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Hepatorenal Reflex Regulating Kidney Function

FLORIAN LANG et al

In anesthetized male rats, infusion of glutamine (2 μmol/min)into the superior mesenteric vein at a rate known to induce liver cell swelling leads to marked decreases in renal glomerular filtration rate, renal para-amino hippurate clearance, and urinary flow rate. Glutamine infused at identical rates into the jugular vein does not elicit any of these effects. The effect of glutamine is mimicked by serine but not by glutamate. Spinal transection, renal denervation, or section of the vagal hepatic nerves abolishes the effect of mesenteric venous glutamine infusion. Mesenteric application of glucagon (1ng/min)or of both glutamine and glucagon enhances glomerular filtration rate and urinary flow rate. Infusion of 1 ng/min glucagon through the jugular vein does not significantly alter the glomerular filtration rate or urinary flow rate. The data disclose a powerful liver-borne mechanism regulating kidney function that is mediated by hepatorenal innervation. (HEPATOLOGY 1991;14:690-594)

Concentrative cellular uptake of certain amino acids, including glutamine, is followed by hepatocyte swelling (1-3), leading to profound alterations of hepatic metabolism (4). This study was performed to elucidate the effects of portal venous glutamine on kidney function. Both clinical (5,6 )and experimental (7, 8)observations suggest the existence of hepatic mechanisms regulating kidney function, and it appeared not unlikely that those mechanisms are triggered by an increase in liver cell volume.
Accordingly, we tested the effect of glutamine infusions on portal circulation. Intestinal venous glutamine infusions markedly decrease glomerular filtration rate (GFR) and urinary output. In contrast, glutamine infused at the same rate into the jugular vein does not significantly alter kidney function.

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MATERIALS AND METHODS

Clearance Experiments. Male Munich Wistar rats (140 to 250 gm; Ivanova's, Kipling, FRG) were anesthetized after an overnight fast with Inaction (120 mg/kg body wt; Byk Gulden Konstanz, FRG). Body temperature was maintained at 37"C. In each group of animals, catheters were placed in both a jugular (or femoral) vein and the superior mesenteric vein draining into the portal vein. The superior mesenteric artery was ligated. An additional catheter was placed in the femoral artery. Initially, the animals were infused at a rate of 20 FVmin through the jugular and superior mesenteric vein, respectively, with a solution containing 150 mmol/L NaCl and 5 mmol/L KCl. In two series (hepatic unilateral renal denervation; see below), 100 mmol/L mannitol was added to the jugular infusate. After 30 min, 50 mmol/L NaCl was replaced with 100 mmol/L glutamine in either the intestinal or jugular venous infusate. Accordingly, identical rates of glutamine infusion (2 FmoVmin)were achieved in the jugular and mesenteric veins. After another 20 min, glutamine was again replaced by NaC1. Considering the portal venous blood flow of 3 ml/min 100 gm body wt (91, the infused rate corresponded to an increase of portal venous glutamine concentration by some 0.4 mmo/L. For comparison, the endogenous portal venous glutamine concentration ranges in fasted rats between 0.3 and 0.6 mmol/L, depending on the acid/base status of the animal (9).

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In additional series, glutamate or serine was infused instead of glutamine through the superior mesenteric vein, or glutamate was infused through the jugular vein. Furthermore, glucagon (1ng/min) was infused with or without glutamine through the superior mesenteric vein, or glucagon without glutamine was infused through the jugular vein. The amount of glucagon thus infused mimicked the glucagon released after protein intake (10, 11)

Additional experiments have been performed to test the involvement of the nervous system. To this end, the effect of mesenteric glutamine infusion has been studied after spinal transection at the cervical thoracic junction, after section of the hepatic branches of the vagus nerve, and after denervation of the left kidney. The latter was achieved by mobilizing the left kidney and stripping and coating the artery with a solution of 10% phenol in 90%alcohol (12).
In the experiments performed after unilateral renal denervation, the urine from the denervated kidney was collected from the ureter with a PE 50 catheter and the urine of the intact kidney was collected with a catheter placed in the bladder. In all other experiments, urine from both kidneys was collected from the bladder.

