Genome-wide Association Studies Identify Genetic Loci Associated with Albuminuria in Diabetes

Elevated concentrations of albumin in the urine, albuminuria, are a hallmark of diabetic kidney disease and associate with increased risk for end-stage renal disease and cardiovascular events. To gain insight into the pathophysiological mechanisms underlying albuminuria, we conducted meta-analyses of genome-wide association studies and independent replication in up to 5,825 individuals of European ancestry with diabetes mellitus and up to 46,061 without diabetes, followed by functional studies. Known associations of variants in CUBN, encoding cubilin, with the urinary albumin-to-creatinine ratio (UACR) were confirmed in the overall sample (p=2.4*10(-10)). Gene-by-diabetes interactions were detected and confirmed for variants in HS6ST1 and near RAB38/CTSC. SNPs at these loci demonstrated a genetic effect on UACR in individuals with but not without diabetes. The change in average UACR per minor allele was 21% for HS6ST1 and 13% for RAB38/CTSC (p=6.3*10(-7) and 5.8*10(-7), respectively). Experiments using streptozotocin-treated diabetic Rab38 knockout and control rats showed higher urinary albumin concentrations and reduced amounts of megalin and cubilin at the proximal tubule cell surface in Rab38 knockout vs. control rats. Relative expression of RAB38 was higher in tubuli of patients with diabetic kidney disease compared to controls. The loci identified here confirm known and highlight novel pathways influencing albuminuria.

Is there an age cutoff to apply adult formulas for GFR estimation in children?

BACKGROUND Estimation of glomerular filtration rate (eGFR) using a common formula for both adult and pediatric populations is challenging. Using inulin clearances (iGFRs), this study aims to investigate the existence of a precise age cutoff beyond which the Modification of Diet in Renal Disease (MDRD), the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI), or the Cockroft-Gault (CG) formulas, can be applied with acceptable precision. Performance of the new Schwartz formula according to age is also evaluated. METHOD We compared 503 iGFRs for 503 children aged between 33 months and 18 years to eGFRs. To define the most precise age cutoff value for each formula, a circular binary segmentation method analyzing the formulas' bias values according to the children's ages was performed. Bias was defined by the difference between iGFRs and eGFRs. To validate the identified cutoff, 30% accuracy was calculated. RESULTS For MDRD, CKD-EPI and CG, the best age cutoff was ≥14.3, ≥14.2 and ≤10.8 years, respectively. The lowest mean bias and highest accuracy were -17.11 and 64.7% for MDRD, 27.4 and 51% for CKD-EPI, and 8.31 and 77.2% for CG. The Schwartz formula showed the best performance below the age of 10.9 years. CONCLUSION For the MDRD and CKD-EPI formulas, the mean bias values decreased with increasing child age and these formulas were more accurate beyond an age cutoff of 14.3 and 14.2 years, respectively. For the CG and Schwartz formulas, the lowest mean bias values and the best accuracies were below an age cutoff of 10.8 and 10.9 years, respectively. Nevertheless, the accuracies of the formulas were still below the National Kidney Foundation Kidney Disease Outcomes Quality Initiative target to be validated in these age groups and, therefore, none of these formulas can be used to estimate GFR in children and adolescent populations.

Risk factors for encephalitis and death from West Nile virus infection: An analysis of hospitalized cases in Houston, Texas from 2002--2008

We conducted a nested case-control study to determine the significant risk factors for developing encephalitis from West Nile virus (WNV) infection. The purpose of this research project was to expand the previously published Houston study of 2002–2004 patients to include data on Houston patients from four additional years (2005–2008) to determine if there were any differences in risk factors shown to be associated with developing the more severe outcomes of WNV infection, encephalitis and death, by having this larger sample size. A re-analysis of the risk factors for encephalitis and death was conducted on all of the patients from 2002–2008 and was the focus of this proposed research. This analysis allowed for the determination to be made that there are differences in the outcome in the risk factors for encephalitis and death with an increased sample size. Retrospective medical chart reviews were completed for the 265 confirmed WNV hospitalized patients; 153 patients had encephalitis (WNE), 112 had either viral syndrome with fever (WNF) or meningitis (WNM); a total of 22 patients died. Univariate logistic regression analyses on demographic, comorbidities, and social risk factors was conducted in a similar manner as in the previously conducted study to determine the risk factors for developing encephalitis from WNV. A multivariate model was developed by using model building strategies for the multivariate logistic regression analysis. The hypothesis of this study was that there would be additional risk factors shown to be significant with the increase in sample size of the dataset. This analysis with a greater sample size and increased power supports the hypothesis in that there were additional risk factors shown to be statistically associated with the more severe outcomes of WNV infection (WNE or death). Based on univariate logistic regression results, these data showed that even though age of 20–44 years was statistically significant as a protecting effect for developing WNE in the original study, the expanded sample lacked significance. This study showed a significant WNE risk factor to be chronic alcohol abuse, when it was not significant in the original analysis. Other WNE risk factors identified in this analysis that showed to be significant but were not significant in the original analysis were cancer not in remission > 5 years, history of stroke, and chronic renal disease. When comparing the two analyses with death as an outcome, two risk factors that were shown to be significant in the original analysis but not in the expanded dataset analysis were diabetes mellitus and immunosuppression. Three risk factors shown to be significant in this expanded analysis but were not significant in the original study were illicit drug use, heroin or opiate use, and injection drug use. However, with the multiple logistic regression models, the same independent risk factors for developing encephalitis of age and history of hypertension including drug induced hypertension were consistent in both studies.^

