Adrenomedullin Stimulates Renin Release and Renin mRNA in Mouse Juxtaglomerular Granular Cells
Abstract The recently discovered peptide adrenomedullin (AM) alters blood pressure through effects on the resistance vessels. Moreover, AM modifies the secretion of corticotropin and aldosterone and could thereby indirectly influence blood pressure through the renin-angiotensin-aldosterone system. Although plasma AM and renin concentrations have been found to directly correlate, a causal linkage between AM and renin has not been shown. The present study tested the influence of AM on renin secretion and renin gene expression by renal juxtaglomerular granular cells. Prominent expression and release of AM by vascular structures has been reported; therefore, we investigated the local expression of AM in juxtaglomerular structures. Renin release from isolated perfused rat kidneys was dose-dependently increased by AM (1 to 30 nmol/L), whereas renal perfusate flow rate increased up to 17% at a constant perfusion pressure of 100 mm Hg. In primary cultures of mouse granular cells, AM augmented renin release, renin mRNA accumulation, and cAMP production in a dose- and time-dependent manner (threshold values in the range 10 pmol/L to 1 nmol/L). By reverse transcription–polymerase chain reaction, significant expression of the AM gene was detected in microdissected rat glomeruli with afferent arterioles and in primary cultures of mesangial and granular cells. We conclude that AM is expressed in juxtaglomerular structures and that it has a direct stimulatory effect on renin secretion and renin mRNA abundance by receptors on juxtaglomerular cells, possibly through increases in cAMP. AM could act as an autocrine/paracrine stimulatory factor in the control of renin secretion and renin gene expression.
Adrenomedullin (AM), a 52–amino acid peptide recently discovered in human pheochromocytoma, is mainly produced in the adrenal medulla, lung, heart, and kidney.1 2 Rich sources of AM peptide release are adrenal chromaffin cells, vascular smooth muscle cells, and endothelial cells, which also contain specific binding sites for AM.3 4 5 6 The AM receptor seems to be shared by calcitonin gene–related peptide with a lower affinity.6 Similar to other vasoactive peptides, AM is found in picomolar concentrations in plasma.1 7 8 9 When administered intravenously, AM potently dilates many vascular beds and lowers blood pressure.1 2 10 Although the physiological role of AM is still unclear, the evidence suggests that AM participates in the regulation of arterial blood pressure and regional blood flow.
Apart from direct effects on the resistance vessels, AM could influence blood pressure indirectly through interference with the renin-angiotensin-aldosterone system at different levels.11 12 13 AM and plasma renin concentrations are elevated in cardiovascular disorders with acute decreases in cardiac output (eg, sepsis and myocardial infarction).7 8 In heart failure, the failing left ventricle secretes AM,14 and the plasma concentrations of AM and renin have been found to directly correlate.9 AM, released by the heart to the circulation, could thus reach the kidneys and influence the renin secretory rate. On the other hand, AM is produced and secreted in a regulated fashion by cells of the vessel wall.4 5 Therefore, a paracrine role of AM in renin control is also possible. AM potently dilates the renal vasculature, including the afferent arterioles, suggesting the presence of AM receptors at this site.10 15 Since juxtaglomerular granular cells are modified smooth muscle cells, it is conceivable that AM receptors are present on these cells as well. A direct link between AM and renin has not yet been investigated, and we aimed to find out whether AM affects the renin-angiotensin-aldosterone system at the rate-limiting level of renin secretion and renin production. To get a first impression of a paracrine role of AM, we investigated AM expression in the juxtaglomerular area using RT-PCR techniques. Our findings confirm that AM is locally expressed and that AM stimulates renin release and renin mRNA, probably via cAMP.
All procedures conformed with guidelines for the care and handling of animals established by the US Department of Health and Public Services and published by the National Institutes of Health (Guidelines for the Care and Use of Laboratory Animals, NIH publication No. 85-23, revised 1985).
