Alterations in glomerular RNA in diabetic rats: Roles of glucagon and insulin

Kidney International, Vol. 20 (/981), pp. 491—499

Alterations in glomerular RNA in diabetic rats: Roles of glucagon and insulin PEDRO CORTES, FRANCIS DUMLER, KUMARAPURAM K. VENKATACHALAM, JOSE GOLDMAN, K. S. SURY SASTRY, HEMA VENKATACHALAM, JAY BERNSTEIN, and NATHAN W. LEVIN Department of Medicine, Division of Nephrology and Richmond W. Smith Research Laboratory, Henry Ford Hospital, Detroit, and the Department of Anatomic Pathology, William Beaumont Hospital, Royal Oak, Michigan

Alterations in glomerular RNA in diabetic rats: Roles of insulin anti glucagon. Incorporation in vivo of labeled orotate into RNA and total nucleotides was measured in isolated glomeruli and whole renal cortex. In 2-day diabetic animals, glomerular RNA was increased, and there was greater incorporation of orotate into total nucleotides and RNA as compared with controls. Insulin reversed the exaggerated incorporation at infusion rates that corrected hyperglucagonemia without reducing plasma glucose and with only minimal changes in insulin concentra-

tions. The addition of glucagon to insulin infusions reproduced the increased incorporation observed in untreated diabetics. Similar changes occurred in renal cortex, where differences in orotate incorpo-

ration into nucleotide precursors seemed to be the main cause for alterations in RNA labeling. Isotope incorporation in glomeruli correlat-

ed positively with plasma glucagon, but not with insulin or glucose concentrations. Although in 7-month diabetic animals orotate incorpo. ration into RNA was less than in controls, probably as a consequence o1 renal disease, 24-hour insulin infusion decreased it further. Our results confirm that in the diabetic kidney, abnormal uracil nucleotide metabo-

lism and increased cellular content of RNA are demonstrable in glomeruli as in the renal cortex. These changes appear to be related

[1, 2]. The increase in GFR is accompanied by morphologic changes of glomerular hypertrophy [3]. The volume of the capillary tuft and the diameter of the capillary lumina are significantly increased, although the number of glomerular cells

remains constant. Direct measurements of isolated glomeruli demonstrate increased size and weight [4, 5]. Anatomic and functional evidence of renal hypertrophy exists also in experimental diabetes [6, 7]. In addition, cortical RNA content, RNA synthesis rate, and the formation of uracil nucleotides from orotate are all increased 2 days after induction of diabetes with streptozotocin in the rat [8]. These metabolic abnormalities resemble some of those observed at the initiation of compensatory renal growth [8, 9]. Shortly after unilateral nephrectomy, there is enhanced cortical synthesis of uracil nucleotides and of RNA and a rapid increase

in RNA content [10]. These biochemical changes and the compensatory growth of the remaining kidney are augmented after unilateral nephrectomy in diabetic animals [8, 9]. The accentuated renal growth in diabetes may be relevant to

directly to hyperglucagonemia. Modifications du RNA glomérulaire chez les rats diabetiques: Roles de l'insuline et du glucagon. L'incorporation in vivo d'orotate marqué dans

the pathogenesis of diabetic glomerular disease. Unilateral

le RNA et les nucléotides totaux a étb mesurée dans des glomérules

nephrectomy accelerates the development of diabetic glomerular lesions in the remaining kidney of rats injected with strepto-

isolés et l'ensemble du cortex renal. Chez des animaux diabetiques de 2 jours Ic RNA glomerulaire était augmenté et il y avait une plus grande incorporation de l'orotate dans les nucléotides totaux par comparaisort aux contrOles. L'insuline a renversé cette augmentation de l'incorpora-tion a des debits de perfusion qui corrigent l'hyperglucagonémie sans diminuer Ic glucose plasmatique et avec seulement des modifications

zotocin [II]. In man, also, compensatory renal growth may accelerate the appearance of diabetic lesions. In diabetic recipients of transplanted kidneys, characteristic glonierular lesions have been found to develop in the organ undergoing compensa-

minimes de Ia concentration d'insuline. L'addition de glucagon a La perfusion d'insuline reproduit l'augmentation d'incorporation observée chez les diabétiques traités. Des modifications semblables sont obser. vées dans Ic cortex renal oh des differences de l'incorporation d'orotate dans les precurseurs des nucléotides semblent être Ia cause principale des modifications du marquage du RNA. L'incorporation d'isotopes

tory growth earlier than would be expected from the natural course of the renal complications of the disease [12—141. Altered

glomerular microcirculatory dynamics during compensatory

growth have been proposed as enhancing the severity of diabetic glomerular lesions [11]. Alternatively, the additive effects of compensatory growth and diabetes on specific bio-

dans les glomCrules est positivement corrClée avec le glucagon plasma.

tique mais pas avec les concentrations d'insuline et de glucose. Bien que chez les animaux diabetiques de 7 mois I'incorporation d'orotate dans Ic RNA soit moindre que chez les contrôles, probablement en fonction de l'atteinte rdnale, La perfusion d'insuline sur 24 heures La diminue encore. Nos résultats confirment que dans le cortex de rein diabetique un metabolisme anormal des nucléotides et une augmentation des contenus cellulaires en RNA peuvent étre mis en evidence. Ces modifications paraissent être liées directement a l'hyperglucagonemie.

