Rh proteins mediated ammonia excretion in the skin of zebrafish larvae: evidence of active ammonia transport by ionocytes

Abstract

Rhesus (Rh) glycoproteins, Rhbg and Rhcg play a critical role in ammonia transport in mammalian renal collecting ducts. In the model animal, zebrafish, Rhcg1was found in the apical membrane of skin ionocytes (H+‐ATPase‐rich cells; HR cells) which is similar to α‐type intercalated cells in mammalian collecting ducts. However, the cellular distribution and role of Rhbg in zebrafish larvae has not been well investigated. In this study, Rhbg was localized to both apical and basolateral membrane of skin keratinocytes by using in situ hybridization and immunohistochemistry. A scanning ion‐selective electrode technique (SIET) was applied to measure NH4+ flux at the apical surface of keratinocytes and HR cells in intact larvae. Knockdown of Rhbg with morpholino oligonucleotides suppressed ammonia excretion by keratinocytes, and induced compensatory ammonia excretion by HR cells. To test if HR cells can excrete ammonia against higher external ammonia level comparing to keratinocyte, “reversal concentration (RC)” of ammonia excretion was determined by measuring zero NH4+ fluxes in a serial concentration of external NH4+. Results showed that the RC of HR cells was higher than that of keratinocytes demonstrating that HR cells can excrete ammonia against higher external ammonia levels. Either knockdown the expression of Rhcg1 or H+‐ATPase in HR cells suppressed the RC of HR cells to the level of keratinocytes, suggesting that Rhcg1 and H+‐ATPase are involved in the active ammonia excretion of HR cells.