Isolated Perfused Rat Kidney
Male Sprague-Dawley rats (weight, 250 to 300 g) (Charles River, Sulzfeld, Germany) with free access to standard rat chow and tap water were used. Kidney perfusion was performed in a recycling system according to the technique of Schurek and Alt16 with minor modifications as described in detail previously.17 The excised kidney was perfused at a constant pressure (100 mm Hg). For this purpose, the renal artery pressure was monitored by a strain-gauge transducer (model P23Db, Statham), and the pressure signal was used for control of a peristaltic pump. The perfusion circuit was closed by draining the renal venous effluent via a metal cannula back into a reservoir (200 to 220 mL). The basic perfusion medium, which was taken from the thermostat-regulated reservoir (37°C), consisted of a modified Krebs-Henseleit solution that contained (mmol/L) Na+ 140, K+ 5.0, Ca2+ 1.25, Mg2+ 2.0, Cl− 120, HCO3− 27.5, and HPO42− 0.7. The perfusate was enriched with all physiological amino acids in concentrations between 0.2 and 2.0 mmol/L and contained additionally (mmol/L) glucose 8.7, pyruvate 0.3, lactate 2.0, alfa-ketoglutarate 1.0, l-malate 1.0, creatinine 0.15, and urea 6.0 as well as 6 g/100 mL bovine serum albumin, 1 mU/100 mL vasopressin 8-lysine, and freshly washed human red blood cells (10±2 hematocrit). Ampicillin (3 mg/100 mL) and flucloxacillin (3 mg/100 mL) were added to inhibit bacterial growth. To improve the functional preservation of preparations, the perfusate was continuously dialyzed against a 25-fold volume of similar composition but lacking erythrocytes and albumin. For oxygenation of the perfusate, the dialysate was equilibrated with a prewarmed and moistened 96% O2/4% CO2 gas mixture. Perfusate flow rates were obtained from the revolutions of the peristaltic pump, which was calibrated before each experiment. Renal perfusion rate and pressure were continuously monitored on a potentiometric recorder (model REC 102, Pharmacia LKB). After the reperfusion loop was established, perfusion flow rates usually stabilized within 15 minutes. Stock solutions of AM were dissolved in freshly prepared dialysate and infused into the arterial limb of the perfusion circuit directly before the kidneys at exactly 1% of the rate of perfusate flow (perfusion apparatus adapted from Fresenius). For determination of renin concentration, aliquots (0.2 mL) were taken at 2-minute intervals from the arterial limb of the circulation and the renal venous effluent, respectively. Samples were centrifuged (4°C) at 1500g for 15 minutes in a benchtop centrifuge (5413, Eppendorf), and the supernatants were immediately assayed for renin concentration.
Isolation and Primary Culture of Mouse Juxtaglomerular Granular Cells
Mouse juxtaglomerular cells were isolated as described in detail previously.18 In brief, after 6- to 8-week-old mice (C57BL, Charles River, Sulzfeld, Germany) were decapitated, the kidneys were quickly removed, decapsulated, minced with a scalpel blade, and digested for 70 minutes at 37°C with a trypsin/collagenase mixture. The resulting cell suspension was filtered through a 22.4-μm nylon filter, washed two times, and centrifuged at 27 000g for 30 minutes at 4°C in a 30% isosmotic Percoll density gradient (Pharmacia). Four cell layers with different specific renin activities were obtained. The cellular layer (density, 1.07 g/mL) with the highest specific renin activity was used for cell culture. The cell band was recovered, and cells were washed and resuspended in RPMI-1640 (Biochrom) containing 0.66 U/mL insulin, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2% FCS. Each mouse yielded 3 to 4 mL of final cell suspension. Cells were seeded in 100-μL aliquots in 96-well cell culture plates. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2 in air. After 20 hours of primary culture, medium was removed, and the cultures were washed once with 100 μL RPMI-1640 medium containing 2% FCS. Then 100 μL of fresh and prewarmed culture medium with the chemicals to be tested was added.
Isolation and Primary Culture of Rat Mesangial Cells
Mesangial cells were obtained from male Sprague-Dawley rats (weight, 70 to 100 g) by outgrowth from isolated glomeruli essentially as described.19 Renal cortical tissue was consecutively passed through polyamide screens with pore sizes of 180 and 100 μm onto a screen of 50-μm pore size. Preparations of glomeruli from the 50-μm screen were confirmed under phase-contrast microscopy to be free of contaminating single cells and tubular or arteriolar attachment. Aliquots of the glomeruli were placed in 75-cm2 Falcon tissue flasks with 15 mL RPMI-1640 identical to the solution used for the granular cells except for the addition of 10% FCS. The flasks were incubated at 37°C in a humidified atmosphere containing 5% CO2. The medium was changed every third day. Only cells in primary culture were used for the experiments. The cells were usually confluent after 3 weeks and were then harvested for RNA extraction. Mesangial cells were starved for 24 hours before harvest of RNA by a reduction in medium FCS concentration from 10% to 2%.
