfusion and spontaneous resumption of blood perfusion. Colloid-free perfusion resulted in a uniform inhibition of proximal reabsorption (absolute and fractional). Despite identical techniques, substitution of isoncotic and hyperoncotic perfusates resulted, on average, in unchanged and increased rates of reabsorption, respectively. These findings of direct linear changes in reabsorption in response to changes in postglomerular protein concentrations usually occurred in the absence of significant changes in filtered load, and were nearly always found to be reversible within minutes of cessation of experimental perfusion. Given this evidence of a causal relationship between postglomerular oncotic pressure and proximal reabsorption, we undertook to determine whether this relationship is responsible for the parallel adjustments in proximal reabsorption that follow changes in GFR (glomerulotubular balance). Using a separate group of hydropenic rats, proximal reabsorption was studied, initially during partial aortic constriction (during which renal perfusion pressure, single nephron GFR, absolute proximal reabsorption, and calculated filtration fraction all were reduced below levels prior to constriction), and again while adjacent efferent arteriolar and peritubular capillary protein concentrations, but not GFR, were restored to normal (preconstriction) levels by microperfusion with 9-10 g/100 ml albumin-Ringers solution. During this dissociation of GFR and postglomerular protein concentration, absolute and fractional proximal reabsorption nearly always increased in parallel with the changes in the latter, thereby demonstrating that glomerulotubular balance is mediated, at least in part, by changes in postglomerular oncotic pressure brought about by changes in filtration fraction.

Mechanisms of the Puromycin-Induced Defects in the Transglomerular Passage of Water and Macromolecules

To investigate the mechanism(s) of increased filtration of serum proteins after glomerular injury, polydisperse samples of uncharged [(3)H]dextran (D) or anionic [(3)H]dextran sulfate (DS) were infused into 14 control and 16 puromycin aminonucleoside- (PAN) treated Munich-Wistar rats. Fractional clearances of D or DS ranging in radius from 18 to 42A were determined in these rats, together with direct measurements of the forces governing the glomerular filtration rate of water. Whole kidney and single nephron glomerular filtration rates were approximately 40% lower in PAN-treated rats, relative to controls, due mainly to a marked reduction in the glomerular capillary ultrafiltration coefficient and, to a lesser extent, to a small reduction in glomerular plasma flow rate as well. In PAN-treated rats, as in normal controls, inulin was found to permeate the glomerular capillary wall without measurable restriction, and both D and DS were shown to be neither secreted nor reabsorbed. Fractional clearances of uncharged D were reduced after PAN administration, falling significantly for effective D radii from 22 to 38A. Utilizing a theory based on macromolecular transport through pores, these results indicate that in PAN-treated rats, effective pore radius is the same as in controls, approximately 44A. In PAN nephrosis, however, the ratio of total pore surface area/pore length, a measure of pore density, is reduced to approximately one-third that of control, due very likely to a reduction in filtration surface area. In contrast to the results with uncharged D, fractional clearances of DS were found to increase after PAN administration for all DS radii studied. These results with D and DS suggest that proteinuria in PAN nephrosis is due, not to an increase in effective pore radius or number of pores, but rather to a diminution of the electrostatic barrier function of the glomerular capillary wall, thereby allowing increased passage of polyanions such as DS and albumin.

Mechanism of Reduced Glomerular Filtration Rate in Chronic Malnutrition

To determine the physiological basis for the low glomerular filtration rate in chronic malnutrition, micropuncture studies were performed in Munich-Wistar rats chronically pair-fed isocaloric diets of either low (group 1, nine rats) or high protein content (group 2, nine rats). Despite the absence of hypoalbuminemia, average values for single nephron and total kidney glomerular filtration rate were nearly 35% lower in group 1 than in group 2. Mean values for glomerular capillary and Bowmans space hydraulic pressures were essentially identical in the two groups, thereby excluding glomerular transcapillary hydraulic pressure difference as the cause for the low filtration rates in group 1 animals. On the other hand, average glomerular capillary plasma flow rate and glomerular capillary ultrafiltration coefficient were significantly lower (by approximately 25 and approximately 50%, respectively) in group 1 than in group 2. The fall in glomerular capillary plasma flow rate was the consequence of increased afferent and efferent arteriolar resistances. Plasma and erythrocyte volumes were found to be equal in five additional pairs of group 1 and group 2 rats. Thus, the substantial alterations in the ultrafiltration coefficient, glomerular capillary plasma flow rate, and renal arteriolar resistances responsible for the low filtration rate in group 1 animals were not merely a consequence of decreased circulating blood or plasma volumes. Mean values for glomerular cross sectional area were significantly lower in group 1 than in group 2 despite similar values for kidney weight in the two groups. This reduction in glomerular cross sectional area in group 1 rats is presumed to reflect a decrease in effective filtration surface area and therefore likely accounts, at least in part, for the decline in ultrafiltration coefficient observed in this group.Finally, since the daily caloric intake of group 2 animals was restricted because of pair feeding requirements tied to the group 1 rats, we studied a third group of seven rats (group 3) allowed an ad lib. intake of the same high protein diet as given to group 2 rats. Average values for single nephron glomerular filtration rate and its determinants were found to be indistinguishable between groups 2 and 3. These results suggest that low protein intake, rather than calorie deficiency per se, is primarily responsible for the reduction in filtration rate seen in this experimental model of chronic malnutrition.

