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1.  Urine Proteome Analysis in Murine Nephrotoxic Serum Nephritis 
American Journal of Nephrology  2009;30(5):450-458.
Background
Urine contains serum proteins filtered by the glomerulus or secreted by the renal tubules and proteins produced locally by the urinary tract. Proteomic analysis of urine holds the potential as a noninvasive means of studying or monitoring disease activity. In mice, large concentrations of albumin and lipocalins have complicated the ability to identify urinary biomarkers in disease models.
Methods
Passive nephrotoxic serum nephritis was induced in mice. Urine proteins were identified and quantified by iTRAQ and MALDI-TOF mass spectrometry. Results were compared to Western blotting and multiplex immunoassays.
Results
Large concentrations of major urinary proteins dominate the urine proteome of mice even in the context of acute nephritis. Increased proteinuria caused by nephrotoxic serum nephritis is transient and includes increased albumin excretion. There were no alterations in chemokine excretion. Altered hepcidin excretion was identified, most likely reflecting local production and renal retention.
Conclusion
Proteomic analysis of mouse urine remains challenging due to the abundance of a limited subset of proteins. iTRAQ analysis does not circumvent these challenges, but can provide information on post-translational processing of some proteins. Hepcidin is identified as a potential urinary marker of nephritis and its role in disease pathogenesis warrants further study.
doi:10.1159/000242430
PMCID: PMC3712807  PMID: 19776558
Glomerulonephritis; Hepcidin; IP-10; iTRAQ; Nephrotoxic serum; Proteomics
2.  The Association of African Ancestry and Elevated Creatinine in the Coronary Artery Risk Development in Young Adults (CARDIA) Study 
American Journal of Nephrology  2009;31(3):202-208.
Whether genetic factors account for differences in early kidney disease among blacks in a young healthy population is not well known. We evaluated the association of self-reported race and genetic African ancestry with elevated creatinine (≥1.3 mg/dl for men, ≥1.1 mg/dl for women) among 3,113 black and white participants in the Coronary Artery Risk Development in Young Adults (CARDIA) study, ages 38–50 years. We estimated individual African ancestry using 42 ancestry informative markers. Blacks were more likely to have elevated creatinine than whites, and this effect was more pronounced in men: adjusted odds ratio (AOR) for black versus white men = 7.03, 4.15–11.91; AOR for women = 2.40, 1.15–5.02. Higher African ancestry was independently associated with elevated creatinine among black men (AOR = 1.53,1.08–2.16 per SD increase in African ancestry), but not women. A graded increase in odds of elevated creatinine by African Ancestry was observed among black men compared with white men: AOR = 4.27 (2.26–10.06) for black men with 40–70% African ancestry; AOR = 8.09 (4.19–15.61) for black men with 70–80% African ancestry; AOR = 9.05 (4.81–17.02) for black men with >80% African ancestry. Genetic factors common to African ancestry may be associated with increased risk of early kidney dysfunction in a young, healthy population, particularly among black men.
doi:10.1159/000268955
PMCID: PMC3487144  PMID: 20029176
Ancestry; Creatinine; Race; Kidney
3.  CYP3A4 and CYP3A5 Polymorphisms and Blood Pressure Response to Amlodipine among African-American Men and Women with Early Hypertensive Renal Disease 
American Journal of Nephrology  2009;31(2):95-103.
Purpose
To explore the association between CYP3A4 and CYP3A5 gene polymorphisms and blood pressure response to amlodipine among participants from the African-American Study of Kidney Disease and Hypertension Trial randomized to amlodipine (n = 164).
Methods
Cox proportional hazards models were used to determine the risk of reaching a target mean arterial pressure (MAP) of ≤107 mm Hg by CYP3A4 (A–392G and T16090C) and CYP3A5 (A6986G) gene polymorphisms, stratified by MAP randomization group (low or usual) and controlling for other predictors for blood pressure response.
Results
Women randomized to a usual MAP goal with an A allele at CYP3A4 A–392G were more likely to reach a target MAP of 107 mm Hg. The adjusted hazard ratio (AA/AG compared to GG) with 95% confidence interval was 3.41 (1.20–9.64; p = 0.020). Among participants randomized to a lower MAP goal, those with the C allele at CYP3A4 T16090C were more likely to reach target MAP: The adjusted hazard ratio was 2.04 (1.17–3.56; p = 0.010). After adjustment for multiple testing using a threshold significance level of p = 0.016, only the CYP3A4 T16090C SNP remained significant. CYP3A5 A6986G was not associated with blood pressure response.
Conclusions
Our findings suggest that blood pressure response to amlodipine among high-risk African-Americans appears to be determined by CYP3A4 genotypes, and sex specificity may be an important consideration. Clinical applications of CYP3A4 genotype testing for individualized treatment regimens warrant further study.
doi:10.1159/000258688
PMCID: PMC2853591  PMID: 19907160
Pharmacogenetics; Hypertension; Amlodipine; Renal failure; CYP3A polymorphisms; AASK; African-Americans
4.  Changes of Renal AT1/AT2 Receptors and Structures in Ovine Fetuses following Exposure to Long-Term Hypoxia 
American Journal of Nephrology  2009;31(2):141-150.
