Vallabh O. Shah

Genome-wide scans for diabetic nephropathy and albuminuria in multiethnic populations: The Family Investigation of Nephropathy and Diabetes (FIND)

The Family Investigation of Nephropathy and Diabetes (FIND) was initiated to map genes underlying susceptibility to diabetic nephropathy. A total of 11 centers participated under a single collection protocol to recruit large numbers of diabetic sibling pairs concordant and discordant for diabetic nephropathy. We report the findings from the first-phase genetic analyses in 1,227 participants from 378 pedigrees of European-American, African-American, Mexican-American, and American Indian descent recruited from eight centers. Model-free linkage analyses, using a dichotomous definition for diabetic nephropathy in 397 sibling pairs, as well as the quantitative trait urinary albumin-to-creatinine ratio (ACR), were performed using the Haseman-Elston linkage test on 404 microsatellite markers. The strongest evidence of linkage to the diabetic nephropathy trait was on chromosomes 7q21.3, 10p15.3, 14q23.1, and 18q22.3. In ACR (883 diabetic sibling pairs), the strongest linkage signals were on chromosomes 2q14.1, 7q21.1, and 15q26.3. These results confirm regions of linkage to diabetic nephropathy on chromosomes 7q, 10p, and 18q from prior reports, making it important that genes underlying these peaks be evaluated for their contribution to nephropathy susceptibility. Large family collections consisting of multiple members with diabetes and advanced nephropathy are likely to accelerate the identification of genes causing diabetic nephropathy, a life-threatening complication of diabetes.

Association between Albuminuria, Kidney Function, and Inflammatory Biomarker Profile in CKD in CRIC

BACKGROUND AND OBJECTIVES Increased risk of mortality in patients with CKD has been attributed to inflammation. However, the association between kidney function, albuminuria, and biomarkers of inflammation has not been examined in a large cohort of CKD patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS This study measured the plasma levels of IL-1β, IL-1 receptor antagonist (IL-1RA), IL-6, TNF-α, TGF-β, high-sensitivity C-reactive protein (hs-CRP), fibrinogen, and serum albumin in 3939 participants enrolled in the Chronic Renal Insufficiency Cohort study between June 2003 and September 2008. An inflammation score was established based on plasma levels of IL-1β, IL-6, TNF-α, hs-CRP, and fibrinogen. Estimated GFR (eGFR) and serum cystatin C were used as measures of kidney function. Albuminuria was quantitated by urine albumin to creatinine ratio (UACR). RESULTS Plasma levels of IL-1β, IL-1RA, IL-6, TNF-α, hs-CRP, and fibrinogen were higher among participants with lower levels of eGFR. Inflammation score was higher among those with lower eGFR and higher UACR. In regression analysis adjusted for multiple covariates, eGFR, cystatin C, and UACR were strongly associated with fibrinogen, serum albumin, IL-6, and TNF-α. Each unit increase in eGFR, cystatin C, and UACR was associated with a -1.2% (95% confidence interval, -1.4, -1), 64.9% (56.8, 73.3) and 0.6% (0.4, 0.8) change in IL-6, respectively (P<0.001). CONCLUSIONS Biomarkers of inflammation were inversely associated with measures of kidney function and positively with albuminuria.

Genome-Wide Scan for Estimated Glomerular Filtration Rate in Multi-Ethnic Diabetic Populations The Family Investigation of Nephropathy and Diabetes (FIND)

OBJECTIVE— Diabetic nephropathy, the most common cause of end-stage renal disease, aggregates in families and specific ethnic groups. Deconstructing diabetic nephropathy into intermediate, quantitative phenotypes may increase feasibility of detecting susceptibility loci by genetic screens. Glomerular filtration rate (GFR), which characterizes diabetic nephropathy, was employed as a quantitative trait in a preliminary whole-genome scan. RESEARCH DESIGN AND METHODS— Estimated GFR (eGFR) was calculated for 882 diabetic sibpairs (mean age 57 years) of African-American (25.6% of total), American Indian (8.6%), European-American (14.2%), and Mexican-American (51.6%) descent enrolled in the initial phase of the Family Investigation of Nephropathy and Diabetes (FIND). A whole-genome scan was performed using 404 microsatellite markers (average spacing 9 cM) and model-free linkage analysis. RESULTS— For all ethnicities combined, strong evidence for linkage was observed on chromosomes 1q43 (P = 3.6 × 10−3), 7q36.1 (P = 2.1 × 10−4), 8q13.3 (P = 4.6 × 10−4), and 18q23.3 (P = 2.7 × 10−3). Mexican-American families, who comprised the major ethnic subpopulation in FIND, contributed to linkage on chromosomes 1q43, 2p13.3, 7q36.1, 8q13.3, and 18q23.3, whereas African-American and American-Indian families displayed linkage peaks on chromosomes 11p15.1 and 15q22.3, respectively. CONCLUSIONS— We have demonstrated multiple chromosomal regions linked to eGFR in a multi-ethnic collection of families ascertained by a proband with diabetic nephropathy. Identification of genetic variants within these loci that are responsible for the linkage signals could lead to predictive tests or novel therapies for subsets of patients at risk for diabetic nephropathy.