Diabetic nephropathy in African-Americans with type 1 diabetes: The new Jersey 725 study

Diabetic nephropathy is the most common cause of end-stage renal disease (ESRD) in the United States. African-Americans and patients with type 1 diabetes (T1D) are at increased risk. We studied the rate and factors that influenced progression of glomerular filtration rate (GFR) in 401 African-American T1D patients who were followed for 6 years through the observational cohort New Jersey 725 study. Patients with ESRD and/or GFR<20 ml/min were excluded. The mean (SD) baseline GFR was 106.8 (27.04) ml/min and it decreased by 13.8 (mean, SD 32.2) ml/min during the 6-year period (2.3 ml/min/year). In patients with baseline macroproteinuria, GFR decreased by 31.8 (39.0) ml/min (5.3 ml/min/year) compared to 8.2 (mean, SD 27.6) ml/min (1.3 ml/min/year) in patients without it (p<0.00001). Six-year GFR fell to <20 ml/min in 5.25% of all patients, but in 16.8% of macroproteinuric patients.^ A model including baseline GFR, proteinuria category and hypertension category, explained 35% of the 6-year GFR variability (p<0.0001). After adjustment for other variables in the model, 6-year GFR was 24.9 ml/min lower in macroproteinuric patients than in those without proteinuria (p=0.0001), and 12.6 ml/min lower in patients with treated but uncontrolled hypertension compared to normotensive patients (p=0.003). In this sample of patients, with an elevated mean glycosylated hemoglobin of 12.4%, glycemic control did not independently influence GFR deterioration, nor did BMI, cholesterol, gender, age at diabetes onset or socioeconomic level.^ Taken together, our findings suggest that proteinuria and hypertension are the most important factors associated with GFR deterioration in African-American T1D patients.^

Correlation of breath ammonia with blood urea nitrogen and creatinine during hemodialysis

We have spectroscopically determined breath ammonia levels in seven patients with end-stage renal disease while they were undergoing hemodialysis at the University of California, Los Angeles, dialysis center. We correlated these measurements against simultaneously taken blood samples that were analyzed for blood urea nitrogen (BUN) and creatinine, which are the accepted standards indicating the level of nitrogenous waste loading in a patient's bloodstream. Initial levels of breath ammonia, i.e., at the beginning of dialysis, are between 1,500 ppb and 2,000 ppb (parts per billion). These levels drop very sharply in the first 15–30 min as the dialysis proceeds. We found the reduction in breath ammonia concentration to be relatively slow from this point on to the end of dialysis treatment, at which point the levels tapered off at 150 to 200 ppb. For each breath ammonia measurement, taken at 15–30 min intervals during the dialysis, we also sampled the patient's blood for BUN and creatinine. The breath ammonia data were available in real time, whereas the BUN and creatinine data were available generally 24 h later from the laboratory. We found a good correlation between breath ammonia concentration and BUN and creatinine. For one of the patients, the correlation gave an R2 of 0.95 for breath ammonia and BUN correlation and an R2 of 0.83 for breath ammonia and creatinine correlation. These preliminary data indicate the possibility of using the real-time breath ammonia measurements for determining efficacy and endpoint of hemodialysis.

