Long-term enalapril and verapamil in rats with reduced renal mass

Kidney International, Vol. 36 (1989), pp. 969—977

Long-term enalapril and verapamil in rats with reduced renal mass FELIX P. BRUNNER, GILBERT THIEL, MONIKA HERMLE, H. ANDREAS BOCK, and MICHAEL J. MIHATSCH Departments of Internal Medicine, Research and Pathology, University of Basle, CH-4031 Basle, Switzerland

Long-term enalapril and verapamil in rats with reduced renal mass. The effect of long-term treatment with either enalapril or high dose verapamil on survival, proteinuria, blood pressure and renal morphology was studied in female Wistar rats with markedly reduced renal

decreasing intraglomerular pressure and nephron perfusion [3—5]. Although a low protein diet decreases proteinuria, it has

no effect on systemic hypertension in the rat renal ablation model [3, 5]. Systemic hypertension by itself may cause or worsen progressive glomerular injury as shown in a variety of experimental circumstances such as "post-salt" hypertension El and E2 were treated with enalapril, groups VI and V2 with in rats [6], the unclamped Goldblatt kidney [7, 8] and DOCAmass. Four weeks were allowed for remnant kidney hypertrophy before determining the response to renal ablation of individual animals regarding proteinuna and hypertension. At this time, five groups of 18 rats were formed with equal levels of proteinuria and hypertension. Groups

verapamil, and one group served as control. The daily food allowance was 14 g/rat of a standard rat diet, containing 30% protein and 100 mmol NaClIkg food in groups El and VI. NaCI content was reduced to 20 mmol/kg food in groups E2, V2 and control. The drugs were added to the drinking water, enalapril at a dose of 0.1 glliter, verapamil at 0.5 to 0.7 g/liter. Drug intake thus amounted to 10 to 25 mg/kg for enalapril and 50 to 140 mg/kg for verapamil, Treatment was continued for 15 weeks.

Three of the 18 control rats did not survive up to 15 weeks. Mortality was lower in the enalapril treated groups with a single nonsurvivor in group El. In contrast, mortality was higher in the verapamil treated animals with seven nonsurvivors in group VI and eight in group V2. Blood pressure control was excellent in both enalapril treated groups, and proteinuria decreased in most animals of group El and all of group E2. Glomerulosclerosis did not develop in the majority of the enalapril treated animals. Despite the high dose, verapamil barely lowered blood pressure. However, proteinuria rose markedly in response to verapamil causing hypoalbuminemia and hyperlipidemia. Increased proteinuria was associated with a higher percentage of sclerotic glomeruli in the verapamil treated groups. Kidney weight was highly significantly cor-

related with proteinuria. Kidney enlargement thus appeared to be caused by tubular dilatation and filling with proteinaceous casts depend-

ing on the level of proteinuria. The favorable effect of enalapril on remnant kidney function and morphology was confirmed. In contrast, verapamil worsened proteinuria, tended to promote glomerulosclerosis and shortened survival of rats with reduced renal mass.

salt hypertension [8]. Protection from systemic hypertension by

clamping one renal artery in DOCA-salt hypertension prevented glomerular injury in the clamped kidney [8]. Furthermore, systemic hypertension worsens progressive glomerular injury of experimental glomerulonephritis in salt-sensitive DahI S rats [9], and antihypertensive therapy may reduce glomerular injury, as shown recently in rats with experimental glomerulonephritis [10, 11], diabetes mellitus [12], and after renal ablation [13, 14]. The purpose of the present study was to compare an angiotensin converting enzyme inhibitor to a calcium antagonist

in their effects on hypertension, proteinuria and progressive glomerulosclerosis in rats with hypertrophied remnant kidneys on a moderately high protein intake. Methods

Renal ablation was performed in a total of 125 female Wistar rats over a five week period. Once weekly a group of 25 animals weighing 120 g were anesthetized with Nembutal 40 mg/kg body wt i.p. The left renal pedicle was carefully dissected through a midline abdominal incision. Two to three upper branches of the

left renal artery were tied off using prolene 5-0 suture. One week later, the right kidney was removed after ligating the right

After extensive renal ablation, the remaining normal renal tissue undergoes functional and morphological hypertrophy. In animal models this process is associated with proteinuria and hypertension [1, 2] followed by progressive glomerulosclerosis, and ultimately leading to renal destruction and death in uremia. This deleterious response to renal ablation can be delayed by

feeding a low protein diet, which is thought to act through

renal pedicle from a flank incision under ether anesthesia. Another four weeks later, the rats were placed in individual metabolic cages for two days. The 24 hour urine of the second day was used to determine proteinuria and creatinine excretion. Blood was obtained from the cut end of the tail. Arterial blood pressure was measured by tail plethysmography in the awake rat. Since the range of the plethysmograph used did not exceed 200 mm Hg, pressures over 200 were recorded as "205" mm Hg for calculation of Spearman rank correlation coefficients. The

animals were then allocated to five treatment groups with Received for publication February 18, 1987 and in revised form July 7, 1989 Accepted for publication July 18, 1989

