DNPEP is not the only peptidase that produces SPAK fragments in kidney.

SPAK (STE20/SPS1-related proline/alanine-rich kinase) regulates Na+ and Cl- reabsorption in the distal convoluted tubule, and possibly in the thick ascending limb of Henle. This kinase phosphorylates and activates the apical Na-Cl cotransporter in the DCT. Western blot analysis reveals that SPAK in kidney exists as a full-length protein as well as shorter fragments that might affect NKCC2 function in the TAL. Recently, we showed that kidney lysates exerts proteolytic activity towards SPAK, resulting in the formation of multiple SPAK fragments with possible inhibitory effects on the kinase. The proteolytic activity is mediated by a Zn2+ metalloprotease inhibited by 1,10-phenanthroline, DTT, and EDTA. Size exclusion chromatography demonstrated that the protease was a high-molecular-weight protein. Protein identification by mass-spectrometry analysis after ion exchange and size exclusion chromatography identified multiple proteases as possible candidates and aspartyl aminopeptidase, DNPEP, shared all the properties of the kidney lysate activity. Furthermore, recombinant GST-DNPEP produced similar proteolytic pattern. No mouse knockout model was, however, available to be used as negative control. In this study, we used a DNPEP-mutant mouse generated by EUCOMM as well as a novel CRISPR/cas9 mouse knockout to assess the activity of their kidney lysates towards SPAK. Two mouse models had to be used because different anti-DNPEP antibodies provided conflicting data on whether the EUCOMM mouse resulted in a true knockout. We show that in the absence of DNPEP, the kidney lysates retain their ability to cleave SPAK, indicating that DNPEP might have been misidentified as the protease behind the kidney lysate activity, or that the aspartyl aminopeptidase might not be the only protease cleaving SPAK in kidney.