Laminin Human ELISA Kit

Background: Angiotensin-II (Ang II) mediates progression of autosomal-dominant polycystic kidney disease (ADPKD) and other chronic kidney diseases (CKD). However, markers of kidney Ang II activity are lacking. We previously defined 83 Ang II-regulated proteins in vitro, which reflected kidney Ang II activity in vivo. Methods: In this study, we developed selected reaction monitoring (SRM) assays for quantification of Ang II-regulated proteins in urine of ADPKD and CKD patients. We demonstrated that 47 of 83 Ang II-regulated transcripts were differentially expressed in cystic compared to normal kidney tissue. We then developed SRM assays for 18 Ang II-regulated proteins overexpressed in cysts and/or secreted in urine. Methods that yielded CV <= 6 % for control proteins, and recovery similar to 100 % were selected. Heavy-labeled peptides corresponding to 13 identified Ang II-regulated peptides were spiked into urine samples of 17 ADPKD patients, 9 patients with CKD predicted to have high kidney Ang II activity and 11 healthy subjects. Samples were then digested and analyzed on triple-quadrupole mass spectrometer in duplicates. Resluts: Calibration curves demonstrated linearity (R-2 > 0.99) and within-run CVs < 9 % in the concentration range of 7/13 peptides. Peptide concentrations were normalized by urine creatinine. Deamidated peptide forms were monitored, and accounted for < 15 % of the final concentrations. Urine excretion rates of proteins BST1, LAMB2, LYPA1, RHOB and TSP1 were significantly different (p < 0.05, one-way ANOVA) between patients with CKD, those with ADPKD and healthy controls. Urine protein excretion rates were highest in CKD patients and lowest in ADPKD patients. Univariate analysis demonstrated significant association between urine protein excretion rates of most proteins and disease group (p < 0.05, ANOVA) as well as sex (p < 0.05, unpaired t test). Multivariate analysis across protein concentration, age and sex demonstrated good separation between ADPKD and CKD patients. Conclusions: We have optimized methods for quantification of Ang II-regulated proteins, and we demonstrated that they reflected differences in underlying kidney disease in this pilot study. High urine excretion of Ang II-regulated proteins in CKD patients likely reflects high kidney Ang II activity. Low excretion in ADPKD appears related to lack of communication between cysts and tubules. Future studies will determine whether urine excretion rate of Ang II-regulated proteins correlates with kidney Ang II activity in larger cohorts of chronic kidney disease patients.

BACKGROUND: Epigenetic variability is hypothesized as a regulatory pathway through which prenatal exposures may influence child development and health. OBJECTIVE: We sought to examine the associations of residential proximity to roadways at birth and epigenome-wide DNA methylation. We also assessed associations of differential methylation with child cognitive outcomes. METHODS: We estimated residential proximity to roadways at birth using a geographic information system (GIS) and cord blood methylation using Illumina's HumanMethylation450-array in 482 mother child pairs in Project Viva. We identified individual CpGs associated with residential proximity-to-roadways at birth using robust linear regression [false discover), rate (FDR) <0.05]. We also estimated association between proximity-to-roadways at birth and methylation of the same sites in blood samples collected at age 7-11 y = 415). We ran the same analyses in the Generation R Study for replication (N =641). In Project Viva, we investigated associations of differential methylation at birth with midchildhood cognition using linear regression. RESULTS: Living closer to major roadways at birth was associated with higher cord blood (and-more weakly-midchildhood blood) methylation of four sites in LAMB2. For each halving of residential-proximity-to-major-roadways, we observed a 0.82% increase in DNA methylation at cg05654765 [95% confidence interval (CI): (0.54%, 1.10%)], 0.88% at cg14099457 [95% CI: (0.56%, 1.19%)], 0.19% at cg03732535 [95% CI: (0.11%, 0.28)], and 1.08% at cg02954987 [95% CI: (0.65%, 1.51%)]. Higher cord blood methylation of these sites was associated with lower midchildhood nonverbal cognitive scores. Our results did not replicate in the Generation R Study. CONCLUSIONS: Our discovery results must be interpreted with caution, given that they were not replicated in a separate cohort. However, living close to major roadways at birth was associated with cord blood methylation of sites in LAMB2 a gene known to he linked to axonal development-in our U.S. cohort. Higher methylation of these sites associated with lower nonverbal cognitive scores at age 7-11 y in the same children.