© 1989 by the International Society of Nephrology

similar degrees of proteinuria and hypertension. Of every 25 rats that underwent renal ablation simultaneously, a few animals died immediately after surgery or developed uremic symptoms soon thereafter and were excluded. We thus obtained five subgroups of three or four animals each, which were housed in

969

970

Brunner et a!: Enalapril, verapamil and remnant kidney

Table 1. Renal function and systolic blood p ressure before starting treatment with enalapril (E) or verapamil (V) Control

N = 18

Group mean

SEM

mean

SEM

4

72

1Cr tmo1e/liter

El

E2

V2

VI

N=

18

N=

18

N=

18

N = 18

73

5

73

4

68

5

73

3

Cr mi/mm 100 g body wt

0.32

0.02

0.30

0.02

0.31

0.02

0.32

0.02

0.29

0.01

Proteinuria mg/mol

2.16

0.30

2.14

0.30

2.16

0.33

2.03

0.26

2.06

0.28

mean

Cr

SEM

Median systolic BP mm Hg (N rats with BP 200 mm Hg)

185 (7)

190

185

193

(6)

(6)

(7)

group cages with good access to food and with tap water ad libjtum. Over five weeks a total of 18 rats were started in each experimental group (groups El and E2 on enalapril, groups Vi and V2 on verapamil) and the control group. Enalapril and verapamil were added to the drinking water, enalapril at a

180 (7)

tology without knowledge of the treatment the rats had received. Results are expressed as means

SEM,

or the medians are

given with the range. Mann-Whitney's U-test, Wilcoxon's

paired test, Fisher's exact test and the Spearman rank correlaconcentration of 0.1 g/liter, and verapamil at a concentration of tion were used as appropriate for statistical evaluation. 0.5 g/liter during the first week and 0.7 g/Iiter thereafter. Rats on Results enalapril drank between 10 and 25 ml/i00 g body wt and were Our technique of tying off two to three upper branches of the thus receiving 10 to 25 mg/kg body wt of the drug. Fluid intake varied between 10 and 20 ml/l00 g body wt in the verapamil left renal artery caused infarction of about half to two-thirds of

treated groups, resulting in a drug intake of 50 to 140 mg/kg that kidney. Together with the removal of the right kidney, body wt. The animals were offered 14 glrat/day of a standard renal mass was reduced by at least 4/5. Most animals survived ground rat diet (Klingentalmuhle AG, Basel, Switzerland) con- the procedure and thereafter gained weight normally. At the taining 30% protein (casein IA) and 100 mmol NaCI per kg food time of forming the four experimental and the control groups, before separation into the five groups. Groups El and Vl mean body weight had risen to 180 g. Mean values for renal remained on this diet, whereas salt intake was restricted in function and proteinuria as well as median blood pressure groups E2, V2 and the control group by reducing dietary salt before commencing the different regimens are shown in Table 1. content to 20 mmol/kg food. Left-over food was weighed. Proteinuria is expressed in mg per jmol urinary creatinine to Every five weeks, the animals were placed into the metabolic minimize variability due to collection errors. The range of cages for two days to determine proteinuria and creatinine proteinuria was extremely wide but again quite similar between clearance followed by phlethysmographic measurement of groups (Fig. 1). The extremes of 0.5 and 5 mg/imol creatinine blood pressure in the afternoon of the third day. Rats that corresponded to daily protein excretion rates of 15 and 150 looked sick were killed with ether, exsanguinated for determi- mg/l00 g rat/day. There was a highly significant correlation nation of plasma creatinine, and the kidneys removed for between proteinuria and blood pressure (Spearman r = 0.68, P histological examination. After 15 weeks all the surviving rats <0.00001). For practical reasons drugs were administered with the were sacrificed, and hearts and kidney remnants weighed after exsanguination. A Beckman creatinine analyzer was used to drinking water. The approximate daily drug intake was between measure creatinine in blood and urine, and the central hospital 10 and 25 mg/kg enalapril and 50 to 140 mg/kg verapamil. laboratories' multichannel Hitachi analyzer for determining Animals of groups El, E2 and control consumed their daily plasma electrolytes and lipids. Plasma albumin was measured food allowance of 14 g almost always without any left-overs. A colorimetrically with bromcresol green [151. Urinary protein few of the verapamil treated animals were eating less, which led content was determined by tannic acid precipitation with Fe3 + to slightly lower mean body weight at 10 and 15 weeks in those colorimetry as described by Yatzidis [161. For histological surviving the whole experimental period. Survival curves are shown in Figure 2. A single animal had to examination, the kidneys were fixed in phosphate-buffered 4% formalin. Three m sections were stained with hematoxylin be killed in group El at day 64, while all the 35 other rats on eosin, periodic acid/Schiff, periodic acid/silver methenamine enalapril were surviving up to sacrifice at 15 weeks. Survival and chromotrope anilin-blue. Fifty glomeruli were examined in was intermediate in the control group where one rat died each kidney for the presence or absence of segmental or global spontaneously and two had to be killed after 33 to 59 days. For glomerular sclerosis, and 10 to 20 sections of arterioles or the two verapamil groups mortality was significantly higher interlobular arteries for exsudative or proliferative hypertensive than control (42% vs. 16%, Fisher's exact test, P < 0.05). Seven vasculopathy. Care was taken to evaluate renal histology in rats of group V 1 and eight of group V2 did not survive, with two intact hypertrophied parts of the remnant kidneys outside of animals in each group dying spontaneously and the others scarred regions close to infarcted areas. Mean glomerular having to be killed mostly between 30 and 100 days after being diameter was determined for each kidney by measuring the started on verapamil. All but three of these 15 nonsurvivors had baseline proteindiameter of 10 nonsclerosed glomeruli above the papilla halfway between the corticomedullary junction and the cortical uria and blood pressure above the median values of the 90 rats surface. The pathologist (M.J. Mihatsch) evaluated renal his- studied. Higher than median initial proteinuria worsened in all

