Inflammation is a suspected cause of kidney damage; and we were interested in testing associations of genes involved in the inflammatory response with urinary albumin excretion. We describe here the results of this work carried out with adults who participated in NHANES III, a population-based sample survey that is nationally representative of the U.S. population. We restricted analyses to albuminuria outcomes instead of chronic kidney disease, since the genes that influence renal function may differ from those contributing to renal damage and proteinuria [51
]. Many of the included genes are involved in other cellular or physiologic pathways, such as lipid metabolism (e.g., APOE
), hemostasis (e.g., F2
), and oxidative stress/nitric oxide production (e.g., PON1
It is unknown whether polymorphisms in inflammation genes affect the normal variation in urinary albumin excretion in humans, or whether such genetic variation plays a role only in the susceptibility to renal damage once some sort of ischemic or toxic insult is acquired. Our results only minimally support a role for such polymorphisms in the establishment of normal urinary albumin excretion, since all but one individual genotype and few haplotypes were significantly associated with continuous log(ACR) after adjustment for multiple testing. We do show that increasing minor allele copy number of polymorphisms in TNF in non-Hispanic whites (crude and age-adjusted models) and IL1B in Mexican Americans (both multivariable models) were associated with albuminuria as defined by sex-specific criteria. However, TNF rs1800750 failed the test for Hardy-Weinberg proportions in non-Hispanic whites, so this result should be interpreted cautiously. No polymorphisms were associated with sex-specific albuminuria in codominant models in any race/ethnic group. For comparison, we also analyzed data for albuminuria defined by a single threshold for both men and women. These analyses yielded results similar to those for sex-specific albuminuria, though among Mexican Americans, genetic variants in CRP and NOS3 were also significantly associated with ACR ≥ 30 mg/g under additive models. This CRP variant (rs1800947) was also the only polymorphism significantly associated with single-threshold albuminuria in codominant models after correcting for multiple testing (FDR-P < 0.0001). The significance of this variant only after full multivariable adjustment may be due to instability of the regression model because of the low allele frequency of rs1800947 in Mexican Americans. CRP haplotypes were not associated with single threshold albuminuria in Mexican Americans, though rare CRP haplotypes as a group were marginally associated (uncorrected P value < 0.05) with sex-specific ACR. The C-T haplotype of NOS3 was found to be significantly associated with both definitions of albuminuria in Mexican Americans, strongly implicating this gene in the susceptibility to kidney damage. We found no evidence of an effect of other haplotypes on the odds of albuminuria after adjusting for multiple testing.
We calculated the power of our study to detect associations of individual polymorphisms with log(ACR) and sex-specific albuminuria within each race/ethnic group, using a two-sided α set at 0.05 and assuming a design effect of 1.2. For log(ACR), we calculated power to detect effect sizes consistent with an R2
of 0.5%. In non-Hispanic whites, we have 86% power to detect changes in the log(ACR) beta coefficient of 0.41 and 0.25 at minor allele frequencies (MAFs) of 10% and 40%, respectively. In non-Hispanic blacks, our power is 70% to detect changes in the beta of 0.33 and 0.20 at the same MAFs. For Mexican-Americans, changes in the beta coefficient of 0.45 (at 10% MAF) and 0.28 (at 40% MAF) could be detected with 72% power. For sex-specific albuminuria, we calculated the study power for an odds ratio of 1.3, which is indicative of effects found both in candidate gene studies [54
] and in genome-wide association studies [55
]. At MAFs of 10% and 40%, the power to detect an OR = 1.3 in non-Hispanic whites is 32% and 64%, respectively. In non-Hispanic blacks, power is 18% and 36%, respectively, at these MAFs. For the Mexican American population, study power to detect an OR = 1.3 is 24% and 49% at MAFs of 10% and 40%, respectively.
Few published studies have assessed the association of inflammation-related genes with albuminuria. No association with albuminuria was found for the TGFB1
Pro10Leu (rs1800470) variant in non-diabetic Chinese [56
]. Neither -509C > T (rs1800469) nor rs1800470 was found associated with urine albumin levels or albuminuria in hypertensive Italian men [57
], though the TC and CC genotypes of the Pro10Leu variant was found associated with albuminuria and urine albumin excretion in a separate study of Italian hypertensive patients [58
-786T > C (rs2070744), but not Asp298Glu (rs1799983), was positively associated with increased ACR and higher risk of albuminuria in diabetic and nondiabetic European American family members [59
]. Neither variant was associated with urine albumin excretion or albuminuria in a smaller study of hypertensive white men [60
]; nor was the Asp298Glu variant associated with urinary albumin excretion in healthy Venezuelans [61
]. Neither NOS3
variant was associated with ACR in a family-based study of Mexican Americans [62
], a finding not completely supported by our study. The AA genotype of PON1
variant rs662 (Gln192Arg) was recently found associated with albuminuria in a large study of Japanese, but only among women [63
]. Genetic variation in the CCL5
gene has been associated with ACR in a non-diabetic Japanese population, but the -95C > G variant (rs2280788) included in our analyses was not studied [64
]. The Pro12Ala variant (rs1801282) of PPARG
has been associated with urinary albumin excretion in a type 2 diabetic population [65
]. The well studied APOE
variants rs429358 and rs7412, from which the APOE
ε2, ε3, and ε4 alleles (isoforms) are composed, were weakly associated with glomerular filtration rate (a measure of kidney function) in non-Hispanic whites and non-Hispanic blacks in NHANES III, but kidney damage was not assessed separately [39
]. In one study that assessed progression of chronic kidney disease, APOE
genotypes were not significantly associated with macroalbuminuria (ACR ≥ 300 μg/mg) [66
]. Studies of MBL2
variants have been limited primarily to type 1 diabetic populations, in which there are conflicting data on the role of MBL2
polymorphisms in diabetic nephropathy [67
]. To our knowledge, there have been no published genetic association studies of urinary albumin excretion or albuminuria that included variants in many of the genes in which we found polymorphisms or haplotypes significantly associated with any outcome in at least one race/ethnic group (e.g., CRP, IL1B, TNF