Four control clearance periods were followed by four experimental clearance periods and four recovery clearance periods(5min each). Urine dead space was accounted for in the calculations. Inulin (0.4 mg/min) and, where indicated, para amino hippurate (PAH; 0.2 mg/min), was infused throughout the experiments, beginning at least 90min before the clearance periods. Inulin clearance was taken as a measure of renal GFR and PAH clearance was taken as an indicator of renal plasma flow. To this end, urinary and plasma inulin (13)and (14) concentrations were determined photometrically. Blood samples were taken at the beginning of the control period and at the end of the recovery period. The average difference of inulin concentrations between the two samples was 9%+-3%; the difference in PAH concentrations was 3%k 2%. Absolute values for GFR and PAH were determined by interpolation of the plasma concentrations. No correction was made for transient changes of plasma concentrations in response to altered GFR or PAH clearance. Thus the respective alterations are slightly underestimated.

In one series, glutamine was infused into either the jugular or mesenteric vein for 3.5hr. Where indicated, femoral arterial blood pressure was monitored continuously with an electronic pressure transducer (Hellige, Gottingen, FRG).

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Perfusion of Isolated Liver, Livers of male Wistar rats (140 I to220gmbodywt) were perfused as described previously(15, 16) in an open (nonrecirculating) system at 37" C with a solution composed of (in mmol/L): NaCl, 116; KC1, 5.9; Solutions were equilibrated with 96% 0, and 4% CO, The perfusion flow rate was approximately4 ml/min/gm liver and was kept constant throughout the individual perfusion experiments. Potassium activity in the hepatic venous effluent was continuously monitored with K+-selective electrodes (Radiometer Copenhagen, Copenhagen, Denmark). Liver weight was determined continuously during the experiments with a specially designed balance pan (16). After a control period of 40 min, glutamine (2 mmol/L) or glucagon (100 pmol/L) were added to the perfusate. Cellular water space (Vc) was determined from the distribution of P'CIurea: livers were equilibrated with C"C1urea and [sHlinulin containing perfusate. Subsequently, the livers were perfused with tracer-free perfusate and the tracers were washed out. Apparent Vc was calculated from:

VC= M^l4C/[^l4C]- M^ЗW[^ЗH]

where M"C andM3H are the amounts of the respective tracers washed out and [l4C1and T3H1are the respective tracer concentrations in the effluent perfusate during steady-state perfusion. During the washout, samples were taken each 30 to 60 sec, and washout was considered complete when the tracer concentrations decreased to less than 0.1%of the radioactivity found at the beginning of the washout.
Stati.tics. Applicable data are expressed as arithmetic mean +- S.E.M.Statistical analysis was made by Student's t-test where appropriate. A p-value <0.05 was considered significant.

RESULTS

As shown in Figure 1 and Table 1, infusion of glutamine (2 μmol min) into the superior mesenteric vein led within 20 min to a decline of renal GFR and urinary flow rate 0. These changes are paralleled by a proportional decrease of PAHclearanceby 67% +- 14%. The ratio of PAH clearance/GFR remained virtually constant The respective values were 3.68 +- 0.42before and 3.50 +- 0.62 (n = 4) during mesenteric glutamine infusion. The effects of mesenteric glutamine infusion were partially reversible(Fig. l), but were sustained for 3 hr (Table 1). During superior mesenteric venous infusion of glutamine, systemic blood pressure increased by 3 & 1mm Hg (n = 4).

In contrast to mesenteric infusion of glutamine, infusion of identical amounts(2 μmol/min)of glutamine into the jugular vein did not significantly interfere with any of the kidney function parameters tested (Fig. 1, Table 1).
Mesenteric infusion of serine (2μmol/min) decreased GFRandV, as did mesenteric infusion of glutamine(Fig. 2, Table 1).In contrast, mesenteric venous infusion of glutamate (2 μmol/min) increased GFR slightly but significantly without significant alteration of V. Jugular venous infusion of glutamate (2μmol/min) did not significantly alter GFR or V. When glutamine (2μmo/min) was infused together with glucagon (1ng/min) into the superior mesenteric vein, GFR and V increased transiently. Glucagon (1ng/min) infused alone into the superior mesenteric vein led to a more sustained increase of GFR and V. Glucagon (1ng/min) infused into a jugular vein did not significantly alter renal function (Fig. 3).

