Changes in some chromatin and cytoplasmic enzymes of perinatal rat hepatocytes during culture.

Hepatocytes prepared from rats at various perinatal stages were cultured in selective medium that does not allow fibroblastic cell growth. Cell population remained homogeneous during the culture. Hepatocytes undergo divisions for a period, which varies according to the stage of development of the rat. Light and electron microscope observations showed the presence of numerous cytoplasmic organelles; moreover, hydrocortisone-induced structures similar to bile canaliculi. Chromatin protein kinase decreased rapidly during culture except in samples prepared from 17-day fetuses in which it remained unchanged for 2 days and decreased to a lesser extent afterwards. Chromatin nonhistone proteins were incubated with (gamma-32P) ATP and the phosphorylation pattern analyzed on polyacrylamide gels. Many radioactive peaks were observed in chromatin proteins from 17-day fetuses; they were much lower in proteins than 19-day fetuses. The phosphorylation pattern was analyzed in hepatocytes after 2 days of culture. Many radioactive peaks were observed with proteins from hepatocytes taken from 17-day fetuses; no radioactivity was observed in proteins from 19-day fetuses. This is in contrast with the absence of radioactive peaks in chromatin proteins from adult rat hepatocytes. In cytoplasm, aldolase and pyruvate kinase specific activities varied according to the age of the rat. They strongly decreased during culture except in hepatocytes and 15- and 17-day fetuses, in which they remained stable for a least 5 days. The stability of chromatin and cytoplasmic enzymes in hepatocytes from 17-day fetuses could result from their ability to be regulated by hormones that are secreted at this stage of development.

Relevance of enzyme evaluations in 24h urine to rat kidney injury caused by i.v. cephaloridine injection.

Male rats were housed singly in metabolic cages, injected i.v. with cephaloridine, 24 h urine samples collected successively; then the rats were killed for obtaining the kidneys of corresponding animals. The concentrations of protein, aminopeptidase (AP), alkaline phosphatase (aPP), lactic dehydrogenase (LDH), and aldolase (ALD) were determined in urine and the percentages of injured proximal tubules counted in sections stained for aPP. The results from individual animals were: (1) After placing animals singly in metabolic cages large but not systematic changes of urinary enzyme concentrations occurred. After 6-10 days the enzymes reached steady state levels. (2) After a single injection of cephaloridine a dose dependent injury of proximal tubules was observed, the urinary LDH content correlating best with the tubular injury (r greater than 0.93) and giving up to 1,000 fold increases above normal values. (3) A circadian rhythm of the susceptibility of rat kidney for cephaloridine was observed, the smallest response was seen when the animals were injected at 7 a.m. and the largest after injection at 7 p.m. (4) In subacute toxicity studies urinary LDH was increased on day 2 above the extent after a single dose, but declined on day 3 to reach normal levels after 8 to 10 days (time of sacrifice). The kidneys revealed practically normal histology. The other enzymes studied had also returned to normal values. This indicates some adaptation mechanism.

Probenecid and the rat kidney: investigations by renal enzyme excretion technique.

Probenecid in doses of 640 mg/kg was administered to rats by the oral route, and the changes in five important enzymatic activities of urine were recorded thereafter for two days. The resluts exclude that probenecid impairs tubular reabsorption of low molecular weight protein, as urinary muramidase activity was not found increased. On the other hand, increased activities were encountered in those enzymatic activities in urine which derive from the renal tubular cells (ALD, G-6-PDH, LDH). These observations point towards a nephrotoxic effect of probenecid, which, however, is only of very low degree, as other "standard" enzymatic activities of urine, such as alkaline phosphatase, remained unchanged.