, or VDR
Studies such as ours are complicated by the fact that the assessment of albuminuria in any population is not straightforward. Though the gold standard for the quantitative evaluation of proteinuria is a 24-hr urine collection, this method is inconvenient and is prone to errors [28
]. For the screening of adults for proteinuria, various organizations recommend measurement of an untimed (i.e., spot) urine sample using a ratio of albumin to creatinine [69
]. This use of a ratio corrects for variability due to hydration, diuretics, osmotic diuresis, and concentrating defects [28
] and has been shown to correlate well with 24-hr urine albumin excretion measurements [28
]. Most often, albuminuria is defined as urinary ACR ≥ 30 mg/g for both men and women. Sex-specific criteria have been advocated in order to account for greater creatinine excretion in men than in women [30
], though there are no explicit recommendations for use of such criteria [28
]. Importantly, given the strong predictive value of even low-grade albuminuria for cardiovascular disease, it has been suggested that albuminuria be interpreted as a continuum rather than as threshold cut-off values, as dichotomizing albuminuria results in the loss of important clinical information [5
]. Therefore, we analyzed urinary ACR as both a continuous variable and as dichotomous variables. We did not adjust for hypertension or diabetes in our analyses as these conditions are major risk factors for chronic kidney disease. The genetic associations examined here may have been distorted if the included polymorphisms act, at least partially, via these causal pathways to affect urine albumin excretion.
Statistical analyses were performed for individual polymorphisms under two modes of inheritance. Additive models detect both additive and dominant genetic loci effectively, but perform poorly for recessive alleles [77
]. Codominant models have the best performance when the true inheritance pattern is unknown [77
]. However, codominant models seemed to be less robust in situations of low MAF, such that our estimates may be less stable and our power to identify a recessive effect poor. Furthermore, odds ratios could not be estimated for some codominant models when the MAF was very low.
Our study has several limitations, some of which are specific to analyses in NHANES. First, we included one spot urine sample for the measurement of urinary ACR, as repeat tests were performed only in a small subset of NHANES III participants [1
]. Therefore, we could not differentiate between participants with persistent albuminuria and those with transiently elevated urinary ACR. The use of the single albuminuria measurement could misclassify cases and controls and bias our results since agreement between initial and repeat urine tests was not perfect [1
]. Secondly, because NHANES III is a nationally representative study, there were few participants with severe kidney damage. In fact, only a very small proportion (N = 128; 1.03 ± 0.18%) of persons in our study population displayed clinical proteinuria (ACR ≥ 250 mg/g in men or ≥ 355 mg/g in women). This may have hampered our ability to detect genetic associations with log(ACR) or albuminuria. Also, we did not perform separate analyses for diabetics or hypertensives as the resulting sample size within each race/ethnic group would have been limiting. However, there is some evidence that similar genes may contribute to ACR in both nondiabetic and diabetic individuals [80
]. Furthermore, we were unable to assess population stratification in our analyses, as data from ancestry-informative markers are not yet available for NHANES III. The large sample size of NHANES III did allow for separate analyses within each of the three main race/ethnic groups, self-reported designations that have been shown to correspond with ancestry as derived from ancestry-informative markers for these populations [81
]. These race/ethnic group-stratified analyses enabled us to account at least partially for differences in albuminuria profile, allele frequencies, and linkage disequilibrium patterns between race/ethnicities. We cannot, however, discount that there may be residual population substructure within the race/ethnic groups that may have affected our results. Therefore, caution should be exercised in interpreting our findings, especially in non-Hispanic blacks and Mexican Americans, since these populations have experienced recent genetic admixture. In addition, the lack of "replication" of marginally associated (uncorrected P-value < 0.05) or significantly associated variants across race/ethnic groups may be due to differences in the underlying linkage disequilibrium patterns of the populations, due to untested interactions, or due to lack of power. Indeed, for categorical outcomes, our power to detect a typical effect size (OR = 1.3) in each race/ethnic group was modest. The combined analyses that included all participants and adjusted for race/ethnicity did not affect our overall findings, as the statistical weighting of the race/ethnic groups produced results not materially different than those found in non-Hispanic whites alone.
A couple of limitations are not specific to NHANES. Defining albuminuria with sex-specific criteria does not alleviate all concerns regarding the measurement of urine albumin excretion. It has been proposed that additional urinary ACR thresholds that account for differences by age [86
] and race/ethnicity [30
] may be needed to avoid misclassification of certain demographic groups, though no specific guidelines have been developed. In addition, the use of FDR-adjusted P values to determine statistical significance may be overly conservative, as the assumption of independent tests is not met given that some polymorphisms are correlated owing to linkage disequilibrium.
The NHANES data are a unique resource for epidemiologic analyses in a large, population-based and nationally representative sample of the U.S. population. Our findings are generalizeable to the larger U.S. population and point to a few genes that may play a role in the susceptibility to albuminuria. Ideally, we could have included other variants known to be related to the progression and severity of renal disease, such as those within inflammatory cytokine IL-6
and polymorphisms identified in a recent genome-wide association study as significantly associated with urine albumin excretion [36
]. Such variants, however, have not yet been genotyped in NHANES III. NHANES should be used for additional research into the associations between genetic variants and renal disease and to evaluate the importance of gene-environment interactions.