journal of Internal Medicine 1991; 229: 163-170
ADONIS 09 5468209 100029 JIM
Contrasting effects of lisinopril and nifedipine on albuminuria and tubular transport functions in insulin dependent diabetics with nephropathy H. HOLDAAS, A. HARTMANN, M. G. LIEN, L. NILSEN, J. JERVELL*, P. FAUCHALD, L. ENDRESEN, 0. DJQSELANDI & K. J. I3ERG From the Department of Nephrology and the *Deprtment of Endocrinology. Medical Department B. National Hospitul. Oslo. Norway
Abstract. Holdaas H, Hartmann A, Lien MG, Nilsen L. Jervell J. Fauchald P.Endresen L. Djeseland 0, Berg KJ (Department of Nephrology and Department of Endocrinology, Medical Department B, National Hospital, Oslo. Norway). Contrasting effects of lisinopril and nifedipine on albuminuria and tubular transport functions in insulin dependent diabetics with nephropathy. Journal of Internal Medicine 1991 : 229: 163-1 70. An open, randomized, cross-over study was undertaken to assess the effects of lisinopril
and nifedipine on albumin excretion, renal haemodynamics and segmental tubular reabsorption in overt diabetic nephropathy. The study consisted of a 4-week run-in period, a 3-week active treatment period, a 4-week wash-out period and a second 3-week active treatment period. Twelve patients with type 1 diabetes with albuminuria, mild to moderate hypertension and a serum creatinine level of < 200 pmol I-' were included. Lisinopril reduced albumin excretion from 1343 337 pg min-I to 879 f299 pg min-' (P < 0.01). whereas nifedipine was without effect, 1 4 3 6 f 3 3 6 ,ug m i d vs. 1319 f342 pg min-'. Glomerular filtration rate (GFR) was unchanged by either drug. Both drugs increased effective renal plasma flow (ERPF) by about 20%. No differences between the drugs were observed with regard to their effect on renal haemodynamic parameters. By contrast, nifedipine exerted an inhibitory effect on several proximal tubular transport markers, whereas lisinopril was without effect. The different actions on tubular transport mechanisms exerted by lisinopril and nifedipine may contribute to the observed effect on albumin excretion.
Keywords: ACE inhibitor, albuminuria, calcium-channel blocker, diabetes, renal haemodynamics, tubular function.
Introduction The first clinical sign of diabetic nephropathy is characterized by an increase in urinary excretion of albumin. Preceding overt proteinuria, microalbuminuria has usually been manifest for years. The deterioration of renal function in patients with diabetic disease may be retarded by vigorous control of systemic blood pressure [l.21. Strict metabolic control [3] and reduced phosphate/protein intake [4] may also contribute towards preservation of renal function. The antihypertensive drugs used in longterm prospective studies that demonstrate a favourable effect of good blood pressure control for preservation of renal function include beta-blockers,
vasodilators, diuretics and ACE inhibitors [l,2, 51. These drugs have widely differing effects on renal haemodynamics, an observation which suggests that the beneficial effect of antihypertensive treatment on renal function is related to good control of systemic blood pressure rather than specific intrarenal effects on renal haemodynamics. However, as reviewed by Keane et al. [6], several studies have advocated that ACE inhibitors may possess specific advantages in decreasing proteinuria and slowing progression of diabetic nephropathy. The rationale for this view is based on animal models that have demonstrated favourable alterations in glomerular haemodynamics with ACE inhibition [7].In man the same'beneficial effects of ACE inhibitors have been inferred from 163
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changes in filtration fraction [6]. The data have been interpreted as showing a preferential effect of ACE inhibitors on efferent arterioles [8]. The lack of consistent data reported with regard to the effect of calcium-channel blockers on diabetic proteinuria has been attributed to a non-selective dilation of afferent and efferent arterioles, or a selective reduction of preglomerular resistance [9]. In an attempt to determine the effects of calcium channel blockers and ACE inhibitors on albumin excretion we performed a short-term cross-over study on insulin dependent diabetics with overt nephropathy. We used the ACE inhibitor lisinopril and the calciumchannel blocker nifedipine. Particular emphasis was placed on renal haemodynamics and tubular markers for proximal tubular function.
Subjects and methods Patient The patient sample consisted of 1 2 hypertensive patients (five women and seven men) aged 22-58 years, with insulin dependent diabetes of 1 8 4 5 years duration. Patients with heart failure or coronary heart disease were excluded from the study. Informed consent was obtained according to the Declaration of Helsinki, and the study was approved by the Regional Ethical Review Board of the University of Oslo. The patients were selected on the basis of an increase in urinary albumin excretion rate to > 3 0 0 p g min-' in two consecutive 24-h urine samples, provided that urine culture was negative. Serum creatinine levels had to be < 200 pmol I-'. Mild to moderate hypertension was defined as diastolic blood pressure in the range 85115 mmHg. Blood pressure readings were mean values of the last two of three recordings taken with a standard sphygmomanometer with the patient seated for at least 5 min. Diastolic blood pressure was recorded as the disappearance of the Korotkoff sound (phase 5). Mean arterial pressure (MAP) was regarded as diastolic blood pressure plus 1 / 3 pulsatile pressure. Study protocol The study was conducted in a randomized two-way cross-over manner, and consisted of a 4-week run-in period, a 3-week active treatment period, a 4-week wash-out period and a second 3-week active treat-
ment period. During the 4-week run-in period, five patients were withdrawn from previous antihypertensive medication. At the end of the run-in period, patients were randomized to one of the two possible sequences for active treatment (lisinopril followed by nifedipine, or nifedipine followed by lisinopril). Lisinopril treatment started with 5 mg daily, while nifedipine treatment started with 10 mg twice a day. After 1 week of treatment, titration was allowed by doubling the dose of medication, and after another week of treatment a final titration was allowed for the lisinopril treatment, if systolic blood pressure was still > 1 2 0 mmHg. The maximal daily dose was 4 0 mg for nifedipine and 2 0 mg for lisinopril. Blood samples and duplicate 24-h urine samples were collected at the end of the run-in period, at the end of the wash-out period and at the end of the two treatment periods. Renal clearance examinations were performed on the last day of the run-in period and on the last day of each treatment period. The evening before each clearance study, each subject ingested 18 mmol lithium citrate in order to obtain a plasma lithium concentration of about 0.3 mmol I-'. The subjects were hydrated orally with tap water, 20 ml kg-'. Renal haemodynamic parameters were assessed by para-amino-hippurate (PAH) and inulin clearances for the estimation of ERPF and GFR, respectively. Priming doses of inulin (10%solution) and PAH (20% solution) were administered to obtain plasma concentrations of approximately 200300 mg I-' and 2 0 4 0 mg 1-', respectively. Infusions of inulin and PAH were administered in order to maintain initial plasma concentrations. After a 60mins equilibration period, four carefully timed urine collections of 25-35 min each were obtained by spontaneous voiding. Blood samples were drawn at approximately the midpoints of the collection periods. The clearance studies were not initiated until the urine osmolality was < 200 mosm kg-'. Biochemical analyses Sodium and potassium levels in serum and urine were determined by a standard autoanalyser technique. Urinary albumin was determined with a radioimmunoassay kit (Pharmacia A.B., Uppsala, Sweden). Total protein in the urine was determined by the biuret method. Serum and urine 8-2-microglobulin was assayed by a commercial radioimmunoassay kit (Pharmacia A.B., Uppsala, Sweden). Urine samples for 8-2-microglobulin were
ALBUMINURIA IN INSULIN DEPENDENT DIABETICS stored at pH > 6. The urinary excretion rate of B-2microglobulin in healthy controls was 1 37 f66 pg 24 h-'. Creatinine was determined by a ratedependent modiEcation of the Jaffe reaction using a creatinine analyser, Bechman creatinine analyser model 2. Urinary N-acetyl-beta-glucosaminidase (NAG) was determined by a fluorometric method of Price et al. [lo]. Activity was expressed as units pmol-' creatinine. The mean value in healthy controls was 5.4 f2.3 units pmol-' creatinine. Urinary alkaline phosphatase (ALP) was determined spectrophotometrically by a commercial kit (Boehringer, GmbH Diagnostica, Mannheim, Germany), and the activity was expressed as units 24 h-'. The mean HbA, level was determined spectrophotometrically after ion-exchange chromotography. Plasma renin activity and plasma aldosterone were determined using commercial radioimmunoassay kits, and lithium was determined by flame emission photometry. The inulin concentration was determined according to Schreiner [ l 11. High concentrations of interfering glucose in diabetic patients were removed using glucose oxidase. PAH was determined according to Smith et al. [12].
