Scand. J. cliii. Lab. Invest. 37, 477-486, 1977.

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Studies on renal tubular protein reabsorption: partial and near complete inhibition by certain amino acids C. E. M O G E N S E N & K . S 0 L L I N G Second and First University Clinic of Internal Medicine, Kommunehospitalet, DK-8000 Aarhus C , Denmark

Mogensen, C.E. & Sdling, K. Studies on renal tubular protein reabsorption: partial and near complete inhibition by certain amino acids. Scand. J. clin. Lab. Invest. 37, 477-486, 1977. Urinary excretion of albumin, free light chains of immunoglobulins and beta-2microglobulin was measured after injection of certain amino acids and derivatives. Substances with a positively charged group located terminally in the molecule (ornithine, lysine, arginine, and the almost not metabolized epsilon-aminocaproic acid, and Cyclocaprone@)proved to inhibit instantaneously the tubular protein reabsorption. Lysine is the most effective molecule tested. We hypothesize that the initial event in the normal tubular protein reabsorption is a binding between a free positive amino- or guanidino-group in the protein molecule and a negative site on the tubular cell surface. In an attempt to achieve complete inhibition of tubular protein reabsorption, large doses of lysine were injected intravenously. The maximal protein clearance rose from the exceedingly low baseline excretion to values probably representing near complete inhibition of protein reabsorption. The minimal value of albumin concentration in the glomerular filtrate was calculated to 2.6 mg/l. Key-words: albumin, amlno acids, beta-2-microglobulin, kidney function test, kidney glomerulus, kidney tubule, light chains, proteinuria Dr C . E. Mogensen, Second University Clinic of Iuternal Medicine, Kommmehospitalet, DK-8000 Aarhus C, Denmark

The proteins of the blood plasma are generally retained by the glomerular capillaries but a very small part is filtered through the glomerulus and subsequently reabsorbed almost completely by the proximal tubular cells [3, 81. However, the mechanisms governing tubular protein reabsorption in the kidney are poorly understood, and neither the rate of reabsorption nor glomerular filtration of plasma proteins has been quantitated. A year ago we observed that injection of arginine in man resulted in greatly increased

urinary protein excretion, returning to normal levels within 1 h [13]. As the excretion of the small beta-2-microglobulin molecule, freely filtered by the glomerulus, was much more pronounced than the excretion of albumin, it was clear that the mechanism behind the increase after arginine administration was not due to changes in glomerular filtration of proteins but to inhibition of the tubular protein reabsorption. Starting from this observation we have carried out a series of experiments with a number of amino acids and derivatives, and 477

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C. E. Mogensen C K . Solling

have obtained results that justify the putting forward a general hypothesis about the primary event in tubular protein reabsorption. The normal rate of glomerular filtration and tubular reabsorption of plasma proteins is not known. These important rates could be determined if complete inhibition of reabsorption were possible by amino acids. The present report includes experiments with large doses of lysine used in an attempt to obtain complete inhibition. It appears that the transport rates are very large compared to the basal urinary excretion.

MATERIAL A N D METHODS Experimental design

The studies were performed in the morning on non-fasting, young male subjects (medical students and doctors) examined in the supine position except when voiding. To promote urine output 250 ml of tap water was given every 20 min throughout the experiments, starting 14 h before the first control urine collection period. The studies are presented in two sections, one dealing with the molecular configuration required for an effect on protein reabsorption, and the other describing experiments where complete blocking of reabsorption was attempted by large lysine injection. Molecular configuration studies. The following substances were tested by intravenous administration: dibasic amino acid and derivatives : ornithine, lysine, arginine, epsilon amino caproic acid (EACA), tranexamic acid (Cyclocapronem) and citrulline. Other amino acids : glycine, alpha-alanine, leucine, aspartic acid, methionine, phenylalanine, valine, proline, histidine and tryptophan. The L form of amino acids was used in all cases. Osmotically active substances: mannitol and urea. The effect of these compounds on protein excretion was studied by determining the urinary excretion of albumin, free light chains of immunoglobulins and beta-2-microglobulin [131. Eight amino acids and two osmotically active substances (ornithine, citrulline, lysine, arginine, EACA, leucine, cyclocaprone, alpha-alanine, mannitol and urea) were tested in four sets of experiments, each one being performed on five

subjects. In each set of experiments three of the ten substances were tested and two substances were tested twice. To establish baseline conditions urine was collected in two periods of 20 min. Thereafter the first compound to be examined was given by intravenous injection over a period of 2 min. Following the injection urine was collected for two or three periods of 20 min each. Then equimolar doses of two other compounds were injected successively, according to the same schedule (see Figs 1-3, and Table I). Equimolar amounts of nine other amino acids (valine, proline, aspartic acid, phenylalanine, histidine, glycine, methionine, tryptophan and arginine) were tested in only two sets of experiments, each again being performed on five subjects. The experimental set-up was as above. Some of the amino acids are less soluble and the series of substances were therefore given in smaller amounts (Table 11). In the methionine and tryptophan experiments, 5 min injection time and 10 min collection periods were used (Table 111). A modified design was used to study the earliest events after administration of an amino acid (Table IV). Lysine was chosen for this purpose and three subjects were examined. The experiments started as usual with two 20 min baseline periods. 5 min later 3 g lysine were injected intravenously in the course of 1 min. 2 min later (due to the renal transit time) urine was voided, resulting in an 8 min period. This was followed by four periods of 2 min each, and finally by two periods of 20 min each. lZ5Ilabelled iothalamate (10 pC) was injected together with lysine in these experiments as a filtration marker, not being reabsorbed in the tubules [12]. Large dose lysine injection experiments. Urine

samples were collected every 20 min startingwith three control periods. Increasing amounts of lysine chloride as a 33 % solution were injected intravenously in each experiment in an attempt to obtain complete inhibition of tubular protein reabsorption. Two or three of the following doses of 16.5, 24.8, 33.0, 41.3 g of lysine were given with intervals between 40 and 100 min. 10 ml of the solution was given per minute late in a 20 min period. A blood sample was taken in the middle of each period for analysis of the substances indicated below.