chemical changes common to both conditions offer a metabolic basis for the rapid development of glomerular disease. It is not known if the metabolic alterations observed in the whole diabetic renal cortex occur also in the glomerulus, where

the abnormalities induced by long-term diabetes have their

Insulin-dependent diabetes in man is characteristically associated with an increased GFR and renal size that are evident at the onset of the disease and that persist for as long as 18 years 491

Received for publication July 17, 1980 and in revised form February 4, 1981

0085-2538/81/0020-0491 $01.80

© 1981 by the International Society of Nephrology

492

Cortes et a!

greatest clinical implications. In addition, the factors mediating

the biochemical changes in the kidney in the early stages of experimental diabetes are not defined. Strict control of diabetes from its onset corrects the increased renal size and GFR in man [15] and reverses the metabolic changes in the renal cortex of

the diabetic rat [7, 8]. But, the relative importance of individual hormonal abnormalities or of hyperglycemia as mediators of the renal changes remains unknown. This study represents the first investigation of the metabolism

of RNA in diabetic glomeruli. Glomerular RNA content and incorporation of exogenous precursors into RNA have been studied in vivo in short- and long-term diabetic rats, and the glomerular metabolic changes compared with those occurring simultaneously in the whole renal cortex of short-term diabetic animals. The roles of insulin, glucagon, and glucose have been evaluated separately as factors influencing glomerular metabolism in diabetes. Methods

Charles River CD-F male rats weighing 160 to 200 g were used. Cannulae were inserted into the left femoral vein [8, 10], and animals were left to recover from surgery for 14 days before any experiments were carried out. After this period, they had

resumed a normal rate of weight increase. Animals had free access to food and water throughout the duration of the studies to avoid adverse effects of dietary restrictions on renal growth [16]. Diabetes was induced by the i.v. injection of streptozotocm (50 mg/kg; U-9889, a gift from the Upjohn Co., Kalamazoo,

MI) dissolved in 0.15 M sodium chloride acidified to a pH of 4.0 with 0.1 M citrate buffer [17]. Controls were age-matched and

injected with the same volume of acidified 0.15 M sodium chloride. The following experimental groups were studied. Group I. To compare purine and pyrimidine metabolism, we infused control and 48-hour diabetic animals with [5-3H]-orotic acid or [2-H]adenine. Treated diabetic animals received a continuous infusion of insulin for the last 38 hours of the experiment. Group2. To study the effects of glucagon, we treated separate groups of 48-hour diabetic animals with a continuous infusion of insulin or insulin plus glucagon for the last 38 hours of the experiment and infused them with [5-3FT]-orotic acid. Group 3. To study the effects of long-term diabetes, we infused control and 7-month diabetic animals with [5-3HJ-orotic acid, Diabetic animals treated with insulin received a continuous infusion for the last 24 hours of the experiment. For the measurement of glomerular

due to surface adsorption were minimized. All infusions were given to unanesthetized animals at a rate of 3 p.1/mm. Sodium orotate or adenine hydrochloride and their corresponding radiolabeled compounds were added to the infusates

during the last 2 hours of the infusion period. Each animal received 60 ii.Ci of [5-3H]-orotic acid (specific activity, 20 Ci/ mmoie) or 89.4 p.Ci of [5-3H]-adenine (specific activity, 16.6 Ci! mmole) and a total of 0.1 p.moles of the appropriate precursor. At the end of the infusion period, both kidneys were removed and rapidly immersed in 0.15 M sodium chloride at 2° C. A portion of renal cortex from each animal in group 3 was fixed in buffered formaldehyde for light-microscopic examination of PAS-stained sections. In some animals in group 2, the left kidneys were exteriorized through a lumbar incision. Before ligation of the renal pedicle and removal of the kidney for glomerular isolation, a sample of cortex was obtained with immediate freezing in liquid nitrogen

as previously described [10]. Contaminating blood and any medullary portion included in the samples were scraped from the cortex under liquid nitrogen. Tissue samples weighing 200 to 300 mg were pulverized in a pre-cooled stainless-steel mortar

and homogenized in 0.2 M perchloric acid at 2° C. The acidsoluble fraction was used for the quantitative analysis of uridine triphosphate (UTP) by ion-exchange high-pressure liquid chromatography [18]. As an internal standard, 0.25 p.Ci of [2-14CJUTP was added to each sample for assessment of UTP recovery. RNA and DNA were extracted from the acid-insoluble fraction by alkaline hydrolysis in 0.3 M potassium hydroxide and by acid hydrolysis in I M perchloric acid, respectively [19]. At the termination of each experiment, blood samples were obtained by cardiac puncture using heparinized plastic syringes. Plasma was immediately separated at 2° C and portions taken for the measurement of glucose, immunoreactive insulin (IRI), and urea nitrogen. A sample of blood was mixed with a kallikrein-trypsin inhibitor (500 U/mi, Trasylol, Farbenfabriken Bayer, New York, New York) for plasma immunoreactive glucagon (IRG) measurements. Renal cortical tissue from animals in each experimental group