Background/Aims
The present study tested the hypothesis that chronic hypoxia adversely affects renal development in the ovine fetus.
Methods
Kidneys were collected from near-term fetuses of pregnant ewes maintained at sea level or high altitude (3,801 m, PaO2: approx. 60 mm Hg) for 110 days (n = 6 for each group).
Results
Long-term high altitude hypoxia reduced the fetal kidney/body weight ratio. Histological analysis showed a significant enlargement in the Bowman's space and swelling of tubule epithelial cells in the kidney of the hypoxic fetus. The histological alterations were limited to the cortical, but not medullary, zone. These alterations were associated with an increase in serum creatinine and a decrease in the BUN-to-creatinine ratio in hypoxic fetuses. Angiotensin II receptors (AT1R and AT2R) were detected in the glomerular and tubular regions of the kidney. Chronic hypoxia caused a significant increase in AT1R and a decrease in AT2R protein and mRNA abundance, resulting in a large increase in the AT1R/AT2R ratio in the fetal kidney.
Conclusion
The results demonstrate an adverse effect of chronic hypoxia on renal AT1R and AT2R expression and functions in the fetus, suggesting a possible role of fetal hypoxia in the programming of renal diseases in fetal origins.
doi:10.1159/000259901
PMCID: PMC2853592  PMID: 19923800
Hypoxia; Angiotensin II receptors; Ovine fetus
5.  Effect of Community Characteristics on Familial Clustering of End-Stage Renal Disease 
American Journal of Nephrology  2009;30(6):499-504.
Background
Lower socioeconomic status is generally associated with an increased risk of end-stage renal disease (ESRD). The relationship between community characteristics reflecting socioeconomic status and familial aggregation of common forms of ESRD has not been studied.
Methods
Demographic data and family history of ESRD were collected from 23,880 incident dialysis patients in ESRD Network 6 between 1995 and 2003. Addresses were geocoded and linked to the 2000 census 5-digit zip code-level database that includes community demographic, social and economic characteristics. Clustering of patients having a family history of ESRD at the community level was accounted for using a generalized estimating equations (GEE) model. Multivariate analysis estimated associations between family history of ESRD and community-level characteristics.
Results
Twenty-three percent of patients reported a family history of ESRD. After adjusting for individual demographic characteristics, multivariate analyses failed to reveal statistically significant relationships between a family history of ESRD and indicators of community socioeconomic status such as median household income, percentage high school graduates, percentage vacant housing units or ethnic composition.
Conclusions
Although select community measures of lower socioeconomic status may contribute to the familial clustering of ESRD, non-socioeconomic factors, potentially inherited, appear to be more important contributors to familial aggregation of the common forms of ESRD.
doi:10.1159/000243716
PMCID: PMC2853588  PMID: 19797894
End-stage renal disease; Environment/neighborhood; Familial aggregation; Geocode; Socioeconomic status
6.  Sex and Age Result in Differential Regulation of the Renal Thiazide-Sensitive NaCl Cotransporter and the Epithelial Sodium Channel in Angiotensin II-Infused Mice 
American Journal of Nephrology  2009;30(6):554-562.
Background/Aims
We determined the effects of age and sex on the blood pressure (BP) response to angiotensin II (Ang II) infusion and evaluated the potential mechanistic role of the thiazide-sensitive NaCl cotransporter (NCC) and the epithelial sodium channel (ENaC).
Methods
Male and female mice (∼3 or 21 months of age) were infused with Ang II or control for 7 days.
Results
Males had a greater BP response to Ang II, somewhat enhanced by aging. Mean systolic BPs (at 7 days) were (mm Hg): 161, 143, 172, and 157 in young male, young female, old male, and old female mice, respectively. Immunoblotting changes in the whole kidney that supported this BP profile included a 51 and 52% increase in NCC band density in the old females and old males (as compared to sex-respective controls) with Ang II infusion, while the young males and young females showed an increase of 40 and 0%, respectively. Young males also had a greater reduction in major bands of β- and γ-ENaC, than did young female mice. The natriuretic response to hydrochlorothiazide supported an increase in activity of NCC with Ang II in aged mice only.
Conclusions
Increased sensitivity to Ang II in aging and male mice may involve overactivity of NCC.
doi:10.1159/000252776
PMCID: PMC2853589  PMID: 19844087
Hypertension; Gender differences; Epithelial sodium channel; NaCl cotransporter
7.  Validation of Endogenous Internal Real-Time PCR Controls in Renal Tissues 
American Journal of Nephrology  2009;30(5):413-417.
Background
Endogenous internal controls (‘reference’ or ‘housekeeping’ genes) are widely used in real-time PCR (RT-PCR) analyses. Their use relies on the premise of consistently stable expression across studied experimental conditions. Unfortunately, none of these controls fulfills this premise across a wide range of experimental conditions; consequently, none of them can be recommended for universal use.
