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Kidney International, Vol. 51(1997), pp. 1100—1105

Increased atrial natriuretic mRNA expression in the 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 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, 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 . 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 - 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 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 >< 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]. USA). The cycle for the amplification of ANP mRNA was repeated 30 times for renal cortex and outer medulla, 22 times for Radioimmunoassay atria and ventricles as in our previous study [21]. The amplified ANP-like immunoreactivity of plasma and urine were deter- products were then electrophoresed on 1.8% agarose gels andmined by a specific radioimmunoassay (RIA; Peninsula Labora- transferred to nylon membranes (Schleicher & Schuell Gmbl-I,tories, Inc., Belmont, CA, USA) after extraction as in our previous Dassel, Germany). The blots were hybridized with [32P] labeled,report [7, 21]. PRA was also measured using an RIA kit (Du Pont randomly-primed, 426 bp rat ANP cDNA prepared by PCRCompany, Boston, MA, USA). cloning of the rat atria extracts for 16 hours at 65°C, according to the standard technique. After each hybridization, the blots were Statistical analysis washed twice in a solution containing 0.1% SDS and 2 X SSC for Data were expressed as mean SEM. To test the differences 15 minutes at 37°C, then twice in 0.1% SDS and 0.1 X SSC atamong the three groups, analysis of variance was performed. If a 65°C. Blots were exposed to Kodak XAR (Eastman Kodakdifference was found, unpaired Student's t-test was used for Company, Rochester, NY, USA) film at —70°C. A radioisotope-comparison between two groups. Linear regression analysis was labeled probe for /3-actin used as an internal control was alsoused to test the correlation between the urine ANP excretion rates made by the primer extension method. After autoradiography, theand urine sodium excretion rates and urine volume in normal x-ray film was scanned by a laser densitometer (Molecular Dy-control rats and diabetic rats on days 2, 4, 7, 14 and 42. A P namics, Sunnyvale, CA, USA), and the data analyzed by MDvalue < 0.05 was considered statistically significant. ImageQuant software release version 3.22. The value obtained for each ANP PCR product was normalized by its /3-actin levels. To Results control the accuracy of RT-PCR followed by Southern hybridiza- The characteristics of NC, UT-DM and IT-DM on the 42nd day tion, serially diluted total RNA of heart and renal tissues wereare shown in Table 1. The mean blood glucose levels and kidney subjected to RT-PCR amplification. Moreover, an intra-assayweight/body wt ratio in UT-DM were significantly increased, variation of semiquantitative RT-PCR followed by Southern blotwhereas body wt were significantly decreased as compared to NC. analysis from renal tissues was tested. Plasma ANP levels in untreated diabetic rats were increased, 1102 Shinet al: ANP mR/VAindiabetic rat kidney