was dissected from medullae and pooled. The tissue was minced, forced through a 120-mesh sieve, and the glomeruli were separated by graded sieving at 2°C [18, 20]. Storage of the samples for up to 3 hours at this temperature does not affect the

recovery of glomerular RNA and DNA [18]. The purity and glomerular concentration of each isolate was assessed by lightmicroscopic examination of the final suspensions. A purity of RNA content in an adequate number of glomerular prepara- 95% or greater was obtained. Two samples, each containing tions, noninfused normal and 48-hour diabetic animals were about 64 x l0 glomeruli, were obtained from each glomerular used. suspension and stored at —40° C until processed. Glomerular In experimental groups 1 and 3, control and diabetic animals yield per renal cortex was approximately 10.6 x l0. received a continuous i,v, infusion of 0.15 M sodium chloride. Glomerular samples, suspended in 3 ml of 0.15 M sodium To this solution, 173.6 to 463 mU/mI insulin (Regular Iletin®, E. chloride, were homogenized at 2° C in all-glass homogenizers. Lilly Co., Indianapolis, Indiana) was added in the treated Free nucleotides were separated from nucleic acids and prodiabetic groups. In group 2, control and diabetic animals were teins by precipitation of the latter in 0.34 M perchloric acid at 2° infused with 0.07 M sodium chloride solution containing 1% C after which RNA and DNA were extracted by methods bovine serum albumin (fraction V, Sigma Chemical Co., St. identical to those described above for the renal cortical samLouis, Missouri), and 0.05 M sodium phosphate buffer (pH, ples. RNA and DNA were quantified in triplicate by their 7.40). To this, insulin (115.7 to 254.6 mU/mi) with or without pentose content as determined by the orcinol and diphenylglucagon (200-400 ng/ml; E. Lilly Co.) were added in the treated amine reactions, respectively [21, 22]. With these methods, 0.6 diabetic animals. Infusates containing insulin in albumin solu- p.g of ribose is equivalent to I p.g of yeast RNA (type I, Sigma), tion delivered greater amounts of the hormone because losses and 0.13 p.g of deoxyribose is equivalent to 1 p.g of calf thymus

493

Glucagon and glomerular RNA changes in diabetic rats

DNA (type I, Sigma). Total protein was measured by the method of Lowry et al [23] with crystalline bovine serun were quantified by the glucose oxidase method using the Beckman glucose analyzer-Il and with the Dupont automated clinical analyzer, respectively. Plasma immunoreactive insulin and glucagon were measured by double-antibody radioimmunoassay methods [24, 251 with rat insulin and porcine glucagon as standards (Novo Research Institute, Bagsvaerd, Denmark).

:.:

4.0

albumin as standard (Sigma). Plasma glucose and urea nitrogen a)

0 0

0 2.0

200

Total free nucleotides in acid-soluble samples, collected chromatographic fractions, and extracts of RNA were analyzed for radioactivity in a liquid-scintillation counter (LS-350, Beck-

C 100

man Instruments, Fullerton, California). Where required, the perchloric acid in the samples was neutralized with I M Tris

C

0

base buffer before counting. A xylene-based commercial scintillator for aqueous samples was used (ACS. Amersham, Arling-

ton Heights, Illinois). Efficiency of counting was from 35 to 300

38%.

RNA and DNA values are expressed as their pentose equiva-

0,

lents. Incorporation values are presented as fractions of the disintegrations per minute (dpm) infused per 200 g of body

C

weight to correct for the small changes in mean weight present between animals in different experimental groups in the studies on short-term diabetics. In experiments on long-term diabetic

0

animals, the amount of labeled precursor to he infused was based on body weight because there were large differences

0

0i' 200

Control

5.2 2.6 0 Rate of insulin infusion, mu/kg/rn/n

6.95

between diabetic and control animals (control) 332 12 g (N = Fig. 1. Plasma glucose, immunoreactive insulin and immunoreactive glucagon concentrations in 48-hour diabetic animals: Effect of insulin 7), diabetic 160 6 g (N = 15). The amount of UTP incorporatinfusion. Values are means SEM in groups of 8 to 12 diabetic (closed ed into RNA was calculated as dpm incorporated into RNA per circles) or control (open circles) animals. one half of the specific activity of UTP obtained at the end of