Alterations in the regulation of androgen-sensitive Cyp 4a monooxygenases cause hypertension

Hypertension is a leading cause of cardiovascular, cerebral, and renal disease morbidity and mortality. Here we show that disruption of the Cyp 4a14 gene causes hypertension, which is, like most human hypertension, more severe in males. Male Cyp 4a14 (−/−) mice show increases in plasma androgens, kidney Cyp 4a12 expression, and the formation of prohypertensive 20-hydroxyarachidonate. Castration normalizes the blood pressure of Cyp 4a14 (−/−) mice and minimizes Cyp 4a12 expression and arachidonate ω-hydroxylation. Androgen replacement restores hypertensive phenotype, Cyp 4a12 expression, and 20-hydroxy-arachidonate formation. We conclude that the androgen-mediated regulation of Cyp 4a arachidonate monooxygenases is an important component of the renal mechanisms that control systemic blood pressures. These results provide direct evidence for a role of Cyp 4a isoforms in cardiovascular physiology, establish Cyp 4a14 (−/−) mice as a monogenic model for the study of cause/effect relationships between blood pressure, sex hormones, and P450 ω-hydroxylases, and suggest the human CYP 4A homologues as candidate genes for the analysis of the genetic and molecular basis of human hypertension.

Genetic determinants of human hypertension.

Hypertension is a common trait of multifactorial determination imparting an increased risk of myocardial infarction, stroke, and end-stage renal disease. The primary determinants of hypertension, as well as the factors which determine specific morbid sequelae, remain unknown in the vast majority of subjects. Knowledge that a large fraction of the interindividual variation in this trait is genetically determined motivates the application of genetic approaches to the identification of these primary determinants. Success in this effort will afford insights into pathophysiology, permit preclinical identification of subjects with specific inherited susceptibility, and provide opportunities to tailor therapy to specific underlying abnormalities. To date, mutations in three genes have been implicated in the pathogenesis of human hypertension: mutations resulting in ectopic expression of aldosterone synthase enzymatic activity cause a mendelian form of hypertension known as glucocorticoid-remediable aldosteronism; mutations in the beta subunit of the amiloride-sensitive epithelial sodium channel cause constitutive activation of this channel and the mendelian form of hypertension known as Liddle syndrome; finally, common variants at the angiotensinogen locus have been implicated in the pathogenesis of essential hypertension in Caucasian subjects, although the nature of the functional variants and their mechanism of action remain uncertain. These early findings demonstrate the feasibility and utility of the application of genetic analysis to dissection of this trait.

Effects of pathophysiological concentrations of albumin on NHE3 activity and cell proliferation in primary cultures of human proximal tubule cells

The progression of renal disease correlates strongly with hypertension and the degree of proteinuria, suggesting a link between excessive Na+ reabsorption and exposure of the proximal tubule to protein. The present study investigated the effects of albumin on cell growth and Na+ uptake in primary cultures of human proximal tubule cells (PTC). Albumin (1.0 mg/ml) increased cell proliferation to 134.1 +/- 11.8% (P < 0.001) of control levels with no change in levels of apoptosis. Exposure to 0.1 and 1.0 mg/ml albumin increased total Na-22(+) uptake to 119.1 &PLUSMN; 6.3% (P = 0.005) and 115.6 &PLUSMN; 5.3% (P < 0.006) of control levels, respectively, because of an increase in Na+/H+ exchanger isoform 3 (NHE3) activity. This was associated with an increase in NHE3 mRNA to 161.1 +/- 15.1% (P < 0.005) of control levels in response to 0.1 mg/ml albumin. Using confocal microscopy with a novel antibody raised against the predicted extracellular NH2 terminus of human NHE3, we observed in nonpermeabilized cells that exposure of PTC to albumin (0.1 and 1.0 mg/ml) increased NHE3 at the cell surface to 115.4 &PLUSMN; 2.7% (P < 0.0005) and 122.4 +/- 3.7% (P < 0.0001) of control levels, respectively. This effect was paralleled by significant increases in NHE3 in the subplasmalemmal region as measured in permeabilized cells. These albumin-induced increases in expression and activity of NHE3 in PTC suggest a possible mechanism for Na+ retention in response to proteinuria.