Calculations Rates of clearance of various substances were calculated according to standard clearance formulas [13]. The Eltration fraction was calculated as GFR/ERPF, and renal vascular resistance was taken to be MAP/ERPF. Using lithium as a marker of proximal sodium reabsorption, segmental sodium handling was analysed using the following equations : FL,, =GFR x P,,/lOOO (mmol min-') DD,, =C,, x P,,/lOOO (mmol min-') APR,, = FL,, - DD,, (mmol min-') ADR,, =DD,, - U,,V (mmol min-') where FL,, is filtered sodium, P,, is the plasma sodium concentration, DD,, is distal sodium delivery, C,,, is lithium clearance, APR,, is absolute proximal reabsorption, and U,,V is sodium excretion [14].
Statistical analyses Data were analysed using the appropriate methology for two-period cross-over trials as described by Grizzle [15]. Analysis of the cross-over data was performed in two steps. In the Erst step the sequence effect was
165
Table 1. Blood pressure responses to lisinopril and nifedipine Lisinopril
Nifedipine
Baseline 3 weeks
Baseline 3 weeks
Systolic blood 146*3 pressure (mmHg) Diastolic blood pressure (mrnHg) Mean blood pressure (mmHg)
89k2
137*4*
146k5 134*3*
83f3'
109i-2 102*3*
91k3
78k2'
110k3
96*2*
* P < 0.01 vs. baseline. Table 2. Renal haemodynamic responses to lisinopril and nifedipine Baseline
Ltsinopril
Nifedipine
95f12 551f57*
94k11 521*36*
GFR (ml min-')
95k12
ERPF (mlmin-')
466f32
Filtration fraction
0.20k0.02 0.16kO.02t 0.17k0.027 0.26f0.02 0.20f0.02t 0.20k0.02t
RVR (mmHg min-' ml-')
CFR = glomerular Bltration rate. ERPF = effective renal plasma flow,RVR = renal vascular resistance. P < 0.05 vs. baseline, t P c 0.01 vs. baseline.
tested at a level of a = 0.10 ; if no significant sequence effect was detected, the treatment and period effects were tested subsequently at a level of a = 0.05. The results are presented as mean valuesfSEM. The mean value for the two 24-h urine collections in each period was used for comparison of the urine data, and the values in each treatment period were compared with those for the preceding control period. For the evaluation of inulin, PAH and lithium clearances, data from each treatment period were compared with the 6rst control period. The data for the two drug periods were also compared with each other. A t-test was used :for statistical evaluation, all statistical tests were two-tailed and significance was defined as P < 0.05.
Results Eflects on blood pressure Lisinopril and nifedipine significantly reduced both systolic and diastolic blood pressure (Table 1).The mean decrease in diastolic blood pressure was slightly, but not significantly, larger during nifedipine treatment than during lisinopril treatment. In both sequence groups the nifedipine dose reached a
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Table 3. Blood biochemical responses to lisinopril and nifedipine Lisinopril
Nifedipine
Baseline
3 weeks
Baseline
3 weeks
10.2f0.3 139f1
11.2f0.7
Renin (nmol I-' h-l)
10.6f0.5 140*2 4.5f 0.1 36f1 113f9 0.4 0.1
Aldosterone (pmol I-')
303 f 63
232f68t
10.5&0.4 139f 1 4.3k0.1 37f2 122 f 9 1.7f 0.6 515f123
HbA,
(%I
Na (mmol I-')
K (mmol I-') Albumin (g I-') Creatinine (pmol I-')
P < 0.01 vs. baseline,
4.5k0.2 36f1 120* 1 1 4.7f1.7.t
140f 1 4.5f0.1 36*1 119f9 0.9f 0.3 365f 72
t P < 0.01 vs. nifedipine.
--
4000
C ._ E
CF
-3 3000 c
._ + El
2
5
zoo0
5 e
a
lo00
Fig. 1. Twenty-four-hour albumin excretion in response to lisinopril and nifedipine. Mean values fSEh4
are shown.
n v Control
maximal level of 40 mg d-'. For the lisinopril treatment, four patients, two in each sequence group, were not titrated to the maximum dosage. The mean final dosage of lisinopril was 16.25 mg d-'. No serious adverse side-effects were observed during treatment in either group. Effects on renal haemodynamics The data are summarized in Table 2. GFR, estimated as inulin clearance, was remarkably stable throughout the study. Neither lisinopril nor nifedipine induced significant changes in GFR. However, both drugs significantly increased ERPF from baseline levels. Consequently, the baseline Eltration fraction of 0.20 f0.02 was reduced to 0.16 f 0.02 by lisinopril and to 0.17 f 0.02 by nifedipine. A substantial and equal decrease to 0.20 f 0.02 mmHg min-' ml-' of estimated renal vascular resistance occurred in response to both lisinopril and nifedipine, from a
Lisinopril
Control
Nifedipine
baseline value of 0 . 2 6 f 0 . 0 3 mmHg min-' ml-'. Thus there was no difference in the effects of lisinopril and nifedipine on renal haemodynamic parameters. Effects on blood biochemical parameters A summary of the results is given in Table 3. No change in plasma electrolyte concentrations was recorded. Plasma albumin, HbA, and creatinine levels were stable throughout the study. Short-term therapy with lisinopril was, as expected, associated with a marked increase in plasma renin activity. Nifedipine had no effect on plasma renin activity and aldosterone concentration. Effects on 24-h-urine biochemical parameters The most striking finding was the consistent reduction in albumin excretion in all 12 patients during lisinopril therapy, as illustrated. in Fig. 1. Albumin excretion decreased from 1343 f337 pg min-' to
ALBUMINURIA IN INSULIN DEPENDENT DIABETICS
167
Table 4. Twenty-four-hoururine responses to lisinopril and nifedipine Lisinopril
Nifedipine
3 weeks
Baseline
3 weeks
Baseline
Na (mmol 2 4 h-')
182f 17
167f21
181 f 14
1 8 0 f 19
K (mmol 2 4 h-')
87+9
75k6
90f 7
87k 7
3.4 f 0 . 7
3.4 f 0.7
603 f250
237*69t$
468 f 1 5 0
623 f 2 0 4
7 . 3 k 1.2
7.5f 1.5
7.3f 1.2
8.3f l . l t
21.3k2.9
19.0* 2 . 0
18.3f2.1
22.8f 3.2.
