Downregulation of Renin Gene Expression by Interleukin-1
Abstract The As4.1 cell line was established from a mouse kidney tumor by transgene-targeted tumorogenesis. These cells express high levels of renin mRNA from their endogenous renin gene and release approximately eightfold-more prorenin than active renin in culture. Levels of renin mRNA in As4.1 cells are decreased in a dose-dependent manner by the addition of physiological concentrations of cytokine interleukin-1 to the media. Stability of renin mRNA and initial rates of release of active renin and prorenin were not significantly altered by interleukin-1. In contrast, transcription initiated from a construct that consisted of 4.1 kilobases of renin 5′ flanking sequence fused to a reporter gene (chloramphenicol acetyltransferase) was markedly inhibited by interleukin-1. On the basis of our findings, we conclude that downregulation of renin synthesis caused by interleukin-1 occurs primarily at the level of transcription and that DNA sequence or sequences mediating that effect are positioned within 4.1 kilobases upstream of the renin gene. The physiological relevance of this regulation is related to the events that occur during septic shock, characterized by hypotension, cardiovascular collapse, multiple organ failure, and high mortality. Unexpectedly, hypotension associated with septic shock does not lead to activation of the renin-angiotensin system. The hypotension in septicemia is believed to be mediated by the combined action of many modulators including cytokines, and data presented here suggest direct involvement of interleukin-1 in this process.
Septic shock syndrome, which results from severe infection or trauma, is characterized by severe hypotension that leads to cardiovascular collapse, multiple organ failure, and often death. Death from septic shock may be related to hypotension caused by a decrease in systemic vascular resistance and a large reduction in cardiac output. The hypotension in septicemia is believed to be mediated by the combined action of many modulators, including cytokines, prostaglandins, and complement components. An as-yet-unexplained feature of the hypotension associated with septic shock is that it does not lead to activation of the RAS. Cytokines, especially alpha and beta types of IL-1α and IL-1β, interleukin-6, and tumor necrosis factor, have been implicated as primary mediators of the physiological response to septicemia. Many in vivo findings point toward a pivotal role of IL-1β in septic shock–induced hypotension.1 A major side effect observed in IL-1β clinical trials is dose-limiting hypotension.2 Also, specific blockade of IL-1β at the receptor level with an IL-1β receptor antagonist (IL-1ra) has been found to eliminate septic shock–induced hypotension in rabbits.3 Moreover, mice with both chromosomal copies of the IL-1β–converting enzyme gene inactivated were resistant to endotoxic shock.4
Here we present evidence that suggests that IL-1β may have direct actions on the RAS, namely downregulation of renin gene expression. Since renin is a key participant in the regulation of systemic blood pressure and electrolyte balance through its fundamental role in the RAS, this downregulation could prevent the maintenance of blood pressure (ie, RAS response) and thus result in uncorrected hypotension.
To determine direct effects of IL-1β on renin-expressing cells we used a clonal cell line, As4.1 (AmericanType Culture Collection [ATCC] No. CRL2193), of renal origin. This cell line was established from a kidney tumor of mice with a Ren-2 5′ flank/Simian virus 40 (SV40) T-antigen transgene.5 Tissue-specific expression of the transgene was confirmed in transgenic animals.6 7 8 The As4.1 cell line maintains high expression of its endogenous renin gene in addition to the transgene over long-term culture and demonstrates several features that are characteristic of kidney juxtaglomerular cells, including the ability to store and release active renin.5 9 The endogenous renin gene expressed in the As4.1 cell line is Ren-1c. Thus, As4.1 cells may represent a unique model of juxtaglomerular cells with which to study in greater detail the mechanism of IL-1β effects on renin expression during septicemia. In addition, the results from the present study will provide a basis for further studies of the effects of cytokines on renin synthesis in juxtaglomerular cells in vivo.
