Claudin-12 Knockout Mice Demonstrate Reduced Proximal Tubule Calcium Permeability

Abstract

The renal proximal tubule (PT) is responsible for the reabsorption of approximately 65% of filtered calcium, primarily via a paracellular pathway. However, which protein(s) contribute this paracellular calcium pore is not known. The claudin family of tight junction proteins confers permeability properties to an epithelium. Claudin-12 is expressed in the kidney and when overexpressed in cell culture contributes paracellular calcium permeability (P_Ca). We therefore examined claudin-12 renal localization and its contribution to tubular paracellular calcium permeability. Claudin-12 null mice (KO) were generated by replacing the single coding exon with β-galactosidase from Escherichia coli. X-gal staining revealed that claudin-12 promoter activity colocalized with aquaporin-1, consistent with the expression in the PT. PTs were microperfused ex vivo and P_Ca was measured. P_Ca in PTs from KO mice was significantly reduced compared with WT mice. However, urinary calcium excretion was not different between genotypes, including those on different calcium containing diets. To assess downstream compensation, we examined renal mRNA expression. Claudin-14 expression, a blocker of P_Ca in the thick ascending limb (TAL), was reduced in the kidney of KO animals. Thus, claudin-12 is expressed in the PT, where it confers paracellular calcium permeability. In the absence of claudin-12, reduced claudin-14 expression in the TAL may compensate for reduced PT calcium reabsorption.

Keywords: calcium permeability; claudin-12; proximal tubule.

Figure 1

Claudin-12 knockout mouse model. (A) Claudin-12 gene deletion targeting strategy. (B) Relative claudin-12 mRNA expression normalized to GAPDH, from kidneys of wild type (WT) or claudin-12 knockout mice (KO). (C) Genotyping reactions of wild type (+/+), heterozygous (+/−), or claudin-12 knockout (−/−) mice using specific primers for the coding exon of claudin-12 (WT reaction) or the β-galactosidase coding sequence from E. coli (KO reaction). (D) Sequencing results of the gDNA sequence of claudin-12 WT or KO mice.

Figure 2

X-gal staining of kidney sections from wild type (A) and heterozygous (B) mice. β-galactosidase is expressed in the renal cortex in knockout kidney slices and not in wild type ones. Higher power image (C).

Figure 3

Co-staining of cortical renal tubule markers with X-gal on kidney slices from claudin-12 heterozygous mice. X-gal staining representing claudin-12 promoter expression (Cld12) co-stained with: (A) aquaporin-1 (AQP1), (B) sodium–potassium–chloride cotransporter 2 (NKCC2), (C) sodium–chloride cotransporter (NCC), or (D) carbonic anhydrase II (CAII); markers of the proximal tubule (PT), thick ascending limb (TAL), distal collecting tubule (DCT) and collecting duct respectively. The box in the right lower corner is a digitally magnified image from the smaller area depicted with the same shape.

Figure 4

Microperfused proximal tubules from claudin-12 knockout (KO) and wild type (WT) mice. (A) Representative image of a proximal tubule perfused ex vivo. (B,C) Representative recording traces from original experiments in proximal tubules from WT (B) and KO (C) animals. Basolateral addition of ouabain, and solutions containing a low sodium or high calcium concentration altered the trace by first reducing transcellular transport, and then generating diffusion potentials. (D–F) Transepithelial resistance (R_te), transepithelial voltage (V_te), and short-circuit current (I_sc) before and after the addition of ouabain.

Figure 5

Ion permeabilities in proximal tubules from claudin-12 knockout (KO) and wild type (WT) mice. (A) Sodium–chloride and calcium–sodium permeability ratios. (B) Absolute permeabilities to sodium, chloride, and calcium. *—p < 0.05.

Figure 6

Claudin-12 wild type (WT) and knockout (KO) mouse renal cortical protein expression. (A,C) Claudin-10 and (B,D) Claudin-2. * p < 0.05.

Figure 7

Claudin-12 wild type (WT) and knockout (KO) mouse renal mRNA expression of key genes involved in Ca²⁺ transport. (A) Calcium transport mediators: transient receptor potential cation channel subfamily V member 5 (TRPV5), calbindin 1 (Calb1), plasma membrane calcium ATPase 1b (PMCA1b), and calcium sensing receptor (CaSR). (B) Sodium transporters: sodium–potassium–chloride cotransporter 2 (NKCC2), sodium–chloride cotransporter (NCC), sodium-hydrogen exchanger 3 (NHE3), sodium–calcium exchanger (NCX), and epithelial sodium channel (ENaC). (C) Vitamin D related genes: vitamin D receptor (VDR), 25-hydroxyvitamin D 1 alpha hydroxylase (1aOHase), 24-hydroxylase (24aOHase), and klotho. (D) Renal claudins involved in cation transport normalized to 18S as house-keeping gene: claudins 1, 2, 3, 8, 10a, 14, 15, 16, and 19. n = 26 and 35, respectively. *—significant with Benjamini–Hochberg critical value for false discovery rate of 0.05. ° represents values that are above or below 1.5 times the interquartile range.

Figure 8

Examples of cTAL (thinner tubule) attached to the straight portion of the pars recta of the proximal tubule (thicker tubule).