J. Physiol. (1977), 266, pp. 103-121 With 6 text-ftguree Printed in Great Britain

103

SUBSTRATE-LIMITED FUNCTION AND METABOLISM OF THE ISOLATED PERFUSED RAT KIDNEY: EFFECTS OF LACTATE AND GLUCOSE BY JULIUS J. COHEN, YOUNG JOHNG KOOK* AND JOHN R. LITTLEt From the Department of Physiology, University of Rochester, Rochester, New York 14642, U.S.A.

(Received 14 June 1976) SUMMARY

1. The objective of this study was to determine the separate contributions of exogenous substrate and of kidney tissue to the support of function and metabolism in the isolated perfused rat kidney. The effects of the addition of L( +) [U-14C]lactate or D[U-4(C]glucose either to a specially prepared substrate-free albumin (SFA) or to Fr. V bovine serum albumin (Fr. V-BSA) were compared. The Fr. V-BSA has significant quantities of lactate, citrate and free fatty acids associated with it. 2. Perfusion of the rat kidney with the Krebs-Ringer bicarbonate solution containing SFA, without addition of exogenous substrate, resulted in a lower % Na+ reabsorption (- 43 %) than when the perfusions contained Fr. V-BSA (- 80 %). Thus, kidney tissue can support at most 45 % of Na+ reabsorption, while the substrates associated with the Fr. V-BSA can support 30 % of Na+ reabsorption. When the initial concentration of L( + )lactate in the perfusate containing SFA was progressively raised from 0 to 10 mm, % Na+ reabsorption increased to between 85 and 90 %. 3. The apparent Km (0.59 mM) and the Vmax (0 67 ,mole g-1. min-') for lactate oxidation in the presence of SFA were both significantly lower than when Fr. V-BSA was present (Km, = 2 0 mM; Vmax = 1 Iumole g-. min-'). The lower Km is interpreted as being due to the removal of substances from the Fr. V-BSA which competitively inhibit either the uptake or oxidation of lactate; the lower Vmax is considered to be related to the lower rate of Na+ reabsorption when SFA is present. * Present address: Department of Pharmacology, Chonnam University Medical School, Kwangju, Republic of Korea.

t Present address: University of Maryland, School of Medicine, Baltimore, Maryland, U.S.A.

104 J. J. COHEN, Y. J. KOOK AND J. R. LITTLE 4. Addition of glucose enhanced glomerular filtration rate in the presence of both types of albumin. The resulting increase in the filtered load of Na+ in the presence of glucose was associated with either no change (Fr. V-BSA) or an increase (SFA) in fractional Na+ reabsorption. Although absolute Na+ reabsorptive rate was greater in the presence of glucose than in the presence of lactate, the oxidation rate of glucose, on a carbon-atom basis, was less than 50 % of the oxidation rate of lactate. 5. The metabolism of glucose may regulate the permeability characteristics of the glomerulus and the tubular epithelium: by contrast, the high oxidation rate of lactate suggests it can provide direct support for a major fraction of the Na+ actively absorbed. INTRODUCTION

The isolated rat kidney preparation, perfused with a cell-free solution, is a useful model for determining the colloid (Little & Cohen, 1974), substrate and hormonal requirements for the function and metabolism of the intact kidney (Cohen & Little, 1976; Bowman, 1970; Trimble & Bowman, 1973; Ross, Epstein & Leaf, 1973; Bertermann, Franke, Huland & Weiss, 1973; Franke, Huland, Weiss & Unisicker, 1971; Schurek, Brecht, Lohfert & Hierholzer, 1975). In those studies in which Fraction V bovine serum albumin (Fr. V-BSA, 6-7 5 %) was the colloid in the perfusate, and without any added substrate, from 70 to 85 % of the filtered load of Na+ was reabsorbed. When glucose (Trimble & Bowman, 1973; Ross et al. 1973) or lactate (Cohen & Little, 1976) was added to the perfusate a modest increase in per cent Na+ reabsorption (°/, i-NNa+) to, or slightly above 90 % resulted. By contrast, addition of other substrates such as butyrate (Ross et al. 1973) had no effect on % Na+ reabsorption. From such observations it was suggested that metabolic support for reabsorption of the 75 % of the filtered Na+ which occurs in the presence of Fr. V bovine serum albumin (Fr. V-BSA) but in the absence of any added substrate, is derived from the catabolism of kidney tissue (Trimble & Bowman, 1973; Ross et al. 1973; Cohen & Little, 1976). Another possible source of substrate for the perfused kidney is the substrate mixture associated with the Fr. V-bovine serum albumin as obtained from several commercial sources, contains variable, and often significant quantities of free fatty acids (Chen, 1967), lactate, citrate as well as small quantities of pyruvate (Hanson & Ballard, 1968). All these substrates are known to be utilized by the kidney (Cohen & Barac-Nieto, 1973; Cohen & Kamm, 1976). Further, the presence of the substrates associated with the Fr. V-BSA would tend to obscure the specific qualitative and quantitative effects on renal function or metabolism produced by the addition of an exogenous substrate.