The renin-expressing As4.1 cell line (ATCC No. CRL2193) was cultured at 37°C in 10% CO2 atmosphere in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (GIBCO-BRL). At the beginning of the experiments cells were 90% confluent. Approximately 1 mL of media was used per 1×106 of cells in culture. Recombinant IL-1β was purchased from R&D Systems and actinomycin D from Fisher.
Plasmid p-117CAT was constructed by inserting a renin promoter fragment (+6 to −117, relative to the major transcription start site) into the Xba I site adjacent to the CAT coding sequences of plasmid pCAT-basic (Promega) as previously described.10 Plasmid R1C-4.1CAT was constructed by inserting the 5′ flanking sequences (−4100 to −118) of the BALB/c Ren-1c gene isolated from a lambda clone, λBALB-1, into a BamHI site present at position −117 of the renin promoter in p-117CAT plasmid.11 Plasmid DNA was prepared by the triton-lysis method12 and two sequential cesium chloride/ethidium bromide equilibrium centrifugation steps.
As4.1 cells were transfected by electroporation13 using a Cell Porator and electroporation chambers with 0.4 cm electrode spacing (Gibco-BRL). Conditions for electroporation were as follows: 1×107 cells were resuspended in 1 mL of 1× HeBS (25 mmol/L HEPES, pH 7.05, 140 mmol/L NaCl, 5 mmol/L KCl, 0.75 mmol/L Na2HPO4, 6 mmol/L glucose) with DNA concentration of 25 to 50 μg/mL, plus 250 μg/mL of sonicated salmon sperm DNA, added as carrier. The cells were then exposed to a single electric impulse of 300 V at a capacitance setting of 1180 μF.
To correct CAT values in each experiment for transfection efficiency, cells were cotransfected with 5 μg of plasmid containing RSV promoter driving β-galactosidase (RSV–β-gal). Transfection of promoterless CAT plasmid, pCAT-basic (Promega) into As4.1 cells, used to determine the background in these assays, did not result in any significant CAT activity (data not shown).
Cells were harvested 48 to 72 hours post-transfection and resuspended in 0.25 mol/L Tris, pH 8.0. The extract was subjected to three freeze-thaw cycles, and prior to CAT assays extracts were heated to 60°C for 10 minutes. The protein concentration was determined by the method of Bradford14 using bovine serum albumin as standard. CAT activity was determined as described by Gorman et al15 except that n-butyryl CoA was substituted for acetyl-CoA in the assay. Equal amounts of protein (35 μg) were assayed for CAT activity in 0.25 mol/L Tris, pH 8.0, 0.125 μCi of [14C]-chloramphenicol (55 mCi/mmol, 1 Ci=37 GBq) obtained from Amersham. Amount of butyrylated [14C]-chloramphenicol generated in the reaction was determined by thin-layer chromatography. CAT activities were given as percent conversion of chloramphenicol obtained in the enzyme assay with 35 μg of cellular protein. Values in each experiment were corrected for transfection efficiency by measuring β-galactosidase activity in the same lysates. All radioactive signals were quantified by phosphorimagery using Imagequant software (Molecular Dynamics).
β-Galactosidase activity was determined using Galacto-Light Plus chemiluminescence reporter assay (Tropix) according to the manufacturer’s instructions. Chemiluminescence was measured on a Monolight 2010 Luminometer (Analytical Luminescence Laboratory). Results were expressed as relative light units per microgram of cell lysate.
