Role of TASK2 Potassium Channels Regarding Volume Regulation in Primary Cultures of Mouse Proximal Tubules

Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wild-type and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using β-galactosidase staining. TASK2 was only localized in PCT cells. K+ currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K+ currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 μM 293B, but blocked by 500 μM quinidine and 10 μM clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K+ currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K+ channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules

In vivo Cre/loxP mediated recombination in mouse Clara cells.

In small airways, Clara cells are the main epithelial cell type and play an important physiological role in surfactant production, protection against environmental agents, regulation of inflammatory and immune responses in the respiratory system. Thus, Clara cells are involved in lung homeostasis and pathologies like asthma, Chronic Obstructive Pulmonary Diseases (COPD) or cancers. To date, Clara cells implication in these pathological processes remains largely enigmatic. The engineering of a transgenic strain mouse allowing specific gene invalidation in Clara cells may be of interest to improve our knowledge about the genes involved in these diseases. By using the Cre/loxP strategy we report the engineering of a transgenic mouse strain with expression of Cre recombinase under the control of the Clara Cell Secretory Protein (CCSP) promoter. Specific staining and immuno-histochemistry performed after breeding with reporter mice revealed that CCSP drives a functional Cre expression specifically in Clara cells. This mouse strain is a powerful tool for Cre-loxP-mediated conditional recombination in the lung and represents a new tool to study Clara cell physiology

Specific Cre/Lox recombination in the mouse proximal tubule.

The present work reports for the first time the construction of a transgenic mouse strain with specific expression of Cre recombinase in the kidney proximal tubule. A Cre/loxP strategy was developed using sglt2 promoter to drive Cre recombinase expression in transgenic mice. The mouse sglt2 5' region consisting of the first exon, the first intron, and part of the second exon was cloned upstream of a nucleotide sequence encoding the Cre recombinase. Transgenic mice were generated by pronuclear injection, and tissue specificity of Cre expression was analyzed using reverse transcription-PCR. The iL1-sglt2-Cre mouse line scored positive for kidney transcription of Cre but not for the other tissues analyzed. Within the kidney, Cre transcripts were demonstrated to be restricted to the proximal tubule only. iL1-sglt2-Cre mice were bred with ROSA26-LacZ reporter mice that contained a loxP-flanked stop sequence upstream of the LacZ gene. X-gal staining and immunohistochemistry using specific antibodies (anti-megalin, anti-Tamm-Horsfall, anti-NaCl co-transporter, and anti-aquaporin 2) revealed that sglt2 drives Cre functional expression specifically in proximal tubules. The iL1-sglt2-Cre mouse therefore represents a powerful tool for Cre-LoxP-mediated conditional expression in the renal proximal tubule

CFTR-dependent and -independent swelling-activated K+ currents in primary cultures of mouse nephron.

The role of CFTR in the control of K(+) currents was studied in mouse kidney. Whole cell clamp was used to identify K(+) currents on the basis of pharmacological sensitivities in primary cultures of proximal (PCT) and distal convoluted tubule (DCT) and cortical collecting tubule (CCT) from wild-type (WT) and CFTR knockout (KO) mice. In DCT and CCT cells, forskolin activated a 293B-sensitive K(+) current in WT, but not in KO, mice. In these cells, a hypotonic shock induced K(+) currents blocked by charybdotoxin in WT, but not in KO, mice. In PCT cells from WT and KO mice, the hypotonicity-induced K(+) currents were insensitive to these toxins and were activated at extracellular pH 8.0 and inhibited at pH 6.0, suggesting that the corresponding channel was TASK2. In conclusion, CFTR is implicated in the control of KCNQ1 and Ca(2+)-sensitive swelling-activated K(+) conductances in DCT and CCT, but not in proximal convoluted tubule, cells. In KO mice, impairment of the regulatory volume decrease process in DCT and CCT could be due to the loss of an autocrine mechanism, implicating ATP and adenosine, which controls swelling-activated Cl(-) and K(+) channels