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Amino acids and tubular protein reabsorption

Glomerular filtration rate (GFR) and renal plasma flow (RPF) were determined by constant infusion technique throughout the experiments as described previously [12]. Results were corrected to 1.73 m2 body surface. Albumin, light chains, beta-2-microglobulin, transferrin, ceruloplasmin and IgG as well as lysine, sodium, potassium, creatinine, urea, calcium and phosphate were determined on all the plasma and urine samples. Total CO, and total protein were measured on plasma samples and pH was estimated in the urine samples. ANALYTICAL METHODS Urinary proteins. Albumin, free light chains and beta-2-microglobulin were measured by radioimmunoassay [2, 11, 181. No crossreactivity with the effective amino acids was noted. The beta-2-microglobulin assay was modified using filtration by 0.65 pm Micropore filters (DAWP 04700) for separation. Transferrin, ceruloplasmin and IgG were measured by Rocket-immunoelectrophoresis a.m. Laurell [6]. Before electrophoresis the urine was concentrated in an Amicon ultrafiltration cell using a PM 30 or a UM 10 membrane. Albumin, measured by radioimmunoassay before and after concentration, was used as concentration index. The urine was concentrated from 21 to 1210 times. Furthermore the urinary protein pattern after large lysine injections was investigated by electrophoresis on cellulose acetate using barbitate buffer, pH 8.6. Serum proteins. Beta-2-microglobulin and light chains were measured by radioimmunoassay as mentioned above [2, 181. Albumin, transferrin, ceruloplasmin and IgG were measured by Rocket-immunoelectrophoresis a.m. Laurell [61.

Estimation of Iyine in serum and urine was performed on a Technicon@Sequential MultiSample Analyser. Other substances: sodium, potassium, creatinine, urea, phosphate, calcium in urine and serum, total-protein and ‘total CO,’ in serum, and pH in urine was determined by standard methods. Spearman’s test for correlation analysis and paired t test were used as statistical methods.

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RESULTS MolecuIar configuration studies

Table I shows the effect on urinary albumin, light chain and beta-2-microglobulin excretion of (a) dibasic amino acids (ornithine, lysine, and arginine), of (b) substances with a terminal amino group, but without an alpha-amino group (EACA and cyclocaprone), and of (c) substances of related structure (citrulline and leucine). The figures on Table I only indicate the values obtained during the injection periods. Figs 1-3 shown examples of complete experiments. It appears from Table I and Figs 1-3 that ornithine, lysine, arginine, EACA and cyclocaprone are all effective in producing increased protein excretion. The most pronounced effect was found on the excretion of beta-2-microglobulin ; when small doses were used, 1 g EACA and equimolar doses of leucine and cyclocaprone, no effect on albumin excretion was found. A significant increase in light chain excretion is found in all cases with increase in beta-2-microglobulin excretion, but quantitatively the excretion of the latter substance is most pronounced. Lysine is the most effective substance tested, especially with regard to effect on beta-2-microglobulin excretion, where it is three to six times more effective than ornithine, arginine and EACA. Tables I1 and I11 show beta-2-microglobulin excretion after injection of the smaller amounts of a number of other amino acids. Arginine was used as control for effectiveness of the applied molar dose. Only arginine proved active. By the small doses of amino acids used in this series of experiments no effect of the amino acids, including arginine, was noted on albumin and light chain excretion. Alphaalanine, mannitol, and urea were tested together and no effect was demonstrated on excretion of any of the three proteins. Table IV shows the instantaneous appearance of [ Z51]iothalamate and increased excretion of beta-2-microglobulin after lysine administration along with [‘Z51]iothalamate. Large dose lysine injection studies

Figure 4a-f, shows the relationship between the lysine concentration in serum and the protein clearances as per cent of G F R for six

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TABLE I. Albumin, light chains, and beta-2-microglobulin excretion in pg/min (with SD in parentheses) after injection of equimolar doses of amino acids in each series ( n = 5 ) . Only peak-values shown

Albumin Light chains Beta-2-microglobulin

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Ornithine (2.4 g) 5.8 (2.4) NS 20.9 (9.6)** 2.37 (1.7)* Arginine (6 g) 24.6 (12.1)* 21.2 (5.2)*** 7.3 (4.6)** EACA (1 g) 4.9 (1.7) NS 27.7 (6.0)* 0.331 (0.190)*

Baseline 6.2 (3.2) 5.9 (1.5) 0.050 (0.029) Baseline 9.5 (4.7) 4.1 (2.7) 0.084 (0.037) Baseline 4.6 (1.4) 11.0 (4.9) 0.059 (0.02)

Albumin Light chains Beta-2-microglobulin

Albumin Light chains Beta-2-microglobulin

NS, not significant; *, 2 P

Studies on renal tubular protein reabsorption: partial and near complete inhibition by certain amino acids.

Scand. J. cliii. Lab. Invest. 37, 477-486, 1977. Scand J Clin Lab Invest Downloaded from informahealthcare.com by University of Sussex Library on 01/...
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