Total protein (g 2 4 h-') b2-microglobulin (pg 24 h-')
3.2f0.8
ALP units (mmol 2 4 h-') NAG units (pmol-' creatiine)
1.9f0.7tS
ALP = alkaline phosphatase, NAG = N-acetyl-beta-glucosaminadase. P c 0.05 vs. baseline, t P c 0.01 vs. baseline, $ P < 0.01 vs. nifedipine. Table 5. Segmental electrolyte transport in response to nifedipine and lisinopril Baseline
Lisinopril
2 3 . 4 f 1.9 72.8f2.7
21.1 f 1.5
29.2 f 2.0*$
75.9 f 2 . 0
66.0f 3.6t5
12.9 f 1 . 4
13.1 f 1.4
12.8f1.4
APR,, (mmol min-')
9.6f1.4
10.2f1.5
ADR,, (mmol min-')
2.9k0.3
2.6k0.2
U,,V (mmol min-')
0.4f 0.05
0.3f0.02
Li clearance (ml min-') Li fractional reabsorption (%) FL,,
(mmol min-')
Nifedipine
8.8f1.5.t 3.6f0.2*$ 0.4k 0.05
FL,, = filtered sodium. APR,, = absolute proximal sodium reabsorption. ADR,, = absolute distal sodium reabsorption. U,,V excretion. 'P < 0.05 vs. baseline. t P c 0.01 vs. baseline. P c 0.05 vs. lisinopril. 5 P c 0.01 vs. lisinopril.
= sodium
*
89 7 f299 pg min-' during treatment with lisinopril, a reduction of 35%. By contrast, nifedipine treatment had no effect on albumin excretion, there was a decrease in albumin excretion in seven patients, whereas in Eve patients there was an increase in albuminuria during the drug phase. Albumin excretion was 1436 f336 p g min-' prior to and 1319 f342 pg min-' during nifedipine treatment. The changes in albumin excretionwere also reflected by parallel changes in total urinary protein excretion, as shown in Table 4. During nifedipine therapy, urinary P-2-microglobulin excretion increased from 486 f 150 pg 24 h-' to 623 f 204 pg 2 4 h-l, while lisinopril treatment decreased excretion from 603 f 250 pg 24 h-' to 2 3 7 f 69 pg 2 4 h-' (P < 0.01). The rate of sodium excretion was high, but remained unchanged throughout the study. As shown in Table 4, the proximal brush-border enzyme marker ALP showed a significant increase in activity from 7.3f 1. 2 to 8. 3f1 .1 units 24 h-' during the nifedipine treatment period. This was accompanied by a corresponding increase in excretion of the proximal
lysosomal enzyme NAG from 1 8 . 3 f 2 . 1 to 22.8+ 3.2 units pmol-I creatinine (P < 0.05). Lisinopril treatment had no significant effect on urinary ALP or NAG.
Effects on segmental electrolyte transport The effects of both drugs on segmental electrolyte handling are shown in Table 5. During nifedipine treatment, a significant increase in lithium clearance was observed. The fractional reabsorption of lithium was substantially reduced during nifedipine therapy from 72.8f2.7% to 6 6 .0 f 3 .6 % ( P < 0.01). Although lisinopril treatment tended to increase fractional reabsorption of lithium and reduce lithium clearance, none of the changes were significantly different. The proximal reabsorption of sodium, estimated by lithium clearance, was significantly reduced during nifedipine therapy from 9.6 & 1.4 mmol min-' to 8.8 f 1.5 mmol min-', indicating that nifedipine had a n inhibitory effect on proximal transport. However, as can be seen from Table 5, the
168
H. HOLDAAS et al.
decrease in proximal ion transport was almost totally compensated for by an increase in electrolyte reabsorption in the distal tubules.
Discussion The most important finding in this study is that the ACE inhibitor lisinopril reduces urinary albumin excretion in insulin dependent patients, whereas nifedipine has no such effect. The albumin-sparing effect of lisinpril was also confirmed by a significant reduction in excretion of 8-2-microglobulin and total urinary protein. Previous reports on the effect of lisinopril on albumin excretion are scarce; Heeg and co-workers have demonstrated a protein-sparing effect in patients with predominantly non-diabetic renal disease [16, 171. In contrast to the present study, the protein-sparing effect in non-diabetic patients appears to be dependent on adequate restriction of sodium, as the reduction in albumin excretion was abolished by increasing the rate of sodium excretion from 50 to 200 mmol d-' [17]. In our study sodium intake was not regulated and, despite a sodium excretion rate of 180 mmol d-', reflecting a standard diet, a consistent effect on albuminuria was found. The uniform effect of lisinopril treatment on blood pressure and albumin excretion in our study is at variance with the results of August et al. [18], who felt the need to subdivide patients on lisinopril therapy into responders and non-responders. Moreover, in their study, in neither responders nor non-responders could any effect on protein excretion be detected. The reason for these discrepancies may be related to the degree of stimulation of the renin-angiotensin system, any effect of ACE inhibitors being more easily detected when the system is stimulated. However, as shown in our study, even under conditions of high sodium excretion and consequently low renin, a consistent effect was detected. The effect of lisinopril treatment on urinary albumin excretion observed in our study is consistent with previous reports of beneficial effects of other ACE inhibitors on albumin excretion in patients with diabetic and non-diabetic nephropathy. A comprehensive review has been given on this topic [6]. The consensus seems to be that ACE inhibitors are effective antihypertensive agents with a favourable effect on albumin excretion in most of patients with diabetic and non-diabetic renal diseases [6]. Previous results concerning the effect of calcium channel-blockers in short-term studies on protein/
albumin excretion in patients with nephropathy have been conflicting. In studies that have reported an unfavourable effect on albumin excretion in diabetic patients, the results have been explained by a detrimental effect of calcium-channel blockers on renal haemodymanics [19, 201. However, both beneficial effects and a total lack of effect on albumin excretion during calcium-channel-blocker treatment have been reported. Baba et al. [21] demonstrated a beneficial effect of nicardipine on microalbuminuria in type I1 diabetes. Ikeda et al. were unable to demonstrate any effect on albumin excretion during treatment with a dihydropyridine-derived calciumchannel blocker in patients with non-diabetic renal disease [22]. In our study the reduction in mean blood pressure was more pronounced with nifedipine. The observed lack of effect of nifedipine treatment on albumin excretion thus cannot be ascribed to an inadequate systemic blood pressure-lowering effect compared to lisinopril therapy. Furthermore, there were no differences in the changes induced by either drug on ERPF, FF and RVR. No change in GFR was induced by either drug. This does not support the concept of differential intrarenal vascular effects of the two agents applied. In man, such a hypothesis has been primarily inferred from the observed changes in filtration fraction [6]. Although both ACE inhibitors and calcium-channel blockers have been employed in numerous trials in man, only a few studies have reported data on RBF and GFR. thus allowing calculation of the filtration fraction. A review of the filtration fraction data in patients with diabetic and non-diabetic disease reveals no consistent change in FF fraction when ACE inhibitors [6] or calcium channel blockers [23] have been applied. In addition, Navar and his co-workers have advocated the view that changes in filtration fraction are not necessarily indicative of selective alteration in segmental vascular resistance [24]. As long as renal haemodynamic parameters are changed to an equal extent by two different drugs, a difference in the effects of the drugs on. pre- and postglomerular vascular resistance is difficult to envisage. The lack of effect of the calcium-channel blocker nifedipine on albumin excretion in our study may be explained by an inhibitory effect of the drug on proximal tubular reabsorption mechanisms. Such a specific intratubular effect of nifedipine could offset the albumin-sparing effect that has been demonstrated in diabetic nephropathy during treatment with other antihypertensive drugs [l,2, 5. 211.