971

Brunner et al: Enalapril, verapamil and remnant kidney El

Control

Vi

E2

V2

20.010.0-

5.0

2.01.0-

I

0.5-

O.2 0

5

10

15

0

5

10

15 0

5

10

15

0

5

10 150

5

10 15

Time, weeks after starting treatment

Fig. 1. Proteinuria of individual animals at 0, 5, 10 and 15 weeks after starting treatment with enalapril (E) or verapamil (V) compared to control.

+ denotes nonsurviving rat.

SU—

1.0-

'IL ¼

.75 -

>

"IS..I....

n .50-

C

0

U U-

.25 -

I

I

I

25

50

75

I 100

Time, days on treatment

Fig. 2. Survival curve showing higher mortality for 36 rats on verapamil (groups VI + V2) and lower mortality for 36 rats on enalapril (groups El + E2) as compared to the 18 control rats.

kidney remnants available for histological examination (serum creatinine 190, 350 and 530 imo1Iliter at sacrifice). For rats surviving the entire experimental period, results are summarized in Tables 2 and 3. Enalapril significantly lowered arterial blood pressure in groups El and E2. In contrast, blood pressure remained elevated in groups Vl and V2 despite the relatively high dose of verapamil. Poor control of blood pressure was substantiated by a higher heart weight in group Vi, while both enalapnl groups had significantly smaller hearts than control (Table 3). No statistically significant differences between groups were obtained regarding creatinine clearance, but plasma creatinines were significantly lower at five weeks in the verapamil treated groups, and tended to be lower than control at five and 10 weeks in the enalapril treated groups (Table 2). In contrast, proteinuria was influenced markedly by the two drugs and in opposite direction (Fig. 1). Enalapril decreased proteinuria at five weeks in all but four of the 36 rats. Only three rats of group El with marked baseline proteinuria excreted rising

amounts of urinary protein, while proteinuria continued to decrease or remained at the same low level in the other 15 group El rats as well as in the entire salt-restricted group E2. Just the

opposite occurred in the verapamil treated groups, where

proteinuria rose continuously in all but one rat. In the control group proteinuria continued to increase in nine, stabilized in values between 5 and 20 mgI.tmol creatinine at 5 or 10 weeks five and decreased in four rats. The sequelae of proteinuria, that (Fig. 1). Plasma creatinine concentration at time of death or one is, hypoalbuminemia, hypercholesterolemia and hypertriglycerday earlier averaged 202 37 iimol/liter in 13 nonsurvivors of idemia, were conspicuous in the control group and significantly the verapamil treated groups (no plasma available in two more pronounced in the verapamil treated groups when comanimals) and ranged between 120 and 210 tmo1Iliter in the three pared to groups El and E2 (Table 3). Remnant kidney size and histology differed between groups nonsurvivors in the control group. Renal histology of these nonsurviving rats differed little between groups and disclosed after 15 weeks of treatment (Table 4). Mean kidney wet weight marked glomerulosclerosis involving 30 to 90% of glomeruli. of enalapril treated rats was comparable to control. In contrast, Only in the very early deaths in the verapamil treated groups the majority of the verapamil treated rats had much larger was the degree of glomerulosclerosis inferior, with 20% or kidneys at sacrifice. Kidney weight correlated well with profewer glomeruli showing segmental sclerosis in three of four teinuria in all groups except group E2, where all animals had

