1 Department of Internal Medicine I, University Hospital Dijkzigt, Dr. Molewaterplein 40, Rotterdam, The Netherlands and 2 Steno Diabetes Center, Niels Steensensvej 2, Gentofte, Denmark
Correspondence and offprint requests to: Jaap Deinum, Department of Internal Medicine I, University Hospital Dijkzigt, Room L253, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands.
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Abstract |
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Methods. We studied plasma renin, prorenin, and four polymorphic markers of the renin gene in 199 patients with IDDM and DN, and in 192 normoalbuminuric IDDM controls matched for age, sex, and duration of diabetes. Plasma renin and total renin were measured by immunoradiometric assays. Genotyping was PCR-based.
Results. Plasma renin was increased in patients with nephropathy (median (range), 26.3 (5.2243.3) vs 18.3 (4.2373.5) µU/ml in the normoalbuminuric group, P<0.0001). Prorenin levels were elevated out of proportion to renin levels in nephropathic patients (789 (885481) vs 302 (362226) µU/ml, P<0.0001). Proliferative retinopathy had an additive effect on plasma prorenin, but not on renin. DN was associated with a BglI RFLP in the first intron of the renin gene (bb-genotype: n=106 vs 82 in DN and normoalbuminuric patients respectively, P=0.037), but not with three other polymorphisms in the renin gene. A trend for association of higher prorenin levels with the DN-associated allele of this renin polymorphism was observed in a subgroup of patients with DN (bb vs Bb+BB, P=0.07).
Conclusions. The results indicate that in DN there is an increase in both renin and prorenin levels. A renin gene polymorphism may contribute weakly to DN. Although speculative, one of the renin gene alleles could lead to increased renin gene expression, leading to higher renin and prorenin levels. These may play a role in the pathogenesis of DN.
Keywords: diabetes mellitus; nephropathy; polymorphism; prorenin; renin; retinopathy
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Introduction |
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The favourable effect of ACE inhibition on the development [5] and progression [6] of DN is suggestive evidence that the reninangiotensin system (RAS) is involved in the pathogenesis of DN and that variation at RAS gene loci might determine susceptibility to DN. In many studies, however, plasma renin and angiotensin II are normal or suppressed in diabetics (for review see [7,8]) and reports on association between DN and polymorphic markers have been equivocal for the angiotensinogen gene ([911] positive and [12] negative) and negative for the AT1-receptor [13,14] gene. Three meta-analyses of the numerous studies on association of the ACE I/D polymorphism with DN show that the D-allele may confer an increased risk of DN [1517]. Interaction between the ACE I/D polymorphism and an angiotensinogen allele may also present elevated risk of DN [18]. Similarly, poor metabolic control may interact with the AT1-receptor polymorphism to augment risk of DN [14]. The renin gene that codes for prorenin and renin has not yet been investigated in DN in large study groups. Prorenin is the enzymatically inactive biosynthetic precursor of renin, the rate-determining enzyme in the proteolytic cascade leading to formation of angiotensin II, the effector of the RAS. Several reports have described increased plasma prorenin levels when diabetes is complicated by microvascular disease like DN and retinopathy [1922]. In the present case-control study we measured plasma levels of renin and prorenin and determined genotypes of the renin gene in patients with IDDM complicated by DN and in matched normoalbuminuric controls.
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Subjects and methods |
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Methods
Blood pressure was measured with a Hawksley random zero standard sphygmomanometer after a 10 min rest in the sitting position. The disappearance of Korotkoff sounds (phase V) was used to determine diastolic blood pressure. Retinopathy was assessed by fundus photography after pupillary dilatation. Retinal lesions were categorized as either no, background, or proliferative retinopathy. EDTA-anticoagulated blood for measurement of plasma prorenin and renin was collected in the morning after an overnight fast from an indwelling catheter in a forearm vein after the patient had been in the supine position for at least 15 min. The blood was centrifuged at 3000 g at room temperature and plasma was stored at -80°C. Sixty-nine patients with nephropathy and nine with normoalbuminuria did not want to stop their antihypertensive drugs at least 8 days before the day the investigations were performed.