ALBUMINURIA IN INSULIN DEPENDENT DIABETICS
In order to estimate proximal tubular function, several techniques and markers were applied. For proximal ion and water transport we used the lithium clearance technique. Although the reliability of lithium as a marker for proximal tubular function has been questioned, it is generally agreed that, provided patients are examined sequentially at the same level of GFR during water diuresis, this method represents the best clinical marker of proximal tubular function [25]. Nifedipine caused a marked reduction in fractional lithium reabsorption and a corresponding increase in lithium clearance. The calculated reabsorption of sodium in the proximal tubules was consequently suppressed during nifedipine therapy. ~-2-microglobulin,a freely filtrable protein, has also been widely used as a marker of proximal tubular function [26]. The excretion of a-2-microglobulin was significantly increased during nifedipine treatment compared to lisinopril therapy. NAG and ALP are intracellular and brush-border enzyme markers, respectively, of proximal tubular function [27, 281. The levels of both these enzyme markers increased during nifedipine treatment, thus supporting the view that the drug has a proximal tubular effect. All these results suggest an inhibitory effect of nifedipine on proximal ion and water transport. This concept is consistent with the view that the most likely mechanism for increased sodium excretion of calcium-channel blockers is a direct inhibition of tubular sodium reabsorption [29]. For one calciumxhannel blocker, isradipine, it has been demonstrated that the effect is localized in the proximal tubules [30]. Christensen et al. proposed a proximal tubular effect of nifedipine, based on its effect on the urinary excretion of albumin and /3-2-microglobulin [3 11. Our results, which demonstrated an inhibitory effect of nifedipine on several proximal tubular function markers, support the hypothesis that nifedipine might also inhibit proximal tubular albumin transport mechanisms. In healthy human subjects the estimated minimum capacity for albumin reabsorption in proximal tubules is about 200400 p g min-' [32]. The albumin reabsorption in protein-losing disease is not known, although it may be assumed that it is high and probably at a transport maximum. Even a small decrease in proximal function may result in increased excretion of albumi, since no distal segments transport proteins. Nifedipine may therefore depress albumin reabsorption in proximal tubules in diabetic nephropathy, leading
169
to albuminuria despite the fact that there is a convincing systematic antihypertensive effect. Moreover, it has been shown that a dihydropyridine calcium-channel agonist, Bay K 8644, increases sodium and water reabsorption when infused into the renal artery [33]. Thus data from both animal and human models indicate that manipulation with calcium-channel agonists or antagonists may interfere with proximal tubular transport mechanisms. By contrast, proximal tubular transport function during lisinopril therapy remained unchanged or was even slightly improved, when estimated by the lithium clearance technique or by b-2-microglobulin. The excretion of proximal tubular enzyme markers, the intracellular NAG and the brush-border enzyme ALP, was also unchanged by lisinopril treatment. Thus in our study, no harmful effect of the ACE inhibitor lisinopril on proximal tubular function could be detected. On the basis of the results from our study we propose that the observed difference between lisinopril and nifedipine on albumin excretion may at least in part be explained by different tubular effects. A previous report has also proposed that an increase in urinary excretion of albumin in diabetic patients may be tubular in origin [34]. In conclusion, lisinopril reduced diabetic albuminuria in all patients, whereas nifedipine has no such effect. Lisinopril and nifedipine had similar antihypertensive effects, and similar actions on renal haemodynamics. Nifedipine had an inhibitory effect on markers for proximal tubular function. A potentially favourable effect of nifedipine on albumin excretion might be masked by the suppression of proximal tubular reabsorption mechanisms.
Acknowledgements Lisinopril was provided by Merck Sharp & Dohme International (Rahway, NJ, USA). This study was supported by the Norwegian Research Council for Science and the Humanities, the Norwegian Drug Monopoly, and Merch Sharp & Dohme, Norway.
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3 Viberti. GC. Bilous RW. Mackintosh D. Bending JJ, Keen H. Long term correction of hyperglycemia and progression of renal failure in insulin dependent diabetes. EM\ 1 9 8 3 : 286: 598-602. 4 Zeller KR. Jacobson H. Raskin P. The effect of dietary protein and phosphorus restriction on renal function in diabetic nephropathy-results of a 5 year study (Abstract). Kidney Int 1990: 3 7 : 246. 5 Bjark. S. Nyberg G. Mulec H. Granerus G. Herlitz H. Aurell M. Beneficial effects of angiotension converting enzyme inhibition on renal function in patients with diabetic nephropathy. BMI 1986: 2 9 3 : 4 7 1 4 . 6 Keane WF, Anderson S. Aurell M. de Zeeuw D, Narins RG. Povar G. Angiotensin converting enzyme inhibitors and progressive renal insufficiency. Ann Intern Med 1989; 1 1 1 : 503-1 6. 7 Zatz R. Dunn RB. Meyer TW. Anderson S. Rennke HG. Brenner BM. Prevention of diabetic glomerulopathy by pharmacological amelioration of glomerular capillary hypertension. 1 Clin Invest 1 9 8 6 : 77: 1925-30. 8 Feig PU. Rutan GH. Angiotensin converting enzyme inhibitors: the end of end-stage renal disease. Ann Intern Med 1989: 111:451-2. 9 Loutzenhiser R. Epstein M. EKects of calcium antagonists on renal hemodynamics. Am ] Physiol 1985: 249: F619-29. 10 Price RG. Dance N. The excretion of N-acetyl-betaglucosaminidase and beta-galactosidase following surgery to the kidney. Clin Chim Actn 1970: 7 : 65-72. 1 1 Schreiner GE. Determination of inulin by means of resorcinol. Proc Soc Exp Biol Med 1950: 74: 117-20. 12 Smith HW. Finkelstein N. Aliminose L. Crawford B. Graber M. The renal clearance of substituted hippuric acid derivates and other aromatic acids in dog and man. 1 Clin Invest 1945 : 2 4 : 388-404. 13 Levinsky NG. Levy M. Clearance techniques. In: Handbook oj Physiology. Renal Physiology. Washington I X: American Physiological Society, 1973 : 103-1 8. 14 Boer WH. Koomans HA, Mees EJD. Lithium clearance in mineralocorticoid escape in humans. Am / Physiol 1 9 8 7 : 2 5 2 : F382-F86. 15 Grizzle JE. The two-period change-over design and its use in clinical trials. Biornetrics 1965: 21 : 467-80. 16 Heeg JE. de Jong PE. van der Hem GK. de Zeeuw D. Reduction of proteinuria by angiotensin converting enzyme inhibition. Kidney Int 1 9 8 7 : 32: 78-83. 17 Heeg JE. de Jong PE. van der Hem GK. de Zeew U. H c a c y and variability of the antiproteinuric effect of ACE inhibition by lisinopril. Kidney Int 1989: 36: 272-9. 18 August P. Cody RJ. Sealy JE. Laragh JH. Hemodynamic responses to converting enzyme inhibition in patients with renal disease. Am Hypertens 1989: 2: 599-603. 1 9 Mimran A, Insua A, Ribstein J, Monnier L. Bringer J. M i r o w J. Contrasting effects of captopril and nifedipine in normotensive patients with incipient diabetic nephropathy. / Hypertens 1988: 6 : 919-23. 2 0 Mickisch 0.Scmid M. Mann JF. Helder T. Bergen F. Rauterberg
