Otto W. Neuhaus

Age-dependent changes in the excretion of urinary proteins by the rat.

The excretion of total urinary proteins (TUP) of rats beginning at weaning and extending to 12 months of age was correlated with the output of the sex-dependent alpha2u-globulin and albumin. At puberty, 40 days of age, the excretion of TUP corresponded to the output of alpha2u-globulin. At this age, alpha2u represented 30% of the total while albumin less than 10%. From 100 to 200 days of age, TUP remained constant while the excretion of albumin steadily increased. After 150--180 days of age, the concentrations of alpha2u and albumin in TUP were approximately equal. Thereafter, the excretion of albumin and TUP increased markedly whereas alpha2u excretion remained constant. At 373 days of age, albumin represented over 50--60% of the TUP while alpha2u was only 6--7%. Female rats which excrete little or no alpha2u exhibited a much lower level of proteinuria than the male during the first year. We suggest the existence of two phases of proteinuria in the male rat, namely, an early physiologic or alpha2u-globulinuric phase and a later albuminuric phase during which increasing quantities of plasma proteins, especially albumin, are lost.

Synergistic effect of glucocorticoids and androgens on the biosynthesis of a sex-dependent protein in the male rat

Abstract Castration of male rats reduced the urinary excretion of sex-dependent α 2 u-globulin by 50%. Replacement therapy with testosterone, dihydrotestosterone, androstenedione, and dehydroepiandrosterone increased the urinary excretion of α 2 u. To determine if adrenal androgen contributions to the excretion of α 2 u, especially in castrated rats, some animals were adrenalectomized. Adrenalectomy, however, almost abolished the excretion of the protein, an effect which could be reversed with glucocorticoid but not androgen or aldosterone. When male rats were adrenalectomized and castrated, then the excretion of α 2 u was obtained only following therapy with both androgen and glucocorticoid. It is suggested that the glucocorticoid is required in a synergistic or permissive sense for the androgen-dependent excretion of α 2 u.

Urinary Excretion of α2u-Globulin and Albumin by Adult Male Rats following Treatment with Nephrotoxic Agents

Nephrotoxic agents such as puromycin aminonucleoside (PAN) and sodium maleate (MAL) have been used to induce experimental glomerular proteinuria and tubular disease, respectively. Current studies show that PAN caused a massive loss of albumin in the urine while not affecting the excretion of the smaller, sex-dependent alpha 2 mu-globulin. On the other hand, MAL which inhibits the reabsorption of proteins, increased the loss of both alpha 2 mu and albumin. Both nephrotoxic agents increased the excretion of albumin when administered to female rats. MAL-induced proteinuria was used as a direct measure of the renal load for alpha 2 mu and albumin. Renal loads and excretion of alpha 2 mu and albumin were measured in male rats maintained on 0, 20, and 50% casein diets. On the protein-free diet, the excretion of both alpha 2 mu and albumin was reduced (less than 1 mg daily); their renal loads were almost totally reabsorbed. On a 50% casein diet the reabsorption of alpha 2 mu was reduced from a normal of 60% to 10% of the renal load. Thus the high protein diet increased the excretion of alpha 2 mu while having little effect on the excretion of albumin. We suggest that dietary protein exerts two levels of control on the excretion of protein in the urine of the adult male rat. Protein-deficient diets stimulate the general reabsorption of proteins thereby minimizing the excretion of alpha 2 mu and albumin. High protein diets appear to reduce selectively the reabsorption of alpha 2 mu-globulin, thereby increasing its excretion in the urine.

Further studies on the isolation and characterization of a sex-dependent protein from the urine of male rats☆

Abstract The major urinary protein (α 2u ) of the adult male rat has been isolated by a combination of (NH 4 ) 2 SO 4 fractionation, gel filtration on Sephadex G-100, and isoelectric focusing. The final preparation was carbohydrate-free, had a molecular weight of 20 000–21 000, an N-terminal glutamyl or glutaminyl residue, and consisted of a family of species varying in isoelectric points from pH 5.0 to 5.8. These species were immunologically identical among themselves and also with a product produced by an earlier procedure.

Renal Reabsorption of Low Molecular Weight Proteins in Adult Male Rats: α2u-Globulin

Abstract Urinary proteins are reabsorbed by the renal tubule cells by two processes, the first for high molecular weight (HMW) and the second for low molecular weight proteins (LMW). The purpose of this report is to establish that α2u-globulin, the sex-dependent, major urinary protein of the adult male rat, is reabsorbed in the kidneys by the general mechanism for LMW proteins. Parameters such as clearance rates were determined to show that α2u is reabsorbed by a process comparable to that for lysozyme. The aminoglycoside, gentamicin, was observed to inhibit the reabsorption of α2u in a dose-dependent fashion. It increased the α2u excretion rate from 4.2 to 13.5 μg/min; the clearance was increased from a normal of 0.33 to 0.91 ml/min. The excretion rate for α2u was also increased by the injection of lysozyme from a normal of 7.4 to 18.1 μg/min. The effect of lysozyme was dose-dependent and reversible. Although gentamicin and lysozyme each increased the excretion of α2u, they had no effect on albumin. Both were equally effective as inhibitors of α2u reabsorption and were 80% as effective as sodium maleate. It is suggested that α2u is reabsorbed by a mechanism which is shared with other LMW proteins. Furthermore, this process is independent of the one which serves to translocate HMW proteins such as albumin.