LACTATE AND GLUCOSE: EFFECTS IN RAT KIDNEY 105 Therefore, we have removed the above three substrates associated with the commercially available Fr. V-bovine serum albumin and determined the changes in renal function which result. Then, by adding either L( + )lactate or D-glucose to the perfusate, we have measured the resultant changes in the function and metabolism of the isolated rat kidney and compared them with observations made earlier with Fr. V-BSA as the colloid in the perfusate (Cohen & Little, 1976). METHODS

Perfuaion apparatus The previously described perfusion apparatus (Little & Cohen, 1974), with the modifications introduced for measurement of 14CO2 (Cohen & Little, 1976) was used in all experiments. The glass arterial and venous cannulae were replaced by stainless steel cannulae to reduce breakage, and in the case of the arterial cannula, to increase the internal diameter of the lumen and reduce the resistance of flow. As a result, smaller increments in perfusion pressure were required to compensate for the resistance of the arterial cannula to maintain mean intra-renal arterial pressure at 120 mmHg. Perfu8ion medium Crystalline, dry substrate-free albumin (12 g), was dissolved in 40 ml. glass. distilled water and the pH adjusted to 7-4 with 0-2 N-Na2CO3. The albumin was added to - 150 ml. of a gassed (5 % CO2 -95 % 02) solution of Krebs-Ringer bicarbonate (containing all the salts for a final volume of 200 ml.) and made up to a total volume of 200 ml. with distilled water. The perfusion medium was promptly transferred to the perfusion circuit which included the membrane gas-exchanger (gassed with 5 % CO2 -95 % 02). There was no prior or in-line Millipore filtration of the perfusate. Stock L(+)lactate solution containing L(+)[U_14C]lactate acid or stock D-glucose containing D-[U-14C]glucose was prepared as previously described (Cohen & Little, 1976) and added to the perfusion medium to achieve the nominal initial concentrations of lactate (0-5-10 mM) or glucose (5 mM). No L( + )[U_14C]lactic acid or D-[U-14C]glucose was present in those experiments in which chemical quantities of lactate of glucose were not added to the medium. The final composition of the KrebsRinger bicarbonate solution was (mM): Na+, 145; K+, 5; Ca2+, 2-5; Cl-, 104; HCO3-, 25; S04, 1; phosphate, 2; urea, 7; inulin, 250-30C fg/ml. and albumin, 60 mg/ml. The perfusate pH was between 7-40 and 7-50 during the 75 min of perfusion. Preparation of the aubatrate-free bovine serum albumin. Analysis of substrates in several lots of Pentex (Miles Laboratories, Kankakee, Illinois, U.S.A.) Fr. V.BSA confirmed the previous observations of Chen (1967), and of Hanson & Ballard (1968) (Table 1). Therefore, 300 g quantities of Fr. V-BSA were treated by adsorption with activated charcoal at pH 3-5-4-0 (Chen, 1967) followed by dialysis against glass distilled water (Hanson & Ballard, 1968). The resulting albumin solution was lyophilized and the dry crystalline albumin was then stored at 2-4° C until used. Approximately 15-20 % of the original weight of albumin was lost in this process. The quantities of FFA, lactate and citrate associated with representative lots of albumin before and after the above treatment are shown in Table 1. The weight gain of the kidney perfused with 6 % substrate-free albumin was the same as that of kidneys perfused with 6% Fr. V-BSA. The mean ratio of wet weight of the perfused right kidney (PK) to that of the control, left kidney (CK) when the perfusate contained