EW. Calcium antagonist in chronic renal failure. Undesirable effects on glomerular hemodynamics. Dtsch Med Wochenschr 1988: 113: 1546-8. 21 Baba T, Murabayashi S, Takebe K. Comparison of the renal effects of angiotensin converting enzyme inhibitor and calcium antagonists in hypertensive type 2 (non-insulin-dependent) diabetic patients with microalbuminuria : a randomised controlled trial. Diabetologia 1 9 8 9 : 3 2 : 4 0 4 4 . 2 2 keda T. Nakayama D, Gomi T, Sakurai J, Yamazaki T. Yuhara M. Captopril, and angiotensin I-converting enzyme inhibitor, decrease proteinuria in hypertensive patients with renal diseases. Nephron 1989: 5 2 : 72-5. 2 3 Romero JC, Raij L, Granger JP, Ruilope LM. Rodicio JL. Multiple effects of calcium entry blockers on renal function in hypertension. Hypertension 1 9 8 7 : 10: 140-51. 2 4 Carmines PK. Perry MD. Hazelrig JB. Navar LG. EKects of preglomerular and postglomerular vascular resistance alterations on filtration fraction. Kidney Int 1 9 8 7 : 31: S-229S-32. 25 Koomans, HA. Boer WH, Mees EJD. Evaluation of lithium clearance as a marker of proximal tubule sodium handling. Kidney Int 1989: 36: 2-12. 2 6 Schardijn GHC. van Eps LWS. Beta-2-microglobulin: its significance in the evaluation of renal function. Kidney Int 1 9 8 7 ; 32: 6 3 5 4 1 . 2 7 Kunin CM, Chesney RW. Craig WA. England AC. Deangelis C. Enzymuria as a marker of renal injury and disease : studies of N-acetyl-hlucosaminidase in the general population and in patients with renal disease. Pediatrics 1978: 6 2 : 751-60. 28 Pflederer G, Baier M. Mondorf AW. Stefanescu T. Scherberich JE. Muller H. Change in alkaline phosphatase isoenzyme pattern in urine as a possible marker for renal disease. Kidney rnt 1980: 1 7 : 242-6. 2 9 Man in’t Veld, AJ. Calcium antagonists in hypertension. Am / Med 1989: 8 6 (Suppl. 4A): 6-14. 3 0 Lederballe OL. Krusell LR. Sihm 1. Jespersen LT. Thomsen K. Long-term effect of isradipine on blood pressure and renal function. Am ] Med 1 9 8 9 : 8 6 (Suppl. 4A): 8 6 : 15-8. 31 Christensen CK. Lederballe Pedersen 0, Mikkelsen E. Renal effects of acute calcium blockade with nifedipine in hypertensive patients receiving beta-adrenoceptor-blocking drugs. Clin Pharmacol Ther 1 9 8 2 ; 3 2 : 572-6. 32 Mogensen CE. Selling K. Studies on renal tubular protein reabsorption : partial and near-complete inhibition by certain amino acids. Scand 1 Clin Lab Invest 1977: 37: 477-86. 33 Ichihara. T. Matsumura Y, Shinyama H, Ohyama T. Morimoto S. EKects of Bay K 8 6 4 4 on renal function and renin secretion in anesthetized rats. Lije Sci 1989: 4 4 : 1945-53. 34 Gibb DM. Tomlinson PA, Dalton NR. Turner C. Shah V. Renal tubular proteinuria and microalbuminuria in diabetic patients. Arch Dis Child 1989: 6 4 : 129-34. Received 1 7 June 1990, accepted 14 August 1990. Correspondence : Dr Hallvard Holdaas. .Department of Nephrology. Medical Department B. National Hospital. Pilestredet 32.0027. Oslo 1 . Norway.
ADONIS 09 5468209 100029 JIM
Contrasting effects of lisinopril and nifedipine on albuminuria and tubular transport functions in insulin dependent diabetics with nephropathy H. HOLDAAS, A. HARTMANN, M. G. LIEN, L. NILSEN, J. JERVELL*, P. FAUCHALD, L. ENDRESEN, 0. DJQSELANDI & K. J. I3ERG From the Department of Nephrology and the *Deprtment of Endocrinology. Medical Department B. National Hospitul. Oslo. Norway
Abstract. Holdaas H, Hartmann A, Lien MG, Nilsen L. Jervell J. Fauchald P.Endresen L. Djeseland 0, Berg KJ (Department of Nephrology and Department of Endocrinology, Medical Department B, National Hospital, Oslo. Norway). Contrasting effects of lisinopril and nifedipine on albuminuria and tubular transport functions in insulin dependent diabetics with nephropathy. Journal of Internal Medicine 1991 : 229: 163-1 70. An open, randomized, cross-over study was undertaken to assess the effects of lisinopril
and nifedipine on albumin excretion, renal haemodynamics and segmental tubular reabsorption in overt diabetic nephropathy. The study consisted of a 4-week run-in period, a 3-week active treatment period, a 4-week wash-out period and a second 3-week active treatment period. Twelve patients with type 1 diabetes with albuminuria, mild to moderate hypertension and a serum creatinine level of < 200 pmol I-' were included. Lisinopril reduced albumin excretion from 1343 337 pg min-I to 879 f299 pg min-' (P < 0.01). whereas nifedipine was without effect, 1 4 3 6 f 3 3 6 ,ug m i d vs. 1319 f342 pg min-'. Glomerular filtration rate (GFR) was unchanged by either drug. Both drugs increased effective renal plasma flow (ERPF) by about 20%. No differences between the drugs were observed with regard to their effect on renal haemodynamic parameters. By contrast, nifedipine exerted an inhibitory effect on several proximal tubular transport markers, whereas lisinopril was without effect. The different actions on tubular transport mechanisms exerted by lisinopril and nifedipine may contribute to the observed effect on albumin excretion.
Keywords: ACE inhibitor, albuminuria, calcium-channel blocker, diabetes, renal haemodynamics, tubular function.
Introduction The first clinical sign of diabetic nephropathy is characterized by an increase in urinary excretion of albumin. Preceding overt proteinuria, microalbuminuria has usually been manifest for years. The deterioration of renal function in patients with diabetic disease may be retarded by vigorous control of systemic blood pressure [l.21. Strict metabolic control [3] and reduced phosphate/protein intake [4] may also contribute towards preservation of renal function. The antihypertensive drugs used in longterm prospective studies that demonstrate a favourable effect of good blood pressure control for preservation of renal function include beta-blockers,
vasodilators, diuretics and ACE inhibitors [l,2, 51. These drugs have widely differing effects on renal haemodynamics, an observation which suggests that the beneficial effect of antihypertensive treatment on renal function is related to good control of systemic blood pressure rather than specific intrarenal effects on renal haemodynamics. However, as reviewed by Keane et al. [6], several studies have advocated that ACE inhibitors may possess specific advantages in decreasing proteinuria and slowing progression of diabetic nephropathy. The rationale for this view is based on animal models that have demonstrated favourable alterations in glomerular haemodynamics with ACE inhibition [7].In man the same'beneficial effects of ACE inhibitors have been inferred from 163
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changes in filtration fraction [6]. The data have been interpreted as showing a preferential effect of ACE inhibitors on efferent arterioles [8]. The lack of consistent data reported with regard to the effect of calcium-channel blockers on diabetic proteinuria has been attributed to a non-selective dilation of afferent and efferent arterioles, or a selective reduction of preglomerular resistance [9]. In an attempt to determine the effects of calcium channel blockers and ACE inhibitors on albumin excretion we performed a short-term cross-over study on insulin dependent diabetics with overt nephropathy. We used the ACE inhibitor lisinopril and the calciumchannel blocker nifedipine. Particular emphasis was placed on renal haemodynamics and tubular markers for proximal tubular function.
