1 Unit of Endocrinology and 2 Unit for Atherosclerosis and Thrombosis, Scientific Institute Casa Sollievo della Sofferenza San Giovanni Rotondo (FG), Italy, 3 Unit for Metabolic Medicine, KCL School of Medicine, Guy's Hospital, London, UK, 4 Diabetes Unit, Hospital Brotzu, Cagliari, Italy, 5 Chair of Metabolic Medicine, Padua, Italy, 6 Institute of Internal Medicine, Turin, Italy, 7 Institute of Internal Medicine, Endocrine and Metabolic Disease, University of Catania, Garibaldi Hospital, Catania, Italy and 8 Department of Clinical Science, University "La Sapienza", Rome, Italy
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Abstract |
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Methods. Type 1 diabetic patients either with (n=159) or without (n=122) nephropathy were evaluated in a cross-sectional study. DN was defined as the presence of microalbuminuria or persistent proteinuria in a subject with more than 10-year duration of disease and concomitant diabetic retinopathy, and with no evidence of heart failure or other renal disease. Seventy-five (47 male/28 female) type 1 diabetic patients with nephropathy in whom retrospective information with repeated measurements of serum creatinine was available, were analysed in a longitudinal study.
Results. No association of the PC-1 Q121 variant and the ACE D/D genotype with DN development was observed. However, the ACE DD genotype and the PC-1 Q121 variant were associated, both independently (P=0.02 and P=0.025, respectively) or in combination (P=0.02), with a faster rate of glomerular filtration rate decline. An interaction (P=0.03) was observed between the two genes in increasing the individual patient's risk of being a fast progressor.
Conclusion. Our data suggest that, in type 1 diabetic patients, the ACE and the PC-1 genes interact in increasing the individual risk of having a faster DN progression.
Keywords: albuminuria; end-stage renal failure; genegene interaction; gene polymorphism; type 1 diabetes
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Introduction |
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Insulin resistance characterizes type 1 diabetic patients who develop albuminuria as well as their non-diabetic first degree relatives [3] and underlies many of the alterations of DN, including high blood pressure, lipid abnormalities, increased left ventricular mass, and a family history of hypertension and cardiovascular disease. These observations suggest that insulin resistance may precede and play a role in DN. As for DN, insulin resistance too has genetic determinants [4,5]. One could speculate, therefore, that insulin resistance and DN may share common genetic determinants. We have shown recently that the Q121 variant of PC-1, a gene which encodes for a membrane glycoprotein known to inhibit insulin signalling, is associated with insulin resistance in the general population [69] and with a faster progression of DN in type 1 diabetic patients [10].
The DD genotype of the ACE gene is a marker of higher ACE activity and mortality for coronary heart disease among diabetics [11]. In addition, the ACE DD genotype associates with insulin resistance in hypertensive but not in normotensive subjects [12]. Most patients with DN are hypertensive and the DD genotype has been found to be associated with faster DN progression both in type 1 [13] and in type 2 diabetes [14].
Whether the PC-1 and ACE genes act in combination with respect to DN development and/or progression is unknown. In this study we have examined the combined effect of the PC-1 Q121 variant and ACE DD genotype on the risk of DN development and progression in type 1 diabetic patients.
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Subjects and methods |
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Patients with nephropathy
DN was defined as the presence of microalbuminuria or persistent proteinuria in a subject with more than 10 years duration of disease and concomitant diabetic retinopathy (background or proliferative), but no evidence of heart failure or other renal disease. Microalbuminuria was diagnosed if an AER was greater than 30 but lower than 300 mg/24 h or an albumin/creatinine ratio (ACR) was greater than 2.5 in males and 3.5 mg/mmol in females but lower than 30 mg/mmol on at least two consecutive occasions. Persistent proteinuria was diagnosed if an AER was greater than 300 mg/24 h or an ACR greater than 30 mg/mmol or a urine sample dipstick positive for protein (1+ or more) on at least two consecutive occasions. 159 patients satisfied the inclusion criteria and were genotyped for both ACE and PC-1 genes.