nonsurvivors irrespective of treatment group, and reached

Brunner et a!: Enalapri!, verapamil and remnant kidney

972

Table 2. Renal function, proteinuria and systolic blood pressure in surviving rats before (week 0) and at 5 to 15 weeks of treatment with enalapril (E) and verapamil (V) E2 V2 El Vl Control N = 18 N = 17 N = 11 N 10 Week N = 15

Pc i.unole/liter mean

SEM

0

73 4

5

81

10

15

Ccr mi/mm

100 g body wt

mean SEM

0

0.31

5

0.25

10 15

Proteinuria mg//.unoi Cr median (range)

0

72 74

4 81±3 91±4

0.31

0.02

0.01 0.24 0.01 0.22 0.01 1,56

0.26

0.01 0.01 0.01

1.94 (0.39—4.21)

10

15

0 5

0.25

0.23

0.03 0.01 0.02 0.02

(0.70—5.82)

(0.57—3.02)

1.18

93±12 0.32 0.29 0.24 0.20 1.36

(0.55—2. 17)

362c,h (0.62—10.50)

0.02 0.02 0.02 0.02

2.98C1 (1.66—9.47)

2.40

o.soe

o44e

756C.h

523e,h

(0.18—6.69) 0.50C (0.21—11.50)

(0.23—0.86)

(0.48—16.50)

(2.83—15.90)

(0,24—0.96) 185 (I 10—>200)

(0.40—16.40) 160 (145—>200) 165 (125—>200) 180" (145—>200)

(2.66—19.50)

1.94 175

190

(100—>200)

(135—>200) 140g

180 180 (145—>200)

15

74±3

0.32 0.23 0.22

(0.38—1.21)

4

80±7 84±8 0.35

0.59

3

65 61

0.02 0.26 0.02 0.25 0.01 0.21 0.01 2.08 0.31

1.61 (0.72—3.78) 0.68C (0.33—5.20)

(l40—>200) 10

82±5

5

60

(0.37—7.58) (0.31—11.30) Systolic BP mm Hg median (range)

67±3e

794"

0.02

62

3C

69

71±4c

(0.51—3.72)

5

73 4

5 3

iIS

(100—170)

(105—150)

(100—155)

(100—145)

125 I20

175

(125—>200)

8.8l"

0.43

l58"

1l0

(105—180)

(145—>200)

(100—130)

658c,h 173

(140—>200) 168" (145—>200) 165" (105—180) 158b,h (145—185)

Salt intake was restricted to 20 mmol NaCl/kg food in groups E2, V2 and control.

N_ 9 bN 8

P (Mann-Whitney's U-test) vs. control El vs. VI, E2 vs.

V2 s"'

e.f <0.01,

Table 3. Body weight, wet heart weight, plasma albumin and plasma lipids of surviving rats at sacrifice Control

N=15 Body wt g Heart wt

228 0.33

5

33

1

0.06

g/100 g body wt

P albumin gluier

P cholesterol

El

N=17 225

4

E2

0.24

0.25

VI

N=l8 224 4

N=l1

201 8

0.02w

36

IC

37

IC

0.40

0.08cI

V2

N=lO 210 0.37

31 l

31

0.07"

2.6

0.4

1.8

0.3

1.5

0.lC

3.5 9"

4.0

07b.c.h

0.9

0.2

0.7

0.2

0.5

0.1

1.8

07b

2.0

0.7"

.unoi/liter

P triglycerides

c,d

All values are mean SEM

=8

bN7

P (Mann-Whitney's U-test) vs. control El vs. Vl, E2 vs. V2 C.d <0.05, Cf <0.01,

"