Subjects and methods Patient The patient sample consisted of 1 2 hypertensive patients (five women and seven men) aged 22-58 years, with insulin dependent diabetes of 1 8 4 5 years duration. Patients with heart failure or coronary heart disease were excluded from the study. Informed consent was obtained according to the Declaration of Helsinki, and the study was approved by the Regional Ethical Review Board of the University of Oslo. The patients were selected on the basis of an increase in urinary albumin excretion rate to > 3 0 0 p g min-' in two consecutive 24-h urine samples, provided that urine culture was negative. Serum creatinine levels had to be < 200 pmol I-'. Mild to moderate hypertension was defined as diastolic blood pressure in the range 85115 mmHg. Blood pressure readings were mean values of the last two of three recordings taken with a standard sphygmomanometer with the patient seated for at least 5 min. Diastolic blood pressure was recorded as the disappearance of the Korotkoff sound (phase 5). Mean arterial pressure (MAP) was regarded as diastolic blood pressure plus 1 / 3 pulsatile pressure. Study protocol The study was conducted in a randomized two-way cross-over manner, and consisted of a 4-week run-in period, a 3-week active treatment period, a 4-week wash-out period and a second 3-week active treat-
ment period. During the 4-week run-in period, five patients were withdrawn from previous antihypertensive medication. At the end of the run-in period, patients were randomized to one of the two possible sequences for active treatment (lisinopril followed by nifedipine, or nifedipine followed by lisinopril). Lisinopril treatment started with 5 mg daily, while nifedipine treatment started with 10 mg twice a day. After 1 week of treatment, titration was allowed by doubling the dose of medication, and after another week of treatment a final titration was allowed for the lisinopril treatment, if systolic blood pressure was still > 1 2 0 mmHg. The maximal daily dose was 4 0 mg for nifedipine and 2 0 mg for lisinopril. Blood samples and duplicate 24-h urine samples were collected at the end of the run-in period, at the end of the wash-out period and at the end of the two treatment periods. Renal clearance examinations were performed on the last day of the run-in period and on the last day of each treatment period. The evening before each clearance study, each subject ingested 18 mmol lithium citrate in order to obtain a plasma lithium concentration of about 0.3 mmol I-'. The subjects were hydrated orally with tap water, 20 ml kg-'. Renal haemodynamic parameters were assessed by para-amino-hippurate (PAH) and inulin clearances for the estimation of ERPF and GFR, respectively. Priming doses of inulin (10%solution) and PAH (20% solution) were administered to obtain plasma concentrations of approximately 200300 mg I-' and 2 0 4 0 mg 1-', respectively. Infusions of inulin and PAH were administered in order to maintain initial plasma concentrations. After a 60mins equilibration period, four carefully timed urine collections of 25-35 min each were obtained by spontaneous voiding. Blood samples were drawn at approximately the midpoints of the collection periods. The clearance studies were not initiated until the urine osmolality was < 200 mosm kg-'. Biochemical analyses Sodium and potassium levels in serum and urine were determined by a standard autoanalyser technique. Urinary albumin was determined with a radioimmunoassay kit (Pharmacia A.B., Uppsala, Sweden). Total protein in the urine was determined by the biuret method. Serum and urine 8-2-microglobulin was assayed by a commercial radioimmunoassay kit (Pharmacia A.B., Uppsala, Sweden). Urine samples for 8-2-microglobulin were
ALBUMINURIA IN INSULIN DEPENDENT DIABETICS stored at pH > 6. The urinary excretion rate of B-2microglobulin in healthy controls was 1 37 f66 pg 24 h-'. Creatinine was determined by a ratedependent modiEcation of the Jaffe reaction using a creatinine analyser, Bechman creatinine analyser model 2. Urinary N-acetyl-beta-glucosaminidase (NAG) was determined by a fluorometric method of Price et al. [lo]. Activity was expressed as units pmol-' creatinine. The mean value in healthy controls was 5.4 f2.3 units pmol-' creatinine. Urinary alkaline phosphatase (ALP) was determined spectrophotometrically by a commercial kit (Boehringer, GmbH Diagnostica, Mannheim, Germany), and the activity was expressed as units 24 h-'. The mean HbA, level was determined spectrophotometrically after ion-exchange chromotography. Plasma renin activity and plasma aldosterone were determined using commercial radioimmunoassay kits, and lithium was determined by flame emission photometry. The inulin concentration was determined according to Schreiner [ l 11. High concentrations of interfering glucose in diabetic patients were removed using glucose oxidase. PAH was determined according to Smith et al. [12].
Calculations Rates of clearance of various substances were calculated according to standard clearance formulas [13]. The Eltration fraction was calculated as GFR/ERPF, and renal vascular resistance was taken to be MAP/ERPF. Using lithium as a marker of proximal sodium reabsorption, segmental sodium handling was analysed using the following equations : FL,, =GFR x P,,/lOOO (mmol min-') DD,, =C,, x P,,/lOOO (mmol min-') APR,, = FL,, - DD,, (mmol min-') ADR,, =DD,, - U,,V (mmol min-') where FL,, is filtered sodium, P,, is the plasma sodium concentration, DD,, is distal sodium delivery, C,,, is lithium clearance, APR,, is absolute proximal reabsorption, and U,,V is sodium excretion [14].
Statistical analyses Data were analysed using the appropriate methology for two-period cross-over trials as described by Grizzle [15]. Analysis of the cross-over data was performed in two steps. In the Erst step the sequence effect was
165
Table 1. Blood pressure responses to lisinopril and nifedipine Lisinopril
Nifedipine
Baseline 3 weeks
Baseline 3 weeks
Systolic blood 146*3 pressure (mmHg) Diastolic blood pressure (mrnHg) Mean blood pressure (mmHg)
89k2
137*4*
146k5 134*3*
83f3'
109i-2 102*3*
91k3
78k2'
110k3
96*2*
* P < 0.01 vs. baseline. Table 2. Renal haemodynamic responses to lisinopril and nifedipine Baseline
Ltsinopril
Nifedipine
95f12 551f57*
94k11 521*36*
GFR (ml min-')
95k12
ERPF (mlmin-')
466f32
Filtration fraction
0.20k0.02 0.16kO.02t 0.17k0.027 0.26f0.02 0.20f0.02t 0.20k0.02t
RVR (mmHg min-' ml-')
CFR = glomerular Bltration rate. ERPF = effective renal plasma flow,RVR = renal vascular resistance. P < 0.05 vs. baseline, t P c 0.01 vs. baseline.
tested at a level of a = 0.10 ; if no significant sequence effect was detected, the treatment and period effects were tested subsequently at a level of a = 0.05. The results are presented as mean valuesfSEM. The mean value for the two 24-h urine collections in each period was used for comparison of the urine data, and the values in each treatment period were compared with those for the preceding control period. For the evaluation of inulin, PAH and lithium clearances, data from each treatment period were compared with the 6rst control period. The data for the two drug periods were also compared with each other. A t-test was used :for statistical evaluation, all statistical tests were two-tailed and significance was defined as P < 0.05.
