Anti-Laminin polyclonal antibody

Pierson syndrome is a congenital nephrotic syndrome with ocular and neurological defects caused by mutations in LAMB2, the gene encoding the basement membrane protein laminin beta 2 (Lam beta 2). It is the kidney glomerular basement membrane (GBM) that is defective in Pierson syndrome, as Lam beta 2 is a component of laminin-521 (LM-521; alpha 5 beta 2 gamma 1), the major laminin in the mature GBM. In both Pierson syndrome and the Lamb2(-/-) mouse model for this disease, laminin beta 1 (Lam beta 1), a structurally similar homolog of Lam beta 2, is marginally increased in the GBM, but it fails to fully compensate for the loss of Lam beta 2, leading to the filtration barrier defects and nephrotic syndrome. Here we generated several lines of Lam beta 1 transgenic mice and used them to show that podocyte-specific Lam beta 1 expression in Lamb2(-/-) mice abrogates the development of nephrotic syndrome, correlating with a greatly extended lifespan. In addition, the more Lam beta 1 was expressed, the less urinary albumin was excreted. Transgenic Lam beta 1 expression increased the level of Lam alpha 5 in the GBM of rescued mice, consistent with the desired increased deposition of laminin-511 (alpha 5 beta 1 gamma 1) trimers. Ultrastructural analysis revealed occasional knob-like subepithelial GBM thickening but intact podocyte foot processes in aged rescued mice. These results suggest the possibility that up-regulation of LAMB1 in podocytes, should it become achievable, would likely lessen the severity of nephrotic syndrome in patients carrying LAMB2 mutations.

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.