the infusion with labeled orotate. The mean UTP specific activity for the entire labeling period is equivalent to one-half nucleotides in diabetics was about twofold the amount incorpothe final value because incorporation of orotate into UTP rated in controls (Figs. 2 and 3). Labeled adenine was incorpoincreases linearly during a continuous infusion [81. Final UTP rated to a greater extent than orotate into both glomerular RNA recovery for extraction and quantitation methods in the renal and total nucleotides, but no differences were found between cortical samples was between 66% and 82%. Results were diabetics and controls (Figs. 2 and 3). The continuous administration of insulin for a period of 38 expressed per unit of DNA, because this is the reference of choice to evaluate compositional tissue changes during growth hours to diabetic animals at increasing rates resulted in progres[28] and is the only renal component that remains constant in sive reduction of the exaggerated incorporation of orotate into the total organ during the early stages of both compensatory glomerular RNA (Fig. 2). This effect was evident at infusion and diabetic hypertrophy [7, 16]. Selection of glomerular DNA rates (2.6 and 5.2 mU/kg/mm) insufficient to correct hyperglyceas a reference is equivalent to the expression of metabolic mia or hypoinsulinernia, although the rates were sufficient to decrease IRG concentrations to values similar to or lower than changes per cell because of the uniploidy of glomerular cells. All results are presented as means SEM with number of those in controls (Figs. 1, 2). For example, at the lowest observations in parenthesis. The statistical significance of the infusion rate tested (2.6 mU/kg/mm), there was a small increase differences was determined by Student's t test for nonpaired in plasma IRI (from 9 to 19% of control values) and no decrease variables and by linear regression analysis. The significance of in plasma glucose concentrations, but IRG concentrations were the linearity of the regressions was tested by means of analysis reduced (P < 0.005) from those found in diabetics to control levels (Fig. 1). The excessive incorporation of orotate was of variance. totally prevented by a higher infusion rate of insulin (5.2 mL/kg/ Results mm), which resulted in plasma concentrations of glucose and Incorporation of radiolaheled precursors into glomerular insulin that were between control and diabetic values (Fig. 1). Insulin administration corrected the increased incorporation RNA and total nucleotides in ear/v diabetes: Ejfrcts of insulin (group 1). Forty-eight hours after streptozotocin injection. of orotate into glomerular RNA to a greater degree than it did plasma glucose and immunoreactive glucagon (IRG) were in- into total nucleotides. Only at the highest infusion rates used creased by 127% and 32%, respectively, and plasma immunore- (6.95 mU/kg/mm) was incorporation into total nucleotides reactive insulin (IRI) was reduced to less than 10% of control duced to control values (Fig. 3). values (Fig. 1). The fraction of labeled orotate incorporated at Relative effects of glucose, insulin, and glucagon on the the end of the labeling period into glomerular RNA and total incorporation of labeled orotate into glomerular RNA in short-

494

Cortes et al 3.0

S

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as o3

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1.0

ast°'

8.0

as0,

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,-._ -E, 'E' o8 a;5

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58

a,

H '0.5 "O

6.0 <

.E

Control

0

2.6

5.2

Control

6.95

Rate of insulin infusion, mu/kg/mitt

Fig. 2. Incorporation of radiolabeled orotate and adenine into glomerular RNA in 48-hour diabetic animals.' Effect of insulin infusion. Obser-

vations were carried out in the same groups of animals as in Fig. 1. In every group, each data point represents one or two glomerular samples derived from the pooled renal cortices of 8 to 12 animals. Open circles denote t5-3H1-orotate infusion in controls: closed circles, [5-3H]-orotate infusion in diabetics; open triangles, [2-3H]-adenine infusion in controls; closed triangles, [2-3HJ-adenine infusion in diabetics.

0

I 2.6

5.2

'a

I 6.95

Rate of insulin infusion, mU/kg/rn/n

3. Incorporation of radiolabeled orotate and adenine into free nucleotides in the glomeruli of 48-hour diabetic animals: Effect of Fig.

insulin infusion. Symbols are the same as in Fig. 2.

significant correlation (P < 0.1) was found with IRG values. This correlation was positive and linear (significance of linearity

P < .01) (Fig. 4). Multiple regression analysis of the data term diabetic animals (group 2). The results of experiments in which insulin and glucagon were infused at different rates were combined to study the relationships between changes in plasma IRI, IRG, and glucose concentrations and orotate incorporation into RNA. Experiment I in Table I shows that when plasma IRI was restored to control values with the infusion of 3.8 mU of insulin/kg/mm, hyperglycemia and hyperglucagonemia were fully corrected. In these animals, both incorporation of orotate into RNA and RNA specific radioactivity decreased. When glucagon was added to this insulin infusion at a rate of 3.0 ng/kg/

revealed that only IRG concentration was significantly related to RNA incorporation (P = 0.475, 0.566, 0.036 for glucose, IRI, and IRG, respectively).

Relative effects of glucose, insulin, and glucagon on the incorporation of labeled orotate into RNA, total nucleotides,

and UTP in the whole renal cortex of short-term diabetic animals. Forty-eight hours after the induction of diabetes, the

renal cortical content of UTP and RNA were increased as compared with controls (Table 2).