Methods
To determine which endogenous RT-PCR controls are suitable for analyses of renal tissues altered by kidney disease, we studied the expression of 16 commonly used ‘reference genes’ in 7 mildly and 7 severely affected whole kidney tissues from a well-characterized cystic kidney disease model. Expression levels of these 16 genes, determined by TaqMan® RT-PCR analyses and Affymetrix GeneChip® arrays, were normalized and tested for overall variance and equivalence of the means.
Results
Both statistical approaches and both TaqMan- and GeneChip-based methods converged on 3 out of the 4 top-ranked genes (Ppia, Gapdh and Pgk1) that had the most constant expression levels across the studied phenotypes.
Conclusion
A combination of the top-ranked genes will provide a suitable endogenous internal control for similar studies of kidney tissues across a wide range of disease severity.
doi:10.1159/000235993
PMCID: PMC2818397  PMID: 19729889
Real-time PCR; Kidney disease; Endogenous internal control; Reference gene; Housekeeping gene
8.  Effect of Pioglitazone on Survival and Renal Function in a Mouse Model of Polycystic Kidney Disease 
American Journal of Nephrology  2009;30(5):468-473.
Background/Aims
Cystic epithelia in polycystic kidney disease display features similar to malignant cells. Thiazolidinediones have been shown to have anti-neoplastic properties, therefore we tested the hypothesis that pioglitazone reduces cyst formation, improves renal function, and prolongs survival in a mouse model of polycystic kidney disease.
Methods
PC-Pkd1-KO mice, which have homozygous mutations of the Pkd1 gene in principal cells, were used. On the day after giving birth, mothers were fed standard mouse chow with or without pioglitazone (30 mg/kg chow). After weaning, the assigned diet was continued. At 1 month of age, blood pressure was measured and animals were sacrificed to determine kidney weight, body weight, and serum urea. Kidneys were evaluated for proliferation using Ki-67, apoptosis using TUNEL analysis, and cyst number using MRI. Survival was observed.
Results
Pioglitazone did not alter renal function, cell proliferation, apoptosis, or cyst formation in animals with polycystic kidney disease, however it did increase survival. Pioglitazone reduced blood pressure in PC-Pkd1-KO, but not in controls.
Conclusion
These findings suggest that pioglitazone may have a unique antihypertensive effect in polycystic kidney disease, and that such an effect may promote improved survival.
doi:10.1159/000242432
PMCID: PMC2818398  PMID: 19776560
Polycystic kidney disease; Blood pressure; Pioglitazone
9.  Modeling of Human Anti-GBM Antibody–α3(IV)NC1 Interactions Predicts Antigenic Cross-Linking through Contact of Both Heavy Chains with Repeating Epitopes on α3(IV)NC1 
American Journal of Nephrology  2009;30(5):474-480.
Background/Aims
Patients with anti-glomerular basement membrane diseases produce pathogenic autoantibodies (autoAb) that deposit in the kidney and initiate severe inflammation. Restricted antigenic specificity of the autoAb against 2 regions (with related sequences) within α3(IV)NC1, along with shared idiotypes (i.e. structural determinants), among pathogenic human autoAb suggested that common genetic elements encode the autoAb. The aim of this study was to determine whether the idiotypic relatedness of the autoAb was due to the fact that unique and similar genes were used to encode them, divergent genes were used to produce Ab with similar Ag-binding properties and conformation, or if other mechanisms were operative.
Methods
The encoding V gene sequences of pathogenic human anti-α3(IV)NC1 Ab, derived following immunization of XenoMice® which produce human but not murine IgG, with α3(IV)NC1 were determined. Predicted conformations of autoAb–α3(IV)NC1 interactions were derived using the Ab sequences and molecularmodels of the α3(IV)NC1 structure.
Results
The pathogenic Ab were encoded by multiple, common VH and VL gene families indicating that they were not encoded by a unique subset of genes and that normal individuals have the capacity to produce them. However, modeling of the Ag–Ab interactions suggested that although the contact regions varied for individual Ab, the optimized energy constraints facilitate interaction of both Ab-binding regions with pathogenically relevant epitopes on α3(IV)NC1.
Conclusions
The results suggest that the repetitive nature and relatedness of the α3(IV)NC1 antigenic epitopes facilitate cross-linking of pathogenic Ab, in vivo, by allowing both IgG Fab to bind to the basement membrane. This most likely accounts for the high-affinity Ab binding we and others observed among human anti-α3(IV)NC1 Ab. Based on these observations, we postulate that this interaction provides for the stability of the Ab interaction, resulting in a high-affinity interaction that serves as an ideal scaffold for optimal FcR engagement and complement activation, thereby accelerating inflammation and contributing to the rapidly progressive nature of this disease.
doi:10.1159/000242476
PMCID: PMC2818399  PMID: 19786737
Glomerulonephritis; Anti-glomerular basement membrane disease; α3(IV) collagen
10.  Nebivolol Reduces Proteinuria and Renal NADPH Oxidase-Generated Reactive Oxygen Species in the Transgenic Ren2 Rat 
American Journal of Nephrology  2009;30(4):354-360.