Table 2. Daily urine volume, daily urinary sodium excretion, and 1 2 3 4 urinary AN? excretion of normal control (NC), untreated diabetes 6 x 2.4 ig (UT-DM), and insulin-treated diabetes (IT-DM) rats A 6 6 6 6 6 NC UT-DM IT-DM N=6 N=7 N=6 Urine volume mi/day Day 0 16 2.1 19 2.9 17 1.8 —ANP 2 17 1.0 103 49a38 3.2a 4 15 1.5 165 9.2a 24 2.8c 7 18 1.0 153 7j 19 2.2c 14 22 1.5 147 12.525 3.2c 42 24 3.0 164 10.619 5.2 Urine sodium excretion mmol/day — 3-actin Day 0 1.3 0.12 1.40.05 1.30.10 2 0.80.04 1.40.10'0.9 4 1.00.07 1.6 015b1.30.15 7 1.20.16 1.70.10k1.2 013d 14 1.20.12 1.6 009h1.0 015d 11 2 3 4 5 6 42 1.20.17 1.6 012b1.0 o.l6 5 6x2.4 tg 66B 6 66 66 6 666 6 Urine ANP excretion fmol/day Day 0 17 1.4 26 1 25 2.4 S55 a —ANP 2 25 3.2 580 8988 12.0 4 34 3.72150 30337 54C 7 36 4.93342 32945 3.8c 14 41 5.0 3169 25630 3.1c — — — p— 3-actin 42 35 3.2 2828 22732 33C Values are mean SCM. P < 0.001, 'P< 0.05 vs. NC °P < 0.001, 'P <0.01vs. UT-DM as e — a S—ANP whereas PRA were suppressed. All these changes in the UT-DM were ameliorated in the IT-DM. As shown in Table 2, mean daily — 3-actin urinary sodium excretion and urinary volume were significantly increased day 2 after STZ induction and remained elevated forFig. 1. The quality control of semi-quantitative PCR followed by Southern blot analysis. Serial dilutions (6:1, 6:2, 6:3, 6:4, and 6:5) of 2.4 .rg RNA the entire 42-day study period in diabetic rats as compared to from rat ventricle were subjected to RT-PCR amplification for 22 and 22 those in normal rats. The urinary ANP-Ll excretion rate was also cycles for ANP and /3-actin, respectively. (A) Ethidium bromide stained elevated in diabetic rats and ran parallel to changes in the urinary1.8% agarose gel containing PCR products amplified for 22 and 22 cycles sodium excretion rate and urine volume. Daily urinary ANPfor ANP and j3-actin, respectively. (B) Southern blot analysis of PCR excretion rates were significantly correlated with sodium excretion products from (A). (C) To test intra-assay variation of RT-PCR amplifi- cation followed by Southern blot analysis, 2 j.g RNA (N =6)from rat rate (r0.503, P < 0.05) and urine volume (r =0.799,P < 0.01) cortex were subjected to RT-PCR amplification for 30 and 22 temperature in normal rats, untreated diabetic rats and insulin-treated diabeticcycles for AN? and /3-actin, respectively. rats on day 42. Significant correlation were also found between urinary ANP excretion rates and urinary sodium excretion rates, urine volume on days 2, 4, 7 and 14 in normal and diabetic ratsintra-assaytest of RT-PCR followed by Southern blot analysis in (data not shown). The elevations of daily urine volume, urinaryrenal cortex (Fig. 1) was done and the variation coefficients of sodium excretion rate, and ANP-LI excretion returned to nearANP and J3-actin mRNA were 6.2% and 6.9%, respectively. normal range in insulin-treated diabetic rats. Figure 2 shows the autoradiographs of RT-PCR amplification RT-PCR coupled with Southern blot analysis revealed a 458-bpof ANP and /3-actin mRNA in the renal cortex, outer medulla, product from rat kidney and heart tissue RNA extracts. Toatrium and ventricle from diabetic and control rats on day 42. The determine the relative changes in tissue ANP mRNA expression,relative ratios of the densitometry measures of the PCR products the yield of ANP PCR products was normalized to the amount offor ANP and 13-actin are shown in Figure 3. increases in the /3-actin eDNA amplified from the same RT eDNA of tissuerelative ratio of PCR products for ANP and J3-actin mRNA from samples. This method has been used by several investigators [28,renal cortex and outer medulla in UT-DM rats were found when 29]. The accuracy of the semiquantitative RT-PCR with Southerncompared with those in NC rats. The relative ratio for ANP and blot analysis in this study was tested. Serial dilutions of 2.4 zg/3-actin PCR product in ventricle increased significantly, while that RNA were subjected to RT-PCR amplification for ANP andin atrium decreased significantly in UT-DM rats. No significant J3-actin, respectively. The correlation coefficients between RNAchanges for AN? mRNA expression in the renal medulla, atrium concentrations and corresponding densities were r =0.995andand ventricle were observed between IT-DM rats and NC rats. r =0.996from renal cortex, r =0.967and r =0.949from cardiac Figure 4 represents the autoradiographs of RT-PCR amplifica- ventricles for ANP and 13-actin genes (Fig. 1), respectively. The tion of ANP and /3-actin mRNA in the renal cortex and outer Shin et al: ANP mRNA in diabetic rat kidney 1103 NC UT-DM IT-DM II Cortex s S — a aseSSeeee e — ANP e e a a a a a e ee e e ee fl — actin

—— Medulla a a a afle a e a —ANP tee. fl • — -actin

Atrium —ANP a-pafl —u-pap-re— 3-actin

- Ventricle —a — —n—a as —ANP — — fl

— j3-actin

Fig.2. Autoradiographs of the amplification of ANP and f3-actin mRNA by RT-PCR followed by Southern blot analysis in renal cortex, renal outer medulla, atrium, and ventricle from 5 representative rats of normal controls (NC), untreated diabetic rats (UT-DM) and insulin-treated diabetic rats (IT-DM) on the 42nd day after the administration of citric buffer or streptozotocin. Each lane represent an individual rat.

medulla of NC rats on days 2 and 14 and of UT-DM rats on days 6 2, 4, 7 and 14 day. The relative ratio of the PCR products for ANP and J3-actin mRNA in UT-DM rats significantly increased (P < 5 * 0.01) on the day 2 and remained elevated until day 14. In Table 3, mean GFR value of UT-DM rats was not higher 4 than that of NC rats or that of IT-DM rats. Untreated diabetic + rats had a markedly elevated blood glucose value and decreased + body wt, whereas IT-DM rats had no difference in blood glucose 3 value or body wt as compared to NC rats. There was no difference in mean arterial blood pressure among the three groups. 2