Because the incorporation of labeled orotate into cortical UTP and RNA increases linearly during a 150-mm infusion in

mm, hyperglucagonemia similar in degree to that seen in control and diabetic animals [8], comparisons of quantities nontreated diabetics was induced although plasma IRI and incorporated can be made at any interval during this infusion. glucose concentrations were maintained at values similar to At the end of a 120-mm infusion, the incorporation into total controls. Under these conditions, RNA incorporation and RNA nucleotides, UTP, and RNA, and RNA specific radioactivity specific radioactivity increased substantially (Table I). In the were increased 30, 36, 40, and 20%, respectively, in the diabetic same experiment, further increases in plasma IRG concentra- renal cortex (Table 2). In addition, the amount of UTP incorpotions produced by infusing glucagon at 6.0 ng/kg/min or by rated into RNA during the labeling period was increased also decreasing the rate of the insulin infusion resulted in still greater (control 311 6 [N = 9], diabetic 362 [N 8] nmo!es/mg DNA! incorporation of radioactivity into RNA, although these infu- hour; P < 0.005). sions with less insulin or more glucagon did increase plasma The 38-hour infusion of a small amount of insulin in diabetic animals which did not alter the degree of hyperglycemia (exglucose concentrations (Table 1). In a confirmatory experiment, similar results were obtained periment 2, Table 1), resulted in a small decrease in UTP although the degree of alteration in plasma hormonal concentra- content (10%, P = 0.065) and lesser incorporation of labeled tion in these diabetic animals was somewhat different from that orotate into total nucleotides, UTP, and RNA (Table 2). As a in the previous group (experiment 2, Table 1). The infusion of a consequence of this change in incorporation, UTP and RNA small amount of insulin (1.73 mU/kg/mm) caused an appreciable specific radioactivities were IS and 24% lower, respectively, decrease in plasma IRG concentration, whereas hyperglycemia than in noritreated diabetics (148 6 [N = 9] vs. 175 5 [N = was unchanged and hypoinsulinemia was still present. This 8] x 102 dpm!nmoles UTP; P <0.001) (Table 2). The decrease infusion decreased the incorporation of orotate into RNA and in RNA labeling was not accompanied by a significant decrease consequent RNA specific radioactivity as compared with non- in the amount of UTP incorporated into RNA (343 7 [N = 9] treated diabetic animals, Further, addition of glucagon to this nmoles/mg DNA/hour). The addition of glucagon to the insulin insulin infusion resulted in hyperglucagonemia and reversal of infusion in amounts insufficient to alter plasma concentrations the effects of insulin on RNA incorporation without change in of glucose or insulin (experiment 2, Table 1) resulted in small plasma IRI or decrease in glucose concentrations (experiment increases in the incorporation of orotate into total nucleotides 2, Table 1). As a consequence of the greater incorporation into (P = 0.061) and (JTP (P = 0.069) and a significantly greater RNA, the RNA specific radioactivity in the glomeruli of these incorporation into RNA (Table 2). The consequent RNA specifanimals treated with insulin plus glucagon increased 39% as ic radioactivity was 11% greater. But, this increase was not compared with animals receiving insulin only (experiment 2, associated with greater incorporation of UTP into RNA (337 5 [N 9] nmoles!mg DNA/hour). In animals receiving glucaTable 1). When RNA incorporation was correlated with plasma con- gon, there were no significant changes in UTP content (Table

centrations of glucose, IRI, and IRG in these experiments, a 2).

495

Giucagon and gio,neruiar RNA changes in diabetic rats Table 1. Effect of insulin and glucagon on the incorporation of radiolabeled orotate into glomerular RNA in 48-hour diabetic animals

Plasma concentration

Treatment Insulin Experiment 1' Control (10) Diabetic (9) Diabetic (8) Diabetic (9) Diabetic (9) Diabetic (9)

Experiment 2 Diabetic (8) Diabetic (9) Diabetic (JO)

Glucose mg/mi

Glucagon ng/kg/min

mU//..g/min

— — 3.8 3.8 3.8

— — — 3.0 6.0 3.0

1.73

—

1.70

4.50 1.20 1.16 2.91

4.76

— — 3.0

1.73 1.73

4.30 4.59

4.30

IRG pg/mi

IRI

pU/mI

0.03 0.14 0.04 0.12 0.53 0.16

187 + 55 198 228 +

0.10 0.04 0.10

RNA incorporationh

7

4

t dpm inji,sed/

221 492 223

61 41

364 409

76 107

22

146

18 26 15

66

4

743

59

27

4 5 3

280 166 290

33 12

5! 57

36

Specific activity

ing DNA

dp,n/g RNA

0.120 0.276 0.181 0.226 0.308 0.399

326 483 355

0.196 0.172 0.277

572 584 931 365

28! 393

Values are mean SEM Values represent the mean of two glomerular samples obtained from one pool of renal cortices. Values in parentheses are number of animals.

Incorporation of labeled orotate into glomerular RIVA in ion g-term diabetic animals: Effect of insulin (group 3). Seven

y = 117— 873x

r 0.480

months after the induction of the disease, diabetic animals 4.0

8

showed inadequate growth. All had bilateral cataracts. Microscopic examination showed mild or moderate mesangial thickening in all diabetic animals and arteriolar sclerosis in one-

—

o o 2.0

.2

—

. ______________________________

fourth. Glomerular nodular sclerosis was not observed. No abnormalities were found in the kidneys of age-matched controls. Functional effects of the renal disease in these diabetic animals were suggested by a twofold increase in plasma urea concentration (Table 3). The urea concentration was not rnodifled by the 24-hour infusion of insulin, which induced a moderate increase in body weight (diabetic, 150 5 [N — 81; diabetic + insulin, 170 8 [N — 7] g, P = 0.057) probably as a result of sodium and water retention [271. In the diabetic animals, plasma glucose and IRG concentrations were increased 286% and 89%, respectively, and there

y = 182— 291x r 0.322

was a 78% decrease in plasma IRI as compared with their corresponding controls (Table 3). In these animals, the incorporation of orotate into free nucleotides, RNA. and the resulting RNA specific radioactivity were markedly reduced compared

with control values (free nucleotides in diabetic, 0.63%; in y = —96 + 1884x = 0.895