Microdissection of Rat Glomeruli With Afferent Arterioles
Rats weighing 70 to 100 g were used. The initial steps were identical to the protocol used for isolation of granular cells. Minced cortical tissue was incubated for 15 minutes with enzymes and then was filtered through a 150-μm mesh onto a 50-μm screen. Material captured on the 50-μm screen was thoroughly washed in solution 1 (mmol/L: NaCl 130, KCl 5, CaCl2 2, glucose 10, sucrose 20, and Tris 10, pH 7.4), resuspended, and placed in tissue culture dishes. Microdissection was done with sharpened glass pipettes under a stereomicroscope while the tissue was maintained on ice. Afferent arterioles with attached glomeruli were gently isolated from the adhering tubular structures. No precaution was taken to include efferent arterioles, and because they are very fragile, the adhering vessels predominantly consisted of afferent arterioles. The specimens were transferred directly to RNA extraction solution. Dissection continued for 2 hours and resulted in approximately 200 arterioles with glomeruli, which yielded approximately 5 μg total RNA.
Male Sprague-Dawley rats weighing 140 to 160 g were fed standard chow and allowed free access to tap water. Rats were killed by decapitation, and organs were rapidly removed, frozen in liquid nitrogen, and stored at −80°C until extraction of total RNA. RNAs were extracted from organs, afferent arterioles, and cell cultures basically according to the acid guanidinium/phenol/chloroform protocol of Chomczynski and Sacchi.20 Final RNA pellets were dissolved in diethylpyrocarbonate-treated water and stored at −80°C until further processing. The RNA yield was quantified by spectrophotometry at 260 nm. The quality of extracted RNA was confirmed by the observation of intact 18S and 28S bands after gel electrophoresis in an ethidium bromide–stained agarose gel.
Experiments on Renin Secretion From Cultured Granular Cells
Experiments on renin secretion were performed during various times of incubation. At the end of experiments, supernatants were collected and centrifuged at 10 000g at room temperature to remove cellular debris. The supernatants were then stored at −20°C until assayed for renin concentration. Cells were lysed by adding to each culture well 100 μL of phosphate-buffered saline containing 0.1% Triton X-100 and shaking for 45 minutes at room temperature. The lysates were centrifuged at 10 000g at room temperature, and supernatants were stored at −20°C until further processing. Renin secretion rates were estimated from the appearance rate of renin in the culture medium. To minimize differences among different cell culture preparations, renin secretion rates were calculated as fractional release of total renin (ie, Renin Released/[Renin Released+Renin Remaining in the Cells]). The relative changes of release rates in response to standard stimuli obtained by this measure are identical to those obtained by relating secretory rate to total protein content.18
Determination of Renin Concentration
Renin concentrations in cell-conditioned medium, cell lysates, and perfusate from the isolated perfused kidney were determined by the ability of the samples to generate angiotensin I from the plasma of bilaterally nephrectomized rats. Angiotensin I was measured by radioimmunoassay (Sorin Biomedica).
Measurement of Renin mRNA by Semiquantitative RT-PCR
RT-PCR experiments were performed as described in detail previously.21 To avoid coamplification of genomic DNA coding for renin, two oligonucleotide primers, one spanning the exon 6/exon 7 border (sense, 5′-ATG AAG GGG GTG TCT GTG GGG TC-3′) and the other located on exon 8 of the renin gene (antisense, 5′-ATG TCG GGG AGG GTG GGC ACC TG-3′), were chosen, thus amplifying a 194-bp sequence. The internal standard was co–reverse transcribed and coamplified with mRNA from the granular cells and consisted of a mutated renin cRNA that contained a 60-bp linker fragment insert generated as previously described in detail.21
After RT-PCR, the amplification products originating from renin mRNA and from the internal standard were separated by agarose gel electrophoresis. Band density was assessed with a densitometer (BioProfil, Fröbel Labortechnik) under UV light. The ratio between the intensities of the band from renin mRNA and the corresponding internal standard from control cells was set to 100%, and the ratios obtained from the different experimental conditions were expressed as a percentage of this ratio. This relative measure made it possible to compare experiments with different RNA yields.
