The anti-inflammatory peptide Ac-SDKP is released from thymosin-4 by renal meprin-␣ and prolyl oligopeptidase. Am J Physiol Renal Physiol 310: F1026 -F1034, 2016. First published March 9, 2016 doi:10.1152/ajprenal.00562.2015.-N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural tetrapeptide with antiinflammatory and antifibrotic properties. Previously, we have shown that prolyl oligopeptidase (POP) is involved in the Ac-SDKP release from thymosin-4 (T4). However, POP can only hydrolyze peptides shorter than 30 amino acids, and T4 is 43 amino acids long. This indicates that before POP hydrolysis takes place, T4 is hydrolyzed by another peptidase that releases NH2-terminal intermediate peptide(s) with fewer than 30 amino acids. Our peptidase database search pointed out meprin-␣ metalloprotease as a potential candidate. Therefore, we hypothesized that, prior to POP hydrolysis, T4 is hydrolyzed by meprin-␣. In vitro, we found that the incubation of T4 with both meprin-␣ and POP released Ac-SDKP, whereas no Ac-SDKP was released when T4 was incubated with either meprin-␣ or POP alone. Incubation of T4 with rat kidney homogenates significantly released Ac-SDKP, which was blocked by the meprin-␣ inhibitor actinonin. In addition, kidneys from meprin-␣ knockout (KO) mice showed significantly lower basal Ac-SDKP amount, compared with wild-type mice. Kidney homogenates from meprin-␣ KO mice failed to release Ac-SDKP from T4. In vivo, we observed that rats treated with the ACE inhibitor captopril increased plasma concentrations of Ac-SDKP, which was inhibited by the coadministration of actinonin (vehicle, 3.1 Ϯ 0.2 nmol/l; captopril, 15.1 Ϯ 0.7 nmol/l; captopril ϩ actinonin, 6.1 Ϯ 0.3 nmol/l; P Ͻ 0.005). Similar results were obtained with urinary Ac-SDKP after actinonin treatment. We conclude that release of Ac-SDKP from T4 is mediated by successive hydrolysis involving meprin-␣ and POP.N-acetyl-seryl-aspartyl-lysyl-proline; thymosin-4; meprin-␣; prolyl oligopeptidase; angiotensin-converting enzyme THE ANTI-INFLAMMATORY PEPTIDE N-acetyl-seryl-aspartyl-lysylproline (Ac-SDKP) was identified originally in the bone marrow (24). It has also been widely distributed in mammalian organs, plasma, urine, and circulating mononuclear cells (23,39,46). In animal diseases with hypertension, as well as cardiovascular and kidney disease, Ac-SDKP reduces inflammatory cell infiltration and collagen deposition in the heart, kidney, lung, and liver (14,15,33,34,37,44). Ac-SDKP is hydrolyzed by angiotensin-converting enzyme (ACE) and has a short half-life of 4.5 min in the circulation (16). Treatment with ACE inhibitors significantly increased plasma concentration of Ac-SDKP (2), and some of the ACE inhibitors protective effects were reported to be mediated by the increases in Ac-SDKP concentrations (34, 35).Thymosin-4 (T4) is a major G-actin-sequestering peptide that consists of 43 amino acids. Since it contains the Ac-SDKP sequence in its NH 2 -terminal, T4 is considered to be the precursor of 39). Our group has shown p...