Nephrotic Liver Research Articles

Albumin gene transcription is enhanced in liver of nephrotic rats.

The level of albumin mRNA and the transcription rate of the albumin gene were studied in the liver of control rats and rats with nephrosis induced by injection of the aminonucleoside of puromycin. Total RNA was extracted from liver by the guanidium thiocyanate method. The albumin mRNA level was measured by cDNA-RNA dot-blot hybridization, and the transcription rate of the albumin gene was measured by the "run-on" transcription assay using isolated nuclei. Urinary protein excretion in nephrotic rats was significantly higher than in control rats (258 +/- 132 vs. 12 +/- 2 mg/day), and the serum albumin concentration in nephrotic rats was significantly lower. There was no difference in body weight, liver weight, serum creatinine, or urea nitrogen between the two groups. Both the level of albumin mRNA and the transcription rate of the albumin gene in the nephrotic liver were about twice as high as those in the control liver. There was no difference in the level of beta-actin mRNA between the two groups. Northern blot analysis showed that both putative precursor RNA and the mature form of albumin mRNA were increased in nephrotic rats. We conclude that albumin synthesis is increased in the liver of nephrotic rats and a transcriptional process is responsible for this increase.

Lipoprotein metabolism in experimental nephrosis

Proteinuria, hypoalbuminemia, edema, and hyperlipemia are the main features of the nephrotic syndrome. The proteinuria, which leads to the other characteristics of the syndrome, is caused by increased permeability of the glomerular capillary to plasma proteins. The glomerular filtration apparatus is a complex barrier formed by the surface glycoproteins of endothelial and epithelial cells which enclose the glomerular basement membrane (1). Damage to the filtration apparatus occurs in several human diseases, such as disseminated lupus erythematosus, interstitial glomerulosclerosis (seen in diabetes), idiopathic lipoid nephrosis, and others (2). It can also be produced experimentally by the intravenous injection of heterologous and homologous antibodies to glomerular glycoprotein antigens (3) or by administration of puromycin aminonucleoside (PAN) (4). The glomerular filtration apparatus is ordinarily more permeable to uncharged macromolecules than it is to proteins of the same size carrying a negative charge at pH 7.4. The loss of this charge selectivity, as well as the change in the size-selective barrier, is responsible for the proteinuria in nephrosis (5). Because it is much smaller than most plasma globulins, and because it comprises more than half of the total plasma proteins, plasma albumin is the main urinary protein found in nephrosis. The hyperlipemia in the rat is a reflection of a 4to 8-fold increase in the plasma concentration of all of the lipoprotein density classes (6). With the exception of HDLs , all of the other plasma lipoproteins are too large to escape the damaged glomerular capillary. Human HDL,, with an average diameter of about 70 A is found in the urine of patients with nephrotic syndrome in appreciable amounts, so that human subjects with nephrosis may have normal plasma levels of HDL as a result of increased synthesis (7). In rats, however, most of the HDL is HDL2 with a diameter of about 110 A and its urinary loss is only 1.8 mg of cholesterol, or 10% of the plasma HDL cholesterol pool, per day (8). Gherardi and co-workers (9) reported that in a group of seven children averaging 10 yr of age, and total serum cholesterols of about 580 mg/dl, the plasma concentrations of VLDL, IDL (1.006 < d < 1.019 g/ml), and LDL were, respectively, 6.3, 5.3, and 3.0 times normal. In contrast, HDL2 and HDLs were 0.6 and 0.8 of normal. There were changes in lipid composition in all density fractions; perhaps the most striking changes were in the d < 1.063 g/ml fractions where the phospholipid and esterified cholesterol were increased and the free cholesterol and protein were decreased. In VLDL, cholesteryl esters tended to replace triglyceride. In nephrotic rats, similar but less striking changes have been observed (6, IO). As we shall point out later on, these changes in lipid composition may be related to a decreased lipoprotein lipase and hepatic lipase activity. At about the time the Journal of Lipid Research was born, David L. Drabkin and I put forth the hypothesis (1 1) that the hyperlipemia of the nephrotic syndrome was due to hepatic overproduction of plasma lipoproteins, which were too large to be lost readily in the urine. The evidence in support of hepatic overproduction of lipoproteins was obtained in the isolated perfused rat liver (11). Studies by Radding and Steinberg (12) with liver slices reached the same conclusion. The increased synthesis of the protein moiety drives the hepatocyte to increase de novo lipid synthesis. Increased hepatic synthesis of total fatty acids, cholesterol, and triglycerides (13, 14) has been reported, but not phospholipids (15). In collaboration with Dr. Charles E. Sparks, we have confirmed the findings of increased hepatic synthesis of lipoproteins in nephrosis and extended them to include the individual apolipoproteins (16). Table 1 summarizes these results and compares them to those obtained two decades earlier with a different method of induction of nephrosis (anti-kidney serum) and a different methodology. Perhaps the theory of cancelling errors applies, but it is nevertheless gratifying to note the agreement between the two studies. The studies shown in Table 1, and similar observations reported by Calandra et al. (1 7), point to a far greater percentage increase in apoAI synthesis by nephrotic liver than for the other apoli-

Secretion of lipoproteins, apolipoprotein A-I and apolipoprotein E by isolated and perfused liver of rat with experimental nephrotic syndrome