/ S

600

200

•,'• —. 0.1 0.3 Incorporated into RNA, % dpm/mg DNA

Fig. 4. Relationship between incorporation of radioiabeied orotate into glonierular RNA and the plasma concentrations of glucose, i,nmunoreactive insulin and immunoreactive glucagon in 48—hour diabetic ani—

control, 1.72% infused dpm/mg DNA) (Table 3). But, at the end of a 24-hour infusion of insulin, the incorporation of orotate into free nucleotides and RNA decreased to 86% and 59%, respectively, of that in nontreated diabetic animals (free nucleotides in treated diabetics, 0.55% infused dpm/mg DNA) (Table 3). The

effects of insulin replacement on RNA incorporation were demonstrated even when hyperglycemia was unaltered and hypoinsulinemia still evident (Table 3). But the quantity of insulin given was sufficient to reverse the marked increase in plasma IRG concentrations present in these long-term diabetic animals. Glomerular RNA content in short- and long-term diabetic

animals. The glomerular content of RNA was not altered significantly by the infusion of sodium chloride solution in

either normal control or diabetic animals. Thus, for statistical analysis, RNA/DNA values from noninfused animals were ma/s. Values plotted correspond to those in experiments I and 2 of pooled with values obtained in controls and in infused diabetic animals not receiving insulin in groups 1 and 2 (Table 4). Table I.

Cones et a!

496

Table 2. Effect of insulin and glucagon on the content of UTP and RNA, and on the incorporation of radiolabeled orotate into total nucleotides, UTP and RNA in the renal cortex of 48-hour diabetic animals Cortical incorporationb

Treatment Insulin mU/kg/mm

Control (10) Diabetic (8) Diabetic (9) Diabetic (10)

Cortical contentb UTP nmoles/mg DNA

Glucagon ng/kg/min

— — — 3.0

— — 1.73 1.73

438 497 452

8 18'

RNA mg/mg DNA 1.42 1.87 1.82 1.76

14

490 25r

Total UTP RNA nucleotides -______ % dpm infused/mg DNA

0.03 0.03"

0.02

17.6 22.9 19.4

0.03"

21,8

0.5 0.9"

4.38

l.ld 0.7'

5.03 5.96

5.97

0.13

3.11

0.08 0.20"

0.24d 3.82 0.41" 4.34

0.16d

0.24' 4.36

-

0.l1"-

RNA specific activity5 dpm/p4 2635 3157 2547 2842

114

46" 96d

65

"The samples studied originated from the same animals as in experiment 2 of Table I. Control values correspond to animals in experiment I of Table I. Values in parenthesis are number of animals. b

Values are

mean SEM.

"Significant differences are, diabetic vs. control, I' = 0.01 or less. Significant differences are, diabetic vs. insulin-treated diabetic, P — 0.05 or less. Significant differences are, insulin-treated diabetic vs. insulin and glucagon-treated diabetic, P = 0.025 or less.

Table 3. Effects of 24-hour insulin infusion on the incorporation of radiolabeled orotate into glomerular RNA in long-term diabetic rats Plasma concentration"

Urea mg/mi

Insulin treatment mU/kg/mm

Control (7)" Diabetic (8) Diabetic (6) a Values

— — 6.8

Glucose mg/mi

0.321

0.032

1.62

0.14

0.688 0.617

0.049 0.066

4.64

t).18

4.36

0.64

RNA incorporationh

IRI ILU/mi

209 47 63

33 4 7

IRG pg/mi 334 631

378

9 45 27

dpm infused/mg DNA 0.607 0.325 0.193

Specific activity dpm/i.g RNA 1253

768 401

are mean SEM.

Values represent the mean of two glomerular samples obtained from one pool of renal cortices. Values in parenthesis are number of animals.

In isolated normal glomeruli, the content of RNA per unit DNA was only a fraction of the content in whole renal cortex

for several hours after its administration [8, 17]. Hence, the metabolic changes probably developed 24 to 36 hours after

(Tables 2 and 4). RNA content was slightly increased in

than control values (P < 0.001). But, in other experiments,

diabetes was established. The degree of labeling of RNA with exogenous precursors is the end result of several variables [28]. These include cellular transport, synthesis rate of nucleotide precursors, dilution in endogenous pools, synthesis and breakdown rates of RNA, and possible changes in nucleotide pool compartmentation. Red cells, hepatocytes and renal cortical cells are highly permeable

renal cortical RNA/DNA increased up to 32% (Table 2). Signifi-

to orotate, hence its rate of incorporation into nucleotides

cant differences in glomerular RNA content were not evident when results were expressed as per unit of protein (Table 4). RNA/DNA did not change in 48-hour diabetics after the infusion of insulin at any of the rates tested. Glomerular RNA content in long-term diabetic animals was appreciably greater than in age-matched controls, an increase partially reversed by insulin administration (Table 4). But, statistical analysis of the differences was not possible due to the small number of diabetic animals that survived long periods of the disease and subsequent surgery required for the insertion of chronic intravenous cannulae.