One microgram total RNA from the different organ and cell preparations, 1 μg yeast tRNA, and 0.5 μg oligo(dT) primer(12-18) (GIBCO) were heated at 94°C for 3 minutes in a volume of 8 μL. Then samples were cooled on ice, and each of the following components was added for RT: 4 μL deoxyribonucleotides (2.5 mmol/L); 4 μL RT buffer (supplied with the RT kit), 2 μL dithiothreitol, 0.5 μL RNasin (40 IU/mL, Promega), 0.5 μL bovine serum albumin (20 mg/mL), and 1 μL reverse transcriptase (GIBCO BRL). Samples were then incubated for 1 hour at 37°C, and the reaction was stopped by heating the samples to 95°C for 2 minutes. From the respective cDNA samples, 3 μL was used for PCR.
Primers were based on available cDNA sequence for rat AM.2 Because the rat AM genomic sequence was unknown at the time of the experiments, we could not predict whether our chosen primers spanned an intron. Negative controls without RT were performed for all mRNA preparations to exclude amplification of genomic DNA. Renin primers were identical to those used for the semiquantitative renin-mRNA measurements, and they generate an amplification product similar to that of rat cDNA.
Adrenomedullin. Two oligonucleotide primer sets were generated (Pharmacia, Biotech). Set 1-2: Sense primer, 5′-TTA TTG GGT TCG CTC GC-3′, and antisense primer, 5′-TTC TGC ATT GTG CAG GT-3′, generated a 320-bp product spanning bases 181 to 500 of rat AM cDNA. The primers were generated with restriction sites recognized by BamHI and EcoRI in the 5′ direction. The amplified fragment therefore had a total size of 335 bp. Primer set a-b spanned bases 334 to 585 (sense, 5′-ACA GTC CCG ACC CAG ACT-3′, and antisense, 5′-GCC ATA GCC TTG AGG GCT-3′). The amplified sequence contained 252 bp and with restriction sites, added up to 267 bp.
Rat β-actin. Sense (5′-CCG CCC TAG GCA CCA GGG TG-3′) and antisense (5′-GGC TGG GGT GTT GAA GGT CTC AAA-3′) primers amplified a 286-bp sequence.
Polymerase Chain Reaction
PCR was performed with 3 μL undiluted cDNA. To the cDNA was added 1 μL of each primer (10 pmol), 2 μL desoxyribonucleotides (2.5 mmol/L), and 2 μL PCR buffer (supplied with the Taq polymerase); water was added to a final volume of 20 μL. The mixture was overlaid with one drop of mineral oil, and the samples were denatured at 95°C for 5 minutes followed by annealing at 65°C for 5 minutes, during which 1 U of Taq polymerase (Boehringer Mannheim) was added. Negative controls for the PCR reactions included tubes lacking template (water added instead of cDNA). PCR was performed on at least three different RNA preparations.
Eighteen microliters was withdrawn from each PCR reaction, and after addition of loading buffer, the PCR products were separated on 2% agarose gels stained with ethidium bromide. Results were captured by photography of the gels in UV light.
Verification of cDNA Sequence for the AM PCR Amplification Product
Lung cDNA was used as a template to obtain large amounts of AM amplification products for restriction analysis. cDNA was amplified as described for 34 cycles in 12 PCR reaction cups.
Amplified products were verified on an agarose gel, pooled, and purified with Wizard PCR Preps (Promega). Part of the AM products was digested for 2 hours at 37°C in two cups that each contained 8 μL PCR product, 2 μL One Phor All Buffer (Pharmacia, Biotech), 1 μL Alu I or Bgl II (Pharmacia, Biotech), and 9 μL water. The digestion products were run in an agarose gel and compared with an undigested control. Another part of the PCR products was digested with BamHI and EcoRI for 2 hours in an identical reaction mixture, separated on low-melting-point agarose gels, excised, purified by phenol/chloroform extraction, and ligated for 16 hours at 14°C into the Bam/Eco polylinker site of vector Psp73 (Promega) for heat-shock uptake into Escherichia coli (DH5α). Of eight screened colonies, seven contained a correctly sized insert after digestion of plasmid DNA for 2 hours with BamHI and EcoRI. Positive clones were grown in large quantity, and plasmids were isolated for sequencing by a plasmid purification kit (Qiagen). Inserts were sequenced by the dideoxy-chain termination method in both directions by the use of SP6 and T7 polymerases (Sequiserve).