Glomerular number and size in autopsy kidneys: The relationship to birth weight

Background. In the Southeast United States, African Americans have an estimated incidence of hypertension and end-stage renal disease (ESRD) that is five times greater than Caucasians. Higher rates of low birth weight (LBW) among African Americans is suggested to predispose African Americans to the higher risk, possibly by reducing the number of glomeruli that develop in the kidney. This study investigates the relationships between age, race, gender, total glomerular number (N-glom), mean glomerular volume (V-glom), body surface area (BSA), and birth weight. Methods. Stereologic estimates of N-glom and V-glom were obtained using the physical disector/fractionator combination for autopsy kidneys from 37 African Americans and 19 Caucasians. Results. N-glom was normally distributed and ranged from 227,327 to 1,825,380, an 8.0-fold difference. A direct linear relationship was observed between N-glom and birth weight (r=0.423, P=0.0012) with a regression coefficient that predicted an increase of 257,426 glomeruli per kilogram increase in birth weight (alpha=0.050:0.908). Among adults there was a 4.9-fold range in V-glom , and in adults, V-glom was strongly and inversely correlated with N-glom (r=-0.640, P=0.000002). Adult V-glom showed no significant correlation with BSA for males (r=-0.0150, P=0.936), although it did for females (r=0.606, P=0.022). No racial differences in average N-glom or V-glom were observed. Conclusion. Birth weight is a strong determinant of N-glom and thereby of glomerular size in the postnatal kidney. The findings support the hypothesis that LBW by impairing nephron development is a risk factor for hypertension and ESRD in adulthood.

Practical considerations for the measurement of free light chains in serum

Background: A new immunoassay for free light chain measurements has been reported to be useful for the diagnosis and monitoring of monoclonal light chain diseases and nonsecretory myeloma. We describe experience with and some potential pitfalls of the assay. Methods: The assay was assessed for precision, sample type and stability, recovery, and harmonization of results between two analyzers on which the reagents are used. Free-light-chain concentrations were measured in healthy individuals (to determine biological variation), patients with monoclonal gammopathy of undetermined significance, myeloma patients after autologous stem cell transplants, and patients with renal disease. Results: Analytical imprecision (CV) was 6-11% for kappa and A free-light-chain measurement and 16% for the calculated kappa/lambda ratio. Biological variation was generally insignificant compared with analytical variation. Despite the same reagent source, values were not completely harmonized between assay systems and may produce discordant free-light-chain ratios. In some patients with clinically stable myeloma, or post transplantation, or with monoclonal gammopathy of undetermined significance, free-light-chain concentration and ratio were within the population reference interval despite the presence of monoclonal intact immunoglobulin in serum. In other patients with monoclonal gammopathy of undetermined significance, values were abnormal although there was no clinical evidence of progression to multiple myeloma. Conclusions: The use of free-light-chain measurements alone cannot differentiate some groups of patients with monoclonal gammopathy from healthy individuals. As with the introduction of any new test, it is essential that more scientific data about use of this assay in different subject groups are available so that results can be interpreted with clinical certainty. (C) 2003 American Association for Clinical Chemistry.

Carotid intima-media thickness, cardiovascular risk factors and albuminuria in a remote Australian Aboriginal community

Background: Rates of cardiovascular disease and renal disease in Australian Aboriginal communities are high, as is the prevalence of some 'traditional' cardiovascular (CV) risk factors, such as diabetes and cigarette smoking. Recent work has highlighted the importance of markers of inflammation, such as C-reactive protein (CRP), homocysteine and albuminuria as predictors of cardiovascular risk in urban westernised settings. It is not clear how these factors relate to outcome in the setting of these remote communities, but very high CRP concentrations have been shown in this and other Aboriginal communities. Methods and results: In a cross-sectional survey including 237 adults in a remote Aboriginal community in the Northern Territory of Australia, we measured carotid intima-media thickness (IMT), together with blood pressure, diabetes, lipid levels, smoking and albuminuria, CRP and fibrinogen, serum homocysteine concentration, and IgG titres for Chlamydia pneumoniae, Helicobacter pylori and cytomegalovirus. Median carotid IMT was 0.63 [interquartile range 0.54-0.71] mm. As a categorical outcome, the prevalence of the highest IMT quartile ('increased IMT', greater than or equal to0.72 mm) was compared with the lower three quartiles. Increased IMT was associated in univariate analyses with greater waist circumference, systolic BP, fibrinogen and serum albumin concentrations, urine albumin/creatinine ratio and older age as continuous variables. Associations of increased IMT with some continuous variables were not linear; univariate associations were seen with the highest quartile (versus all other quartiles) of CRP and homocysteine concentration and CMV IgG titre. In a multivariate model age, smoking, waist circumference and the highest quartile of CRP concentrations (greater than or equal to14 mg/l) remained significant predictors of IMT greater than or equal to0.72 mm. Conclusions: Measurement of carotid IMT was possible in this remote setting. Increased IMT (greater than or equal to0.72 mm) was associated with increased CRP concentrations over a range that suggests infection/inflammation may be important determinants of cardiovascular risk in this setting. The associations of IMT with markers of renal disease seen in univariate analyses were explained in this analysis by confounding due to the associations of urine ACR with other risk factors. (C) 2004 Published by Elsevier Ireland Ltd.

