The role of claudin-2 in the proximal tubule and kidney stone disease
Curry, Joshua Nicholas
University of Kansas
Molecular & Integrative Physiology
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The concentration of circulating blood calcium is vital to the function of many cellular processes. As such, it is maintained within a narrow range through the actions of gastrointestinal, bone, kidney, and endocrine tissues. In the kidney, the first portion of the nephron, called the proximal tubule (PT), performs the majority of solute reabsorption including about two-thirds of calcium. In vivo and ex vivo studies of PTs have shown that the major component of calcium reabsorption is passive and tightly linked to sodium reabsorption. This passive transport has long been suspected to be paracellular. The precise mechanisms and molecular facilitators of calcium reabsorption in the PT, however, remain unknown. Claudins are a group of transmembrane tight junction (TJ) proteins that are vital to the regulation of paracellular transport. In the PT, claudin-2 is a highly expressed isoform. Claudin-2 increases the calcium permeability of renal epithelial cells upon overexpression. In addition, it was previously shown that mice with deletion of claudin-2 have increased urinary calcium excretion. I hypothesized that PT calcium reabsorption is facilitated by claudin-2. My overall hypothesis was tested in several ways. First, I examined the patterns of claudin expression in the proximal nephron and found that claudins-2 and -10a are expressed throughout the PT, in both convoluted and straight segments. In contrast, claudin-3 is expressed only within the proximal straight tubule (PST). Furthermore, claudin-2 and claudin-3 expression are found in separate and distinct subpopulations of thin descending limbs. Next, I used co-immunoprecipitation experiments to show that claudin-2 physically interacts with claudin-3. Then I generated renal epithelial cell lines with inducible expression of PT claudins, claudin-2 and either claudin-3 or claudin-10a. I found no effect of claudin-3 overexpression on conductance or sodium permeability (PNa), with or without claudin-2 expression. However, overexpression of both claudins-2 and -10a led to a highly conductive cell monolayer with loss of charge selectivity. The results of calcium permeability assays show that calcium transport was reduced with co-expression of claudins-2 and -10a but unchanged with co-expression of claudins-2 and -3. In order to test the potential contribution of claudin hetero-oligomerization on these permeability properties, I measured permeability of claudin-2/claudin-10a cells with titrated expression of each protein and with blockage of the claudin-2 pore. My results suggest that, rather than forming a hybrid channel with novel characteristics, claudin-2 and claudin-10a form pores in parallel. Next, I tested the hypothesis that deletion of the claudin-2 gene Cldn2 in mice causes nephrocalcinosis similar to human kidney stone disease using micro-computed tomography (micro-CT) and histological analyses. My findings indicate that this papillary pattern of nephrocalcinosis shares striking similarities to human kidney stone disease. I also examined the mechanism of hypercalciuria in these animals and found both a reduction in renal calcium reabsorption and a large increase in intestinal calcium absorption. Our colleagues subsequently identified multiple SNPs in the CLDN2 locus that associate with human kidney stone disease. Importantly, cis-acting expression quantitative trait loci (eQTL) analysis reveals that these same risk variants correlate with reduced claudin-2 mRNA expression in human tissues. My work suggests that proximal delivery of calcium to the loops of Henle is important in the pathogenesis of nephrocalcinosis and kidney stone formation. In addition, claudin-2 expression is an important mediator of calcium transport that is associated with kidney stone disease in humans. These findings have led to a better understanding of renal physiology and paracellular transport and provide a novel treatment target for disorders of calcium balance and homeostasis.
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