Single cell RT-PCR analysis of ClC-2 mRNA expression in ureteric bud tip

Area of Science:

• Developmental Biology
• Molecular Physiology
• Renal Physiology

Background:

• Embryonic epithelia in the ureteric bud (UB) are crucial for kidney development through cell signaling.
• Understanding the membrane conductive proteins in these cells is essential for elucidating early nephrogenesis.

Purpose of the Study:

• To characterize the membrane conductive proteins of embryonic rat ureteric bud tip cells.
• To investigate the role of specific chloride channels, particularly ClC-2, in early kidney development.

Main Methods:

• Patch-clamp electrophysiology was used to measure whole-cell conductance in microdissected embryonic rat UB tip cells (embryonic day 17).
• Single-cell reverse transcription polymerase chain reaction (RT-PCR) was employed to detect the expression of ClC-2 chloride channel mRNA.
• Cytoplasmic harvesting during patch-clamp allowed for simultaneous electrophysiological recording and molecular analysis.

Main Results:

• UB tip cells displayed high whole-cell conductance, with a significant fraction resembling Ca-activated Cl channels.
• A distinct Cl-selective current, suggestive of ClC-2, was identified, activating upon hyperpolarization.
• ClC-2 mRNA was detected in a subset of single UB tip cells, with evidence of alternative splicing.

Conclusions:

• Embryonic UB tip cells possess a distinct electrophysiological phenotype compared to mature kidney cells.
• The ClC-2 chloride channel is likely involved in the critical processes of early nephrogenesis.