To determine the amount of total renin activity, prorenin was activated (converted to active renin) with trypsin treatment (0.3 mg/mL, 1 hour at room temperature) as previously described.16 Samples used to measure only active renin were not treated with trypsin. Prorenin content was estimated by subtraction of active renin activity from total renin activity. After inhibition of trypsin with phenylmethylsulfonyl fluoride (1 mmol/L), renin assays were performed with Angiotensin-I[125I] Radioimmunoassay Kit (Du Pont) according to the manufacturer’s instructions. Activity measurements were made by incubation of samples with bilateral nephrectomized rat plasma as a source of renin substrate angiotensinogen. The amount of released Ang I was determined by the same kit. Renin activity is defined as nanograms of Ang I formed by 1 mL of culture media for 1 hour. The entire experiment was performed within 1 day without freezing the samples. The media also were assayed for renin activity in the presence of specific renin inhibitor (CP-71,362, a gift from Pfizer Inc, New York, NY) (data not shown). The specific renin inhibitor caused full inhibition of both active and total renin activities. To test for nonspecific degradation of the substrate, an assay was performed with the culture media from Ltk− (mouse fibroblast) control cell line. No significant renin activity was detected in this assay (data not shown).
Northern Blot Analysis
Total RNA was isolated from As4.1 cells using the method based on the widely used guanidinium thiocyanate/acid phenol/chloroform procedure in which Ultraspec RNA isolation kit (Biotecx) is used, according to the manufacturer’s instructions. Total RNA samples (30 μg) were analyzed for renin mRNA levels by Northern blot analysis17 with mouse submandibular Ren-2d cDNA probe18 and human GAPDH probe (Clontech). Probes were labeled with dCTPα[P32] (Amersham) by random-priming (kit from Promega). After quantification of renin probe signals by phosphorimagery, blots were stripped of the renin probe and rehybridized with GAPDH probe for determination of GAPDH mRNA levels.
The As4.1 cell continuously expresses high levels of renin mRNA when grown in standard culture media. The addition of IL-1β caused a dose-dependent decrease in renin mRNA levels (Fig 1A⇓). IL-1β affected renin mRNA levels in As4.1 cells at a concentration (ED50 is 30 pg/mL) (Fig 1B⇓) that correlates well with IL-1β levels found in normal human serum and in serum of septic shock patients (2.5±2 and 120±17 pg/mL, respectively).19 IL-1β (1 ng/mL) caused rapid decreases in renin mRNA, approximately 7-fold, within 24 hours (Fig 2⇓). Further increases in IL-1β concentration (Fig 1B⇓) or incubation time (Fig 2⇓) did not result in a further decline in renin mRNA. The observed effect may be specific to IL-1β, since another cytokine elevated during sepsis, namely IL-6, did not significantly affect renin mRNA levels (data not shown). Tumor necrosis factor-α was found to be extremely toxic to the As4.1 cells in the concentrations usually used in cell culture studies (data not shown).
The kinetics of renin mRNA decay do not seem to be affected significantly by IL-1β after transcription is blocked with actinomycin D (Fig 3⇓). In control experiments, actinomycin D added to As4.1 cells in culture inhibited incorporation of [5,6-3H]-uridine into total RNA by more than 95% within 1 hour (data not shown).
As4.1 cells grown in culture media released both active renin and prorenin linearly with time during the 72 hours of the experiment (Fig 4⇓). The amount of prorenin released by As4.1 cells was approximately 8-fold higher (8 ng of Ang I formed/mL of media/h) than active renin (approximately 1 ng of Ang I formed/mL of media/h) after 72 hours in cell culture (Fig 4⇓). Release of active renin and prorenin from As4.1 cells in response to IL-1β was not significantly affected at 1 or 2 hours (data not shown) up through 24 hours (Fig 4⇓). After 24 hours of IL-1β incubation, a decrease in the amounts of both prorenin and active renin was observed.
The As4.1 cells contain a transgene incorporated into their chromosomal DNA. The transgene consists of SV40 Large T antigen gene (an oncogene) under the regulation of 4.6 kb of 5′ upstream region of the Ren-2 gene. If IL-1β affects transcription of the renin gene, and if the responsive DNA regulatory regions mediating this effect are positioned within the 4.6-kb fragment, then the effects on expression of the transgene and endogenous Ren-1c gene in As4.1 cells would be expected to be similar. Indeed, Northern blot analysis revealed that the steady state levels of endogenous renin mRNA and T-antigen mRNA expressed from the transgene (Fig 5⇓) were similarly decreased by IL-1β. In contrast, the levels of control GAPDH mRNA were unaltered by IL-1β (Fig 5⇓).