106

J. J. COHEN, Y. J. KOOK AND J. R. LITTLE

substrate-free albumin was 1-118 ± 0-02 (mean ± s.E., n = 19); while the ratio of the dry weights was 0943 + 0013 (mean ± s.E., n = 19). The wet weight ratio, PK/CK, when the perfusate contained Fr.-V-BSA (Cohen & Little, 1976) was -1X16 + 0-014 (n = 12), and the ratio of the dry weights was 0-952 + 0'018 (n = 12). Thus, the colloid osmotic properties of the albumin after the above treatment was not changed. The albumin concentration of 6 g/100 g in the perfusion medium was chosen as a result of an earlier study (Little & Cohen, 1974). Experimental procedure Male Long-Evans rats weighing between 400 and 500 g (Blue Spruce Farms, Altamont, New York) were allowed to eat Purina rat chow and drink water ad libitum before the experiment. The preparation of the animals, cannulation of the vessels and transfer of the kidney to the thermostated perfusion chamber have been described (Little & Cohen, 1974). The mean perfusion pressure of 120 mmHg was higher than the 100 mmHg used in our previous study (Cohen & Little, 1976). The higher pressure was selected due to the recent observations in our laboratory (Zamlauski & Cohen, 1976) that the mean arterial pressure of the intact LongEvans rat, anaesthetized with Inactin (5 sec-butyl-5-ethyl-2-thiobarbituric acid) is 118±3 mmHg mean±s.E.) (n = 17). A similar mean arterial pressure has also been observed (in the Sprague-Dawley rat (Kaillskog, Lindbom, Ulfendahl & Wolgast, 1975). After a 15 min equilibrium period (Schurek et al. 1975), six consecutive 10 min observation periods followed. A total of twenty-two experiments were done in which SFA was the colloid added to the perfusion medium, four with no added substrate; four experiments each at the nominal initial lactate concentrations of 0 5, 2*5, and 5 mM; three experiments at 10 mm lactate; and three in which glucose (5 mM) was the added substrate. Two experiments were done in which glucose (5 mM) was the added substrate and Fr. V-BSA was the added colloid. The decarboxylation rate of glucose was determined in two of the three experiments with SFA and in both experiments with Fr. V-BSA. All the observations with Fr. V-BSA and lactate as the added substrate or without any substrate added were previously reported (Cohen & Little, 1976) and are presented for comparison with the present observations with SFA.

Analytical Methods Inulin, Na+ and K+ concentrations in the perfusate and urine were measured as previously described (Little & Cohen, 1974). L( + )lactate and D-glucose concentrations in urine and perfusate were determined by the methods of Hohorst (1963) and Bergmeyer & Bernt (1963), respectively, as previously described (Cohen & Little, 1976). Citrate and free fatty acid concentrations in the solutions of the Fr. V-BSA and the substrate-free albumin were determined by enzymatic assay using citrate lyase and malic dehydrogenase (Moellering & Gruber, 1966), and by the titration method of Dole & Meinertz (1960) respectively. The 14CO2 content of the perfusate was measured by a modification (Anderson & Snyder, 1969; Cohen & Little, 1976) of the diffusion flask method (Passman, Radin & Cooper, 1956). The calculations for the metabolism of lactate or glucose, as well as those for renal function, were done as described earlier (Cohen & Little, 1976).

Statistical analysis During the 60 min of observation the lactate concentration in the perfusate decreases from the initial level. Because the concentration of lactate is a determinant of the rate of lactate metabolism (Cohen & Little, 1976), regression analyses were

LACTATE AND GLUCOSE: EFFECTS IN RAT KIDNEY 107 done to evaluate relationships among rates of metabolism, substrate concentrations and functions. Two variables were evaluated at a time. The equation for each regression line was calculated by the least squares technique. It was then determined whether the calculated slope of each regression was significantly different from zero. The significance of the differences between the slopes or intercepts of the regression lines for observations made with substrate-free albumin and Fr. V-BSA was tested as described by Dixon & Massey (1969). The statistical significance of the difference between the observations made in the presence of glucose and either lactate or no substrate was determined by the t test for unequal number of observations (Snedecor & Cochran, 1967). RESULTS

