SINGLE-CELL RT-PCR ANALYSIS OF CLC-2 MESSENGER-RNA EXPRESSION IN URETERAL BUD TIP

Embryonic epithelia at the tip of the ureteric bud (UB) face the inter space between epithelial and mesenchymal cells and are fundamentally i nvolved in reciprocal signaling during early nephrogenesis. To charact erize their membrane conductive proteins, patch-clamp and single cell RT-PCR techniques were applied to embryonic rat UBs [embryonic day 17 (day E17)] microdissected from the outer cortex. Cells at the UB tip h ad a high whole cell conductance (14 +/- 2 nS/10 pF, n = 8). The main fractional conductance resembled that of Ca-activated Cl channels in n onepithelial cells, with its time-dependent activation at depolarizing and inactivation at hyperpolarizing voltages. A second Cl-selective c urrent fraction, by contrast, activated slowly during strong hyperpola rization, suggestive of a ClC-2-mediated conductance. To determine the origin of this current, cytoplasm was harvested into the patch pipett e, RNA was reverse transcribed, and cDNA encoding the glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) housekeeper gene or the ClC-2 Cl chan nel was amplified by polymerase chain reaction (PCR). GAPDH and ClC-2 PCR products were identified in 23 and 8 tout of a total of 57) single cell cDNA samples, respectively. ClC-2 PCR products with two differen t lengths were obtained, which might be due to two alternatively splic ed ClC-2 mRNA isoforms. This first and combined approach by patch-clam p and single cell RT-PCR techniques to embryonic epithelia indicates t hat 1) cells at the UB tip express a phenotype remarkably different fr om that of postembryonic collecting duct principal cells and that 2) C lC-2 is likely to have a key role in early nephrogenesis.