Villin and actin in the mouse kidney brush border membrane bind 1 to and are degraded by meprins : This interaction contributes to 2 injury in ischemia reperfusion 3 4

23 Meprins, metalloproteinases abundantly expressed in the brush border membranes 24 (BBMs) of rodent proximal kidney tubules, have been implicated in the pathology of 25 renal injury induced by ischemia reperfusion (IR). Disruption of the meprin β gene, and 26 actinonin, a meprin inhibitor, both decrease kidney injury resulting from IR. To date, the 27 in vivo kidney substrates for meprins are unknown. The studies herein implicate villin 28 and actin as meprin substrates. Villin and actin bind to the cytoplasmic tail of meprin β, 29 and both meprin A and B are capable of degrading villin and actin present in kidney 30 proteins as well as purified recombinant forms of these proteins. The products resulting 31 from degradation of villin and actin were unique to each meprin isoform. The meprin B 32 cleavage site in villin was Glu-Val. Recombinant forms of rat meprin B and 33 homomeric mouse meprin A had Km values for villin and actin of approximately 1 μM 34 (0.6 – 1.2 μM). The kcat values, varied substantially (0.6 – 128 sec) resulting in 35 different efficiencies for cleavage, with meprin B having the highest kcat/Km values (128 36 Ms x 10). Following IR, meprins and villin redistributed from the BBM to the cytosol. 37 A 37 kDa actin fragment was detected in protein fractions from wild-type, but not in 38 comparable preparations from meprin knockout mice. The levels of the 37 kDa actin 39 fragment were significantly higher in kidneys subjected to IR. The data establish that 40 meprins interact with and cleave villin and actin, and these cytoskeletal proteins are 41 substrates for meprins. 42 43