Background/Aims
Renin-angiotensin-aldosterone system (RAAS) and sympathetic nervous system activation are crucial in the pathogenesis of hypertension, cardiovascular and renal disease. NADPH oxidase-mediated increases in reactive oxygen species (ROS) are an important mediator for RAAS-induced cardiovascular and renal injury. Increased levels of ROS can diminish the bioactivity of nitric oxide (NO), a critical modulator of RAAS effects on the kidney. Thereby, we hypothesized that in vivo nebivolol therapy in a rodent model of activated RAAS would attenuate glomerular damage and proteinuria through its actions to reduce NADPH oxidase activity/ROS and increase bioavailable NO.
Methods
We utilized the transgenic Ren2 rat which displays heightened tissue RAAS, hypertension, and proteinuria. Ren2 rats (6–9 weeks of age) and age-matched Sprague-Dawley littermates were treated with nebivolol 10 mg/kg/day (osmotic mini-pump) for 21 days.
Results
Ren2 rats exhibited increases in systolic blood pressure, proteinuria, kidney cortical tissue total NADPH oxidase activity and subunits (Rac1, p67phox, and p47phox), ROS and 3-nitrotyrosine, as well as reductions in podocyte protein markers; each of these parameters improved with nebivolol treatment along with increases in renal endothelial NO synthase expression.
Conclusions
Our data suggest that nebivolol improves proteinuria through reductions in renal RAAS-mediated increases in NADPH oxidase/ROS and increases in bioavailable NO.
doi:10.1159/000229305
PMCID: PMC2814025  PMID: 19609077
Nebivolol; Proteinuria; NADPH oxidase; Reactive oxygen species
11.  Rate of Kidney Function Decline in Older Adults: A Comparison Using Creatinine and Cystatin C 
American Journal of Nephrology  2009;30(3):171-178.
Background/Aims
The aim of this study was to determine the decline in the estimated glomerular filtration rate (eGFR) in elderly persons and to compare estimates based on creatinine and cystatin C.
Methods
In the Cardiovascular Health Study, GFR changes in an elderly cohort were estimated from serum creatinine and cystatin C measured at baseline, year 3 and year 7 in 4,380 participants (age 72 ± 5 years at entry). Outcomes were mean eGFR decline, incident chronic kidney disease (CKD) and rapid decline in eGFR (annual loss >3 ml/min/1.73 m2).
Results
Mean annual eGFR loss as estimated from creatinine was 0.4 ± 3.6 ml/min/1.73 m2, with 16% of the participants experiencing a rapid decline. Mean eGFR loss as estimated from cystatin C was 1.8 ± 2.6, with 25% of the participants experiencing a rapid decline (p < 0.001 for both). Among participants without baseline CKD, incident CKD was detected at year 7 in 10% (n = 263) using creatinine and 19% (n = 544) using cystatin C (p < 0.001). Increasing age was the strongest predictor of rapid decline; adjusted odds ratios were 1.38 (1.16–1.65), 1.62 (1.31–1.99) and 2.96 (2.28–3.84) for participants aged 70–74, 75–79 and 80+ at baseline, compared with those aged 65–69.
Conclusion
In elderly persons, cystatin C estimated substantially larger declines in kidney function than creatinine did. Defining the optimal measurement of kidney function in elderly persons should be a high priority for future research.
doi:10.1159/000212381
PMCID: PMC2820322  PMID: 19349699
Glomerular filtration rate; Creatinine; Cystatin C; Chronic kidney disease
12.  Role of G Protein-Coupled Receptor Kinase-2 in Peroxisome Proliferator-Activated Receptor Gamma-Mediated Modulation of Blood Pressure and Renal Vascular Reactivity in SHR 
American Journal of Nephrology  2009;30(3):201-208.
Background
Peroxisome proliferator-activated receptor γ (PPARγ), a nuclear transcription factor, modulates the expression/activity of G protein-coupled receptors (GPCRs), but its role in GPCR signaling is not clear. Increased GPCR kinase-2 (GRK-2) activity and receptor desensitization have been reported in hypertension.
Method
In this study we investigated the role of GRK-2 in PPARγ-mediated blood pressure regulation in hypertension. SHR or WKY rats were treated with GW1929, a selective PPARγ ligand (0.5 mg/kg/day), or vehicle for 2 months. Systolic blood pressure (tail cuff plethysmography), whole kidney perfusion (laser scanner) and renal vascular reactivity (isolated perfused kidney) was determined.
Results
GW1929 significantly reduced blood pressure (20 ± 1%) and increased renal perfusion (61 ± 3%) in SHR compared to WKY rats. Vasoconstriction to phenylephrine (100 μg) in the isolated perfused kidney was greater in SHRs (29 ± 1%) compared to WKY rats and this was abolished by GW1929. GW1929 enhanced acetylcholine-induced (30–300 μg) and sodium nitroprusside-induced vasodilatation in SHR by 46 ± 2% (p < 0.05) and 33 ± 2% (p < 0.05), respectively. Isoprenalin-induced (5–30 μg) vasodilatation was 43 ± 2% lower in SHR compared to WKY and GW1929 enhanced this vasodilatation by 55 ± 2%. In SHR kidney, GW1929 enhanced expression of PPARγ mRNA (34 ± 1%) but reduced that of GRK-2 (31 ± 3%).