<0.001

little proteinuria and low kidney weight. Systolic blood pressure segmental or global sclerosis was highest in groups Vi and V2 generally did not correlate with kidney weight. (P < 0.05 for Vi and V2 pooled vs. control, Mann-Whitney's Histologic examination disclosed marked differences be- U-test) and lowest in the salt-restricted group E2 (P < 0.001 vs. tween small and large kidneys. The latter showed a great control; Fig. 4). Mean systolic blood pressure correlated with number of dilated tubules which contained huge proteinaceous glomerulosclerosis in groups El and Vi but not in groups E2

casts. This was particularly conspicuous in the verapamil and V2. Pi-oteinuria showed an excellent correlation with treated groups and typical for animals with the heaviest proteinuria. The difference in kidney weight appeared to be accounted for entirely by proteinaceous casts within dilated tubules (Fig. 3). Typical hypertensive arteriolopathy occurred rarely and was limited to a few vessels of kidneys from each group, although minor vascular alterations were widespread. Fifty glomeruli were examined in each kidney for the presence or absence of sclerosis. The percentage of glomeruli with

glomerulosclerosis in all groups except group E2, where both glomerulosclerosis and proteinuria were minimal in all animals (Table 4). Nonsclerosed glomeruli tended to be smaller in the enalapril treated rats and larger in the verapamil treated groups (Table 4).

However, as shown in Figure 5, there was a definite relation between the glomerular diameters and the percentage of sclerosed glomeruli, which did not differ between the experimental

973

Brunner et al: Enalapril, verapamil and remnant kidney

Table 4. Kidney wet weight and histological findings in surviving rats Control

N—iS Kidney weight

g/100 g body wt Correlation with systolic BP

Proteinuria Glomerulosclerosis Percent glomeruli Correlation with systolic BP proteinuria

Median

range

r

El

N=17

E2

N=18

VI

N=11

0.41

0.39

0.31 —0.65

0.31 —0.49

0.44 —1.42

0.52 —1.98

0.49

0.83

0.20 NS 0.78

<0.01

<0.02

0.93'Ig

0.47

0.15

0.60

<0.05

NS

NS

r

0.78 <0.002

0.85 <0.002

NS 0.06 NS

mean SEM r

P r P

073d.g

0.42

0.33—0.96

P P

V2

N=I0

20.0

6.5

9.4

4.7

O.6

1.2

8.0aC

34.9

578

31.6

0.69 <0.01 0.83

0.65

0.36

0.75

<0.01

NS

<0.02

NS

0.77

0.22

0.87

0.63

<0.002

<0.002

NS

<0.01

<0.05

0.21

Glomerular diameter

.un mean SEM

131

4

5

130

117

Spearman rank correlation was determined with the mean values of systolic blood pressure at 5,

5b 10 and

142

6

150

8b.e

15 weeks with proteinuria immediately

before sacrifice.

a N = 10 P (Mann-Whitney's U-test) vs. control El vs. VI, E2 vs. V2 b.c <0.05,

d.c

<0.01,

<0.001

mass with fully developed hypertrophy of the kidney remnant associated with variable degrees of hypertension and proteinuria. A rather high dietary protein content of 30% was chosen to assure rapid hypertrophy and hyperfiltration. The amount of

food offered to the animals was restricted to 14 glday to minimize differences in food intake between groups and to achieve identical protein loads. The response to renal ablation varied considerably. Proteinuria was minimal in some and 10 times higher—clearly within the nephrotic range—in other animals. Systolic blood pressure was normal or barely elevated in a few rats and exceeded 200 mm Hg in many others. Since previous studies had shown that the level of proteinuria was the most important prognostic factor regarding survival [17], we took great care to evenly allocate rats with similar amounts of proteinuria to the five experimental groups. Since proteinuria correlated well with systolic blood pressure, an even distribu-

tion of blood pressures was also achieved among the five

Fig. 3. Whole kidney sections of enalapril treated animal with proteinuria of 0.35 mg/p.mol creatinine (left) compared to verapamil treated animal with proteinuria of 3.1 mgI j.smol creatinine (right) at sacrifice after 15 weeks of treatment. Note enlargement due to dilated tubular structures with darkly staining proteinaceous casts in verapamil kidney (magnification 4 times, chromotrope anilin blue stain).

groups. Kidneys with little glomerulosclerosis had the same glomerular diameters in all groups, which appeared to be affected neither by treatment with enalapril nor verapamil. Thus, the progressive loss of viable glomeruli by sclerosis was associated with increasing size of nonsclerosed glomeruli irrespective of treatment.

groups. In the control group where salt intake was restricted to 20 mmol NaCl/kg food, proteinuna decreased or stabilized in half and continued to increase in the other half of the rats, three of which did not survive the 15 week experimental period. Compared to this control group, we obtained excellent therapeutic results by enalapril and the opposite, worsening of proteinuna and increased mortality by verapamil. For practical reasons, enalapril and verapamil were administered in the drinking water. Unpublished pilot studies in our

laboratory had shown that enalapnl in a dose of 0.1 g/liter drinking water completely blocks serum converting enzyme levels in female Wistar rats. Anderson et al [14] reported that even half that dose was effective in lowering blood pressure and

decreasing proteinuna and glomerulosclerosis in Munich-

Wistar rats when administered immediately after renal ablation. These elegant studies furthermore demonstrated that convertDiscussion ing enzyme inhibition prevented the rise in glomerular capillary This study aimed at examining the effect of commonly used pressure which occurs in rats with renal ablation fed abundant antihypertensive drugs in a model of markedly reduced renal amounts of protein. The present study demonstrates a benefi-

