Ex Vivo Quantification of pH i in Drosophila Malpighian Tubule Principal Cells Reveals Basolateral H + Equivalent—Coupled Oxalate Transport through Likely Slc26a1 Ortholog

Nephrolithiasis is a painful and costly healthcare complication. The most common kidney stones are composed of calcium oxalate and thus renal handling of oxalate is an important facet of understanding the pathogenesis of nephrolithiasis. Recently, the Drosophila melanogaster Malpighian tubule (MT) has emerged as a robust model of trans‐epithelial ion transport and nephrolithiasis as MTs readily form luminal calcium‐oxalate crystals in the presence of oxalate. Drosophila Prestin (dPrestin, Slc26a6) transports oxalate across the apical surface of the MT into the lumen but a full model of the trans‐epithelial movement of oxalate in the Drosophila MT has been lacking as the basolateral oxalate transporter has remain uncharacterized. The objective of this work was to identify and characterize the Drosophila basolateral oxalate transporter through ex vivo real‐time quantification of intracellular pH (pH i ). pH i was monitored by using the GAL4/UAS system to selectively express pHerry, a pseudo‐ratiometric genetically‐encoded pH indictor (GEpHI) in the cytosol of the principal cells of the MT. Calibration of pHerry in extracted adult MTs yielded a p K a of 7.24. Basolateral perfusion of MTs in CO 2 /HCO 3 − ‐buffered solution produced a large acidification followed by rapid recovery in all three functionally distinct segments of the anterior MT (initial, transitional, and main). Recovery was likely due to HCO 3 − loading by HCO 3 − transporters and this process could be interrupted by basolateral application of 1mM oxalate (in the initial and transitional segments) or 1mM SO 4 2− (in all three segments). The response was most robust in the transitional segment and oxalate induced a more rapid acidification than SO 4 2− ( dpH i / dt = −0.60 ± 0.09 pH units min −1 compared to −0.06 ± 0.01). A putative ortholog of the mammalian renal basolateral oxalate transporter (SAT 1; Slc26a1) was identified through sequence homology. Tissue specific knock‐down of this protein with interference RNA (RNAi) reduced the rate of acid‐loading in the transitional segment of the MT with regard to oxalate but not SO 4 2− ( dpH i / dt = −0.34 ± 0.06 pH units min −1 ; p = 0.03 compared to controls, and −0.06 ± 0.01; p = 0.94 compared to controls for oxalate and SO 4 2− respectively). These data indicate this protein is likely the Drosophila basolateral MT oxalate transporter and the basolateral movement of oxalate is functionally coupled to movement of acid equivalents, potentially as oxalate/HCO 3 − exchange, oxalate/OH − exchange, or oxalate/H + co‐transport. Support or Funding Information Funding: T32‐DK007013 to AJR, GM075148 (JBA), DK092408 and DK100227 to MFR.