Patients without nephropathy
Absence of nephropathy was defined as an AER persistently (on three or more 24 h collections) within the normal range of less than 30 mg/24 h or ACR lower than 2.5 in males and 3.5 mg/mmol in females in a subject with more than 15 years of diabetes. Epidemiological studies suggest that the risk of developing nephropathy in this group of patients is low. One hundred and twenty-two patients satisfied the inclusion criteria and were genotyped for both ACE and PC-1 genes.
The clinical characteristics of the two groups of diabetic patients studied are shown in Table 1. In the DN group, 59 patients had microalbuminuria. No significant difference was seen between DC and DN patients in terms of age and duration of diabetes. As expected, there was a greater representation of males in the DN group, even though this difference did not reach statistical significance. DN patients had higher values of systolic and diastolic blood pressure, glycated haemoglobin, serum creatinine, total cholesterol, and triglycerides (P<0.001 for all). Sixty per cent of DC patients had diabetic retinopathy. There were no significant clinical and biochemical differences between Italian and British patients (data not shown).
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The clinical features of the 75 type 1 diabetic patients with DN studied longitudinally are given in Table 2. There were no significant clinical and biochemical differences between Italian and British patients (data not shown). At the time of DNA sampling patients had a mean age of 42 years and a mean duration of diabetes of 26 years. All patients were receiving antihypertensive therapy of whom 95% were taking ACE inhibitors. As a result of antihypertensive therapy, the AER had fallen below 300 mg/24 h in 20% patients. Median serum creatinine was 97 mmol/l ranging from 55 to 504 mmol/l.
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All subjects gave their informed consent to the study, which was performed according to the Declaration of Helsinki guidelines and with the approval of the local ethic committees.
Genotyping
Determination of PC-1 and ACE gene polymorphisms was performed in a central Laboratory at Scientific Institute Casa Sollievo della Sofferenza San Giovanni Rotondo, Italy, as described previously [6,15].
Statistical analysis
All data are reported as mean±SD, mean (range), or median (range). Mean differences were compared by unpaired Student's t or MannWhitney U tests, as appropriate, for two groups and by one-way ANOVA for more than two groups.
Two-way ANOVA on all measurements in each patient (n=773, with an average of 10.7±1.0 observations/patient) of delta GFR (GFR decline over the time) was used to compare different groups, taking into consideration different length of follow-up and different number of assessment time points in different individuals. Individual delta GFR values, measured at each time point (on average every 8 months), were used to calculate the individual rate of DN progression. Patients enrolled into the longitudinal study were defined as fast or slow progressors according to their individual rate of GFR decline (i.e. above or below the median value of the rate of GFR decline of the whole cohort, respectively). A 2 test was used to compare the distribution of ACE and PC-1 genotypes and the percentages of patients being fast progressors across different genotype combinations.
Univariate and multiple variate analyses were used to correlate independent variables of progression with the dependent variable, rate of GFR decline. For this analysis, AER values were logarithmically transformed. Interaction between genes was tested with GLM Univariate analysis, statistical packaged SPSS version 10 (SPSS Inc., Chicago, IL, USA).
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Results |
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The distribution of the PC-1 genotypes was not different between DC and DN patients and this was also the case for the ACE genotypes, as reported previously [15]. Also, the distribution of concomitant ACE DD genotype and PC-1 Q121 variant was similar in both cases and controls (Table 3).
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Longitudinal study
In the whole cohort, the median rate of GFR decline was 4.2 ml/min/year (range -3.816.6) and correlated with AER (P=0.001) and MAP (P=0.046) but not HbA1c, measured at genotyping. As shown previously in a series comprising most of the patients presented here [10], PC-1 gene Q121 variant carriers (n=21, 19 KQ, two QQ) had a faster GFR decline than K121K (n=54) patients [6.6 (range 0.1616.6) vs 2.6 ml/min/year (range -3.816.0), P<0.001]. In multivariate analysis, the PC-1 genotype was correlated with GFR decline independently (P=0.025) of AER and MAP.