To independently verify the effects of IL-1β on the renin gene transcription, the As4.1 cells were transfected with DNA plasmids containing the Ren-1c 5′ flanking region (from +6 to −4100 base pairs from the transcription start site) controlling transcription of the CAT reporter gene. Rapid growth of As4.1 cells is believed to be mediated by the high levels of SV40 Large T antigen expressed from the transgene. Since IL-1β suppressed T-antigen expression, this downregulation could in turn affect the general rate of transcription in these cells. In an effort to control for this, an RSV promoter driving CAT reporter gene (RSV-CAT) was used to determine nonspecific effects of IL-1β on transcription in As4.1 cells. The results shown in Fig 6⇓ demonstrate that RSV-CAT activity in control cells (87.8±6.0% chloramphenicol converted by 35 μg of cell extracts) was approximately 3-fold higher than in IL-1β–treated cells (29.8±3.5%). On the other hand, IL-1β caused a much more significant decrease in transcription from the −4.1 Ren-1c–CAT reporter gene construct, approximately 22-fold (39±5.2% in control versus 1.8±0.5% after IL-1β treatment) (Fig 6⇓). Since nonspecific effects of IL-1β on transcription as measured with RSV-CAT were 3-fold, and the total effect observed with −4.1 Ren-1c–CAT was 22-fold, it can be calculated that specific inhibition of −4.1 Ren-1c–CAT transcription in response to IL-1β is approximately 7-fold. An effect of this magnitude is consistent with the decrease observed in the Northern blot experiments (Fig 1⇑).
Promoterless plasmid, pCAT-basic, used to determine the background of CAT activity in these assays, did not show any significant CAT activity (data not shown). The transcription initiation site used by transfected Ren-1c–CAT constructs is the same one found for endogenous Ren-1– and Ren-2–gene–derived renal transcripts.10
In the present report, we show that cytokine IL-1β inhibits, at physiological concentrations, transcription of the renin gene, in cultured, renal-derived, renin-expressing As4.1 cells. Our data indicate not only that the IL-1β effect occurs primarily at the level of transcription but also that DNA regulatory region or regions mediating this effect are present on 4.1 kb of the renin gene 5′ regulatory region. The inhibitory effect of the IL-1β on renin expression does not appear to be caused by general inhibition of transcription and proliferation of As4.1 cells.
Our findings are consistent with previously described effects of IL-1β on renin mRNA in primary human decidual cells that express renin.20
The dynamics of renin mRNA decay do not seem to be affected significantly by IL-1β after the addition of actinomycin D (Fig 3⇑). The possibility remains that unknown regulatory proteins, the expression of which is blocked by inhibition of transcription, may affect renin mRNA stability under IL-1β. These putative proteins would have to exert their effects by binding to a six-nucleotide stretch on renin mRNA (from +1 to +6), since only that sequence is shared between endogenous renin mRNA and mRNA transcribed from CAT and T-antigen reporters, which were affected by IL-1β similarly. Whether such an effect underlies the discrepancy in apparent half-life of renin mRNA as estimated from IL-1β treatment alone (Fig 2⇑) compared with that obtained by actinomycin D treatment (Fig 3⇑) remains to be elucidated. In any event, the magnitude of the transcriptional effects observed (Figs 5⇑ and 6⇑) can adequately account for the steady state levels of mRNA found.