After spinal transection, intestinal venous glutamine infusion (2 μmo/min)did not significantly alter GFR or V. Section of the hepatic branches of the vagus nerve completely abolished the effect of mesenteric infusion of glutamine. As illustrated in Figure 4, denervation of the left kidney abolished the effect of glutamine (2 μmo/min)on GFR and V in that kidney, whereas the effect was preserved in the intact kidney of the same animal.

Addition of 1mmoVL glutamine to the portal venous perfusate of an isolated perfused liver increased liver mass by 2.2% +-0.3%(n = 5)and hepatic cellular water space by 5.7% +- 1.0% (n = 3) and led to a release of cellular K' by 0.8 +- 0.3 pmol/gm liver (n = 3). Glucagon (100μmo/L) decreased liver mass by 2.9% +- 0.9% (n= 5) and cellular water space by 6.5% +- 2.5% (n = 6),paralleled by a release of cellular potassium by 0.9 +- 0.2 μmol/gm liver.

DISCUSSION

Our observations clearly show that glutamine delivered to the liver leads to a marked reduction of renal plasma flow, GFR, and V. The effect of glutamine must be due to a signal originating from the liver since identical rates of glutamine delivered to the jugular or femoral vein do not elicit this effect. The depressive effect of glutamine on kidney function contrasts with the effect of dietary proteins, which are known to enhance GFR ( 5 ). The discrepancy is apparently due to intestinal hormones such as glucagon, which are released on ingestion of proteins and do enhance GFR (10, ll). Similar to that of glutamine, the action of glucagon is mediated by the liver (10). As shown in this study, glucagon is indeed able to reverse the liver-mediated effect of glutamine on the kidney.

The effect of glutamine could be either due to a hepatorenal reflex or to a humoral factor. The observation that the effect of mesenteric glutamine is abolished after spinal transection or hepatic or renal denervation strongly suggests the involvement of renal and hepatic nerves. Evidence for hepatorenal reflexes has been presented earlier (17-23). Accordingly, renal nerve activity is enhanced by an increase in intrahepatic pressure (23)and is believed to account for sodium retention in cirrhosis (18-22,24)

The intrahepatic mechanism of glutamine-induced impairment of renal function could involve liver cell swelling: as shown in several previous papers (2,25271, amino acids do increase liver mass; according to current data, glutamine does increase cellular water space. Glutamine-induced cell swelling is the result of concentrative amino acid uptake into the liver cells (2,27,28). A previous study (27)defined the ability of different amino acids to swell liver cells. Glutamine and serine are among the amino acids that swell liver cells, whereas glutamate does not increase liver mass. Half-maximal .swelling is achieved by 0.5 mmol/L glutamine concentration (27), well in the range of physiological glutamine concentration. Glucagon reverses the amino acid-induced increase of liver mass, apparently by stimulation of cellular potassium release. Thus the effect of the amino acids and glucagon on liver cell volume correlates with their ability to alter renal GFR. Liver cell swelling could increase intrahepatic pressure, which has indeed been shown to increase renal nerve activity (23)

Nevertheless, the evidence for the role of liver cell volume in the initiation of glutamine-induced hepatorenal reflex is circumstantial. Furthermore, the effect of glucagon is not necessarily transmitted by the same mechanism as glutamine but could involve a reflex and/or a humoral factor.

The physiological and pathophysiological significance of the hepatorenal reflex described here cannot be defined from the results of this study. Since cell injury leads to cell swelling (291, the hepatorenal reflex could, theoretically, impair renal function in the course of liver disease. As a matter of fact, the lumbar sympathetic block has been shown to improve renal function in liver disease (30), and the elimination of the mechanism described here could well have contributed to this improvement.

In conclusion, glutamine delivered to the liver leads to marked impairment of kidney function, disclosing a potent mechanism of hepatorenal influence. The effect can be antagonized by denervation or by glucagon.

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