Nephrotic syndrome induced in the rat by the administration of puromycin aminonucleoside is accompanied by a hyperlipoproteinemia characterized by an elevation of all plasma lipoproteins, particularly of VLDL (1.006 g/ml) and HDL 1 (1.050–1.090 g/ml). The increase of HDL 1 is due to the accumulation of a lipoprotein species floating mainly in the density interval 1.050–1.090 g/ml, in which apolipoprotein A-I replaces apolipoprotein E as the major constituent peptide. This lipoprotein has been designated nephrotic HDL. The present study was conducted to establish whether nephrotic liver secreted more lipoproteins than the control liver and, in addition, produced a lipoprotein similar to nephrotic HDL found in plasma. Isolated livers from control and nephrotic rats were perfused with a lipoprotein-free medium for 3 h in a recirculating system. Lipoproteins were isolated by ultracentrifugation; apolipoprotein A-I and apolipoprotein E were measured in the whole perfusate at various time intervals. Nephrotic liver secreted twice as much VLDL and HDL 2 and 30% more LDL and HDL 1 than the control liver. This was accompanied by an increased secretion of both apolipoprotein A-I and apolipoprotein E, the levels of which were 6.5- and 2-fold, respectively, of those found in the control perfusates at the end of the perfusion. In view of the increased secretion of apolipoprotein A-I, the apolipoprotein A-I to apolipoprotein E ratio was much higher in the perfusates of nephrotic livers than in those of the controls. The concentration of apolipoproteins A-I and E in plasma of nephrotic rats was 7- and 2-fold, respectively, of that found in the plasma of the controls. In the perfusates of the nephrotic livers, we could not find a HDL 1 (1.050–1.090 g/ml) rich in apolipoprotein A-I similar to that isolated from plasma (nephrotic HDL). We suggest that the latter is formed in the circulation from the intravascular modification of HDL 2 secreted in excess by the liver.

Experimental nephrotic syndrome in the rat induced by puromycin aminonucleoside: hepatic synthesis of lipoproteins and apolipoproteins.

Hepatic synthesis of lipoproteins and apolipoproteins was investigated in male Wistar rats with severe nephrotic syndrome induced by puromycin aminonucleoside by incubating liver slices with a mixture of 14C-amino acids. Labeled lipoproteins were separated by preparative ultracentrifugation from the incubation medium after the addition of carrier plasma. The incorporation of 14C-amino acids into very low density lipoproteins (VLDL) (1.006 g/ml), low density lipoproteins (LDL) (1.006-1.063 g/ml) and high density lipoproteins (HDL) (1.063-1.210 g/ml) was increased in nephrotic liver 6.1-, 5.7- and 5.0-fold, respectively. The measurement of radioactivity associated to apolipoproteins isolated by SDS-PAGE documented an increased incorporation into apolipoprotein E (apoE) of nephrotic VLDL (33.1% vs 20% of the total radioactivity incorporated into VLDL apoproteins) and a markedly increased incorporation into apolipoprotein A-I (apo-A-I) of nephrotic HDL (44.3% vs 16.3% of the total radioactivity incorporated into HDL apoproteins). In nephrotic liver, the total incorporation of amino acids into apolipoproteins (apoVLDL + apoLDL + apoHDL) was increased 12.6 times for apo-A-I, 6,4 times for apoB, 5.0 times for apoE, 4.2 times for apoC + apoA-II and 2.5 times for apoA-IV. We suggest that, in nephrotic liver: (a) the synthesis of VLDL, LDL and HDL is increased, and (b) the total synthesis of apoA-I is selectively increased when compared to that of the other apolipoproteins.

Factors in the Stimulation of Protein Synthesis by Subcellular Preparations from Rat Liver

Abstract The control of protein synthesis has been investigated in subcellular systems from livers of rats with plasma protein overproduction following nephrotoxic serum. The following observations were made. The microsomal fraction from nephrotic liver exhibited an increased synthesis of acid-insoluble protein and of plasma albumin, as measured by amino acid incorporation per mg of ribonucleic acid present. The synthesis of plasma albumin showed a much greater increase than did the synthesis of other proteins. This is in contrast to the situation in regenerating liver, in which the increase in plasma albumin synthesis was of the same order of magnitude as the increase in acid-insoluble protein. Ribosomes or polysomes, prepared from nephrotic liver with deoxycholate, did not differ from normal liver in their ability to synthesize protein. This finding, together with the observation that mixtures of control and nephrotic microsomal fractions behaved like the nephrotic system, suggested that the difference between control and nephrotic microsomes was not the result of any inherent difference in ribosomes but might be due to the presence of additional amounts of messenger RNA. Polysomes prepared from nephrotic liver without the use of deoxycholate but still containing small amounts of attached membrane protein showed slight but significant increases in plasma albumin synthesis, averaging 33%. Cell sap from nephrotic liver was more active than that from controls in promoting protein and plasma albumin synthesis by microsomes. This difference was ascribed to the presence of increased amounts of messenger RNA in this fraction on the basis of indirect evidence.

Stimulation of protein and plasma albumin synthesis in a cell-free system from livers of nephrotic rats

An accelerated hepatic synthesis of plasma proteins in rats with nephrosis, produced by means of anti-kidney sera, has been demonstrated in the intact animal (1), the isolated, perfused liver (2), and in liver slices (3). We have now investigated the incorporation of labeled amino acids into microsomal protein by an anaerobic microsome-supernatant fluid system similar to that employed by Campbell et al (4). The microsome-supernatant fluid system from nephrotic liver has been found to be about twice as active as that from the control. The RNA content of mcirosomes from nephrotic liver was elevated; the ratio of RNA to protein was increased from 0.12 in the control to 0.20 in the nephrotic microsomes. However, the increase in activity of the nephrotic system appeared to be largely due to the 100,000 g supernatant fraction.