Mona Qulali

Estradiol induces class I alcohol dehydrogenase activity and mRNA in kidney of female rats

Rat kidney contains alcohol dehydrogenase (ADH) activity which appears to be similar or identical to the class I ADH expressed in liver. Both tissues contain a 1.6-kb transcript which hybridizes with an ADH cDNA under stringent conditions. Kidney ADH activity is responsive to estradiol. The enzyme activity in the kidneys of sham-operated and ovariectomized animals was the same. Treatment of either group of animals by intramuscular injection of estradiol (1 mg/kg body wt/day) for 10 days induced ADH activity in kidney two- to threefold, whether the activity was expressed as U/g tissue, U/g protein, or U/mg DNA. Estradiol induced kidney ADH mRNA in both ovariectomized and sham-operated rats approximately twofold. Thus, induction of ADH mRNA accounts for the increase in ADH activity. In situ hybridization indicated that the ADH mRNA was present in the inner cortex and medulla of the kidney. Methylation patterns of the ADH gene were examined. The gene resides in a methylated region of chromatin without any of the typical features of a HpaII tiny fragment (HTF) island. Two MspI sites flanking the transcription start site are undermethylated in liver compared with kidney and spleen. This suggests that methylation of this gene may play a role in the tissue-specific expression of ADH.

Estradiol regulates class I alcohol dehydrogenase gene expression in renal medulla of male rats by a post-transcriptional mechanism

Rat kidney contains alcohol dehydrogenase (ADH) activity which appears to be identical to the class I ADH expressed in liver. Treatment of male rats with estradiol for 10 days induced ADH activity and protein in the kidney approximately 3-fold. This was not the result of suppression of testosterone levels by estrogen, as castration did not increase ADH activity. In situ hybridization of kidney sections showed that ADH transcripts were localized to the medulla, that the basal level of mRNA is very low in the male, and that the induction of ADH mRNA by estradiol was approximately 10-fold. As estimated from Northern blot analysis, the induction of the mRNA was approximately 7-fold. Thus, induction of ADH mRNA substantially exceeded the increase of ADH activity and protein. Since the estradiol-treated rats lost weight relative to the oil-injected controls, the effect of starvation on ADH mRNA in kidney was examined. Starvation decreased kidney ADH activity by about 30% but increased mRNA about 2-fold. Time course experiments demonstrated induction of ADH mRNA by estradiol within 1 h with the maximum level achieved by 24 h. The transcription rate of the ADH gene as assessed by nuclear run-on assays performed at 1 and 24 h after treatment with estradiol was unchanged. We conclude that estradiol induces ADH mRNA in kidney by a post-transcriptional mechanism.

STUDIES OF RENAL INJURY : II. ACTIVATION OF THE GLUCOSE TRANSPORTER 1 (GLUT1) GENE AND GLYCOLYSIS IN LLC-PK1 CELLS UNDER CA2+ STRESS

Injury to the renal proximal tubule is common and may be followed by either recovery or cell death. The survival of injured cells is supported by a transient change in cellular metabolism that maintains life even when oxygen tension is reduced. This adaptive process involves the activation of the gene encoding the glucose transporter GLUT1, which is essential to maintain the high rates of glucose influx demanded by glycolysis. We hypothesized that after cell injury increases of cell Ca2+ (Ca2+i) initiate the flow of information that culminates with the upregulation of the stress response gene GLUT1. We found that elevations of Ca2+i caused by the calcium ionophore A23187 activated the expression of the GLUT1 gene in LLC-PK1 cells. The stimulatory effect of Ca2+i on GLUT1 gene expression was, at least in part, transcriptional and resulted in higher levels of GLUT1 mRNA, cognate protein, cellular hexose transport activity, glucose consumption, and lactate production. This response was vital to the renal cells, as its interruption severely increased Ca2+-induced cytotoxicity and cell mortality. We propose that increases of Ca2+i initiate stress responses, represented in part by activation of the GLUT1 gene, and that disruption to the flow of information originating from Ca2+-induced stress, or to the coordinated expression of the stress response, prevents cell recovery after injury and may be an important cause of permanent renal cell injury and cell death.

Endocrine Regulation and Methylation Patterns of Rat Class I Alcohol Dehydrogenase in Liver and Kidney

Alcohol dehydrogenase (ADH) is expressed in a tissue-specific fashion and its activity is modulated by several hormones. Class I ADH activity is largely confined to the liver, with considerably smaller levels in the kidney. Experiments with intact animals indicate that thyroid hormone and testosterone reduce the activity of the enzyme in liver (Mezey and Potter, 1981; Rachamin et al., 1980), and that estrogen and growth hormone increase the activity (Teschke et al., 1986; Mezey and Potter, 1979). In primary hepatocyte cultures, growth hormone produced an increase in ADH activity and mRNA levels, and dihydrotestosterone reduced the activity (Mezey et al., 1986a, 1986b; Potter et al., 1989). Certain hepatoma cell lines express low levels of ADH activity and mRNA, and these can be induced by exposure to glucocorticoids (Wolfla et al., 1988; Dong et al., 1988). The enzyme activity in the kidney is induced by androgens in the mouse (Felder et al., 1988) and by estrogen in the rat (Dembic and Sabolic, 1982). Although the effect of androgens in mouse kidney is known to be mediated by an increase in the transcription of the gene, the mechanism of the effect of estrogens in rat kidney has not been reported.

MODULATION OF HEPATIC AND RENAL ALCOHOL DEHYDROGENASE ACTIVITY AND mRNA BY STEROID HORMONES IN VIVO

Alcohol dehydrogenase (ADH) catalyzes the flux-determining step in the oxidation of ethanol and probably also plays a role in the metabolism of steroid alcohols and retinoids. Five classes of ADH have been discovered to date (Ehrig et al., 1990). The class I enzyme is the major liver enzyme, which is also found in other tissues at lower levels. This class of ADH has been studied in greatest detail. The gene for class I ADH has been cloned from humans (Duester et al. 1986), mice (Zhang et al., 1987), and rats (Crabb et al., 1989). In vitro studies have identified promoter elements important for liver specific expression (C/EBP and HNF1 binding sites (Potter et al., 1991; Stewart et al., 1990a; Stewart et al., 1990b; Stewart et al., 1991; van Ooij et al., 1992)) as well as hormone response elements. There are glucocorticoid response elements in the promoter (Duester et al., 1986), and expression of the gene is activiated in hepatoma cells exposed to dexamethasone (Wolfla et al., 1988; Dong et al., 1988; Winters et al., 1990). Retinoic acid response elements are present in the mouse and human ADH3 gene (Duester et al., 1991), and transfection studies demonstrate retinoic acid responsiveness of the promoter. Androgens induced mouse kidney ADH many fold by an effect that is mainly transcriptional (Ceci et al., 1986; Felder et al., 1988). Other hormone responses are less well understood. Estradiol is reported to induce ADH activity in the kidney (Qulali et al., 1991; Dembic and Sabolic, 1982), but not liver, of rats, and thyroid hormone reduces liver ADH activity (Potter and Mezey, 1983; Mezey and Potter, 1981).