Discussion § In the present study, we demonstrated that the kidney is a site I of ANP synthesis and that renal ANP synthesis is enhanced in diabetic rats, based on the following findings: (1) PCR amplifica- 0 tion identified the presence of ANP mRNA in the rat kidney; (2) Cortex Medulla Atrium Ventricle renal ANP mRNA expression was significantly increased in diabetic rats; (3) urinary ANP-LI excretion was markedly in- Fig. 3. Relative ratios of the densitometiy readings for RT-PCR amplifica- creased in diabetic rats as compared to that of control rats; and (4) tion of AMP and -acrin mRS/A in the renal cortex, Outer medulla, atrium urinary ANP excretion rate correlated well with urine sodiumand ventricle from the normal controls (LI, N =6),untreated diabetic rats excretion and urine volume. (B, N =7)and insulin-treated diabetic rats (E:, N =6)on the 42nd day after the administration of citric buffer or streptozotocin. 'P <0.01,P < Although no measurement of plasma volume was done, the 0.05versus normal controls; 5P <0.001,tp <0.05versus untreated increased plasma ANP level and reduced plasma renin activity indiabetic rats. 1104 Shinet al: AN? ,nRNA in diabetic rat kidney

NC DM DMDMDM NC Table 3. Laboratory data of 6 normal control (NC), 7 untreated 2 2 4 7 14 14 Time, days diabetic (U1-DM) and 5 insulin-treated diabetic (IT-DM) rats during inulin clearance determination done from 4 to 6 weeks after the I II II II II II A injection of STZ or citric buffer 5 a —ANP NC UT-DM IT-DM Number 6 7 5 — f3-actin Body weight g 436 6 276 6" 418 7b Blood glucose mmol/liter 6.2 0.2 38.2 5.4" 5.3 Urine volume mi/day 21 1.6 158 12.8" 26 2.7k' B apan. —a—ANP Mean arterial pressure mm Hg 115 8.2 109 6.9 118 7.3 •—, - GFR mI/mm 1.55 0.26160 0.16 1.640.21 — -actin Values are mean SCM. t•c•.ii "P < 0.001 vs. NC r b Fig.4. Autoradiographs of the amplification of AN? and 13-actin mR/VA by P< 0.001 vs. UT-DM RT-PCR followed by Southern blot analysis in renal cortex (A), outer medulla (B) from normal controls (NC) and untreated diabetic rats (DM) on days 2, 4, 7 and 14 after citric buffer or streptozotocin injection. also found to be significantly correlated with the urinary sodium excretion rate. Taken together with these prior investigations, significantly increased urinary ANP-Ll excretion and enhanced diabetic rats were compatible with another report [221. Therenal AN? gene expression in diabetic rats shown in this study increase of plasma ANP level has been thought to originate fromsuggest that ANP synthesized in the kidney is responsible for the atrium in response to sodium retention and volume expansionsodium and water homeostasis. This is to say that the kidney itself [22-23]. However, our current study shows that diabetic rats havemay sense and regulate fluid and imbalance resulting increased cardiac ventricular ANP mRNA but decreased atrialfrom diabetes by the synthesis of ANP. ANP mRNA levels. Actually, extra-atrial transcription of the Enhanced proximal tubule sodium-glucose cotransport activity, ANP gene has been identified in different organs [5—8],fromosmotic effect of hyperglycemia and blunting of the volume reflex which ANP was released to participate in the regulation of bloodhave been recognized to be contributing factors of increased pressure and body fluid homeostasis [7—8]. In several pathophys-intravascular volume and total body sodium in early diabetes iological conditions, plasma ANP levels correlated much better[34—36]. Circulating ANP levels from the heart therefore in- with the changes in the expression of ANP gene in extra-atrialcreases in a counterregulatory response to increased intravascular tissues when compared to those in the atrium [30, 311]. Thesevolume, and then may mediate glomerular hyperfiltration in observations support the proposal that extra-atrial ANP synthesismoderately hyperglycemic diabetic rats. This hypothesis was re- might play a role in the regulation of volume and electrolytecently confirmed by Zang et a! [34]. By infusion of ANP receptor balance [32]. antagonist (HS-142-1) into diabetic rats, they found a significant In the search of the renal ANP gene message, we found that thereduction of glomerular filtration from markedly elevated to renal ANP mRNA was too low to be measured by Northern blotnear-normal levels. However, in our study, elevated plasma AN? analysis. However, by using RT-PCR coupled with Southern blotconcentration with unchanged glomerular filitration in severely analysis, the presence of renal ANP mRNA message could easilyhyperglycemic diabetic rats were found. This finding is consistent be detected. In this study, we compared the relative amount ofwith another study [22]. Increased plasma ANP level without