Laboratory procedures
All measurements were done by technicians who were unaware of the status of the patients. Urinary albumin excretion rate (AER) was measured from 24 h collections by an enzyme-linked immunoassay [25]. Haemoglobin A1c (HbA1c) was measured by high-performance liquid chromatography (HPLC, Biorad, Diamat, Richmond, CA, USA). The normal range is 4.16.4%. Serum creatinine was measured by autoanalyser.
Plasma prorenin and renin determination
Renin and total renin (i.e. renin+prorenin) were measured by an immunoradiometric assay (IRMA), purchased as a kit from Nichols Institute, Wijchen, The Netherlands [26]. In this assay renin is sandwiched between a biotinylated monoclonal antibody (mAb) R33616 against human renin, immobilized on an avidin-coated polystyrene bead, and a 125I-labelled mAb R1205 against a renin-specific epitope not expressed on prorenin. Therefore the radiolabelled mAb does not recognize native prorenin. Yet prorenin can be measured with this assay, after preincubation with the active-site directed renin inhibitor remikiren, which causes exposure of the renin-specific epitope recognized by mAb R 1205 [26]. The difference between the results with and without remikiren, i.e. the difference between total renin and renin, is a measure of the prorenin concentration. The assay was modified by shortening incubation to 6 h and performing the assay at 37°C. These modifications prevent inadvertent activation of prorenin and therefore exposure of the renin-specific epitope during the assay. Intra- and interassay variation for prorenin (320 µU/ml) is 4 and 13% respectively and for renin (22.2 µU/ml) 8 and 15%. Renin and prorenin of each patient were always measured in the same assay. Results of the renin and prorenin measurements are expressed as µU/ml. Standards in the assay were dilutions of the international reference preparation of human kidney renin 68/356 of the National Institute for Biological Standards and Control (Potters Bar, Hertfordshire, UK). The geometric mean and 95% reference limits for 100 normal subjects (50 women) with an age range of 1962 years was 22.0 (7.754.8) µU/ml for renin and 199 (88.1390) µU/ml for prorenin.
DNA extraction
Genomic DNA was extracted from peripheral leukocytes by a standard technique. It was aliquoted and stored at -80°C until use.
Genotyping
Four polymorphisms were studied at the REN locus (Figure 1). They consist of two restriction fragment length polymorphisms (RFLPs) [27,28] and two microsatellites [29,30]. The TaqI RFLP is located 4063 bases upstream of the start codon and consists of a C-T mutation. The BglI RFLP is a C-T mutation located at base 1161 of intron A. The (ACAG)n microsatellite is located in intron G. The CA-repeat is located downstream of the REN locus. Genotyping was PCR-based. Primers were obtained from Eurogentec (Seraing, Belgium), as were Goldstar Taq polymerase and the restriction enzymes. Except for haplotyping the BglI and TaqI loci (see below), PCR volume was 25 µl, consisting of 200 ng of DNA, 75 mM TrisHCl, 20 mM (NH4)2 SO4, 0.01% Tween 20, 0.5 µM of each primer, dNTP concentrations of 10 µM each and a polymerase activity of 1 U/25 µl PCR volume. Primer sequences and cycling conditions are summarized in Table 1
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Microsatellite loci were amplified in a duplex PCR, with one of each primer pair labelled with the fluorochrome FAM. Labelled PCR products were separated by electrophoresis on a denaturing 5% polyacrylamide gel, with fluorescence detection by an ABI Prism 377 automatic sequencer (PerkinElmer). In each lane a TAMRA-labelled calibration ladder was run. Allele lengths were calculated by Genescan software and checked by eye by two independent observers.
Statistical analysis
Continuous normally distributed data were expressed as mean (standard deviation). AER, creatinine, plasma renin and prorenin were log transformed before statistical analysis in order to obtain normal distribution. They are presented as median (range). Comparisons between groups were performed by Student's t-test or ANOVA. 2 analysis was used to compare the distribution of genotypes and alleles and for comparison between groups for non-continuous variables. Two-sided P values
0.05 were considered significant. All aforementioned analyses were performed using the statistics package Statgraphics (Manugistics, Princeton NJ). Multi-allelic polymorphisms were tested using the CLUMP program. CLUMP employs Monte Carlo simulation and has been developed for use in genetic case-control studies. A description of the program can be found in reference [31].