A progressive increase in the rate of GFR decline was also observed in relation to the ACE genotype in the 75 nephropathic patients: II+ID (n=45, 3.5 ml/min/year (range 0.816)) and DD (n=30, 6.9 ml/min/year (range -3.816.6), P=0.03). The univariate relation between the ACE I/D polymorphism and GFR decline (P=0.02) was no longer significant (P=0.1) after adjusting for both AER and MAP.
To investigate the combined effect of the ACE and PC-1 genes on the rate of DN progression, we divided the whole cohort of 75 patients into four groups, according to the absence/presence of ACE DD genotype and/or PC-1 Q121 variant: group 1 (n=33) patients with ACE II/ID and PC-1 K121K genotypes; group 2 (n=12) patients with PC-1 Q121 variant and ACE II/ID genotypes; group 3 (n=21) ACE DD and PC-1 KK genotype; and, finally, group 4 (n=9) patients with PC-1 Q121 variant and ACE DD genotype. The rate of loss of kidney function increased progressively and significantly from groups 1 to 4 (Figure 1a, P<0.001).
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The effect of the ACE DD genotype and PC-1 Q121 variant on the median value of the rate of GFR decline for each of the four groups seems to be additive. The sum of the net effect of the PC-1 Q121 variant on increased GFR decline (group 2 minus group 1, GFR fall 1.8 ml/min/year) with that of the ACE DD genotype (group 3 minus group 1, GFR fall 3.7 ml/min/year) gives an expected net effect on GFR fall of 5.5 ml/min/year, which is very similar to that actually observed in patients carrying both the Q121 variant and the DD genotype (i.e. group 4). In fact in these patients the net effect of the combined genotypes on the GFR fall in respect to patients carrying the KK/II+ID genotypes (i.e. group 1) is 5.9 ml/min/year (7.3 ml/min/year of group 4 minus 2.4 ml/min/year of group 1). However, when the individual patient's risk of being a fast progressor was taken into account (i.e. rate of GFR decline above 4.2 ml/min/year, the median value of the whole cohort) a clear interaction (P=0.03) between the two genes was observed. The simultaneous presence of both Q allele of PC-1 and DD genotype of the ACE gene (i.e. group 4) was, in fact, needed to significantly increase (P<0.001) the individual risk of being a fast progressor when compared with KK PC-1 and II+ID ACE genotypes (i.e. group 1) (Figure 1b). In contrast, no difference was observed between groups 1, 2, and 3. The genegene interaction was independent of known environmental promoters including AER and blood pressure.
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Discussion |
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The mechanisms through which these two genes affect the progression of diabetic kidney disease are unknown. Patients carrying the ACE DD genotype and PC-1 Q121 variant may share unfavourable metabolic and haemodynamic backgrounds due to the association of these genes with insulin resistance [6,12] and higher plasma ACE levels [12]. Because all patients were on antihypertensive treatment, mostly ACE inhibitors, which affect the rate of progression of DN, it cannot be excluded that our finding is the result of a reduced sensitivity to antihypertensive and/or ACE inhibitor therapy in patients carrying the ACE DD genotype and the PC-1 Q121 variant.
We are aware of the shortcomings of retrospective studies in investigating the rate of disease progression. However, data collection was carefully executed in this cohort of patients and to date no prospective study of the genetic determinants of DN progression is available. We believe, therefore, that properly conducted longitudinal retrospective studies, among which the present is one of the largest, can still provide useful information.
In conclusion, our data suggest that ACE and PC-1 genes interact in modulating progression of DN and the individual risk of being a fast progressor in type 1 diabetic patients. Larger, prospective studies are needed to confirm this preliminary observation.
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Acknowledgments |
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Notes |
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References |
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