Development of an AT2-deficient proximal tubule cell line for transport studies

Angiotensin II is a major regulatory peptide for proximal tubule Na+ reabsorption acting through two distinct receptor subtypes: AT1 and AT2. Physiological or pathological roles of AT2 have been difficult to unravel because angiotensin II can affect Na+ transport either directly via AT2 on luminal or peritubular plasma membranes of proximal tubule cells or indirectly via the renal vasculature. Furthermore, separate systemic and intratubular renin–angiotensin systems impart considerable complexity to angiotensin’s regulation. A transport-competent, proximal tubule cell model that lacks AT2 is a potentially useful tool to assess cellular angiotensin II regulation. To this end, AT2-receptor-deficient mice were bred with an Immortomouse®, which harbors the thermolabile immortalization gene SV40 large-T antigen (Tag), and AT2-receptor-deficient [AT2 (−/−)], Tag heterozygous [Tag (+/−)] F2 offspring were selected for cell line generation. S1 proximal tubule segments were microdissected, and epithelial cell outgrowth was expanded in culture. Cells that formed confluent, electrically resistive monolayers were selected for cryopreservation, and one isolate was extensively characterized for conductance (2 mS/cm2), short-circuit current (Isc; 0.2 μA/cm2), and proximal tubule-specific \( \operatorname{Na} ^{ + }_{3} - \operatorname{succinate} \) (ΔIsc = 0.8 μA/cm2 at 2 mM succinate) and \( \operatorname{Na} ^{ + }_{3} - \operatorname{phosphate} \;cotransport \) (ΔIsc = 3 μA/cm2 at 1 mM phosphate). Light microscopy showed a uniform, cobblestone-shaped monolayer with prominent cilia and brush borders. AT2 receptor functionality, as demonstrated by angiotensin II inhibition of ANF-stimulated cGMP synthesis, was absent in AT2-deficient cells but prominent in wild-type cells. This transport competent cell line in conjunction with corresponding wild type and AT1-deficient lines should help explain angiotensin II signaling relevant to Na+ transport.