Soluble CD14 Levels, Interleukin-6, and Mortality Among Prevalent Hemodialysis Patients

BACKGROUND CD14 is a pattern-recognition receptor that has a central immunomodulatory role in proinflammatory signaling in response to a variety of ligands, including endotoxin. CD14 protein is present in 2 forms: soluble (sCD14) and membrane bound. Here, we studied the implications of increased sCD14 levels in hemodialysis patients. We hypothesized that sCD14 level increase may link to cytokine activation and protein-energy wasting, predisposing to increased mortality risk. STUDY DESIGN Prospective observational study of prevalent hemodialysis patients. SETTING & PARTICIPANTS 211 prevalent hemodialysis patients, median age of 65 years, with 29 months of vintage dialysis time followed up for mortality for a median of 31 months. PREDICTORS Tertiles of baseline circulating sCD14 levels corresponding to less than 2.84, 2.85 to 3.62, and greater than 3.63 microg/mL. OUTCOME The major outcome of interest was all-cause mortality. MEASUREMENTS sCD14 and endotoxin, together with other markers of inflammation and protein-energy wasting. RESULTS Median sCD14 level was 3.2 microg/mL (25th to 75th percentile, 2.7 to 3.9). sCD14 level correlated positively with C-reactive protein, interleukin 6, endotoxin, and pentraxin 3 levels and negatively with serum albumin level, muscle mass, and handgrip strength. Patients with increased sCD14 levels had lower body mass index and increased prevalence of muscle atrophy. Patients within the highest sCD14 tertile had a crude morality hazard ratio of 1.94 (95% confidence interval, 1.13 to 3.32) that persisted after adjustment for multiple confounders (hazard ratio, 3.11; 95% confidence interval, 1.49 to 6.46). In patients with persistent inflammation, the presence of a concurrent sCD14 level increase gradually increased mortality risk, but this effect was less than multiplicative and failed to show a statistical interaction. LIMITATIONS Those inherent to an observational study. CONCLUSIONS sCD14 level is associated with inflammation and protein-energy wasting in hemodialysis patients. It is a strong and independent predictor of mortality that warrants further assessment in the clinical setting regarding its usefulness as a complementary prognosticator to other general inflammatory markers.

Genome-Wide Scan for Estimated GFR in Multi-Ethnic Diabetic Populations: The Family Investigation of Nephropathy and Diabetes

OBJECTIVE— Diabetic nephropathy, the most common cause of end-stage renal disease, aggregates in families and specific ethnic groups. Deconstructing diabetic nephropathy into intermediate, quantitative phenotypes may increase feasibility of detecting susceptibility loci by genetic screens. Glomerular filtration rate (GFR), which characterizes diabetic nephropathy, was employed as a quantitative trait in a preliminary whole-genome scan. RESEARCH DESIGN AND METHODS— Estimated GFR (eGFR) was calculated for 882 diabetic sibpairs (mean age 57 years) of African-American (25.6% of total), American Indian (8.6%), European-American (14.2%), and Mexican-American (51.6%) descent enrolled in the initial phase of the Family Investigation of Nephropathy and Diabetes (FIND). A whole-genome scan was performed using 404 microsatellite markers (average spacing 9 cM) and model-free linkage analysis. RESULTS— For all ethnicities combined, strong evidence for linkage was observed on chromosomes 1q43 (P = 3.6 × 10−3), 7q36.1 (P = 2.1 × 10−4), 8q13.3 (P = 4.6 × 10−4), and 18q23.3 (P = 2.7 × 10−3). Mexican-American families, who comprised the major ethnic subpopulation in FIND, contributed to linkage on chromosomes 1q43, 2p13.3, 7q36.1, 8q13.3, and 18q23.3, whereas African-American and American-Indian families displayed linkage peaks on chromosomes 11p15.1 and 15q22.3, respectively. CONCLUSIONS— We have demonstrated multiple chromosomal regions linked to eGFR in a multi-ethnic collection of families ascertained by a proband with diabetic nephropathy. Identification of genetic variants within these loci that are responsible for the linkage signals could lead to predictive tests or novel therapies for subsets of patients at risk for diabetic nephropathy.