Results Eflects on blood pressure Lisinopril and nifedipine significantly reduced both systolic and diastolic blood pressure (Table 1).The mean decrease in diastolic blood pressure was slightly, but not significantly, larger during nifedipine treatment than during lisinopril treatment. In both sequence groups the nifedipine dose reached a
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Table 3. Blood biochemical responses to lisinopril and nifedipine Lisinopril
Nifedipine
Baseline
3 weeks
Baseline
3 weeks
10.2f0.3 139f1
11.2f0.7
Renin (nmol I-' h-l)
10.6f0.5 140*2 4.5f 0.1 36f1 113f9 0.4 0.1
Aldosterone (pmol I-')
303 f 63
232f68t
10.5&0.4 139f 1 4.3k0.1 37f2 122 f 9 1.7f 0.6 515f123
HbA,
(%I
Na (mmol I-')
K (mmol I-') Albumin (g I-') Creatinine (pmol I-')
P < 0.01 vs. baseline,
4.5k0.2 36f1 120* 1 1 4.7f1.7.t
140f 1 4.5f0.1 36*1 119f9 0.9f 0.3 365f 72
t P < 0.01 vs. nifedipine.
--
4000
C ._ E
CF
-3 3000 c
._ + El
2
5
zoo0
5 e
a
lo00
Fig. 1. Twenty-four-hour albumin excretion in response to lisinopril and nifedipine. Mean values fSEh4
are shown.
n v Control
maximal level of 40 mg d-'. For the lisinopril treatment, four patients, two in each sequence group, were not titrated to the maximum dosage. The mean final dosage of lisinopril was 16.25 mg d-'. No serious adverse side-effects were observed during treatment in either group. Effects on renal haemodynamics The data are summarized in Table 2. GFR, estimated as inulin clearance, was remarkably stable throughout the study. Neither lisinopril nor nifedipine induced significant changes in GFR. However, both drugs significantly increased ERPF from baseline levels. Consequently, the baseline Eltration fraction of 0.20 f0.02 was reduced to 0.16 f 0.02 by lisinopril and to 0.17 f 0.02 by nifedipine. A substantial and equal decrease to 0.20 f 0.02 mmHg min-' ml-' of estimated renal vascular resistance occurred in response to both lisinopril and nifedipine, from a
Lisinopril
Control
Nifedipine
baseline value of 0 . 2 6 f 0 . 0 3 mmHg min-' ml-'. Thus there was no difference in the effects of lisinopril and nifedipine on renal haemodynamic parameters. Effects on blood biochemical parameters A summary of the results is given in Table 3. No change in plasma electrolyte concentrations was recorded. Plasma albumin, HbA, and creatinine levels were stable throughout the study. Short-term therapy with lisinopril was, as expected, associated with a marked increase in plasma renin activity. Nifedipine had no effect on plasma renin activity and aldosterone concentration. Effects on 24-h-urine biochemical parameters The most striking finding was the consistent reduction in albumin excretion in all 12 patients during lisinopril therapy, as illustrated. in Fig. 1. Albumin excretion decreased from 1343 f337 pg min-' to
ALBUMINURIA IN INSULIN DEPENDENT DIABETICS
167
Table 4. Twenty-four-hoururine responses to lisinopril and nifedipine Lisinopril
Nifedipine
3 weeks
Baseline
3 weeks
Baseline
Na (mmol 2 4 h-')
182f 17
167f21
181 f 14
1 8 0 f 19
K (mmol 2 4 h-')
87+9
75k6
90f 7
87k 7
3.4 f 0 . 7
3.4 f 0.7
603 f250
237*69t$
468 f 1 5 0
623 f 2 0 4
7 . 3 k 1.2
7.5f 1.5
7.3f 1.2
8.3f l . l t
21.3k2.9
19.0* 2 . 0
18.3f2.1
22.8f 3.2.
Total protein (g 2 4 h-') b2-microglobulin (pg 24 h-')
3.2f0.8
ALP units (mmol 2 4 h-') NAG units (pmol-' creatiine)
1.9f0.7tS
ALP = alkaline phosphatase, NAG = N-acetyl-beta-glucosaminadase. P c 0.05 vs. baseline, t P c 0.01 vs. baseline, $ P < 0.01 vs. nifedipine. Table 5. Segmental electrolyte transport in response to nifedipine and lisinopril Baseline
Lisinopril
2 3 . 4 f 1.9 72.8f2.7
21.1 f 1.5
29.2 f 2.0*$
75.9 f 2 . 0
66.0f 3.6t5
12.9 f 1 . 4
13.1 f 1.4
12.8f1.4
APR,, (mmol min-')
9.6f1.4
10.2f1.5
ADR,, (mmol min-')
2.9k0.3
2.6k0.2
U,,V (mmol min-')
0.4f 0.05
0.3f0.02
Li clearance (ml min-') Li fractional reabsorption (%) FL,,
(mmol min-')
Nifedipine
8.8f1.5.t 3.6f0.2*$ 0.4k 0.05
FL,, = filtered sodium. APR,, = absolute proximal sodium reabsorption. ADR,, = absolute distal sodium reabsorption. U,,V excretion. 'P < 0.05 vs. baseline. t P c 0.01 vs. baseline. P c 0.05 vs. lisinopril. 5 P c 0.01 vs. lisinopril.
= sodium
*
89 7 f299 pg min-' during treatment with lisinopril, a reduction of 35%. By contrast, nifedipine treatment had no effect on albumin excretion, there was a decrease in albumin excretion in seven patients, whereas in Eve patients there was an increase in albuminuria during the drug phase. Albumin excretion was 1436 f336 p g min-' prior to and 1319 f342 pg min-' during nifedipine treatment. The changes in albumin excretionwere also reflected by parallel changes in total urinary protein excretion, as shown in Table 4. During nifedipine therapy, urinary P-2-microglobulin excretion increased from 486 f 150 pg 24 h-' to 623 f 204 pg 2 4 h-l, while lisinopril treatment decreased excretion from 603 f 250 pg 24 h-' to 2 3 7 f 69 pg 2 4 h-' (P < 0.01). The rate of sodium excretion was high, but remained unchanged throughout the study. As shown in Table 4, the proximal brush-border enzyme marker ALP showed a significant increase in activity from 7.3f 1. 2 to 8. 3f1 .1 units 24 h-' during the nifedipine treatment period. This was accompanied by a corresponding increase in excretion of the proximal
lysosomal enzyme NAG from 1 8 . 3 f 2 . 1 to 22.8+ 3.2 units pmol-I creatinine (P < 0.05). Lisinopril treatment had no significant effect on urinary ALP or NAG.
Effects on segmental electrolyte transport The effects of both drugs on segmental electrolyte handling are shown in Table 5. During nifedipine treatment, a significant increase in lithium clearance was observed. The fractional reabsorption of lithium was substantially reduced during nifedipine therapy from 72.8f2.7% to 6 6 .0 f 3 .6 % ( P < 0.01). Although lisinopril treatment tended to increase fractional reabsorption of lithium and reduce lithium clearance, none of the changes were significantly different. The proximal reabsorption of sodium, estimated by lithium clearance, was significantly reduced during nifedipine therapy from 9.6 & 1.4 mmol min-' to 8.8 f 1.5 mmol min-', indicating that nifedipine had a n inhibitory effect on proximal transport. However, as can be seen from Table 5, the
168
H. HOLDAAS et al.
decrease in proximal ion transport was almost totally compensated for by an increase in electrolyte reabsorption in the distal tubules.
