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1.  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.
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.
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.
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.
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.
PMCID: PMC2749685  PMID: 19153477
African Americans; Albuminuria; Chronic kidney disease; Essential hypertension; HyperGEN study; MYH9 gene
2.  Evaluation of Genetic Association and Expression Reduction of TRPC1 in the Development of Diabetic Nephropathy 
American Journal of Nephrology  2008;29(3):244-251.
The TRPC1 gene on chromosome 3q22–24 resides within the linkage region for diabetic nephropathy (DN) in type 1 (T1D) and type 2 diabetes mellitus (T2D). A recent study has demonstrated that TRPC1 expression is reduced in the kidney of diabetic ZDF- and STZ-treated rats. The present study aimed to evaluate the genetic and functional role of TRPC1 in the development of DN.
Genetic association study was performed with two independent cohorts, including 1,177 T1D European Americans with or without DN from GoKinD population and 850 African-American subjects with T2D-associated end-stage renal disease (ESRD), or with hypertensive (non-diabetic) ESRD, and nondiabetic controls. Seven tag SNP markers derived from HapMap data (phase II) were genotyped. TRPC1 gene expression was examined using real time RT-PCR.
No significant association of TRPC1 DNA polymorphisms with DN or ERSD was found in GoKinD and African-American populations. TRPC1 gene mRNA expression in kidney was found to be trendily reduced in 12-week and significantly in 26-week-old db/db mice.
TRPC1 genetic polymorphism may not fundamentally contribute to the development of DN, while reduction of the gene expression in kidney may be a late phenomenon of DN as seen in diabetic animal models.
PMCID: PMC2698220  PMID: 18802326
TRPC1 gene; Single-nucleotide polymorphism; Diabetic nephropathy; End-stage renal disease; Diabetes types 1 and 2
3.  Tetracycline-Inducible Gene Expression in Conditionally Immortalized Mouse Podocytes 
American Journal of Nephrology  2008;29(3):153-163.
Conditionally immortalized podocytes are valuable research tools but are difficult to efficiently transfect and do not provide graded transgene expression.
Conditionally immortalized mouse podocyte cell lines were established employing a tetracycline-inducible system. Glomerular cells, isolated from transgenic mice bearing two transgenes, NPHS2-reverse tetracycline-controlled transactivator, rtTA (A transgene) and H2-Kb-thermosensitive SV40 T, ts58A (I transgene), were cloned. One clone (AI podocytes) expressing WT1 and synaptopodin was transfected with pBI-EGFP (enhanced green fluorescent protein, G transgene) and separately with ptTS-Neo (transcriptional suppressor, T transgene) to produce stable transformants, AIG podocytes and AIT podocytes.
AIG podocytes expressed EGFP at 33 and 37°C after doxycycline treatment, and retained podocin and rtTA mRNA expression and temperature-sensitive growth regulation. AIT podocytes, transiently transfected with luciferase-BI-EGFP (LG transgene), showed reduced background expression of EGFP and luciferase in the absence of doxycycline. In AITLG podocytes, generated by stable transfection of AIT podocytes with the LG transgene, luciferase expression was tightly regulated by doxycycline in a time- and concentration-dependent manner both at 33 and 37°C, although background expression was not entirely eliminated. These podocytes retained temperature-sensitive growth regulation and expression of podocyte differentiation markers.
Mouse podocytes expressed tetracycline-induced transgenes efficiently while retaining differentiation markers.
PMCID: PMC2698022  PMID: 18753740
Tetracycline-inducible system; Conditional immortalization; Transcription; Gene of interest
4.  Precision of Biomarkers to Define Chronic Inflammation in CKD 
American Journal of Nephrology  2008;28(5):808-812.
Several inflammatory biomarkers have been found to be associated with cardiovascular disease or all-cause mortality in dialysis patients, but their usefulness in clinical practice or as surrogate endpoints is not certain. The purpose of the present study was to determine the intrapatient variation of C-reactive protein, IL-6, fetuin-A and albumin in a population of dialysis patients.
Apparently healthy dialysis patients with either a tunneled dialysis catheter or fistula had monthly assessments of these biomarkers for a total of four determinations, and the intraclass correlation coefficients were calculated as measures of intersubject variance.
Our results showed large within-subject variation relative to the total variation in the measurements (31–46%). Having a tunneled catheter as opposed to a fistula was not significantly associated with mean levels, suggesting that chronic subclinical catheter infection does not explain the variation seen in the biomarkers. In contrast, there was a rapid change in these biomarkers with a clinically apparent acute infection.
These results suggest that these biomarkers have limitations for use as surrogate endpoints in clinical trials due to wide fluctuations, even in apparently clinically healthy individuals.
PMCID: PMC2574778  PMID: 18506106
Biomarkers, precision; Chronic inflammation; Chronic kidney disease; CKD stage 5D; Inflammatory biomarkers, intrapatient variance; Tunneled dialysis catheter
5.  Analyzing Change: A Primer on Multilevel Models with Applications to Nephrology 
American Journal of Nephrology  2008;28(5):792-801.
The analysis of change is central to the study of kidney research. In the past 25 years, newer and more sophisticated methods for the analysis of change have been developed; however, as of yet these newer methods are underutilized in the field of kidney research. Repeated measures ANOVA is the traditional model that is easy to understand and simpler to interpret, but it may not be valid in complex real-world situations. Problems with the assumption of sphericity, unit of analysis, lack of consideration for different types of change, and missing data, in the repeated measures ANOVA context are often encountered. Multilevel modeling, a newer and more sophisticated method for the analysis of change, overcomes these limitations and provides a better framework for understanding the true nature of change. The present article provides a primer on the use of multilevel modeling to study change. An example from a clinical study is detailed and the method for implementation in SAS is provided.
PMCID: PMC2613435  PMID: 18477842
Longitudinal data analysis; Analysis of change; Change over time; Repeated measures; Multilevel modeling; Mixed effects models; Random coefficient models; Hierarchical linear models; Unit of analysis
6.  Angiotensin II Infusion Induces Nephrin Expression Changes and Podocyte Apoptosis 
American Journal of Nephrology  2008;28(3):500-507.
In in vitro studies, angiotensin (Ang) II has been demonstrated to promote podocyte apoptosis. The present study evaluates the effects of Ang II infusion in rats on podocyte nephrin expression and apoptosis and the molecular mechanisms involved in Ang II-induced proteinuria and mesangial expansion.
Sprague-Dawley rats were randomly assigned to receive either normal saline or Ang II (400 ng·kg–1·min–1) by means of a mini-osmotic pump for variable time periods. Systolic blood pressure and urinary protein and albumin excretion rate measurements were carried out on days 7, 14, 21, and 28. The animals were sacrificed on days 14 and 28 and evaluated for serum creatinine, renal pathological changes, podocyte apoptosis, renal nephrin mRNA, and protein expression.
The Ang II-infused rats developed hypertension and proteinuria. On day 14, the Ang II-infused rats showed narrowing of the slit diaphragm, an increase in podocyte nephrin mRNA and protein expression, and alterations in its distribution along the foot processes. On day 28, the Ang II-infused rats demonstrated the presence of apoptotic podocytes and decreased nephrin mRNA and protein expression. There was a negative correlation between nephrin expression and the numbers of apoptotic podocytes (r = −0.63, p < 0.05).
These results suggest that changes in nephrin expression may play a role in the pathogenesis of Ang II-induced podocyte apoptosis.
PMCID: PMC2630486  PMID: 18204248
Angiotensin II; Proteinuria; Nephrin expression; Podocyte; Apoptosis

Results 1-6 (6)