974

Brunner et al: Enalapril, verapamil and remnant kidney

U Group median 80

0 0 0 0

C',

0 0 a, C.,

60

C',

0

0

0

0

0

0

0 0

a,

0

E 40 0

0 —

35

a

0

0

20

$

23—k--

0 0 0

0

0

0

0 El

Control

N=15

200

E2 Nl8

N=17

Vi

N=10

V2

N=iO

Fig. 4. Percent glomeruli with segmental or global glomeruloscierosis of individual kidney remnants at sacrifice after 15 weeks of treatment with enalapril (Ei,E2) or verapamil (Vi, V2) compared to control.

in our rats as well and thereby lowered proteinuria and delayed

—

190 -

or prevented progressive glomerulosclerosis. Converting enzyme inhibition proved particularly successful in reducing proteinuria and glomerulosclerosis when combined with salt restriction (group E2). Salt restriction enhanced the antihyper-

180 -

tensive action of enalapril and may have caused a further reduction in glomerular capillary pressure. Verapamil, like other calcium channel blockers, has excellent antihypertensive properties when given by gavage in doses of 100 to 150 mg/kg daily [19] or added at a concentration of 0.9 g/liter to the drinking water [20] in spontaneously hypertensive rats. The antihypertensive effect is mediated through decreased smooth muscle tone of resistance vessels [211. Hypertensive arteriolopathy and calcium uptake by arterial smooth muscle

170 -

160a'

a

A

6 'a

t 150-

a

'U Co Co

6 1400

a

0) C

cells are effectively prevented by this and a wide variety of other calcium antagonists [22]. Recently low dose verapamil

130-'

0•

A

120 —

.

110 —

100 —

90 —

'I.

-I 0

10

I

I

I

I

20

30

40

50

60

70

80

9f, Olomerulosclerosis

Fig. 5. Mean glomerular diameter of nonsclerosed glomeruli of individual kidney remnants at sacrifice after /5 weeks of treatment with enalapril (Q El, U £2) or verapamil ( Vi, A V2) compared to control (&. The regression lines of the enalapril treated groups (II), of the vcrapamil groups (L) and of the control group (•) have a similar slope.

(0.1 mg/kg twice daily i.m. or s.c.) has been found to ameliorate uremic calcinosis [231 and to slow progressive renal failure [24] in rats with subtotal nephrectomy. At this dose, blood pressure was slightly lowered [25] or unchanged [24]. In view of these reported benefits the finding of increased mortality and glomeruloscierosis in the verapamil treated groups is certainty surprising and difficult to understand. However, there are at least two important differences between this and earlier studies, namely the point in time of starting verapamil and the dosage and route of administration of the drug. Harris et a! [24] started treatment with verapamil immediately after renal ablation, whereas five weeks were allowed for undisturbed hypertrophy of the kidney remnants before administering verapamil in the present study. At the low dose of 0.1 mg/kg injected twice daily by Harris eta!, the elevated blood pressure was not affected at all, Although we intended to administer 100 to 150 mg/kg oral verapamil daily, which is sufficient to normalize blood pressure in spontaneously hypertensive rats [19], many of the group Vl and V2 rats drank

cia! effect of converting enzyme inhibition started five weeks less than anticipated and thus had a lower verapamil intake after reducing renal mass. This is consistent with the findings of which ranged between 40 and 100 mg/kg in many instances. Meyer et a! [18], who reported a reduction of elevated glomer- Hence, the slight reduction of systemic blood pressure meaular capillary pressure and proteinuria when enalapril was sured in the afternoon in some animals did not reach statistical begun eight weeks after renal ablation. We may assume, significance for the group as a whole. We can only speculate therefore, that enalapril reduced glomerular capillary pressure that blood pressure might have been lower during the night

Brunner et a!: Enalapril, verapamil and remnant kidney

975

when the rats were active and, therefore, were receiving the expected larger glomeruli at any level of glomeruloscierosis. better part of their daily verapamil dose dissolved in the The 10% greater mean glomerular diameter for the verapamil drinking water. The bioavailability of verapamil after oral treated groups can easily be explained by the greater number of administration is low, mainly due to removal of over 80% of the verapamil treated animals with high proportions of sclerosed drug during the first pass through the liver (unpublished infor- glomeruli. Verapamil is unlikely, therefore, to have caused mation supplied by Knoll AG, Ludwigshafen, FRG). Assuming excessive kidney enlargement directly by chronic hyperperfuthat only 5% of the administered drug reached the systemic sion. circulation, the regimen of the present study was still supplying 10 to 40 times more verapamil than the one by Harris et a!. It is therefore likely that the large dose used in the present study was hemodynamically effective, and caused decreased afferent renal arteriolar resistance as deduced from increased stop flow pressure and renal blood flow during intravenous infusion of verapamil in our remnant kidney model [261. Subcutaneous vera-