Discussion The most important finding in this study is that the ACE inhibitor lisinopril reduces urinary albumin excretion in insulin dependent patients, whereas nifedipine has no such effect. The albumin-sparing effect of lisinpril was also confirmed by a significant reduction in excretion of 8-2-microglobulin and total urinary protein. Previous reports on the effect of lisinopril on albumin excretion are scarce; Heeg and co-workers have demonstrated a protein-sparing effect in patients with predominantly non-diabetic renal disease [16, 171. In contrast to the present study, the protein-sparing effect in non-diabetic patients appears to be dependent on adequate restriction of sodium, as the reduction in albumin excretion was abolished by increasing the rate of sodium excretion from 50 to 200 mmol d-' [17]. In our study sodium intake was not regulated and, despite a sodium excretion rate of 180 mmol d-', reflecting a standard diet, a consistent effect on albuminuria was found. The uniform effect of lisinopril treatment on blood pressure and albumin excretion in our study is at variance with the results of August et al. [18], who felt the need to subdivide patients on lisinopril therapy into responders and non-responders. Moreover, in their study, in neither responders nor non-responders could any effect on protein excretion be detected. The reason for these discrepancies may be related to the degree of stimulation of the renin-angiotensin system, any effect of ACE inhibitors being more easily detected when the system is stimulated. However, as shown in our study, even under conditions of high sodium excretion and consequently low renin, a consistent effect was detected. The effect of lisinopril treatment on urinary albumin excretion observed in our study is consistent with previous reports of beneficial effects of other ACE inhibitors on albumin excretion in patients with diabetic and non-diabetic nephropathy. A comprehensive review has been given on this topic [6]. The consensus seems to be that ACE inhibitors are effective antihypertensive agents with a favourable effect on albumin excretion in most of patients with diabetic and non-diabetic renal diseases [6]. Previous results concerning the effect of calcium channel-blockers in short-term studies on protein/
albumin excretion in patients with nephropathy have been conflicting. In studies that have reported an unfavourable effect on albumin excretion in diabetic patients, the results have been explained by a detrimental effect of calcium-channel blockers on renal haemodymanics [19, 201. However, both beneficial effects and a total lack of effect on albumin excretion during calcium-channel-blocker treatment have been reported. Baba et al. [21] demonstrated a beneficial effect of nicardipine on microalbuminuria in type I1 diabetes. Ikeda et al. were unable to demonstrate any effect on albumin excretion during treatment with a dihydropyridine-derived calciumchannel blocker in patients with non-diabetic renal disease [22]. In our study the reduction in mean blood pressure was more pronounced with nifedipine. The observed lack of effect of nifedipine treatment on albumin excretion thus cannot be ascribed to an inadequate systemic blood pressure-lowering effect compared to lisinopril therapy. Furthermore, there were no differences in the changes induced by either drug on ERPF, FF and RVR. No change in GFR was induced by either drug. This does not support the concept of differential intrarenal vascular effects of the two agents applied. In man, such a hypothesis has been primarily inferred from the observed changes in filtration fraction [6]. Although both ACE inhibitors and calcium-channel blockers have been employed in numerous trials in man, only a few studies have reported data on RBF and GFR. thus allowing calculation of the filtration fraction. A review of the filtration fraction data in patients with diabetic and non-diabetic disease reveals no consistent change in FF fraction when ACE inhibitors [6] or calcium channel blockers [23] have been applied. In addition, Navar and his co-workers have advocated the view that changes in filtration fraction are not necessarily indicative of selective alteration in segmental vascular resistance [24]. As long as renal haemodynamic parameters are changed to an equal extent by two different drugs, a difference in the effects of the drugs on. pre- and postglomerular vascular resistance is difficult to envisage. The lack of effect of the calcium-channel blocker nifedipine on albumin excretion in our study may be explained by an inhibitory effect of the drug on proximal tubular reabsorption mechanisms. Such a specific intratubular effect of nifedipine could offset the albumin-sparing effect that has been demonstrated in diabetic nephropathy during treatment with other antihypertensive drugs [l,2, 5. 211.
ALBUMINURIA IN INSULIN DEPENDENT DIABETICS
In order to estimate proximal tubular function, several techniques and markers were applied. For proximal ion and water transport we used the lithium clearance technique. Although the reliability of lithium as a marker for proximal tubular function has been questioned, it is generally agreed that, provided patients are examined sequentially at the same level of GFR during water diuresis, this method represents the best clinical marker of proximal tubular function [25]. Nifedipine caused a marked reduction in fractional lithium reabsorption and a corresponding increase in lithium clearance. The calculated reabsorption of sodium in the proximal tubules was consequently suppressed during nifedipine therapy. ~-2-microglobulin,a freely filtrable protein, has also been widely used as a marker of proximal tubular function [26]. The excretion of a-2-microglobulin was significantly increased during nifedipine treatment compared to lisinopril therapy. NAG and ALP are intracellular and brush-border enzyme markers, respectively, of proximal tubular function [27, 281. The levels of both these enzyme markers increased during nifedipine treatment, thus supporting the view that the drug has a proximal tubular effect. All these results suggest an inhibitory effect of nifedipine on proximal ion and water transport. This concept is consistent with the view that the most likely mechanism for increased sodium excretion of calcium-channel blockers is a direct inhibition of tubular sodium reabsorption [29]. For one calciumxhannel blocker, isradipine, it has been demonstrated that the effect is localized in the proximal tubules [30]. Christensen et al. proposed a proximal tubular effect of nifedipine, based on its effect on the urinary excretion of albumin and /3-2-microglobulin [3 11. Our results, which demonstrated an inhibitory effect of nifedipine on several proximal tubular function markers, support the hypothesis that nifedipine might also inhibit proximal tubular albumin transport mechanisms. In healthy human subjects the estimated minimum capacity for albumin reabsorption in proximal tubules is about 200400 p g min-' [32]. The albumin reabsorption in protein-losing disease is not known, although it may be assumed that it is high and probably at a transport maximum. Even a small decrease in proximal function may result in increased excretion of albumi, since no distal segments transport proteins. Nifedipine may therefore depress albumin reabsorption in proximal tubules in diabetic nephropathy, leading
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to albuminuria despite the fact that there is a convincing systematic antihypertensive effect. Moreover, it has been shown that a dihydropyridine calcium-channel agonist, Bay K 8644, increases sodium and water reabsorption when infused into the renal artery [33]. Thus data from both animal and human models indicate that manipulation with calcium-channel agonists or antagonists may interfere with proximal tubular transport mechanisms. By contrast, proximal tubular transport function during lisinopril therapy remained unchanged or was even slightly improved, when estimated by the lithium clearance technique or by b-2-microglobulin. The excretion of proximal tubular enzyme markers, the intracellular NAG and the brush-border enzyme ALP, was also unchanged by lisinopril treatment. Thus in our study, no harmful effect of the ACE inhibitor lisinopril on proximal tubular function could be detected. On the basis of the results from our study we propose that the observed difference between lisinopril and nifedipine on albumin excretion may at least in part be explained by different tubular effects. A previous report has also proposed that an increase in urinary excretion of albumin in diabetic patients may be tubular in origin [34]. In conclusion, lisinopril reduced diabetic albuminuria in all patients, whereas nifedipine has no such effect. Lisinopril and nifedipine had similar antihypertensive effects, and similar actions on renal haemodynamics. Nifedipine had an inhibitory effect on markers for proximal tubular function. A potentially favourable effect of nifedipine on albumin excretion might be masked by the suppression of proximal tubular reabsorption mechanisms.
Acknowledgements Lisinopril was provided by Merck Sharp & Dohme International (Rahway, NJ, USA). This study was supported by the Norwegian Research Council for Science and the Humanities, the Norwegian Drug Monopoly, and Merch Sharp & Dohme, Norway.
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