Plasma metabolomic profiles in different stages of CKD.

BACKGROUND AND OBJECTIVES CKD is a common public health problem. Identifying biomarkers adds prognostic/diagnostic value by contributing to an understanding of CKD at the molecular level and possibly defining new drug targets. Metabolomics provides a snapshot of biochemical events at a particular time in the progression of CKD. This cross-sectional metabolomics study ascertained whether plasma metabolite profiles are significantly different in CKD stages 2, 3, and 4. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS An analysis of plasma metabolites, using gas and liquid chromatography coupled to mass spectrometry, was conducted on 30 nondiabetic men ages 40-52 years, with 10 participants each in CKD stages 2, 3, and 4 based on their estimated GFR (calculated by the Modified Diet in Renal Disease formula). Participants were recruited in late 2008, and plasma samples were tested at Metabolon Inc and analyzed in 2012. RESULTS Comparison of stage 3/stage 2 identified 62 metabolites that differed (P ≤ 0.05), with 39 higher and 23 lower in stage 3 compared with stage 2; comparisons of stage 4/stage 2 identified 111 metabolites, with 66 higher and 45 lower; and comparisons of stage 4/stage 3 identified 11 metabolites, with 7 higher and 4 lower. Major differences in metabolite profiles with increasing stage of CKD were observed, including altered arginine metabolism, elevated coagulation/inflammation, impaired carboxylate anion transport, and decreased adrenal steroid hormone production. CONCLUSIONS Global metabolite profiling of plasma uncovered potential biomarkers of stages of CKD. Moreover, these biomarkers provide insight into possible pathophysiologic processes that may contribute to progression of CKD.

Association of Soluble Endotoxin Receptor CD14 and Mortality Among Patients Undergoing Hemodialysis

BACKGROUND CD14 is a key molecule in innate immunity that mediates cell activation and signaling in response to endotoxin and other bacterial wall-derived components. CD14 protein exists in soluble (sCD14) and membrane-bound forms. The correlates of sCD14 in persons undergoing long-term hemodialysis (HD) therapy are not known. We hypothesized that increased sCD14 levels in HD patients are associated with proinflammatory cytokine activation and increased mortality. STUDY DESIGN Cohort study. SETTING & PARTICIPANTS 310 long-term HD patients who participated in the Nutritional and Inflammatory Evaluation in Dialysis (NIED) Study, a cohort derived from a pool of more than 3,000 HD outpatients during 5 years in 8 DaVita maintenance dialysis facilities in the South Bay Los Angeles, CA, area. PREDICTORS sCD14 levels in serum. OUTCOMES 33-month mortality. RESULTS Mean sCD14 level was 7.24 +/- 2.45 microg/mL. Tumor necrosis factor alpha level was the strongest correlate of sCD14 level (r = +0.24; P < 0.001), followed by interleukin 6 level (r = +0.18; P = 0.002), serum ferritin level (r = +0.21; P < 0.001), total iron-binding capacity (r = -0.19; P < 0.001), body mass index (r = -0.15; P = 0.008), vintage (r = +0.14; P = 0.01), low-density lipoprotein cholesterol level (r = +0.13; P = 0.03), and body fat (r = -0.11; P = 0.06). During the 33-month follow-up, 71 (23%) patients died. Multivariable Cox proportional analysis adjusted for case-mix and other nutritional and inflammatory confounders, including serum tumor necrosis factor alpha, C-reactive protein, and interleukin 6 levels, showed that compared with the lowest sCD14 tertile, sCD14 levels in the third tertile (>7.8 microg/mL) were associated with greater death risk (hazard ratio, 1.94; 95% confidence interval, 1.01 to 3.75; P = 0.04). LIMITATIONS Survivor bias in combined incident/prevalent studies. CONCLUSIONS Increased sCD14 level is related positively to markers of inflammation and negatively to nutritional status and is an independent predictor of mortality in long-term HD patients. Additional studies are needed to examine the usefulness of sCD14 level in risk stratification and the clinical decision-making process in HD patients.