A simple explanation for kidney enlargement becomes appar-

ent by observing the whole kidney sections of Figure 3. Not hypertrophy of viable renal structures but large quantities of proteinaceous material in dilated tubular areas make up the difference in size. It is more likely, therefore, that these dilated

tubular structures belong to dying nephrons which are filled and obstructed by proteinaceous precipitates. Similar morphologipamil twice 0.1 mg/kg daily was recently found not to alter cal changes have been observed in adriamycin-induced nephroafferent arteriolar resistance while decreasing efferent resis- pathy, where massive proteinuria originates from light-microtance and augmenting the glomerular ultrafiltration coefficient scopically normal-looking glomeruli which develop progressive (LpA) of remnant kidneys [271; proteinuria remained un- sclerosis weeks later associated with increasing kidney weights

changed. This small dose was proposed to slow down the [30]. In contrast to the human kidney which shrinks with progression of glomeruloscierosis mainly by preventing or progressive renal failure, the rat kidney appears to enlarge due delaying uremic calcinosis [23, 241, while the larger dose used in to precipitation of proteinaceous material in dilating dying our study may have raised glomerular capillary pressures and nephrons. This different behavior of rat kidneys may be ex-

thus caused marked and continuous worsening of proteinuria in plained simply by the much greater amounts of serum proteins the vast majority of our verapamil treated rats. A tendency to which pass into the tubules of diseased nephrons. In fact, increased proteinuria was also found by Jackson et al [28] six proteinuria in relation to creatinine excretion or to body weight

weeks after subtotal nephrectomy in rats treated with the rose to levels which were at least five times higher in this calcium channel blocker felodipine. remnant kidney model as compared to humans with the neProteinuria was significantly correlated to glomeruloscierosis phrotic syndrome. as well as kidney enlargement at sacrifice, although the causal The sequence of events which lead to progressive glomerurelationships between these parameters are not readily appar- loscierosis has not been clearly established. Hyperfiltration ent. Kidney enlargement could either reflect hypertrophy of with increased glomerular capillary pressure [3, 4, 31] as well as functional renal tissue or could result from storage of material alterations in the mesangial handling of macromolecules [32, 33] within growing nonfunctional structures. Kenner et a! [29] in have been recognized as important and probably interrelated their comprehensive investigation of morphological alterations factors [34, 35]. Proteinuria is a marker of disturbed glomerular after 5/6 nephrectomy, reported a twofold increase in kidney capillary function which appears to be characterized not only weight in rats fed a high protein diet when compared to rats on by increased leakiness of the glomerular capillary filter but also a low protein diet. Similarly to our findings, Kenner et al by increased macromolecule traffic into the mesangium resultascribed the enlargement to "a spongy texture, . . cystic ing in mesangial injury and glomeruloscierosis [34, 35]. Mesanappearance . . and to marked tubular enlargement . . with the gial uptake and clearance of macromolecules is known to be greatest degree of dilation occurring in proximal and collecting altered by angiotensin II both in normal kidneys and in purotubules . . filled with cast material". They suggested these mycin-induced nephrosis of the rat [34, 35]. The effect of tubular and interstitial changes to contribute to the progressive verapamil on mesangial clearance has not been tested, but it is nature of renal failure. In the present study, similarly large conceivable that its vasodilatory effect could increase mesandifferences in kidney enlargement as well as proteinuria were gial uptake of macromolecules similarly to diazoxide [35]. obtained in groups of rats with identical diet and equal protein Vasodilators which increase intraglomerular pressures such as load but undergoing treatment with different vasoactive drugs. hydralazine in Dahi S rats or in glomerulonephritic spontaneAs noted above, intravenous verapamil acutely increased renal ously hypertensive rats [36, 37] may accelerate glomeruloscier-

blood flow and proximal tubular stop flow pressure in our

remnant kidney model despite a fall in systemic arterial blood pressure [26]. Kidney enlargement might thus reflect tissue hypertrophy resulting from chronically increased renal perfusion. Such hypertrophy should have involved vascular, glomerular and tubular structures alike. This, however, was not the case. Although nonsclerotic glomeruli were larger in kidneys with high proportions of sclerotic glomeruli, there was similar enlargement in control, in enalapril and in verapamil treated animals. Hence, glomerular size appeared to be related to the degree of glomeruloscierosis rather than to the type of treatment. Had a direct pharmacological effect of verapamil been responsible for the glomerular hypertrophy, one would have