Measurement of cAMP
For measurement of cAMP accumulation, juxtaglomerular cells were isolated as described.18 The final cell suspension was diluted 1:3 and seeded in 24-well plates in 1-mL aliquots. After 20 hours of primary culture, the medium was removed, and 1 mL of fresh, prewarmed RPMI-1640 with 2% FCS and 0.5 mmol/L of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine were added, together with the chemicals to be tested. After 10 minutes, the cells were placed on ice and the accumulated cAMP was harvested: Medium was removed, and 600 μL ice-cold 95% ethanol with 20 mmol/L HCl was added. The medium was centrifuged at 10 000g for 10 minutes, and after removal of the supernatant, cAMP was extracted from the pellets by addition of 400 μL ethanol-HCl. Cultures and pellets were kept at −20°C for at least 20 hours. After the identical pellets and cells were pooled, the ethanolic solution was evaporated and the samples were stored at −20°C. Samples were resuspended in 700 μL assay buffer (delivered with the assay kit), and the cAMP content was determined after acetylation by a radioimmunoassay kit (Amersham-Biotrak). Each experiment represents the mean of duplicate culture wells.
RPMI-1640 was from Biochrom KG. Rat AM was purchased from the Peptide Institute, Scientific Marketing Associates. Forskolin, 3-isobutyl-1-methylxanthine, ampicillin, vasopressin 8-lysine, isoproterenol, and bovine serum albumin (fraction V, powder) were from Sigma Chemical Co. Pyruvate was obtained from Boehringer Mannheim. Flucloxacillin was from Beecham. Glutamate, urea, sodium acetate, sodium nitrate, sucrose, and alfa-ketoglutarate were obtained from Merck. l-Malic acid and l-lactate as sodium salts were from Serva. l-Amino acids were from Braun (aminoplasmal 5%; pediatric, free of carbon hydrate). AM was dissolved in pure water according to manufacturer’s instructions and stored in 10-μL aliquots (1×10−4 mol/L) at −20°C. Forskolin was dissolved in dimethyl sulfoxide at a final concentration of 1×10−2 mol/L. The dimethyl sulfoxide concentration bathing the cells never exceeded 0.03%. One percent of dimethyl sulfoxide leaves renin release unaltered with the present preparation.
If not otherwise indicated, three to five different kidney preparations were taken for each experimental protocol. In each renin release experiment, four individual wells were assigned per condition, such that each data point represents 12 to 20 single measurements. Data on renin secretion, renin mRNA, and cAMP concentrations are given as mean±SE. Levels of significance were calculated between relevant, selected groups as mentioned in the text. Student’s unpaired t test was used in the cell culture studies, and paired Student’s t test was applied in the isolated, perfused kidney studies. A value of P<.05 was considered significant.
Isolated Perfused Rat Kidney Studies
The basal functional and morphological characteristics of the isolated perfused rat kidney have been extensively described previously.17 To test for an effect on basal renin secretion and renal vascular resistance, we added AM to the perfusates at graded concentrations. Fig 1⇓ illustrates the time course of the renin release response to AM in a single representative experiment. AM at 1 nmol/L significantly stimulated renin release, and at 30 nmol/L, it increased renin secretion rates by threefold from the basal level of 31.5±5 to 98±9 angiotensin I/h per minute. Fig 2⇓ shows the average maximal release rates for each AM concentration. As a positive control and to test for the general cAMP sensitivity of the renin release process, in the present preparation the β-agonist isoproterenol was added at the end of the experiments, and marked stimulation of renin release was noted in all cases.
Basal flow rates through the isolated rat kidneys perfused at a pressure of 100 mm Hg were 14.2±0.9 mL/min per gram (n=5) and remained stable during the experiments. At a perfusion pressure of 100 mm Hg, perfusate flow rates increased 11±2% and 17±3% in response to AM concentrations of 1 and 30 nmol/L, respectively (data not shown).