appears limited only by the rate of its conversion to orotidine 5'phosphate and uridine 5'-phosphate [28—31]. Because orotate and orotidine 5'-phosphate are rapidly metabolized and do not accumulate intracellularly [32], exogenous orotate taken up by the cell will be diluted only in the pool of uracil phosphates.

glomeruli 48 hours after the induction of diabetes (Table 4). This

12% increase was about the same as that observed in random samples of whole renal cortex of the same diabetic and control animals in which RNA/DNA was 1.93 0.02 [N = 13] and 1.77 0.02 [N 101 mg RNA/mg DNA, respectively, or 9% greater

Discussion

This study demonstrates the participation of glomeruli in the metabolic changes that occur in whole renal cortex shortly after the induction of diabetes [18]. The observations reported here were made 48 hours after the injection of streptozotocin, but this drug does not induce hyperglycemia and hypoinsulinemia

The measurement of the endogenous pool of uracil phosphates in isolated glomeruli is probably inaccurate because during the process of glomerular separation the tissue is subject

to ischemia and cooling. These two factors caused marked alterations in the renal contents of nucleotides [33, 34]. The lack

of information on the endogenous precursor pools in vivo prevents an understanding of the reasons for increased incorporation of labeled orotate into glomerular RNA. But, the change in labeling of total nucleotides and RNA appears to be limited to pyrimidine derivatives because no alteration was found in the incorporation of adenine under identical conditions. The absence of greater RNA incorporation of adenine when glomerular RNA content is increased suggests that its degree of

497

Glucagon and glomerular RNA changes in diabetic rats

incorporation does not correlate with the rate of RNA synthesis. In the whole growing renal cortex, calculated ATP incorpcration into RNA after labeling with 3H-adenine is increased (unpublished observations). Possibly, increased glomerular RNA synthesis may not he the primary mechanism for RNA accretion; decreased breakdown of ribosomal RNA could have the same result as that shown in the growing mouse kidney [35]. In the whole renal cortex of 48-hour diabetic animals, the excessive incorporation of labeled orotate into RNA is probably a consequence of: (a) enhanced synthesis of uracil nucleotides, as suggested by the increased incorporation of orotate into UTP

and expansion of the UTP pooi, and (b) increased synthesis cf RNA, as suggested by the greater incorporation of UTP into RNA (if a proportional expansion of all compartmented UTP pools is assumed) [8, 18]. In contrast, the decreased labeling induced by the infusion of small amounts of insulin is probably

Table 4. Glomerular RNA content in diabetic animals

RNA content

ig/ig DNA 2-Month old ratsh Control (14) 2-Day diabetic (14)

0.651

P

9-Month old rats' Control (2)

0.829 1.097 0.902

7-Month diabetic (1) Insulin treated (1) a Values are mean

0.028 0.024 < 0.05

0.73 1

p.g/ing protein

0.4

10.6 11.6

P>

0.7 0.05

7.69 9.37 8.74

SEM.

' Values in parenthesis represent the number of glomerular pools

studied. Two samples were analyzed from each pool and their means used for statistical analysis.

the result only of a change in the incorporation of labeled orotate into uracil nucleotides because the incorporation of spontaneous diabetes mellitus with insulin deficiency [42—44], UTP into RNA remained unchanged. These observations were made under conditions of steady-state plasma hormone concentration because the administration of labeled orotate was car-

but the biochemical effects of glucagon on the diabetic kidney have not been determined. This study demonstrates a direct correlation of the glomerular metabolic changes at the onset of

ried out after 36 to 38 hours of continuous insulin infusion, which is well past the 1- to 3-hour period required to achieve

diabetes with elevated plasma glucagon concentrations of a magnitude similar to those found in diabetic animals. This correlation was independent of the existing plasma concentra-

constant plasma concentrations [36, 37]. The increased incorporation of orotate into glomerular RNA in diabetic animals was partially or totally prevented by the 38hour infusion of insulin. The change induced by a small amount of insulin was similar in whole renal cortex and glomeruli (14%)

when results from both tissues were compared in the same animals (experiment 2, Tables I and 2). In this study, increased RNA content per glomerular cell was found in early and long-term diabetic animals. But, the latter observation was made in only a few animals that survived 7 months of untreated diabetes. Glomerular RNA was not increased in diabetics when expressed according to the protein

content of the sample, probaby because in diabetic, as in compensatory renal growth, there is a concurrent increase in the cellular contents of both RNA and protein [16, 38]. The exaggerated incorporation of orotate was directly related to the diabetic state rather than to nephrotoxic effects occurring after the administration of streptozotocin [39, 40] because optimal insulin replacement reversed the abnormality. The similarities between the renal metabolic alterations of diabetes and those of compensatory growth [9, 10, 41] suggest that the changes found in diabetic glomeruli represent early events of glomerular hypertrophy. These would precede functional alterations because GFR is not increased until day 4 after the injection of streptozotocin in rats [6]. Orotate incorporation into glomerular RNA was less in 7month diabetic animals than it was in age-matched controls,

tions of insulin and glucose.