Changes in renal function on removal of substrates from Fraction V-bovine serum albumin In Fig. 1 are shown the mean values for Na+ reabsorption and renal haemodynamics in four experiments in which SFA, and three experiments in which Fr. V-BSA (Cohen & Little, 1976) was the added colloid and to which no exogenous substrate was added to the perfusate. The filtration rates and perfusate flow rates were similar for perfusions performed with either type of albumin. In contrast, % Na+ reabsorption was considerably lower throughout the perfusion period with substrate-free albumin. These observations suggest that Fr. V-BSA contains substances which provide significant support for tubular reabsorption of Na+. The differences between the changes in renal metabolism and function which occur upon addition of L(+)lactate to perfusates containing either SFA or Fr. V-BSA are consistent with this interpretation. The differences in lactate metabolism between perfusion with Fr. V-bovine serum albumin and substrate-free albumin Net lactate utilization. In the presence of SFA, as lactate concentration was raised, the cumulative net utilization of lactate (Fig. 2A) increased, except perhaps between 5 and 10 mm. However, the slope of the linear regression for concurrent 1/4-LAC vs. 1/ILAC] (Lineweaver-Burk plot, not shown) was not significantly different from zero (1/Q-LAC = 2-181-36 1/[LAC]; n = 78; r = 0-12, P = 0-30) as had also been observed when Fr. V-BSA was the colloid (Cohen & Little, 1976). While the slope of the linear regression for net utilization rate of lactate (Q-LAC) versus lactate concentration ([LAC]) is virtually identical to that observed with Fr. V-BSA (Fig. 3A), the intercept of the regression line for the observations with SFA is significantly lower, indicating that removal of the substances associated with Fr. V-BSA results in a decrease in net lactate utilization. Lactate decarboxylation and glucose production. The cumulative

108 J. J. COHEN, Y. J. KOOK AND J. R. LITTLE decarboxylation of lactate at each nominal lactate concentration was approximately linear with time (Fig. 2B), consistent with the maintenance of a constant specific activity of the lactate during the time course of these observations. In contrast to the absence of a maximal net utilization rate for lactate, a maximal rate for lactate oxidation was apparent (Fig. 3B, C). From the linear regression of 1/Qfjx V8. 1/[LAC] (Lineweaver-Burk), the apparent Vmax (0 67 + 0 05 molee g-1. min-1) and Km (0.59 + 0 18 mm) were calculated. Both are significantly lower than the 24

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35 45 55 65 75 Interval (min) Fig. 1. Comparison of mean values of renal functions during 60 min of perfusion when either substrate-free albumin (SFA) or Fr. V bovine serum albumin (Fr.V) was the colloid in the perfusate. No exogenous substrate was added to the perfusate solutions. The mean % Na+ reabsorption

(77 ± 5 %, n = 9) in the presence of Fr. V-BSA was significantly (P < 0001) higher than when SFA was present (43 ± 2 %, n = 24). There were no significant differences between the mean glomerular filtration rates or mean perfusion flow rates with each type of albumin.

LACTATE AND GLUCOSE: EFFECTS IN RAT KIDNEY 109 values calculated from the observations made with Fr. V-BSA (Vmax = 1 I + 0*2mole g-1. min-' and KmN = 2 9 + 0 7 mM). The constants for the observations made with Fr. V-BSA are somewhat different from those previously reported (Vmax = 1 3 molee g-1. min-; Km = 2.1 mm, Cohen & Little, 1976), due to weighting of the observations as described by Wilkinson (1961). In contrast to the lower net utilization and decarboxylation rates of lactate in the presence of SFA, net glucose production as a function of lactate utilization rate (Fig. 3D) or cumulative glucose production (Fig. 2C) with time, are virtually identical to those observed in the presence of Fr. V-BSA. so

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Over-all, when the perfusate contains SFA rather than Fr. V-BSA, the apparent affinity of lactate for oxidation is increased. Also, although lactate availability is not limiting oxidation, the maximal rate of CO2 production derived from lactate is reduced, which is consistent with the lower rate of