Cultured Juxtaglomerular Cell Studies
To further explore the possibility that AM interacted directly with granular cells in the afferent arterioles, we incubated primary cultures of isolated mouse juxtaglomerular cells for 20 hours in the presence of a range of AM concentrations (1×10−12 to 1×10−6 mol/L). Fig 3⇓ demonstrates that AM stimulated 20-hour renin release from the cell cultures dose dependently with a threshold of 1 nmol/L (P≤.001, n=6) and a putative EC50 value of 7×10−10 mol/L to a maximum 2.8-fold above control after 2×10−6 mol/L. Forskolin, a positive control, increased renin secretion sixfold to nearly 50% of total content. To obtain more information on the time dependency of the stimulation, we examined the effect of AM (2 μmol/L) after different times of incubation (Fig 4⇓). After 45 minutes, AM tended to stimulate release rate, but not significantly. After 1.5 hours, release rate was significantly stimulated (P≤.02, n=4) and remained elevated above control after 3, 6, and 20 hours of incubation. Forskolin induced significant stimulation after 1.5 hours that was maintained throughout (3, 6, and 20 hours).
We also studied the accumulation of renin mRNA in response to AM in primary cultures of granular cells (Figs 5⇓ and 6⇓). Renin mRNA was increased with a threshold at 10 pmol/L AM (P≤.001, n=7) and an EC50 of 2 nmol/L to a maximal level twofold above the control by 2 μmol/L. Forskolin at 3 μmol/L augmented basal renin mRNA 3.5-fold after 20 hours. To elucidate the time course of the renin mRNA response to AM, we incubated granular cells for various intervals. Because renin mRNA is changed with a lag on the order of 1 to 2 hours,21 granular cells were incubated for at least 3 hours with AM. Fig 6⇓ shows that AM and forskolin stimulated renin mRNA abundance after 3 hours (P≤.001, n=6), and the effect was maintained for 6 and 20 hours. Forskolin stimulated renin mRNA accumulation to a level similar to that of AM after 3 and 6 hours, whereas after 20 hours, the stimulation was stronger than observed with AM (356±49% versus 211±27%)
Accumulation of cAMP was assayed 10 minutes after addition of AM to the granular cell cultures (Fig 7⇓). Adenylate cyclase activity was increased in a concentration-dependent way, with a threshold at 0.1 nmol/L (P≤.05, n=8), an EC50 of 7 nmol/L, and a maximal 4.2-fold stimulation after 1 μmol/L.
To obtain information about AM expression in juxtaglomerular structures, we harvested total RNA from a preparation of microdissected rat glomeruli with adherent afferent arterioles, from confluent rat mesangial cells in primary culture, and from granular cell cultures incubated for 24 hours. All PCR experiments were performed on at least three different preparations.
The PCR amplification conditions were optimized on total RNA from lung, adrenal, and kidney, which have the largest basal AM expressions.2 The two primer sets were combined to elucidate the optimal combinations for amplification. Fig 8A⇓ shows a representative gel of the amplification products obtained after PCR on cDNA from lung. This pattern was similar in kidney and adrenals. Primer combinations 1-2, 1-b, and a-2 generated only one significant band, with the largest yield consistently observed with the primer set 1-b (Fig 8A⇓). All subsequent PCR experiments used primers 1-b. The identity of the 1-b amplification product with AM cDNA was demonstrated by the correct size of the band (420 bp) and by the complete digestion into fragments with predicted sizes by two different restriction enzymes (Fig 8B⇓): Alu I (fragments: 131 and 289 bp) and Bgl II (fragments 80 [not seen] and 340 bp). Final cloning and sequencing of the amplification product showed 100% homology with rat AM cDNA.2
On total RNA from microdissected rat glomeruli with afferent arterioles and from primary cultured mesangial and granular cells, RT-PCR for AM resulted in single, significant bands with the predicted size of the AM product (Fig 9⇓). As positive controls, renin and β-actin amplification products were detected with cDNA from the glomerular preparation and from the juxtaglomerular cells, whereas only actin could be detected with cDNA from the mesangial cells. No bands were observed in the absence of cDNA or RT. We conclude that the AM gene is expressed in the juxtaglomerular area.
In the present study, we sought to find out whether the recently discovered hypotensive peptide AM, in addition to interactions with the release of corticotropin12 13 and aldosterone,11 influences renin mRNA and release. To narrow down possible indirect effects initiated by blood pressure changes after systemic administration of AM, we used the isolated perfused kidney and a preparation of cultured juxtaglomerular granular cells for our experiments.