Since inhibition of renin release by IL-1β did not occur until the second day of exposure, the effects on renin release appear to be secondary to the decrease in renin synthesis. Alternatively, if intracellular levels of renin are not altered over the time of experiment, observed changes in the extracellular renin concentration may represent a slow effect on renin release. In this regard, pulse-chase radiolabeling experiments in which 35S-methionine was used9 indicate that turnover of intracellular prorenin is complete within 5 hours. In contrast, turnover of intracellular active renin is much slower (about 72 hours), although precise determination is obscured by the fact that cells continuously grow throughout the experiment. Therefore, while the effect of IL-1β on extracellular prorenin most probably reflects an inhibition of renin synthesis, given the time frame of the experiment, extracellular active renin levels may be influenced by a combination of inhibition of synthesis and slow effects on release. Although the effects of IL-1β on renin accumulation were not directly measured, the observed decrease in transcription of the renin gene in response to IL-1β may completely account for the changes in steady state renin mRNA levels after IL-1β treatment. Thus, while effects of IL-1β on processes other than transcription are possible, they seem less likely to exist or are less pronounced.
IL-1β is synthesized during inflammation and sepsis.1 Sepsis and septic shock are heterogeneous clinical syndromes that are usually triggered by severe microbial infection. Septic shock is defined as severe sepsis accompanied by hypotension.21 Many of the effects of IL-1β are beneficial at the onset of infection, but when produced for extended periods of time or in excessive quantities, this cytokine has been directly linked to the development of hypotension, shock, multiple organ failure, and death.1
IL-1β produced in septicemia causes an increase in the expression of iNOS in peripheral vasculature, which causes a decrease in vascular resistance through relaxation of vascular smooth muscle. NO produced by iNOS has been hypothesized to be responsible for hypotension in septic shock. Inhibition of NOS activity during sepsis has produced conflicting results (see Reference 2121 for review, also References 22 and 2322 23 ) presumably because NO produced by iNOS has also been shown to be beneficial through its antimicrobial activity. Moreover, these studies are further complicated by the fact that most of them have used nonselective NOS inhibitors. Blanket inhibition of all NOS isozymes (inducible and continuously expressed) may have resulted in the side effects observed in these studies.
The diversity of the molecular origins, targets, and actions of NO makes it difficult to anticipate the consequences of inhibiting its production in disease. In some models of sepsis, plasma NO concentration did not correlate with outcome of the septic shock.24 In genetic studies, mice with both chromosomal genes for iNOS inactivated did not show differences in survival rate after induced septic shock compared with the controls.25 Interestingly, other reports on the iNOS knockouts indicated differences in hypotension onset in anesthetized animals.26 This use of different animal systems, as well as the specific states and causes that result in septic shock in patients, may have complicated establishment of a single cause for the hypotension during septicemia, reflecting the fact that blood pressure homeostasis entails interactions among compensatory constellation of factors.
IL-1β produced during septicemia may also have a direct action on the RAS, which is, according to our findings, to decrease synthesis of renin. Such an effect would prevent activation of the RAS, diminish its contribution to the maintenance of blood pressure, and ultimately contribute to the observed hypotension. Indeed, in some patients27 infusion of Ang II caused a reversal of septic shock symptoms. Therefore, at least in some cases of septic shock, impairment of RAS function could be responsible for the development of hypotension.
The use of homogeneous cells in culture allowed us to test for direct effects of cytokines on renin-producing cells. These findings should help us work toward determination of the exact mechanism or mechanisms of cytokine regulation of renin synthesis in As4.1 cells, and should give direction to further in vivo studies aimed at understanding renin gene regulation in the important area of septic shock–induced hypotension.
Selected Abbreviations and Acronyms
|Ang I, II||=||angiotensin I, II|
|GAPDH||=||glyceraldehyde 3-phosphate dehydrogenase|
|IL-1α, 1β||=||interleukin-1α and 1β|
|iNOS||=||inducible nitric oxide synthase|
|NOS||=||nitric oxide synthase|
|RSV||=||Rous sarcoma virus|
This work was supported in part by National Institutes of Health grants HL35792, HL48459, and CA16056 and the Roswell Park Alliance Foundation. C.D. Sigmund is an Established Investigator of the American Heart Association.
- Received July 5, 1996.
- Revision received August 7, 1996.
- Accepted January 16, 1997.
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