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Results |
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In multiple logistic regression analysis, with presence or absence of nephropathy as dependent variable and the renin BglI and TaqI polymorphisms, ACE I/D polymorphism, angiotensinogen M235T and T174M polymorphisms and the angiotensin receptor type 1 A/C polymorphism at position 1166 as independent variables, only the renin bb-genotype, not surprisingly, remained in the final model (P=0.037), but more importantly, no interactions were observed between the bb-genotype and the polymorphisms in the angiotensinogen, ACE and AT1-receptor genes.
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Discussion |
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Although there are other reports of increased renin levels in DN [8,20.32], the general impression has always been that renin levels are low in DN (for a list of references see Ref. [7]). The apparent discrepancy with our finding of increased renin levels could be due to various factors. The first may be the poorly developed measurement methodology for renin [33]. Most studies date from the seventies and early eighties and employed variants of an enzymekinetic, plasma renin activity (PRA) assay, which measures angiotensin I production by renin from endogenous angiotensinogen. The PRA assay depends on both renin and angiotensinogen concentration. Kinetics of the enzymatic reaction of renin with angiotensinogen, often performed at non-physiological pH in PRA assays, may differ in diabetic plasmas, including altered renin reactivity and non-linear angiotensin I generation, but no reports exist that address this problem. The immunological direct assay we employed, measures molecules and is unlikely to be affected by the diabetic state. Another recent study that measured renin by IRMA, also found a trend to higher renin levels in diabetic subjects with microalbuminuriathe early phase of DNcompared to normoalbuminuric diabetics [21]. This suggests again that the cause of the discrepancy may be assay-related. Renin IRMAs may have a problem in co-measurement of prorenin as renin. Our IRMA employs a monoclonal antibody specific for renin. Prorenin may also assume a renin-like conformation by so-called cryoactivation, even at room temperature, which is the temperature of the assay incubation [34]. Although this activation amounts to not more than 12%, renin levels may be overestimated, especially if prorenin levels are high. In our assay co-measurement of prorenin as renin is eliminated by performing the assay for 6 h at 37°C. We are therefore confident that our results on plasma renin in DN are valid. Another explanation for the discrepancy of previous reports with our observation of increased renin may be that study groups were often not homogeneous. Data for NIDDM and IDDM patients were often pooled in earlier studies and it is not known whether this is justified. Moreover, the various stages of DN were not always separated, partly because at the time these stages were not yet discerned. A final possible explanation for our high renin levels is that these are due to previous antihypertensive use. High renin levels may be observed with ACE inhibitors and diuretic use (AT1-receptor antagonists were not yet being prescribed to these patients). Patients had stopped antihypertensive medication at least 8 days before blood-sampling. This is usually sufficient for any effect of ACE inhibition to wear off [35]. After cessation of diuretic use secondary hyperaldosteronism may persist for longer periods than 8 days. However, this, together with a higher blood pressure, is expected to cause decreased renin levels, rather than the increased levels we found. Unfortunately, data on aldosterone that may clarify the issue somewhat, are not available. Finally, nephropathic patients virgin to antihypertensives have somewhat less increase in plasma renin and prorenin levels. This might argue in favour of a carry-over effect of antihypertensives on prorenin and renin levels in the hypertensive subgroup. On the other hand, this normotensive subgroup too does not show suppressed renin levels as is expected from older literature [7], but rather increased levels.
Increased plasma renin and prorenin levels in DN support the hypothesis that the RAS is overactive and possibly even pathogenetic in the development or progression of DN, especially since increase in total renin occurs early [22]. A possible mechanism could be higher intra-glomerular pressure, through angiotensin II-mediated constriction of the efferent arteriole, with resulting proteinuria, but this remains speculative. The origin of elevated renin is most probably the kidney, since no other source of renin has been described [36]. Whether an excess prorenin also originates from the kidney is not known. Franken et al. [20] did not observe an increase in renal vein-to-artery ratio for prorenin in patients with near end-stage diabetic nephropathy and high prorenin, suggesting that there is no markedly increased production of prorenin in the kidney. Prorenin may also originate from extrarenal sites like ovaries, testes, and adrenals [36]. Whether prorenin itself acquires enzymatic activity in DN is an unsolved question. Several mechanisms of prorenin activation have been described [36,37].