Interleukin-6 modulates hepatic and muscle protein synthesis during hemodialysis

Increased demand for amino acids to sustain acute-phase protein synthesis could be the stimulus for the increased muscle protein catabolism during hemodialysis (HD). This could be attenuated by intradialytic amino-acid infusion. To test this, we measured the fractional synthesis rates of albumin, fibrinogen, and muscle protein in eight patients with end-stage renal disease at baseline before dialysis and during HD without or with amino-acid infusion. The percentage change in the fractional synthesis rates of albumin, fibrinogen, and muscle protein from baseline was significantly higher during HD with amino-acid infusion than without amino-acid infusion. Leg muscle proteolysis was significantly increased during unsupplemented HD compared with baseline, but this was not decreased by amino-acid infusion. Arteriovenous balance studies across the leg showed a net efflux of interleukin-6 (IL-6) from the muscle into the vein during HD. The fractional synthesis rate of albumin, fibrinogen, and muscle protein correlated with each other and with the IL-6 efflux from the leg. Leg muscle protein catabolism was positively related to IL-6 release from the leg and not associated with amino-acid availability. Our results show that intradialytic cytokine activation and not amino-acid depletion is the major protein catabolic signal during HD.

Genome-Wide Association and Trans-ethnic Meta-Analysis for Advanced Diabetic Kidney Disease: Family Investigation of Nephropathy and Diabetes (FIND).

Diabetic kidney disease (DKD) is the most common etiology of chronic kidney disease (CKD) in the industrialized world and accounts for much of the excess mortality in patients with diabetes mellitus. Approximately 45% of U.S. patients with incident end-stage kidney disease (ESKD) have DKD. Independent of glycemic control, DKD aggregates in families and has higher incidence rates in African, Mexican, and American Indian ancestral groups relative to European populations. The Family Investigation of Nephropathy and Diabetes (FIND) performed a genome-wide association study (GWAS) contrasting 6,197 unrelated individuals with advanced DKD with healthy and diabetic individuals lacking nephropathy of European American, African American, Mexican American, or American Indian ancestry. A large-scale replication and trans-ethnic meta-analysis included 7,539 additional European American, African American and American Indian DKD cases and non-nephropathy controls. Within ethnic group meta-analysis of discovery GWAS and replication set results identified genome-wide significant evidence for association between DKD and rs12523822 on chromosome 6q25.2 in American Indians (P = 5.74x10-9). The strongest signal of association in the trans-ethnic meta-analysis was with a SNP in strong linkage disequilibrium with rs12523822 (rs955333; P = 1.31x10-8), with directionally consistent results across ethnic groups. These 6q25.2 SNPs are located between the SCAF8 and CNKSR3 genes, a region with DKD relevant changes in gene expression and an eQTL with IPCEF1, a gene co-translated with CNKSR3. Several other SNPs demonstrated suggestive evidence of association with DKD, within and across populations. These data identify a novel DKD susceptibility locus with consistent directions of effect across diverse ancestral groups and provide insight into the genetic architecture of DKD.

Epidemic of Diabetic and Nondiabetic Renal Disease among the Zuni Indians: The Zuni Kidney Project

There is an epidemic of renal disease among the Zuni Indians. The prevalence of end-stage renal disease among the Zuni Indians is 18.4-fold and 7.4-fold higher than among European Americans and American Indians/Alaskan Natives, respectively. In contrast to other American Indian tribes, nondiabetic renal disease accounts for a significant percent of the renal disease burden among the Zuni Indians. To explore this hypothesis, a community epidemiologic study of the Zuni Pueblo was conducted. A questionnaire was administered, blood and urine samples were collected, and BP, height, and weight were measured. Neighborhood household clusters were used as the sampling frame to maximize ascertainment and minimize bias. Age and gender distributions in the sample (n = 1483) were similar to those of the eligible Zuni population (n = 9228). The prevalence, age-adjusted and gender-adjusted to the Zuni population, of incipient (0.03 < or = UACR < 0.3) albuminuria (IA) (15.0% [95% confidence interval, 13.1 to 16.9%]), and overt (UACR > or = 0.3) albuminuria (OA) (4.7% [3.6 to 5.8%]) was high. The prevalence estimates for IA and OA were higher among diabetic participants (IA: 33.6% [27.6 to 39.7%]; OA: 18.7% [13.7 to 23.7%]) than nondiabetic participants (IA: 10.8% [9.0 to 12.6%]; OA: 1.8% [1.0 to 2.5%]). However, there were more nondiabetic participants; therefore, they comprised 58.0% [51.4 to 64.6%] and 30.9% [20.0 to 41.7%] of participants with IA and OA, respectively. In contrast to most other American Indian tribes, nondiabetic renal disease contributes significantly to the overall burden of renal disease among the Zuni Indians.