osis by flooding the glomerular mesangium with macromolecules, a mechanism which might explain the deleterious effect of verapamil in the present study. In contrast, enalapnl could protect the glomerular mesangium either by reducing intraglomerular pressures or by blocking angiotensin 11-dependent alterations in mesangial macromolecule traffic. The macromolecular species which are particularly harmful to the glomerular mesangium appear to be lipoproteins, and hyperlipidemia has been recognized as an important factor in progressive glomeruloscierosis [38—40]. The verapamil treated

animals, in contrast to the enalapril groups, tended to have higher cholesterol levels at sacrifice, probably as a consequence of their more pronounced proteinuria and hypoalbuminemia.

976

Brunner et a!: Ena/april, verapamil and remnant kidney

Unfortunately plasma lipids were not measured early in the course of the experiments. Hence, the role of hyperlipidemia or mesangial uptake of lipoproteins in mediating glomerular injury cannot be determined in the present study.

In summary, the beneficial effect of converting enzyme

BRENNER BM: Prevention of diabetic glomerulopathy by pharmacological amelioration of glomerular capillary hypertension. J Gun Invest

77: 1925—1930, 1986

13, PURKERSON ML, HOFFSTEN PB, KLAHR S: Pathogenesis of the glomerulopathy associated with renal infarction in rats. Kidney ml 9:407—417, 1976

inhibition by enalapnl on progressive glomerulosclerosis in rats 14. ANDERSON S, MEYER TW, RENNKE HG, BRENNER BM: Control of glomerular hypertension limits glomerular injury in rats with rewith reduced renal mass was confirmed. Proteinuria and arterial duced renal mass. J C/in Invest 76:612—619, 1985 blood pressure were particularly well controlled by combined 15. DORMAS BT, WATSON WA, BIGGS HG: Albumin standards and the

enalapril and salt restriction. The calcium channel blocker verapamil, administered at a relatively high dose worsened

measurement of serum albumin with bromcresol green. C/in Chim

proteinuria and tended to promote glomerulosclerosis. Further studies are necessary to conclusively determine whether effec-

16. YATZIDJS H: New colorimetric method for quantitative determination of protein in urine. C/in Chem 23:811—812, 1977 17. BRUNNER FP, HERMLE M, MIHATSCH Mi, THIEL 0: Effect of

tive control of blood pressure by calcium channel blockers equal to that obtained by enalapril in the present study will also adversely affect progressive glomerulosclerosis and survival in rats with renal ablation.

Acta 31:87—96, 1971

cyclosporine in rats with reduced renal mass. Gun Nephrol

25:

1986 18. MEYER TW, ANDERSON S, RENNKE HG, BRENNER BM: ConvertSl48—S154,

ing enzyme inhibitor therapy limits progressive glomerular injury in rats with renal insufficiency. Am J Med 79, S 3C:31—36, 1985 19. VoN WITZLEBEN H, FREY M, KEIDEL J, FLECKENSTEIN A: Nor-

malization of blood pressure in spontaneously hypertensive rats by long-term oral treatment with verapamil and nifedipine. (abstract) Pflugers Arch S R9:384, 1980

Acknowledgments This study was supported by Swiss National Science Foundation Grant no. 3897-0.83. Enalapril was supplied by Merck & Co., USA and verapamil by Knoll AG, FRO. The technical assistance of S. Zurbrugg and J. Violante, and the secretarial skills of Miss E. Oral are gratefully acknowledged.

20. LEDERBALLE PEDER5EN 0, MIKKELSEN B, JESPERSEN LT: Treat-

ment with verapamil reduces blood pressure and tends to normalize

vascular responsiveness and ion transport in the spontaneously hypertensive rat. J Cardiovasc Pharmacol 4:294—297, 1982 21. GRUN G, FLECKENSTEIN A: Die elektro-mechanische Entkoppelung

der glatten Gefässmuskulatur als Grundprinzip der Coronardilatation durch 4-(2'-Nitrophenyl)-2,6-dimethyl-1 ,4-dihydro-pyridin-3,5dicarbonsäure-dimethylester (Bay a 1040, Nifedipin). Arzneim For-

Reprint requests to Professor Felix Brunner, Kantonsspita! Base!, 4031 Basel, Switzerland.

sch 22:334—344, 1972 22. FLECKENSTEIN A, FREY M, ZORN J, FLECKENSTEIN-GRON G:

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