Permselectivity of the glomerular capillary wall. Studies of experimental glomerulonephritis in the rat using dextran sulfate.

To determine whether the increased filtration of serum proteins after glomerular injury is the consequence of altered electrostatic properties of the glomerular capillary wall, we measured fractional clearances of the anionic polymer, dextran sulfate, in nine Munich-Wistar rats in the early autologous phase of nephrotoxic serum nephritis (NSN). In agreement with previous studied from this laboratory, whole kidney and single nephron glomerular filtration rates were normal in NSN rats despite histological evidence of glomerular injury, and despite a marked reduction in the glomerular capillary ultrafiltration coefficient to approximately one-third of normal. In the companion study (9), it was shown that in NSN rats the mean fractional clearances of neutral dextrans over the range of effective molecular radii from 18 to 42 A were reduced, compared to normla. In contrast, in the present study the mean fractional clearances for dextran sulfate over the same range of molecular radii were significantly greater than those found previously for normal Munich-Wistar rats. The fractional clearance of dextran sulfate molecules of the same molecular radius as serum albumin (approximately 36 A) was increased markedly, from 0.015 +/- 0.005 (SEM) in nonnephritic controls to 0.24 +/- 0.03 in NSN (P less than 0.001). The sialoprotein content of glomeruli, estimated by the colloidal iron reaction, was reduced in NSN rats as compared to normal controls. It is concluded that the abnormal filtration of anionic serum proteins, such as albumin, seen in glomerulopathies is, at least in part, the consequence of loss of fixed negative charges from the glomerular capillary wall.

Permselectivity of of the glomerular capillary wall. Studies of experimental glomerulonephritis in the rat using neutral dextran.

Polydisperse [3h] dextran was infused into eight Munich-Wistar rats in the early autologous phase of nephrotoxic serum nephritis (NSN), thereby permitting direct measurements of pressures and flows in surface glomeruli and fractional clearances for dextrans [(U/P) dextran/(U/P) inulin] ranging in radius from 18 to 42 A. Despite glomerular injury, evidenced morphologically and by a marked reduction in the glomerular capillary ultrafiltration coefficient, the glomerular filtration rate remained normal because of a compensating increase in the mean net ultrafiltration pressure. In NSN rats, as in normal controls, inulin was found to permeate the glomerular capillary wall without measurable restriction, and dextrans were shown to be neither secreted nor reabsorbed. For dextran radii of 18, 22, 26, 30, 34, 38, and 42 A, (U/P) dextran/(U/P) inulin in NSN and control rats, respectively, averaged 0.90 vs. 0.99, 0.81 vs. 0.97, 0.63 vs. 0.83, 0.38 vs 0.55, 0.20 vs. 0.30, 0.08 vs. 0.11, and 0.02 vs. 0.03. Using a theory based on macromolecular transport through pores, the results indicate that in NSN rats, effective pore radius is the same as in controls, approximately 50 A. In NSN, however, the ratio of total pore surface area to pore length, a measure of the number of pores, is reduced to approximately 1/3 that of control, probably due to a reduction in capillary surface area. These results suggest that proteinuria in glomerular disease is not due simply to increases in effective pore radius or number of pores, as previously believed. Using a second theoretical approach, based on the Kedem-Katchalsky flux equations, dextran permeability across glomerular capillaries was found to be slightly lower, and reflection coefficient slightly higher in NSN than in control rats.