Effects of chronic hypoxia on renal renin gene expression in rats

Frank Schweda, Friedrich C. Blumberg, Annette Schweda, Martin Kammerl, Stephan R. Holmer, Günter A. J. Riegger, Michael Pfeifer and Bernhard K. Krämer

Klinik und Poliklinik für Innere Medizin II, Klinikum der Universität Regensburg, Regensburg, Germany

Correspondence and offprint requests to: Dr Bernhard K. Krämer, Klinik und Poliklinik für Innere Medizin II, Klinikum der Universität Regensburg, D-93042 Regensburg, Germany.



   Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. The effects of hypoxia on renin secretion and renin gene expression have been controversial. In recent studies, we have demonstrated that acute hypoxia of 6 h duration caused a marked stimulation of renin secretion and renal renin gene expression. This hypoxia-induced stimulation of the renin–angiotensin system might contribute, for example, to the progression of chronic renal failure and to the development of hypertension in the sleep-apnoea syndrome. For this reason, we were interested in the more chronic effects of hypoxia on renal renin gene expression and its possible regulation.

Methods. Male rats were exposed to chronic normobaric hypoxia (10% O2) for 2 and 4 weeks. Additional groups of rats were treated with an endothelin ETA receptor antagonist, LU135252, or a NO donor, molsidomine, respectively. Systolic blood pressure and right ventricular pressures were measured. Renal renin, endothelin-1 and endothelin-3 gene expression were quantitated using RNAase protection assays.

Results. During chronic hypoxia, haematocrit increased to 72±2%, and right ventricular pressure increased by a mean of 26 mmHg. Renal renin gene expression was halved during 4 weeks of chronic hypoxia. This decrease was reversed by endothelin receptor blockade (105 or 140% of baseline values after treatment for weeks 3–4 or 1–4). Furthermore, there was a trend of increasing renal endothelin-1 gene expression (to 173% of baseline values) after 4 weeks of hypoxia. Systolic blood pressure increased moderately during 4 weeks of chronic hypoxia from 129±2 to 150±4 mmHg. This blood pressure increase was higher in rats treated for 4 weeks with an endothelin receptor antagonist (196±11 mmHg).

Conclusions. Chronic hypoxia (in contrast to acute hypoxia) suppresses renal renin gene expression. This inhibition presumably is mediated by endothelins.

Keywords: chronic hypoxia; endothelin; nitric oxide; renal renin expression



   Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aldosterone secretion consistently has been shown to be reduced by hypoxia, due to direct inhibition of secretion from adrenal glomerulosa cells [1]. However, the effects of hypoxia on renin secretion (as reflected by plasma renin activity) and renin gene expression are less clear [211]. The influence of hypoxia may be modulated by sodium balance [3,12], since most studies demonstrated that acute hypoxia causes natriuresis, which may be reversed or even decreased during prolonged exposure to hypoxia [7,13,14]. Such sodium responses may be affected on the one hand by natriuretic mechanisms, for example a fall in aldosterone secretion or decreased proximal tubular sodium reabsorption, and on the other hand by antinatriuretic mechanisms, for example a fall in renal perfusion pressure, increased angiotensin II or a stimulated sympathetic nervous system [7,9,13,14]. We demonstrated in recent studies that acute inspiratory [8% O2], and tissue [0.1% CO] hypoxia caused a marked stimulation of renin secretion and renal renin gene expression in rats within 6 h [10,11]. This stimulation of the renin system was not due to direct effects at the renal juxtaglomerular cell, but might be due to circulating catecholamines [10,11]. In our present study, we were interested in investigating more chronic effects of hypoxia on the renin system, since a hypoxia-induced stimulation of the renin system might contribute to progression of chronic renal failure, to induction of hypertension in the sleep-apnoea syndrome or might play a role in pulmonary hypertension [1517].



   Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animal experiments were conducted according to NIH and national guidelines. Male Wistar rats weighing 250–300 g were exposed to normobaric hypoxia [10% O2, 90% N2] in transparent plastic chambers for 2 weeks (n=5) and 4 weeks (n=5). Two further groups of rats were exposed to hypoxia for 4 weeks and were treated with an endothelin ETA receptor antagonist (LU135252, Knoll AG, Mannheim, Germany; 50 mg/kg/day), one group from the start to the end of hypoxia (weeks 1–4; n=5) and the other (n=5) from week 3 to week 4 of hypoxia. Two additional groups (n=5 each) of rats were exposed to hypoxia for 4 weeks and were treated with the NO donor molsidomine (Hoechst AG, Bad Soden, Germany; 15 mg/kg/day), one group from the start to the end of hypoxia (weeks 1–4; n=5) and the other from week 3 to week 4 of hypoxia. Relative humidity within the chamber was kept at <70% with anhydrous CaSO2. Boric acid was used to keep NH3 levels within the chamber at a minimum. The percentage of CO2 within the chamber was measured daily and did not exceed 0.3%. Normoxic rats were housed in identical cages adjacent to the chambers in the same room while breathing room air. All rats had access to standard chow and tap water ad libitum. Animals were maintained on a 12 h–12 h light–dark cycle. Every second day the chamber was opened to allow for cage cleaning and replenishment of food and fresh water. Immediately after the haemodynamic measurements, the animals were killed by decapitation. Blood was collected from the carotid arteries for determination of haematocrit. Kidneys were removed rapidly, weighed, cut in half, frozen in liquid nitrogen and stored at -80°C until isolation of total RNA.

Measurements of haematocrit, right ventricular and systolic blood pressure
Haematocrit was measured by a standard laboratory test and systolic blood pressure by means of the tail-cuff method in the conscious rat outside the cage. Right ventricular pressure was measured as previously reported [18,19]. Briefly, each rat was taken from its cage and anesthetized immediately with thiopental (50 mg/kg i.p.). The rats were ventilated artificially with room air using a rodent respirator (Animal respirator TSE, Kronberg, Germany). The right jugular vein was cannulated and a catheter was introduced into the right ventricle. Right ventricular pressure was measured within 5 min of introduction of the catheter using a TP120 Statham pressure transducer.

Isolation of RNA
Total RNA was isolated according to the protocol of Chomczynski and Sacchi [20]. Kidneys were homogenized in 10 ml of solution D [guanidine thiocyanate (4 mM) containing 0.5% N-lauryl-sarcosinate, 10 mM EDTA, 25 mM sodium citrate and 700 mM ß-mercaptoethanol] with a polytron homogenizer. Sequentially, 1 ml of sodium acetate (2 mM, pH 4), 10 ml of phenol (water saturated) and 2 ml of chloroform were added to the homogenate, with thorough mixing after the addition of each reagent. After cooling on ice for 15 min, samples were centrifuged at 4°C (15 min, 10000 g) and the supernatant precipitated with an equal volume of isopropanol at -20°C for 1 h. After centrifugation, RNA pellets were resuspended in 0.5 ml of solution D, precipitated with an equal volume of isopropanol at -20°C, dissolved in diethylpyrocarbonate-treated water and stored at -80°C.

Determination of preprorenin mRNA
Renin mRNA was determined by RNAase protection assay in rat kidneys using 20 µg of RNA as described in detail previously [10]. Briefly, a preprorenin cRNA probe containing 296 bp of exons I and III, from a pGEM-4 vector carrying a PstI–KpnI restriction fragment of a rat preprorenin cDNA, was generated by transcription with SP6 RNA polymerase (Amersham, UK). Transcripts were labelled routinely with [32P]GTP (Amersham) and purified on a Sephadex G-50 spin column. For hybridization, total kidney RNA was dissolved in a buffer containing 80% formamide, 40 mM PIPES, 400 mM NaCl and 1 mM EDTA (pH 8). Kidney RNA was hybridized in a total volume of 50 µl at 60°C for 12 h with 5x105 c.p.m. of radiolabelled renin probe. RNAase digestion was carried out with RNAase A and T1 at 20°C for 30 min and terminated by incubation with proteinase K (0.1 mg/ml) and SDS (0.4%) at 37°C for 30 min. Protected preprorenin fragments were purified by phenol–chloroform extraction, ethanol precipitation and subsequent electrophoresis on a denaturating 10% polyacrylamide gel. After autoradiography of the dried gel at -80°C for 1 day, bands representing protected renin mRNA fragments were excised from the gel, and radioactivity was counted with a liquid scintillation counter. Netto c.p.m. of renin mRNA were related to netto c.p.m. of the respective actin signal.