Conclusion
We suggest that downregulation of PPARγ but upregulation of GRK-2 increases blood pressure and impaired renal vascular reactivity in SHR and that PPARγ-mediated improvement in hypertension may involve transcriptional regulation of GRK-2 function.
doi:10.1159/000218061
PMCID: PMC2820323  PMID: 19420904
Peroxisome proliferator-activated receptor γ; Hypertension; G protein-coupled receptor kinase-2; G protein-coupled receptor; Vascular reactivity; Renal perfusion
13.  Reverse Cholesterol Transport Pathway in Experimental Chronic Renal Failure 
American Journal of Nephrology  2009;30(2):147-154.
Background
Chronic renal failure (CRF) causes oxidative stress, inflammation, oxidation of lipoproteins, impaired maturation of HDL and accelerated atherosclerosis. Uptake of oxidized lipoproteins by macrophages via scavenger receptors (scavenger receptor class A type I – SR-AI, and lectin-like oxidized LDL receptor – LOX-1) leads to foam cell formation and atherosclerosis. HDL mitigates atherosclerosis by retrieving surplus cholesterol via ATP binding cassette transporter A1 (ABCA1) and ABCG1 transporters whose expression is regulated by liver X receptor (LXR). Free cholesterol reaching the surface of HDL is esterified by lecithin-cholesterol acyltransferase (LCAT) and sequestered in the core of HDL, thereby maximizing cholesterol uptake. In the liver, lipid-rich HDL unloads its lipid contents via reversible binding to SR-BI while lipid-poor HDL is degraded by the holo-receptor (ATP synthase β-chain).
Methods
Expression of the above molecules involved in reverse cholesterol/lipid transport was assessed in rats 8 weeks after 5/6 nephrectomy (CRF) or sham operation.
Results
CRF caused heavy accumulation of neutral lipids, upregulation of SR-AI, LOX-1, LXRα/β, ABCA1 and ABCG1 in the aorta, reduction in LCAT in the plasma and no significant change in either SR-BI or β-chain ATP synthase in the liver.
Conclusions
Lipid accumulation despite upregulation of the efflux (LXR, ABCA1, ABCG1) system in the aorta in CRF is largely due to upregulation of influx (SR-AI and LOX-1) pathway and LCAT deficiency.
doi:10.1159/000210020
PMCID: PMC2813810  PMID: 19321994
Oxidative stress; Atherosclerosis; Cardiovascular disease; Lipid disorders; Cholesterol
14.  Interaction of Aldosterone and Extracellular Volume in the Pathogenesis of Obesity-Associated Kidney Disease: A Narrative Review 
American Journal of Nephrology  2009;30(2):140-146.
Obesity and obesity-associated kidney injuries have played an important role in the rising prevalence of chronic kidney disease (CKD). The link between obesity and kidney disease begins with obesity's well-known associations with diabetes and hypertension, the two leading etiologies of CKD. However, a growing body of evidence suggests that elevated aldosterone levels and expanded extracellular volume are key components of obesity-induced renal disease via aldosterone's non-epithelial effects on the kidney. Highlighting these blood pressure- and diabetes-independent mechanisms of kidney injury in obesity allows an exploration of whether mineralocorticoid receptor blockade, coupled with weight loss and salt restriction, is an optimal treatment for overweight CKD patients.
doi:10.1159/000209744
PMCID: PMC2909635  PMID: 19299892
Aldosterone; Extracellular volume; Obesity; Chronic kidney disease
15.  Vascular Leak in a Rat Model of Preeclampsia 
American Journal of Nephrology  2009;30(1):26-33.
Background/Aims
Preeclampsia is a hypertensive disorder which develops de novo in women during pregnancy. The urinary excretion of the cardiotonic steroid, marinobufagenin (MBG), is increased prior to the development of hypertension. Preeclamptic patients are volume expanded but much of the excess salt and water appears to be located primarily in the interstitial space. Therefore, ‘capillary leak’ syndrome has been postulated in this disorder.
Methods
We evaluated the vascular leakage in normal rats following MBG injection and in a rat model of human preeclampsia. We measured the changes in light intensity comparing that in the intravascular to the extravascular space by assessing ‘leak’ of fluorescein-labeled albumin (FITC-albumin) from mesenteric postcapillary venules.
Results
FITC-albumin extravasation continued to increase in a time-dependent fashion after MBG infusion and was significant (p < 0.05) at 60 min of observation when compared to sham rats. We also observed a significant difference in ‘vascular leakage’ in preeclamptic rats compared to control non-pregnant and normal pregnant groups starting at 20 min after the FITC-albumin infusion.
Conclusion
We propose that MBG is involved in the production of a ‘vascular leak’ in our rat model of preeclampsia.
doi:10.1159/000193220
PMCID: PMC2821446  PMID: 19194101
Marinobufagenin; Capillary leak; Preeclampsia; Vascular permeability
16.  Polymorphisms in the Nonmuscle Myosin Heavy Chain 9 Gene (MYH9) Are Associated with Albuminuria in Hypertensive African Americans: The HyperGEN Study 
American Journal of Nephrology  2009;29(6):626-632.