The effect of dietary protein on the excretion of α2u, the sex-dependent protein of the adult male rat

Abstract Adult male rats were maintained on normal (20% casein), protein-free (0% casein), high protein (50% casein), deficient protein (20% zein), and a supplemented, deficient protein (20% zein plus L -lysine L -tryptophan) diets. Rats on a protein-free diet excreted approximately 1 mg α2u/24 h compared with a normal of 10–15 mg/24 h. Depleted rats placed on a 20% casein diets showed a rapid restoration of the normal α2u excretion as well as total urinary proteins. Accumulation of α2u in the blood serum was measured in nephrectomized rats. Rats on a 0% casein diet accumulated only 30% of the α2u compared to normals. On a 50% casein diet, rats excreted 30–50 mg α2u/24h. However, the accumulation was normal in the serum of nephrectomized rats. A high protein diet did not stimulate α2u synthesis but probably increased the renal loss of all urinary proteins. The excretion of α2u on a zein diet was reduced to the same degree as with the protein-free diet. Supplementation with lysine and tryptophan restored the capacity to eliminate α2u to near normal levels. Accumulation of α2u in the serum of nephrectomized rats kept on the zein diets showed that the effect was to suppress the synthesis of the α2u. Supplementation restored the biosynthesis of α2u. We conclude that the effect of dietary protein on the excretion of urinary proteins in the adult male rat is caused in large part by an influence on the hepatic biosynthesis of α2u. The biosynthesis of this protein, which represents approximately 30% of the total urinary proteins, is dependent on an adequate supply of dietary protein.

Sequential passage of α2u-globulin through the hepatic endoplasmic reticulum and Golgi apparatus during secretion

Studies on the synthesis and secretion of the sex-dependent urinary protein, alpha2mu-globulin, have been extended by establishing its sequential passage from the rough to the smooth endoplasmic reticulum and Golgi-rich fractions of the liver of adult male rats. After injection of 14C-labeled amino acids, the maximum radioactivity of alpha2mu occurred at 20 min in the rough, 25 min in the smooth microsomes and 30 or 35 min in the Golgi-rich fractions. Radioactive alpha2mu-globulin appeared in the bloodstream and kidneys after a lag of 20--25 min. Results indicate that alpha2mu-globulin follows a secretory pathway similar to that of serum albumin.: Studies on the synthesis and secretion of the sex-dependent urinary protein, alpha2mu-globulin, have been extended by establishing its sequential passage from the rough to the smooth endoplasmic reticulum and Golgi-rich fractions of the liver of adult male rats. After injection of 14C-labeled amino acids, the maximum radioactivity of alpha2mu occurred at 20 min in the rough, 25 min in the smooth microsomes and 30 or 35 min in the Golgi-rich fractions. Radioactive alpha2mu-globulin appeared in the bloodstream and kidneys after a lag of 20--25 min. Results indicate that alpha2mu-globulin follows a secretory pathway similar to that of serum albumin.

Multiple forms of α2u, a sex-dependent urinary protein of the adult male rat

Abstract A sex-dependent protein ( α 2u ) which exists as multiple isoelectric forms in the urine of adult male rats has also been partially purified from the liver, blood serum and kidneys. Only one electrophoretic form of the protein was observed in liver and serum, which corresponded to the major urinary component α 2u (5.2). The kidneys, on the other hand, contained several forms of α 2u , one of which is not found in the urine. In addition, at least one urinary species of the protein has not been identified in the kidneys. A possible explanation for the differences in the multiple forms of α 2u is discussed.

The Role of Stimulated Amino Acid Transport in Promoting Glycogenesis in the Irradiated Rat

KILBERG, M., AND NEUHAUS, O. W. The Role of Stimulated Amino Acid Transport in Promoting Glycogenesis in the Irradiated Rat. Radiation Res. 66, 597-608 (1976). Whole-body irradiation of rats stimulates an amino acid transport system in the liver. Another phenomenon observed after exposure to ionizing radiations is the accumulation of hepatic glycogen. The data presented here relate the increased hepatic uptake of amino acids to glycogenesis. Male rats were exposed to two doses of 7y rays, 2500 and 1500 R. Following exposure to 2500 R, the hepatic free amino acids were elevated during the first 48 hr accompanied by a decline in serum levels. At 72 hr the hepatic amino acids diminished to the control levels while the serum increased abruptly. By contrast, 72 hr after exposure to 1500 R the serum amino acid levels increased only 27% and the hepatic amino acid values increased 52%. These results are explained on the basis of the changes in AIB transport previously reported. The incorporation of 14C from labeled L-alanine into hepatic glycogen was maximal 48 hr postexposure to 2500 R but declined to below control values at 72 hr. On the other hand, exposure to 1500 R resulted in maximal incorporation of 14C at both 48 and 72 hr. We propose that transport of amino acids into liver cells is stimulated by the elevated blood levels of amino acids released from the degradation of protein. The transport increases the levels of hepatic free amino acids, and therefore, is a key factor in regulating postirradiation glycogenesis.

A single, stimulatable transport system in rat liver shared by certain amino acids following whole-body gamma-irradiation.

Exposure of rats to whole-body γ irradiation stimulated the hepatic transport of α-aminoisobutyric acid, N-methyl α-aminoisobutyric acid, cycloleucine, L-methionine, and glycine by a common Na+-dependent transport system. This was demonstrated using reciprocal inhibition transport studies involving liver slices from normal and irradiated rats. The five amino acids studied were mutually inhibitory of each others transport in both normal and irradiated animals. Although the hepatic accumulation of L-phenylalanine was not stimulated by γ irradiation, it effectively inhibited the stimulated transport of AIB, methionine, cycloleucine, and glycine. In normal rats it inhibited the Na+-dependent systems for AIB, cycloleucine, and L-methionine to different degrees.