Some controversy exists concerning the relationship between retinopathy and plasma prorenin. We observed increased prorenin levels with proliferative retinopathy. This is in agreement with Franken et al., who found an increase of plasma prorenin with increasing severity of retinopathy [20]. However, contrary to their findings, we did not find a difference between patients without and those with background retinopathy in the normo-albuminuric group. This is similar to the result obtained by Allen et al. [22], who did not observein a longitudinal surveyan increase in plasma prorenin when patients progressed from no to background retinopathy. Thus, apparently retinopathy and nephropathy have an additive effect on plasma prorenin levels, suggesting that the increase in plasma prorenin is an aspecific marker for microvascular disease as proposed previously [19]. Yet probably only the advanced, proliferative stage of retinopathy influences plasma prorenin. This is in contrast with DN, where plasma prorenin increases very early in the course of the disease [22].
The derangement in renin and prorenin in DN could be connected to a polymorphism in the renin gene, if this polymorphism is associated with DN. DN is weakly associated with the bb-genotype of a BglI RFLP in the first intron of the renin gene. This suggests a deleterious recessive effect of the b-allele. The first intron is involved in renin gene transcription regulation [38] and the BglI polymorphism may be linked to this regulatory sequence. This may provide a mechanism that explains the association of DN with this marker. It could also explain the findings of Daneman et al. [21], who described a genetic influence on plasma prorenin levels in diabetic patients with microalbuminuria, although no genotyping of the renin gene was performed in this study. From this observation we anticipated a relation between BglI renin genotype and plasma renin or prorenin level. However, we could not demonstrate such an association, although a trend towards higher prorenin levels was observed in bb homozygotes. This trend may have failed to reach significance because only prorenin levels in the subgroup of patients not on antihypertensives were included in our analysis.
There was no interaction of the BglI marker with variants of other genes of the RAS, although the power of the study was not sufficient to detect any interactions but very strong ones. Three other markers in the renin gene were not associated with nephropathy. There is only one report about polymorphic markers in the renin gene in diabetic patients with nephropathy and their controls [39]. This report did not show an association of renin gene polymorphisms with diabetic nephropathy. However, the report was based on a small group (40 patients) and diabetes duration was shorter (11 years), so that future nephropathic patients may have been present among control subjects.
The polymorphic marker that was first studied in the present study group, the ACE gene I/D polymorphism, was not in HardyWeinberg equilibrium in the control group [23], which might cast doubt on any conclusion on the genetics of DN in this study group. However, polymorphic markers in the renin, angiotensinogen [10], and AT1 receptor [13] genes were in HardyWeinberg equilibrium in the control group, which makes selection bias of controls unlikely. The absence of HardyWeinberg equilibrium in the ACE gene may be caused by misclassification of alleles [40], or may be incidental, the ACE-gene not being linked to the other RAS genes.
Our control group turned out to have better metabolic control. Individuals with the bb-genotype of the renin gene may have been protected from DN if this better metabolic control has been long-term. Whether this may have weakened the association between DN and a renin gene polymorphic marker, we do not know. In subgroup analyses according to glycated hemoglobin levels either matching was lost or groups became very small.
Our study group is unique in that now the renin, angiotensinogen, ACE, and AT1R genes have been studied for association with DN. We can conclude that in this group of all RAS-components [10,13,23] plasma renin and prorenin and possibly the renin gene BglI polymorphism are associated with DN. Plasma ACE has also been found to be increased in DN in this study group [23], but the ACE gene I/D polymorphism was not associated with DN in these patients. Recent meta-analyses, however, showed that the ACE gene D-allele may confer an increased risk for DN (excess risk up to 56%, depending on ethnic background and reliability of genotyping method) [1517].
Since the effect of the ACE I/D polymorphism and, in our group, the renin BglI polymorphism is small and since there is no major interaction between the RAS-gene variants we must assume that the main part of the genetically determined susceptibility to DN lies outside the genes that code for RAS components. Still, in view of our results on plasma renin and prorenin measurements and in view of the favourable reaction to ACE inhibitors, a pathogenetic mechanism of DN in which the RAS, in particular renin and prorenin, is involved, remains likely.
In conclusion, a direct, validated assay showed that both prorenin and renin are increased in plasma of IDDM patients with diabetic nephropathy. In the present patient group a renin gene variant is the sole genetic factor within the RAS that may contribute, albeit weakly, to the genetic susceptibility to diabetic nephropathy. Renin and prorenin could well play a direct pathogenetic role in DN.
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References |
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