Background
MYH9 is a podocyte-expressed gene encoding nonmuscle myosin IIA that is associated with idiopathic and human immunodeficiency virus-associated focal segmental glomerulosclerosis (FSGS) and hypertensive end-stage renal disease in African Americans.
Methods
Four single nucleotide polymorphisms comprising the major MYH9 E1 risk haplotype were tested for association with estimated glomerular filtration rate (eGFR) and urine albumin:creatinine ratio (ACR) in 2,903 HyperGEN participants (1,458 African Americans (AA) in 895 families and 1,445 European Americans (EA) in 859 families) to determine the role of MYH9 in subclinical nephropathy. Association analyses employed general linear models in unrelated probands and generalized estimating equations in families. Adjustment was performed for age, sex, diabetes, BMI, medications, and mean arterial pressure separately in each race.
Results
Mean (SD) eGFR and ACR were 74.3 (16.0) ml/min/1.73 m2 and 20.3 (119.9) mg/g in EA, and 88.6 (20.9) ml/min/1.73 m2 and 76.8 (394.5) mg/g in AA (both p < 0.0001 across ethnicities). Urine ACR was associated with rs3752462 (p = 0.01) and rs4821481 (p = 0.05) in unrelated AA and with rs4821481 (p = 0.03), rs2032487 (p = 0.04) and the E1 3224 haplotype (p = 0.013) in AA families. Single nucleotide polymorphisms and the haplotype were not associated with ACR in EA or with eGFR in either ethnic group.
Conclusions
MYH9 variants are associated with albuminuria in hypertensive AA. The strength of the association was weaker than that in FSGS and hypertensive end-stage renal disease. MYH9 risk variants appear to be associated with primary FSGS with secondary hypertension, although nephrosclerosis may develop in response to hypertension in subjects homozygous for the MYH9 E1 risk haplotype.
doi:10.1159/000194791
PMCID: PMC2749685  PMID: 19153477
African Americans; Albuminuria; Chronic kidney disease; Essential hypertension; HyperGEN study; MYH9 gene
17.  Association of Serum Total Iron-Binding Capacity and Its Changes Over Time with Nutritional and Clinical Outcomes in Hemodialysis Patients 
American Journal of Nephrology  2009;29(6):571-581.
Serum transferrin, estimated by total iron-binding capacity (TIBC), may be a marker of protein-energy wasting (PEW) in maintenance hemodialysis (MHD) patients. We hypothesized that low TIBC or its fall over time is associated with poor clinical outcomes. In 807 MHD patients in a prospective 5-year cohort, associations of TIBC and its changes over time with outcomes were examined after adjustment for case-mix and markers of iron stores and malnutrition-inflammation including serum interleukin-6, iron and ferritin. Patients with serum TIBC ≥250 mg/dl had higher body mass index, triceps and biceps skinfolds and mid-arm muscle circumference and higher serum levels of iron but lower ferritin and inflammatory markers. Some SF-36 quality of life (QoL) components were worse in the lowest and/or highest TIBC groups. Mortality was incrementally higher in lower TIBC levels (p-trend <0.001). Adjusted death hazard ratio was 1.75 (95% CI: 1.00–3.05, p = 0.05) for TIBC <150 compared to TIBC of 200–250 mg/dl. A fall in TIBC >20 mg/dl over 6 months was associated with a death hazard ratio of 1.57 (95% CI: 1.04–2.36, p = 0.03) compared to the stable TIBC group. Hence, low baseline serum TIBC is associated with iron deficiency, PEW, inflammation, poor QoL and mortality, and its decline over time is independently associated with increased death risk.
doi:10.1159/000191470
PMCID: PMC2818472  PMID: 19136818
Transferrin; Total iron-binding capacity; Chronic kidney disease; Hemodialysis; Protein-energy wasting
18.  Increased Cardiovascular Risk Associated with Reduced Kidney Function 
American Journal of Nephrology  2009;29(6):620-625.
Background
Individuals with chronic kidney disease (CKD) are at substantial risk for cardiovascular mortality, but the risk associated with specific glomerular filtration rates (GFRs) is unknown. The objective of this study was to investigate the relationship between level of kidney function and the risk of cardiovascular mortality in a diverse population.
Methods and Results
This was a nonconcurrent cohort study of 34,982 ambulatory patients. Kidney function was entered into the model as a time-dependent variable to minimize misclassification and allow for improved estimate of the effect of decreasing GFR on cardiovascular mortality. The adjusted hazard ratio for cardiovascular mortality was 1.00 (95% CI 0.93–1.06) with an estimated GFR (eGFR) of 45–59; 1.77 (95% CI 1.65–1.89) with an eGFR 30–44; 3.75 (95% CI 3.47–4.06) with an eGFR 15–29, and 3.83 (95% CI 3.40–4.33) with an eGFR <15.