Kidney volume, blood pressure, and albuminuria: Findings in an Australian Aboriginal community

Background. Australian Aborigines are experiencing epidemic proportions of renal disease, marked by albuminuria and, variably, hematuria. They also have high rates of low birth weight, which have been associated with lower kidney volumes and higher blood pressures. The authors evaluated relationships between kidney volume, blood pressure, albuminuria, and hematuria in 1 homogeneous group. Methods Forty-three percent (672 of 1,560) of the population in a remote coastal Australian Aboriginal community aged 4.4 to 72.1 years participated in the study. Results: Kidney size correlated closely with body size. Systolic blood pressure (SBP) was correlated inversely with kidney length and kidney volume, after adjusting for age, sex, and body surface area (BSA); a 1-cm increase in mean kidney length was associated with a 2.2-mm Hg decrease in SBP, and a 10-mL increase in mean kidney volume was associated with a 0.6-mm Hg decrease in SBP (P = 0.001). Mean kidney volume explained 10% of the variance in SBP in a multivariate model containing age, sex, and BSA. In addition to higher SBP, adults who had the lowest quartiles of kidney volume also had the highest levels of overt albuminuria (P = 0.044). Conclusion: Smaller kidneys predispose to higher blood pressures and albuminuria in this population. The lower volumes possibly represent kidneys with reduced nephron numbers, which might be related to an adverse intrauterine environment. Susceptibility to renal disease could be a direct consequence of reduced nephron numbers; the higher blood pressures with which they are associated could also contribute to, as well as derive from, this association.

Determinants of glomerular volume in different cortical zones of the human kidney

Enlarged glomerular size is a feature of focal segmental glomerulosclerosis, obesity-related glomerulopathy, diabetic nephropathy, and hypertension. The distribution of glomerular volumes within different cortical zones and glomerular volume alterations with age and obesity may contribute to understanding the evolution of these diseases. We analyzed the distributions of volumes of individual glomeruli in the superficial, middle, and juxtamedullary cortex of normal human kidneys using the disector/Cavalieri method. Volumes (V-glom) of 720 nonsclerotic glomeruli (30 per kidney, 10 per zone) were estimated in autopsy kidneys of 24 American men, 12 aged 20 to 30 yr and 12 aged 51 to 69 yr. Black and white individuals were represented equally. The range of individual V-glom within subjects varied from two- to eight-fold. There were no significant zonal differences in V-glom in the young or those with body surface area (BSA) <= 2.11 m(2). In contrast, superficial glomeruli in the older age group, in those with BSA > 2.11 m(2), and in white subjects were significantly larger than juxtamedullary glomeruli. Black subjects tended toward larger V-glom than white subjects, and this difference was significant and most marked in the juxtamedullary zone and independent of age, BSA, and glomerular number. There is a wide range in individual V-glom in adults. BSA, race, and age independently influence V-glom different zones of the renal cortex. These findings might reflect processes of aging and susceptibility factors to renal disease.

Lessons in ethnonephrology

Only recently has the nephrology community moved beyond a fairly singular focus on terminal kidney failure to embrace population-based studies of earlier stages of disease, its markers and risk factors, and of interventions. Observations in developing countries, and in minority, migrant, and disadvantaged groups in westernized countries, have promoted these developments. We are only beginning to interpret renal disease in the context of public health history, social and health transitions, changing population demography, and competing mortality. Its intimate relationships to other health issues are being progressively exposed. Perspectives on the multideterminant etiology of most disease and the pedestrian nature of most risk factors are maturing. We are challenged to reconcile epidemiologic patterns with morphology in diseased renal tissue, and to consider structural markers, such as nephron number and glomerular size, as determinants of disease susceptibility. New work force models are mandated for population-based studies and intervention programs. Intervention programs need to be integrated with other chronic disease initiatives and nested in a matrix of systematic primary care, and although flexible to changing needs, must be sustained over the long term. Cross-disciplinary collaboration is essential in designing those programs, and in promoting them to health-care funders. Substantial expansion and restructuring of the discipline is needed for the nephrology community to participate effectively in those processes.