Maintaining water balance is a real challenge for amphibians in terrestrial environments. Our previous studies with toad Bombina maxima discovered a pore-forming protein and trefoil factor complex βγ-CAT, which is assembled under tight regulation depending on environmental cues. Here we report an unexpected role for βγ-CAT in toad water maintaining. Deletion of toad skin secretions, in which βγ-CAT is a major component, increased animal mortality under hypertonic stress. βγ-CAT was constitutively expressed in toad osmoregulatory organs, which was inducible under the variation of osmotic conditions. The protein induced and participated in macropinocytosis in vivo and in vitro. During extracellular hyperosmosis, βγ-CAT stimulated macropinocytosis to facilitate water import and enhanced exosomes release, which simultaneously regulated aquaporins distribution. Collectively, these findings uncovered that besides membrane-integrated aquaporin, a secretory pore-forming protein can facilitate toad water maintaining via macropinocytosis induction and exocytosis modulation, especially in responses to osmotic stress.

Animals

Toads (B. maxima) were captured in the wild and raised at room temperature by feeding with live Tenebrio molitor. Toads with an average weight of 19 ± 5 g were used in experiments after fasting in isotonic Ringer’s solution for 3 days. All the procedures and the care and handling of the animals were approved by the Institutional Animal Care and Use Committee at Kunming Institute of Zoology, Chinese Academy of Sciences (Approval ID: IACUC-OE-2021-05-001).

Animal experiment

Formulation of isotonic Ringer’s solution (111.2 mM NaCl, 1.9 mM KCl, 1.1 mM CaCl₂, 2.4 mM NaHCO₃, 1.6 mM MgCl₂) was modified based on a previous report⁴⁸. After fasting, toads in the hypertonic group were placed in hypertonic Ringer’s solution (Ringer’s solution with 222.4 mM NaCl) for 3 hours and their weight changes were recorded. Toads in the hypertonic/isotonic group were then transferred from hypertonic Ringer’s solution to isotonic Ringer’s solution for 3 hours and their weight changes were recorded. Weight changes of toads in isotonic Ringer’s solution were recorded for 6 hours as a reference, termed the isotonic group. The skin of some toads was electrically stimulated (4.5 V DC, pulse duration 10 ms) for 3 minutes to deplete skin secretions, and the animals were placed in isotonic Ringer’s solution for 30 minutes. Their survival rates were then recorded after 48 hours in isotonic and hypertonic solutions.

Cell culture

Toad epithelial cells were obtained by tissue digestion. Specifically, the UB, skin, and kidney tissue were dissected from five toads whose spinal cord had been destroyed. Tissues were further rinsed and stripped in Ringer’s solution to remove residual blood, mucus and other impurities. They were then cut into pieces and washed twice in Ringer’s solution, followed by oscillatory digestion with trypsin at room temperature for 40 minutes. The cells were separated with a 200-mesh sieve. In addition, toad peritoneal cells were extracted from the peritoneal fluid of B. maxima. All cells were centrifugally enriched with 2,000 rpm for 5 min at 4℃. The digestive cells in toad skin and UB tissues contain around 90% and 60% of epidermal cells, respectively, by flow cytometry using an anti-pan cytokeratin AE1/AE3 monoclonal antibody (Thermo Fisher Scientific, Rockford, IL, USA).