Conclusion
We demonstrate a graded risk of cardiovascular mortality with decreasing GFR, with a marked increase with an eGFR <45 ml/min/1.73 m2. These data also suggest that the availability of eGFR to physicians has had little impact on reducing the cardiovascular risk facing individuals with CKD. Our findings further highlight the public health significance of CKD and the importance of its early identification and management to reduce cardiovascular mortality.
doi:10.1159/000194455
PMCID: PMC2818473  PMID: 19151549
Chronic kidney disease; Glomerular filtration rate; Cardiovascular mortality
19.  Polymorphisms in the Nonmuscle Myosin Heavy Chain 9 Gene (MYH9) Are Associated with Albuminuria in Hypertensive African Americans: The HyperGEN Study 
American journal of nephrology  2009;29(6):626-632.
Background
MYH9 is a podocyte-expressed gene encoding nonmuscle myosin IIA that is associated with idiopathic and human immunodeficiency virus-associated focal segmental glomerulosclerosis (FSGS) and hypertensive end-stage renal disease in African Americans.
Methods
Four single nucleotide polymorphisms comprising the major MYH9 E1 risk haplotype were tested for association with estimated glomerular filtration rate (eGFR) and urine albumin:creatinine ratio (ACR) in 2,903 HyperGEN participants (1,458 African Americans (AA) in 895 families and 1,445 European Americans (EA) in 859 families) to determine the role of MYH9 in subclinical nephropathy. Association analyses employed general linear models in unrelated probands and generalized estimating equations in families. Adjustment was performed for age, sex, diabetes, BMI, medications, and mean arterial pressure separately in each race.
Results
Mean (SD) eGFR and ACR were 74.3 (16.0) ml/min/1.73 m2 and 20.3 (119.9) mg/g in EA, and 88.6 (20.9) ml/min/1.73 m2 and 76.8 (394.5) mg/g in AA (both p < 0.0001 across ethnicities). Urine ACR was associated with rs3752462 (p = 0.01) and rs4821481 (p = 0.05) in unrelated AA and with rs4821481 (p = 0.03), rs2032487 (p = 0.04) and the E1 3224 haplotype (p = 0.013) in AA families. Single nucleotide polymorphisms and the haplotype were not associated with ACR in EA or with eGFR in either ethnic group.
Conclusions
MYH9 variants are associated with albuminuria in hypertensive AA. The strength of the association was weaker than that in FSGS and hypertensive end-stage renal disease. MYH9 risk variants appear to be associated with primary FSGS with secondary hypertension, although nephrosclerosis may develop in response to hypertension in subjects homozygous for the MYH9 E1 risk haplotype.
doi:10.1159/000194791
PMCID: PMC2749685  PMID: 19153477
African Americans; Albuminuria; Chronic kidney disease; Essential hypertension; HyperGEN study; MYH9 gene
20.  Home Blood Pressure Measurements for Managing Hypertension in Hemodialysis Patients 
American Journal of Nephrology  2009;30(2):126-134.
Home blood pressure (BP) monitoring serves as a practical method to detect changes in BP instead of ambulatory BP monitoring in hemodialysis patients. To evaluate the relationship of reduction in home BP compared to interdialytic ambulatory BP measurements we analyzed the data from the dry-weight reduction in hypertensive hemodialysis patients (DRIP) trial in which 100 patients had their dry weight probed based on clinical sign and symptoms and 50 patients served as controls. We measured home BP 3 times a day for 1 week using a validated oscillometric monitor on 3 occasions at 4-week intervals after randomization. Changes from baseline in home, predialysis BP and postdialysis BP were compared to interdialytic 44-hour ambulatory BP. Home and ambulatory BP monitoring was available in 141 of 150 (94%) patients. Predialysis systolic BP was not as sensitive as ambulatory BP in detecting change in BP with dry-weight reduction. Whereas postdialysis BP was capable of detecting an improvement in systolic BP in response to probing dry weight, by itself it does not provide evidence that change in postdialysis BP persists over the interdialytic period. Home BP reliably detected changes in ambulatory BP, albeit with less sensitivity at 4 weeks. However, at 4 and at 8 weeks, changes in home systolic BP were most strongly related to changes in interdialytic ambulatory systolic BP compared to predialysis and postdialysis BP. The reproducibility of BP measurements followed the order home > ambulatory >> predialysis > postdialysis. These data provide support for the use of home BP monitoring for the management of hypertension in hemodialysis patients.
doi:10.1159/000206698
PMCID: PMC2786027  PMID: 19246891
Home blood pressure monitoring; Ambulatory blood pressure monitoring; Hemodialysis; Hypertension
21.  Low Aerobic Capacity and High-Fat Diet Contribute to Oxidative Stress and IRS-1 Degradation in the Kidney 
American Journal of Nephrology  2009;30(2):112-119.
Background/Aims
Insulin receptor (IR-α and IR-β) is reduced in the kidney of insulin-resistant rodents. It is unknown if there are also reductions in insulin receptor substrate (IRS)-1 or if these effects are due to metabolic injury. Thereby, we hypothesized intrinsically high aerobic fitness would protect against high-fat diet (HFD)-induced reactive oxygen species (ROS) and IRS-1 degradation.