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LACTATE AND GLUCOSE: EFFECTS IN RAT KIDNEY 111 net lactate utilization. These differences in the renal metabolism of lactate are associated with definitive changes in renal tubular function. The effects on renal function of raising the concentration of L( + ) lactate in the presence of substrate-free albumin or Fr. V-BSA Changes in renal function with time of perfusion (Fig. 4). In the absence of prior or in-line Millipore filtration of the perfusate, the glomerular filtration rates were similarly low in both the present and previous (Cohen & Little, 1976) groups of observations, being from 15-45 % of the values observed in vivo. Mean % Na+ reabsorption (% TP-Na+, Fig. 4A), glomerular filtration rate (Fig. 4B) decreased with time at all lactate concentrations. Although filtration rate increased slightly as lactate A %T-Na+

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J. J. COHEN, Y. J. KOOK AND J. R. LITTLE metabolism increased, glomerular filtration rate was not significantly correlated with either lactate oxidation rate (GFR = 222- 17-5 QLAC) r = 0*05; P = 0*5; n = 87) or lactate utilization rate (GFR = 208-9 9 QLACTATE; r = 0*06; P = 0 5; n = 102). Mean perfusate flow rate decreased with time (Fig. 4B) at all initial lactate concentrations except at 10 mM; at this latter lactate concentration, flow rate was maintained relatively constant throughout the period of perfusion. Lactate oxidation and changes in Na+ reabsorption. The slope of the linear regression of the individual observations for %!t-Na+ upon the concurrent lactate oxidation rate is significantly different from zero (Fig. 5A). The differences between both the slopes and the intercepts for the observations made with SFA, compared to those made with Fr. V-BSA, are highly significant. The slope of the linear regression for the absolute rate of Na+ reabsorption versus lactate oxidation rate (Fig. 5B) in the presence of SFA is also significantly different from zero. While the intercept for the latter regression is significantly lower (P < 0 001) than the intercept for the observations made in the presence of Fr. V-BSA, the slopes of the two groups of observations are not significantly different from each other. Thus, in the absence of the substrates associated with Fr. V-BSA, at any lactate oxidation rate, absolute !P-Na+ and % P-Na+ are significantly reduced. However, increases in lactate concentration have more pronounced effects on fractional Na+ reabsorption in the presence of SFA than when the perfusates contain Fr. V-BSA. 112

The difference in the effects on renalfunction between adding lactate or glucose to perfusates containing SFA or Fr. V-BSA (Table 2) Whether the perfusate contained SFA or Fr. V-BSA, addition of glucose (5 mM) increased mean glomerular filtration rate significantly above that observed either when no substrate had been added or when 5 mm lactate had been added. With Fr. V-BSA as the colloid, the mean renal vascular resistance in the presence of lactate or glucose was not significantly different; in the presence of SFA the mean renal vascular resistance with 5 mm glucose was higher (P < 0.01) than with 5 mm lactate. Thus, the increase in filtration rate associated with the addition of glucose is not dependent upon a decrease in total renal vascular resistance, and occurs whether the substances associated with the Fr. V-BSA are present or not. By contrast, a difference between the effects of 5 mm glucose and 5 mm lactate on Na+ reabsorption is manifest only in the presence of SFA (Table 2). The addition of glucose to SFA raised the mean % Na+ reabsorption significantly from 43 to 58 % (P < 0.001), while addition of glucose or lactate to Fr. V-BSA had no significant effect on mean % Na+

LACTATE AND GLUCOSE: EFFECTS IN RAT KIDNEY 113 1001 A SFA Fr. V

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114 J. J. COHEN, Y. J. KOOK AND J. R. LITTLE reabsorption. Addition of 5 mm lactate to SFA increased mean % Na+ reabsorption to 68 %, which was significantly (P < 0 001) higher than the % Na+ reabsorption observed with 5 mm glucose, indicating that each substrate has different quantitative effects on the per cent of the filtered Na+ which is reabsorbed.

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Substrate-limited function and metabolism of the isolated perfused rat kidney: effects of lactate and glucose.

J. Physiol. (1977), 266, pp. 103-121 With 6 text-ftguree Printed in Great Britain 103 SUBSTRATE-LIMITED FUNCTION AND METABOLISM OF THE ISOLATED PERF...
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