Some of the cortical metabolic changes of early diabetes seemed to be associated also with hyperglucagonemia. In whole renal cortex, glucagon probably increased the labeling of RNA

by enhancing the incorporation of orotate into the nucleotide precursor pool with a consequent change in its specific radioactivity. The effects of hyperglucagonemia were more prominent in glomeruli than in whole renal cortex. Although RNA incorpo-

ration in the glomeruli was increased 61% after a 38-hour infusion of insulin to which glucagon was added, the corresponding change in the renal cortex of the same animals was 13% (experiment 2, Tables I and 2). Absolute hyperglucagonemia in short-term diabetic animals was corrected by the continuous infusion of insulin at rates insufficient to restore plasma glucose and insulin concentrations

to control values. Although insulin is a potent suppressor of glucagon release by the a2-cell [45, 46], complete reversal of hypergiucagonemia in human diabetes is difficult to achieve

with conventional insulin therapy [47]. It is likely that a constant supply of insulin is more effective in suppressing glucagon secretion than the same total dose given intermittently [48].

In long-term diabetic animals, in which hyperglucagoriemia was most marked, 24-hour infusion of insulin at rates insufficient to correct hyperglycemia decreased plasma glucagon to control values. This was accompanied by a decrease in RNA

probably as a consequence of the degree of renal disease and total free nucleotide incorporation of precursor. The present in these long-term diabetic animals. The lower rate of incorporation was still excessive for these diseased glomeruli, as demonstrated by the further decrease induced by insulin replacement. Thus, the glomerular metabolic changes of early diabetes probably persist for long periods of the disease although they may be difficult to demonstrate because of concurrent renal damage. Hyperglucagonemia, absolute or relative to plasma glucose concentrations, is present in most forms of experimental and

changes in incorporation after reversal of absolute hyperglucagonemia suggest that, as in early diabetes, glucagon excess may be a factor in the glomerular metabolic alterations of long-term diabetes. Short-term infusion of pharmacological doses of glucagon (200 to 600 ng/kg/min) in experimental animals results in increased GFR, RPF, and filtration fraction [49—511. In normal man, a 2-hour infusion of glucagon at a rate of 10 ng/kg/min causes a steady fourfold increase in plasma glucagon concentra-

Cortes et

498

tion within 30 mm and a 9% increase in GFR and filtration fraction [52]. The quantities of glucagon infused in this study were lower than those required to induce these functional changes. But, in well-controlled juvenile diabetics a short-term infusion (20 to 60 mm) of low doses of glucagon (4 to 5 ng/kgl mm) results in twofold increase in its plasma concentration and a 6% greater GFR [53]. It is not known if these acute functional changes persist after prolonged infusions, In the present studies, a linear correlation was obtained between orotate incorpo-

ration into glomerular RNA and plasma concentrations of glucagon from 75 to 336% of control values, which are well within the physiologic and diabetic range. Thus, the glomerular

alterations were probably due to a direct metabolic effect of glucagon, but the biochemical consequences of preceding acute functional changes induced during the infusion of this hormone are unknown. Increased plasma concentration of growth hormone has been hypothesized to be a determinant of increased GFR and renal mass in diabetes and as a causative factor in diabetic glomeru-

losclerosis [54, 55]. But, the concentration of plasma growth hormone is decreased in rats with streptozotocin-induced diabetes [8], and there is no correlation between concentrations of this hormone and GFR in young patients with recent and shortterm diabetes [56].

The synthesis of uracil nucleotides from orotate can be enhanced by glucagon through increased formation of 5-phosphoribosyl 1-pyrophosphate (PRPP). The concentration and bioavailability of PRPP are some of the major determinants of the regulation of pyrimidine synthesis de novo 157, 58]. Glucagon stimulates PRPP synthesis in liver cells in vivo and in vitro [59, 60]. In addition, the bioavailability of PRPP increases in the rat renal cortex shortly after the induction of diabetes [61]. The increased synthesis of uracil nucleotides and subsequent

expansion of their endogenous pools may be factors in the development of diabetic glomerular disease in addition to their

participation in compensatory and diabetic renal growth. Chronic orotate administration enhances the formation of renal

cortical uridine triphosphate and induces thickening of the glomerular basement membrane in normal rats [62].

It is proposed that hyperglucagonemia, independently of plasma insulin and glucose concentrations, may be a factor in the development of glomerular growth and basement membrane

thickening in diabetes. These effects of glucagon could be mediated by alterations in uracil nucleotide metabolism. Acknowledgments This work was presented at the 1st International Symposium on the

Glomerular Basement Membrane, Vienna 1980. This research was supported in part by research grants from the Kidney Foundation of Michigan and from the Juvenile Diabetes Foundation (Grant 76R0l7) and by a grant from William Beaumont Hospital Research Institute. Mr. K. Adams gave technical assistance. Reprint requests to Dr. Pedro Cones, Division of Nephrology, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, Michigan 48202, USA.

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