ANP mRNA expression in renal tissues. Our results have shownglomerular hyperfiltration in severely hyperglycemic diabetic rats that serial dilution of RNA followed by RT-PCR and Southernmay be explained by markedly increased renal arteriolar resis- blot analysis can detect relatively small changes in the mRNAtance [37]. An increase of vasoconstrictive substances have been message. Our current study has also shown that renal ANPfound [38] and may counteract the direct action of natriuretic mRNA was enhanced in diabetic rats as compared to that inpeptides on renal hemodynamics in severely hyperglycemic dia- normal rats. betic rats. In other words, we may speculate that ANP may be very In addition to the changes of cardiac ANP mRNA levels, ourimportant in the maintenance of GFR in the face of vasoconstric- results have surprisingly showed that ANP mRNA expression intor effects in severely hyperglycemic diabetic rats. renal cortex and outer medulla and urinary ANP-LI excretion Osmotic diuresis due to hyperglycemia are the undisputed were markedly enhanced after the induction of diabetes. Severalfinding observed immediately after the development of diabetes. recent studies have demonstrated that the kidney is also a site ofThe unreabsorbed glucose, acting as an osmotic substance, inhib- ANP synthesis [9—12] and that renal synthesized ANP is specu-its the reabsorption of both water and sodium. Therefore, osmotic lated to be involved in the regulation of renal sodium excretiondiuresis has been thought as the most important contributing [12]. Several investigations indicate that circulating ANP is not afactor to mediate diuresis and natriuresis in diabetes. Because direct regulator of renal sodium excretion [13—18, 33]. In humanexogenous AN? administration may elicit a pronounced natri- studies, renal natriuretic peptide (urodilatin) closely parallelsuretic effect mediated by increased filtered load and by decreased renal sodium excretion [16—18], while circulating ANP levels dotubule sodium reabsorption [3]. The increase of plasma ANP level not correlate with the degree of natriuresis [13—16] in the variousprovides us to hypothesize that ANP may also be related to conditions that influence body fluid regulation. More recently, wenatriuresis and diuresis in diabetes. Zang et al [34] demonstrated have also demonstrated that renal ANP mRNA expression andthat the acute administration of the ANP receptor antagonist urinary ANP excretion rate were markedly increased in DOCA-significantly reduced diuresis and natriuresis in diabetic rats. salt rats [21]. In DOCA-salt rats, urinary ANP excretion rate wasTheir results confirm that plasma ANP, in addition to osmotic Shin et al: ANP mRNA in diabetic rat kidney 1105 dialysis, may contribute to natriuresis arid diuresis in early diabe- 17. GOETZ K, DRUMMER C, ZHU JL, LEADLEY RJ, FIEDLER F, GERZER R: tes. However, this does not exclude the possible contribution of Evidence that urodilatin, rather than ANP, regulates renal sodium renal-synthesized ANP or other natriuretic to diuresis or excretion. JAm Soc Nephrol 1:867—874, 1990 18. HEER M, DRUMMER C, BAISCH F, GERZER R: Long-term elevations of natriuresis in diabetic rats. Specific studies to evaluate these issues dietary sodium produce parallel increases in the renal excretion of are currently in progress in our laboratory. urodilatin and sodium. Iflugers Arch 425:390—394, 1993 In conclusion, the present study has showed that the rat kidney 19. BRENNER BM, BALLERMANN BJ, GUNNING ME, ZEIDEL ML: Diverse is a site of ANP synthesis and that renal ANP mRNA expression biological actions of atrial natriuretic peptide. Physiol Rev 70:665— 699, is strongly enhanced in diabetic rats. Whether renal synthesized 1990 20. KOLER K, GOEDDEL DV: Molecular biology of natriuretic peptides ANP is one of factors to handle the regulation of water and and their receptors. Circulation 86:1081—1088, 1992 sodium balance in diabetic rats is worthy of further investigation.21. LEE YJ, SHIN SJ, TAX MS. HSIEH TJ, T5AI JH: Increased renal atrial natriuretic peptide synthesis in rats with deoxycorticosterone acetate- Acknowledgments salt treatment. Am J Physiol (in press) 22. ALLEN TJ, COOPER ME, O'BRIEN RC, BACH LA, JACKSON B, JERUMS This study was supported by grants (NSC-82-0412-B037-045 and NSC- G: Glomerular filtration rate in streptozotocin-induced diabetic rats. 84-2331 -B037-069) from the National Science Council, Taiwan. We are Role of exchangeable sodium, vasoactive , and insulin grateful to Ms. Penelope Chen, Ms. Yuh-Chih Tzeng, Ms. Suh-Ju Lee and Mr. Nan-Chiun Horng for their technical assistance. therapy. Diabetes 39:1182—1190, 1990 23. 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