Increased Atrial Natriuretic Peptide Mrna Expression in the Kidney of Diabetic Rats
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Kidney International, Vol. 51(1997), pp. 1100—1105 Increased atrial natriuretic peptide mRNA expression in the kidney of diabetic rats SHYI-JANG SHIN, YAU-JIUNN LEE, MIAN-SHIN TAN, TusTY-JIuAN HSJEH, and JuEI-HsIuNG TSAI Division of Endocrinology and Metabolism, Graduate Institute of Medicine, Kaohsiung Medical College, Kaohsiung, Taiwan Increased atrial natriuretic peptide mRNA expression in the kidney of transcription-polymerase chain reaction (RT-PCR) followed by diabetic rats. To investigate whether renal synthesis of atrial natriuretiC Southern blot analysis, we recently demonstrated that renal ANP peptide (ANP) is influenced in diabetes, we measured renal ANP mRNA levels, urine volume, urinary ANP and sodium excretion rates in strepto- mRNA levels and urinary ANP excretion rates markedly in- zotocin (STZ)-induced diabetic rats. By using reverse transcription- creased in rats with deoxycorticosterone acetate-salt (DOCA-salt) polymerase chain reaction (RT-PCR) followed by Southern blot analysis, treatment [21]. The urinary ANP excretion rate was also found to we found that renal cortical and outer medullary ANP mRNA levels in be well correlated with urinary sodium excretion rate in DOCA- untreated diabetic rats were markedly increased as early as the second day salt rats. These observations suggest that renal-synthesized natri- after the onset of hyperglycemia and remained elevated for the entire 42-day study period. Plasma AMP concentrations in untreated diabetic uretic peptide, in addition to circulating ANP, may have a role in rats were increased on the 42nd day, whereas plasma renin activity were the renal regulation of sodium homeostasis. suppressed. The urine volume, urinary ANP and sodium excretion rates in In animals with experimental diabetes, increased plasma vol- untreated diabetic rats were also significantly elevated on the second dayume and exchangeable sodium have been found [22] and have and remained elevated for the entire 42-day study period. Urinary ANP excretion rates were well correlated with urine volume, and urinarybeen thought to lead to changes in plasma ANP concentration sodium excretion rate in normal rats and diabetic rats on days 2, 4, 7, 14 [22—24] and cardiac ANP mRNA expression [25] in the early stage and 42. Our results indicate that renal ANP mRNA expression isof diabetes. In an attempt to find out whether intrarenal ANP enhanced in diabetic rats, and that renal-synthesized AMP as one of synthesis is influenced in severely hyperglycemic diabetic rats, we regulators to handle water and sodium balance in diabetic rats is worthy of measured renal ANP mRNA levels, urinary ANP excretion rates further investigation. and circulating ANP levels in STZ-induced diabetic rats. Methods Atrial natriuretic peptide (ANP), a peptide with potent di- Animal experiments uretic, natriuretic, vasorelaxant, and aldosterone-inhibitory activ- Male Wistar rats weighing 260 to 320 g were individually ities [1—3], is secreted primarily by the cardiac atria [4]. With thehoused in metabolic cages. Diabetes was induced by a single immunocytochemical technique and the availability of cDNAperitoneal injection of 55 mg/kg STZ (Sigma Chemical Co., St. probes, the presence of proANP-like immunoreactive peptide andLouis, MO, USA). Twenty-four hours later, induction of diabetes ANP mRNA were demonstrated in extra-atrial tissues [5—8].was confirmed by the measurement of their tail blood glucose More recently, several studies have provided evidences suggestinglevels (Yellow Springs Instrument Co., Inc., Yellow Springs, OH, that the kidney is also a site of ANP synthesis [9—12] and thatUSA). Rats with blood glucose levels > 19.4 mmol/liter were renal synthesized ANP may contribute to the regulation of renalincluded. Diabetic rats were randomly assigned to one of two sodium excretion [12]. Several investigators have indicated thatgroups: untreated diabetic rats (UT-DM, N =24)and insulin- circulating ANP concentration does not correlate with the degree treated diabetic rats (IT-DM, N =11).Rats in the IT-DM group of natriuresis in the physiological, and pathophysiological condi-received insulin (heat-treated bovine ultralente insulin; Novo- tions [13—16]. In contrast, urine urodilatin (ANP-95 to 126), aNordisk, Copenhagen, Denmark) designed to achieve blood natriuretic peptide of renal origin, excretion closely parallels renalglucose levels between 4.5 and 8.3 mmol/liter. Eighteen body wt sodium excretion under various conditions that influence bodyand age-matched rats were studied as normal controls (NC). In fluid regulation [16—18]. There have also been reports showingExperiment 1, rats (NC, N =6;UT-DM, N =7;IT-DM, N =6) that most of the circulating ANP was degraded in the kidney, aswere sacrificed by decapitation to collect blood for the measure- the proximal tubule contained potent ANP degrading enzyme andments of plasma ANP and plasma renin activity (PRA) on day 42 the natriuretic peptide receptor C subtype (NPR-C) were distrib-after STZ or citric buffer injection, and atria, ventricles, kidney uted over the vascular endothelium [19, 20]. Using reversecortex and outer medulla were immediately removed for ANP mRNA analysis. Urine sodium, and ANP immunoreactivity were also measured on days 2, 4, 7, 14, and 42 in three groups. In Received for publication July 11, 1996 Experiment 2, rats from the UT-DM group were sacrificed on and in revised form November 11, 1996 days 2 (N =4),4 (N =2),7 (N =2),and 14 (N =2),and from Accepted for publication November 12, 1996 the NC group on days 2 (N =3)and 14 (N =3)for renal AMP © 1997 by the International Society of Nephrology mRNA determination. In Experiment 3, for the measurement of 1100 Shin et al: At'/P mRNA in diabetic rat kidney 1101 glomerular filtration rate, seven rats from the UT-DM group, five Table 1. Characteristics of normal control (NC), untreated diabetes from the IT-DM group and six from the NC group were studied (UT-DM), and insulin-treated diabetes (JT-DM) rats on the 42nd day after the administration of citric buffer or streptozotocin from weeks 4 and 6 after injection of STZ or citric buffer. NC UT-DM IT-DM RNA isolation and reverse transcription Number 6 7 6 Total RNA was extracted from the renal cortex, outer medulla,Change in body weight % 43.0 1.5 —9.7 2.0° 42.8 2.8° atrium and ventricle by a modified guanidium isothiocyanateBlood glucose mmol/liter 6.80.2 40.8 1.2° 8.3 1.3° Kidney/body weight ratio 3.60.08 6.3 0.14° 3.7 O.O7 method [26]. Two micrograms of total RNA from each sample ><io- were reverse transcribed by incubating with 20 jkl reverse tran-Plasma ANPfmol/nzl 13.62.1 27.6 2.8' 16.3 5.5 scription mixture containing: 20 pmole oligo (dT)18 primer, 50 mvt Plasma renin activity_ng/ml/hr 3.3 0.72 0.4 0.06! 3.1 0.22° Tris-HCI (pH 8.3), 75 mist KCI, 3 mist MgCl2, 20 units of Rnase Values are mean SEM. inhibitor, 0.5 mist dNTPs, and 200 U of MMLV reverse transcrip- °P <0.001, hp<0.01 vs. NC ° tase (Clontech Laboratories Inc., Palo Alto, CA, USA) at 42°C for p< 0.001, dP< 0.01 vs. UT-DM one hour. The reverse transcriptase was inactivated by heating for five minutes at 94°C. Glomerular filtration rate study Polymerase chain reaction amplification and Southern Rats were anesthetized intraperitoneally with 50 mg/kg pento- hybridization barbital sodium (Abbott Laboratories, Chicago, IL, USA) and Primers for the PCR were designed to flank at least onetracheostomized. A polyethylene (PE-50) catheter was inserted putative intron site for rat ANP and -actin. For ANP, the senseinto the left femoral artery for blood sampling and continuous primer (5 '-GGCTCCTTCTCCATCACCAA-3') corresponded toarterial blood pressure monitoring with a pressure transducer and base pair 4 to 23, and the antisense primer (5'- TGTTATCTTCG-a writing recorder (Gould, Valley View, OH, USA). Catheters GTACCG-3') corresponded to bp 445 to 461 of rat ANP eDNA.were also placed in the left and right femoral veins for infusion of For f3-actin, the sense primer (5'-CGTAAAGACCTCTATGC-inulin and plasma. Inulin (7%) in normal saline was infused via a CAA-3') corresponded to bp 2748-2767, and the antisense primersyringe pump (Stoelting, Wood Dale, IL, USA) at a rate of 1.2 (5 '-AGCCATGCCAAATGTCTCAT-3') corresponded to bpmi/hr. To replace blood loss from surgery, rat plasma from 3203-3222 of rat f3-actin gene. The size of expected PCR productsdiabetic or normal rats, equivalent in volume to 1% of body wt, was 458 bps for ANP and 349 bps for -actin. For amplification,was infused over 50 minutes, followed by a sustained infusion rate PCR was performed at a final concentration of 1>< PCR buffer (10 of 0.6 mI/hr with supplementary isotonic saline to replace urinary mM Tris-HC1, pH 8.3; 50 mist KCI; 1.5 mM MgCI2), 0.2 mM dNTP, losses. Urine was collected from the left ureter by PE-lO cannu- 0.4 j.rM sense and antisense oligos, 2.0 units of Taq DNA poly-lation. After a 60-minute equilibration period, urine was collected merase (Boehringer Mannheim, Indianapolis, IN, USA) to a totalfrom the left ureteral catheter over 30-minute periods, and 200 j.rl volume of 50 p1. The amplification cycles were 45 seconds at 94°C, blood samples were collected for plasma inulin measurement at 45 seconds at 60°C and 90 seconds at 72°C in a Perkin-Elmerthe midpoint of each urine collection. The inulin in plasma and Cetus 9600 thermocycler (Perkin-Elmer Cetus, Norwalk, CT,urine was measured by the thiourea-resorcinol method [27].