Borgards, L.; Voss, H. L.; Tautges, S.; Siebels, B.; Pylaeva, E.; Spangenberg, P.; Siemes, D.; Krisp, C.; Schmitz, J.; Cinkul, L.; Damilola, A.; Hilger, A.; Szymczak, P.; Aust, A.; Braesen, J. H.; Szczurek, E.; de la Fuente-Nunez, C.; Jablonska, J.; Soehnlein, O.; Dobrindt, U.; Schlueter, H.; von Vietinghoff, S.; Wagenlehner, F.; Engel, D. R.; Shevchuk, O.

Antimicrobial peptides (AMPs) are key effectors of host defence, however, their functional deployment across renal tissue and urine in pyelonephritis (PN) remains incompletely understood. Here, we integrate kidney and urine proteomics with urinary peptidomics and computational prediction to define AMP organisation and function. Proteomic analysis indicated coordinated induction of multiple AMPs in infected kidneys. These patterns were recapitulated in the urinary proteome, where AMP abundance correlated with leukocyte counts. Multiplex immunofluorescence microscopy localised these AMPs to myeloid cells, identifying them as central effector sources. Importantly, analysis of the urine peptidome revealed multiple encrypted AMPs (EPs), which arise from proteolytic processing of precursor proteins. To systematically assess their relevance for host defence, we applied an ensemble of machine learning-based predictors to prioritise candidates with activity in the urinary environment. This approach identified several potential EPs, among which the S100A12-derived peptide Calcitermin was confirmed in patient urine. Furthermore, it exerts antibacterial activity against uropathogenic E. coli (UPEC) and modulates myeloid cell responses. Together, these findings define a coordinated and compartmentalised AMP defence programme in human PN that extends beyond increased peptide expression, highlighting EPs as functionally relevant effectors with therapeutic potential.