Renal ion transporters are critical to maintain fluid and electrolyte homeostasis and are primary targets for (patho)physiology studies and drug discovery. Transporter abundance and covalent modifications are determined by immunoblots. Our standard protocol is quick kidney removal and homogenization in buffer containing 0.2 mM PMSF, 0.9 ug/ml aprotinin, and Sigma P0044 phosphatase inhibitors followed by isolation of a low speed supernatant (the homogenate). Collaborations with other labs requires shipping frozen kidneys. We tested whether flash freezing (FZ) Sprague Dawley (SD) kidneys impacts ion transporter recovery compared to quick removal without freezing (QR) defined as 100% recovery; assessed by immunoblot with specific antibodies. FZ and ~ 3 d storage before homogenization in inhibitors significantly reduced recoveries of proximal tubule (PT) NHE3 and megalin to 20% and Cldn2 to 40%. Medullary NHE3 and NKCC2 remained at 100%, DCT/CD proteins were reduced ~ 10-60% after FZ vs. QR. Renal perfusion pre-FZ further reduced recovery of all transporters vs. non-perfused kidneys. To test whether Fz activates proteases not inhibited in our standard buffer, Roche protease inhibitor (minitabs) were added to homogenization buffer. Adding minitabs to FZ kidneys increased NHE3 recovery to 85% and Cldn2 recovery to 100% of QR, NKCC2 and ENaC-beta to 85% and NCC to100%. Lastly, we tested, in male and female (M,F) SD rats, whether minitab addition to QR renal cortex homogenates followed by Fz prevents proteolysis vs. minitab addition to QR homog without Fz. In F, minitab addition with Fz preserved 100% recovery of transporters: NHE3, Cldn2, AQP1, megalin, NKCC2, NCC, ENaC-beta, Cldn-7 and AQP2. In M, minitab addition with Fz preserved 100% recovery of only AQP1, villin, Cldn7 and AQP2; all other M transporters exhibited ~40% less recovery. In conclusion, flash freezing kidney tissue differentially activates proteolysis of ion transporters - up to 80% in cortex and not at all in medulla - despite inclusion of standard inhibitors in homogenizing buffers. Strong sex differences are evident: addition of minitabs prevents proteolysis of F not M cortical ion transporters. Single cell ‘omics approaches may be useful to identify tubule and sex specific proteases. NIH: DK083785, DK134695. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.