Determination of endothelin-1 and endothelin-3 mRNA
Endothelin mRNAs were determined by RNAase protection assay in rat kidneys using 10 µg of RNA as described in more detail previously [21]. Briefly, continuously labelled antisense RNA transcripts were generated by in vitro transcription using SP6 polymerase and [32P]GTP from the riboprobe templates (rat ET-1, genomic fragment comprising 155 bp of exon 2 with adjacent intron 5; rat ET-3, genomic fragment comprising 140 bp of exon 2 with adjacent intron 5). For the assays, total RNA was dissolved in 50 µl of hybridization buffer, hybridized with 5x105 c.p.m. of the ET probe and treated further as described for the renin RNAase protection assay. As there is no cross-reactivity between the ET riboprobes, RNA samples were probed with ET-1 and ET-3 riboprobes simultaneously. The dried gel was subjected to autoradiography for 2 days. The protected fragments were quantified by measuring the radioactivity of excised bands of the dried gel with a liquid scintillation counter and were related to the respective signal of actin mRNA.

Determination of actin mRNA
Actin mRNA was determined by RNAase protection assay as previously described [22].

Statistics
Levels of significance were estimated by analysis of variance (ANOVA) and Bonferroni's correction for multiple testing. Data are given as mean±SEM if not stated otherwise. P<0.05 is considered as statistically significant.



   Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Body weight, haematocrit and haemodynamic measurements
Haematocrit increased from 40±1% for controls to 60±2 or 72±2% after 2 and 4 weeks of hypoxia, and haematocrit after 4 weeks of hypoxia was unaffected by either LU135252 or molsidomine. These increases in haematocrit clearly demonstrate the effectiveness of the chronic hypoxia model used in the present study. Right ventricular pressure increased from 20±1 mmHg for controls to 36±2 or 46±2 mmHg after 2 and 4 weeks of hypoxia, and was decreased after 4 weeks of hypoxia by either LU135252 or molsidomine by 8–10 mmHg. Systolic blood pressure was 129±2 mmHg at baseline, 139±4 mmHg after 2 weeks of hypoxia and 150±4 mmHg after 4 weeks of hypoxia in control animals (P<0.05 vs baseline), 139±5 mm or 141±4 mmHg after 14 or 29 days of chronic hypoxia and molsidomine treatment, 180±4 mmHg after 14 days of chronic hypoxia and endothelin receptor antagonism from week 1 (P<0.05 vs baseline and hypoxic controls) and 174±7 mmHg or 196±11 mmHg after 28 days of chronic hypoxia and endothelin receptor antagonism from week 3 or from week 1 (each P<0.05 vs baseline and vs hypoxic controls), respectively.

Renal renin gene expression
Renal renin gene expression tended to decrease after 2 weeks of inspiratory hypoxia to 76% of control and after 4 weeks to 49% of control (P<0.05). Blocking of endothelin ETA receptors with LU135252 completely abolished this suppression of renin gene expression (105 or 140% of control after 4 weeks of hypoxia, and 2 weeks or 4 weeks of ETA blockade) (Figure 1Go). Also treatment with the NO donor molsidomine partially reversed the inhibition of renin gene expression by chronic hypoxia (70 or 94% of control after 4 weeks of hypoxia, and 2 weeks or 4 weeks of molsidomine treatment).



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Fig. 1. Renal renin gene expression in rats during chronic hypoxia at 10% O2 for 2 and 4 weeks (A), and in rats during chronic hypoxia at 10% O2 for 4 weeks with or without treatment with the endothelin receptor antagonist LU135252 for weeks 1–4 (B). Data are given as mean±SEM. *P<0.05 vs control; #P<0.05 vs 4 weeks of hypoxia.

 
Renal endothelin-1 and endothelin-3 gene expression
Renal ET-1 gene expression tended to increase during chronic hypoxia (120 or 173% of control after 2 or 4 weeks of hypoxia; not statistically significant). This increase of ET-1 gene expression during hypoxia appeared to be unaffected by molsidomine (174 or 167% of control after 4 weeks of hypoxia, and 2 or 4 weeks of molsidomine treatment) or ETA receptor blockade (160 or 145% of control after 4 weeks of hypoxia, and 2 or 4 weeks of ETA blockade). ET-3 gene expression, however, was completely unchanged during hypoxia, as well as during treatment with the endothelin receptor antagonist LU135252 and the NO-donor molsidomine (data not shown).



   Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In recent studies, a marked stimulation of renin secretion and renal renin gene expression was demonstrated after both acute inspiratory and tissue hypoxia (6 h of hypoxia) [10,11]. This stimulatory effect of hypoxia on the renin system was in accordance with most but not all reports in the literature [211]. These contradictory data may be due to different study protocols, for example a different duration of hypoxia. In the present study, a clear suppression of renal renin gene expression during chronic (2 and 4 weeks) hypoxia was demonstrated, explaining some of the discrepancies in the literature.

The decrease in renin gene expression during hypoxia might in part be secondary to the increasing arterial blood pressure induced by hypoxia. Nevertheless, from our data using an endothelin receptor antagonist, it may be inferred that suppression of the renin system during chronic hypoxia is influenced by the endothelin system, since blockade of ETA (and possibly also ETB, because at higher doses LU135252 is thought to block both endothelin receptor subtypes [23]) receptors markedly stimulated the renal renin gene expression. Endothelins are known inhibitors of the renin system in in vitro studies [24]. The results of the present study are in contrast to normal rats and rats with unilateral renal artery stenosis, where the mixed ETA/ETB endothelin receptor antagonist bosentan was without effect on renal renin gene expression [22]. This discrepancy may be explained by differences in stimulation of the endothelin (and counteracting NO) system in hypoxia and normoxia. A role for the renal endothelin system in suppression of renin gene expression during chronic hypoxia is also supported by the increase in renal ET-1 gene expression during acute [21,25] and chronic hypoxia (present study). ET-3, in contrast to ET-1, appeared not to be affected by hypoxia, and also not to be regulated differentially as had been shown with renal endothelin gene expression in, for example, renal artery stenosis [26].

Another cause for the suppressed renin system during chronic hypoxia could be suppression of NO formation, since NO is a known stimulator of renin secretion and renin gene expression in vitro and in vivo [27,28]. However, short-term hypoxia (6 h) has been shown to stimulate NO synthase (eNOS, bNOS) gene expression in, for example, the kidney and the lung [29]. Also, in more chronic models of hypoxia, an up-regulation of eNOS gene expression has been reported [30]. In contrast, Ni et al. suggested that NO production is impaired in hypoxic rats, thus contributing to hypertension [31]. Furthermore, chronic hypoxia (10–12 days) has been shown to be associated with decreased NO production and eNOS expression in piglets [32]. The rather minor effects of continuously supplying NO by means of the NO donor molsidomine on renal renin gene expression in our study argues against a major deficit in NO formation or alternatively suggests that NO does not exert its stimulatory effects on renin secretion and renin release during hypoxia. Further studies are needed that specifically look at these questions, for example at the stimulatory effect of NO on renin secretion during hypoxia in primary cultures of isolated juxtaglomerular cells, and at the renin secretion and renin gene expression when using inhibitors of NO synthase (e.g. L-NAME) during chronic hypoxia in vivo.

Another remarkable result of our study was that systolic blood pressure increased markedly in rats treated with the endothelin receptor antagonist from the start of hypoxia in comparison with a moderate increase of blood pressure in hypoxic control rats and in rats treated with the NO donor molsidomine. This unexpected increase in systemic blood pressure during long-term endothelin receptor antagonism (despite the well-known antihypertensive properties of these compounds [22,33]) might be due to disinhibition of the renin system during chronic hypoxia. Moderate increases in systemic blood pressure in experimental and clinical states of hypoxia-induced pulmonary hypertension have been reported; however, there are no published data available with regard to the effects of endothelin receptor antagonism on systemic blood pressure in experimental animals with pulmonary hypertension due to chronic hypoxia [34,35].

In summary, the present study clearly demonstrated a suppression of renal renin gene expression during chronic hypoxia (in contrast to acute hypoxia) in rats, due to inhibitory actions of endothelins on the renin system and presumably also due to a compensatory response to increasing blood pressure induced by hypoxia. The unexpected further increase in systolic blood pressure during chronic hypoxia and chronic endothelin receptor blockade is suggested to be due to up-regulation of the renal renin system.



   Acknowledgments
 
This study was supported by grants from the Else Kröner-Fresenius-Stiftung, the Doktor Robert Pfleger-Stiftung and the Deutsche Forschungsgemeinschaft. The authors thank Prof. Armin Kurtz, Physiologie, University of Regensburg for critical reading of the manuscript and helpful suggestions.



   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 8. 2.99
Accepted in revised form: 25. 6.99