AM caused a modest but fast and maintained rise in renin secretion from the isolated perfused rat kidney. As already shown by Hirata et al,15 AM leads to a stable increase in renal perfusate flow. In studies with comparable doses of AM administered directly in one renal artery of anesthetized dogs, a marked renal vasodilation was observed, but plasma renin activities were unchanged.22 23 This discrepancy could be due to the different ways of renin measurement (plasma renin concentration versus renal arteriovenous difference), or tachyphylaxis of the AM receptors could be involved because they are constantly exposed to AM plasma concentrations of 2 to 4 pmol/L in the dog kidney in situ. Finally, species differences could play a role. In response to systemic AM infusions, during which blood pressure decreases, plasma renin concentration consistently increases.13 24 Although urine flow and sodium excretion increase in response to AM,15 23 and this would tend to inhibit renin secretion through the macula densa mechanism,25 the integrated response is an augmented renin release rate. The stimulus for renin secretion could have several components: the renal baroreceptor mechanism, which is activated when renal perfusion pressure drops; renal sympathetic nerve activity, which is increased; and finally, a putative direct cellular effect of the peptide. Our data from the isolated perfused kidney suggest that in the absence of changes in perfusion pressure or renal nerve activity, AM stimulates intrarenal renin release, probably directly via juxtaglomerular granular cells. This suggestion was confirmed on isolated cultured granular cells, which responded to AM with an increase in renin release. Threshold values for significant stimulation of secretory rate were on the same order of magnitude in the two preparations (1 nmol/L), but a longer incubation time was needed for detection of an effect in the isolated cells.
Renin secretion and renin mRNA are often regulated in parallel,21 but no data on renin mRNA in response to AM are yet available. Renin mRNA abundance was increased by very low concentrations of AM, and the effect was dose dependent. AM could exert the effect on renin mRNA through an increase in renin gene transcription rate or through a specific mRNA-stabilizing action, which are mRNA regulatory mechanisms that both operate in granular cells.21 26 Altogether, the findings suggest that AM interacts directly with receptors on granular cells to enhance renin secretion and mRNA.
The stimulatory action of AM on renin secretion is common to other hypotensive peptides, such as calcitonin gene–related peptide and vasoactive intestinal peptide. Both peptides are thought to be neurotransmitters present in renal nerve endings that increase cAMP in target cells.27 28 In the present study, AM evoked cAMP production in granular cells at threshold and EC50 concentrations similar to or lower than those of other target cells.1 2 6 We conclude that granular cells contain receptors for AM that couple in a stimulatory fashion to adenylate cyclase. In agreement, intrarenal infusion of AM has been shown to increase urinary excretion of cAMP.15 Whether these receptors are specific for AM or are shared by calcitonin gene–related peptide, as suggested by other researchers,6 is at present unknown.
The approximate similarities between the threshold and EC50 values for cAMP production and stimulation of renin release and renin mRNA transcript abundance are consistent with a causal linkage between activation of adenylate cyclase by AM and renin secretion and mRNA abundance. The larger responses induced by forskolin are reflected in a much larger ability to generate cAMP in granular cells. Moreover, AM has been reported to lower intracellular calcium in target cells,29 30 which could contribute to the present results because renin release and mRNA are inversely coupled to intracellular calcium.25 AM is likely to stimulate renin secretion and mRNA through adenylate cyclase activation, with a potential contribution from changes in intracellular calcium. Calcium and cAMP are classic second messengers in the control of renin secretion.25
Although adrenals, kidney, and heart express high amounts of AM and the failing heart releases AM, no granular storage site of AM peptide has yet been identified.31 Cellular release of AM seems to be constitutive.4 The dispersed expression and release of AM by cells of the vessel wall and the colocalization of specific binding sites in these structures could implicate a primary autocrine/paracrine role of the peptide in the local control of vascular function, as suggested by other researchers.4 5 15 30 Moreover, AM mRNA levels can rapidly be increased severalfold in vivo,32 which suggests transcriptional regulation of AM production and secretion. We therefore investigated AM expression in renal juxtaglomerular structures.