Methods
We investigated the effects of HFD on triglyceride content, ROS production and IRS-1 degradation in the kidney of high-capacity (HCR)/low-capacity (LCR) rats, a model of intrinsic high and low aerobic capacity. Eighteen-week-old HCR and LCR rats were placed on a HFD or normal chow diet for 7 weeks. Intraperitoneal glucose tolerance, ROS, IR-β, total IRS-1 and ubiquitination were measured.
Results
The HCR displayed greater insulin sensitivity and were resistant to HFD-induced insulin resistance. In the LCR kidney, HFD increased ROS potential, and reduced total IR-β and IRS-1 without altering triacylglycerol content. IRS-1 ubiquitination was higher in the LCR than HCR kidney, increased after HFD.
Conclusions
Our data support that HFD-mediated kidney ROS is associated with reductions in IRS-1 and systemic insulin resistance. Further, high intrinsic aerobic capacity protects against IRS-1 degradation in the kidney following exposure to HFD.
doi:10.1159/000204362
PMCID: PMC2786026  PMID: 19229113
IRS-1; Reactive oxygen species; Aerobic capacity; Insulin resistance
22.  High Ambient Glucose Augments Angiotensin II-Induced Proinflammatory Gene mRNA Expression in Human Mesangial Cells: Effects of Valsartan and Simvastatin 
American Journal of Nephrology  2009;30(2):99-111.
Background
Hyperglycemia may potentiate the adverse renal effects of angiotensin II (AII). In the kidney, the major target of AII action is the glomerular mesangial cell, where its hemodynamic and proinflammatory action contributes to renal injury. AII action is mediated by several types of cell receptors. Among those, the AT1 receptor has been best studied using specific AII receptor blockers (ARBs). These agents have emerged as major new modalities in the prevention and amelioration of renal disease where the ARB renoprotective anti-inflammatory properties could be more important than previously appreciated. Like the ARBs, statins may also modulate inflammatory responses that are renoprotective and complement their cholesterol-lowering effects.
Aim
The aim of this project was to (i) identify a repertoire of proinflammatory mesangial cell AII-inducible mRNAs; (ii) determine if the AII-induced proinflammatory mRNA responses depend on ambient glucose, and (iii) test the anti-inflammatory effectiveness of an ARB, valsartan, either alone or in combination with a statin, simvastatin.
Results/Conclusions
Using high-density microarrays and real-time PCR we identified several AII-inducible proinflammatory mesangial genes that exhibited augmented mRNA responses in high-glucose milieu. Valsartan blocked the AII-induced mRNA expression of proinflammatory genes (i.e. MCP-1, LIF and COX-2) maintained in normal and high glucose. These observations add to the mounting evidence that ARBs have anti-inflammatory effects in the kidney, a beneficial effect that may be more important in protecting renal function in diabetic patients. While simvastatin inhibited expression of some mRNAs encoding chemokines/cytokines, it enhanced expression of mRNA encoding COX-2, a key mediator of inflammation. Thus, the non-cholesterol effects of statins on inflammatory responses appear complex.
doi:10.1159/000203619
PMCID: PMC2786028  PMID: 19225232
Angiotensin II; Hyperglycemia; Glomerular mesangial cell; Proinflammatory action
23.  Treatment of Established Peritoneal Fibrosis by Gene Transfer of Smad7 in a Rat Model of Peritoneal Dialysis 
American Journal of Nephrology  2009;30(1):84-94.
Background/Aims
It has been shown that blockade of TGF-β1 signaling with Smad7 prevents experimental peritoneal fibrosis. The present study investigated whether Smad7 has a therapeutic effect on established peritoneal fibrosis associated with peritoneal dialysis (PD).
Methods
A rat model of peritoneal fibrosis was induced by a daily intraperitoneal infusion of 4.25% Dianeal. After peritoneal fibrosis had been established on day 14, groups of 6 rats were treated intraperitoneally with gene transfer of Smad7 or control plasmids using an ultrasound-microbubble-mediated system for 2 weeks until day 28. In addition, a group of 6 diseased rats was euthanized on day 14 before treatment as the basal disease control.
Results
Compared to the control-treatment animals on day 28, real-time PCR, Western blot, and confocal microscopy revealed that Smad7 gene transfer significantly attenuated the increased peritoneal fibrosis including the thickening of fibrotic peritoneum, accumulation of α-SMA and collagen I, and an improvement in peritoneal dysfunction (all p < 0.05). Importantly, Smad7 treatment also improved the severity of peritoneal fibrosis and functional impairment when compared to those on day 14 before treatment (all p < 0.05). Inhibition of the established peritoneal fibrosis by Smad7 was associated with an abrogation of TGF-β signaling and upregulation of TGF-β1 and PAI-1.
Conclusions
Smad7 gene therapy is able to inhibit established peritoneal fibrosis in a rat model of PD. Results from this study suggest that Smad7 may be a therapeutic agent for the treatment of peritoneal fibrosis associated with PD.
doi:10.1159/000203362
PMCID: PMC2786025  PMID: 19223683
Gene therapy; Smad7; Peritoneal fibrosis; Peritoneal dialysis; TGF-β1; Ultrasound

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