AM transcripts were detected both in freshly isolated afferent arterioles and in cultured mesangial cells and granular cells. Our findings would suggest that in addition to the renal artery,30 the renal microvasculature constitutively expresses AM in vivo. The present experiments do not resolve which cell type in this preparation expresses AM. Granular cells and mesangial cells are likely production sites, along with renin-negative smooth muscle cells and endothelial cells in the afferent arterioles.4 5 A high level of constitutive AM peptide secretion by vascular smooth muscle cells has been detected with preparations obtained from different vessels.4 5 It is an intriguing possibility that the afferent arterioles in vivo actively synthesize and release AM. Both AM of systemic origin and locally produced AM could be of significance for the control of granular cell function. Demonstration of a clear role of endogenous AM for renin secretion and renin mRNA in physiology and pathophysiology would require the appearance of specific AM receptor antagonists and also elucidation of the factors that regulate AM expression and peptide release.
Our data suggest that the afferent arteriole is both a source and target for AM, which could have a tonic stimulatory effect on renin release and renin mRNA potentially in an autocrine/paracrine way. Further studies on the regulation of adrenal and renal AM and renin gene expression are currently in progress.
Selected Abbreviations and Acronyms
|FCS||=||fetal calf serum|
|PCR||=||polymerase chain reaction|
This study was supported by grants from the University of Copenhagen, by Statens Sundhedsvidenskabelige Forskningsraad, by Danske Laegers Forsikring under Codan Forsikring (B.L.J.), and by a grant from Deutsche Forschungsgemeinschaft (ku/859 2-2) (A.K.). The expert technical and graphical assistance of Karl-Heinz Götz and Marlies Hamann and the secretarial help provided by Hannelore Trommer are gratefully acknowledged.
Reprint requests to Boye L. Jensen, MD, PhD, Institut für Physiologie I, Universität Regensburg, Postfach 101042, D-93040 Regensburg, FRG.
- Received August 9, 1996.
- Revision received August 11, 1996.
- Accepted October 29, 1996.
Hirata Y, Hayakawa H, Suzujki Y, Suzuki E, Ikenouchi H, Kohmoto O, Kimura K, Kitamura K, Eto T, Kangawa K. Mechanisms of adrenomedullin-induced vasodilation in the rat kidney. Hypertension. 1995;25:790-795.
Schurek HJ, Alt JM. Effect of albumin on the function of the perfused rat kidney. Am J Physiol. 1981;240:F569-F576.
Della Bruna R, Kurtz A, Corvol P, Pinet F. Renin mRNA quantification using polymerase chain reaction in cultured juxtaglomerular cells. Circ Res. 1995;73:639-648.
Jougasaki M, Wie CM, Aarhus LL, Heublein DM, Sandberg SM, Burnett JC. Renal localization and actions of adrenomedullin: a natriuretic peptide. Am J Physiol. 1995;268:F657-F663.
Fukuhara M, Tsuchihashi T, Abe I, Fujishima M. Cardiovascular and neurohumoral effects of intravenous adrenomedullin in conscious rabbits. Am J Physiol. 1995;269:R1289-R1293.
Hackenthal E, Paul M, Ganten D, Taugner R. Morphology, physiology, and molecular biology of renin secretion. Physiol Rev. 1990;70:1067-1116.
Chen M, Schnermann J, Smart AM, Brosius FC, Killen PD, Briggs JP. Cyclic AMP selectively increases renin mRNA stability in cultured juxtaglomerular granular cells. J Biol Chem. 1993;32:24138-24144.
Porter JP, Reid IA, Said SI, Ganong WF. Stimulation of renin secretion by vasoactive intestinal peptide. Am J Physiol. 1982;234:F306-F310.
Kurtz A, Muff R, Born W, Lundberg JM, Millberg BE, Gnädiger MP, Uehlinger DE, Weidmann P, Hökfeldt T, Fischer JA. Calcitonin gene-related peptide is a stimulator of renin secretion. J Clin Invest. 1988;82:538-543.
Nishikimi T, Kitamura K, Saito Y, Shimada K, Ishimitsu T, Tamiya M, Kangawa K, Matsuo H, Eto T, Omae T, Matsuoka H. Clinical studies for the sites of production and clearance of circulating adrenomedullin in human subjects. Hypertension. 1994;24:600-604.
Wang X, Yue TL, Barone FC, White RF, Clark RK, Willette RN, Sulpizio AC, Aiyar NV, Ruffolo RR, Feuerstein GF. Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage. Proc Natl Acad Sci U S A. 1995;92:11480-11484.