Meprins, metalloproteinases abundantly expressed in the brush-border membranes (BBMs) of rodent proximal kidney tubules, have been implicated in the pathology of renal injury induced by ischemia-reperfusion (IR). Disruption of the meprin β gene and actinonin, a meprin inhibitor, both decrease kidney injury resulting from IR. To date, the in vivo kidney substrates for meprins are unknown. The studies herein implicate villin and actin as meprin substrates. Villin and actin bind to the cytoplasmic tail of meprin β, and both meprin A and B are capable of degrading villin and actin present in kidney proteins as well as purified recombinant forms of these proteins. The products resulting from degradation of villin and actin were unique to each meprin isoform. The meprin B cleavage site in villin was Glu(744)-Val(745). Recombinant forms of rat meprin B and homomeric mouse meprin A had K(m) values for villin and actin of ∼1 μM (0.6-1.2 μM). The k(cat) values varied substantially (0.6-128 s(-1)), resulting in different efficiencies for cleavage, with meprin B having the highest k(cat)/K(m) values (128 M(-1)·s(-1) × 10(6)). Following IR, meprins and villin redistributed from the BBM to the cytosol. A 37-kDa actin fragment was detected in protein fractions from wild-type, but not in comparable preparations from meprin knockout mice. The levels of the 37-kDa actin fragment were significantly higher in kidneys subjected to IR. The data establish that meprins interact with and cleave villin and actin, and these cytoskeletal proteins are substrates for meprins.

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 (T␤4). However, POP can only hydrolyze peptides shorter than 30 amino acids, and T␤4 is 43 amino acids long. This indicates that before POP hydrolysis takes place, T␤4 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, T␤4 is hydrolyzed by meprin-␣. In vitro, we found that the incubation of T␤4 with both meprin-␣ and POP released Ac-SDKP, whereas no Ac-SDKP was released when T␤4 was incubated with either meprin-␣ or POP alone. Incubation of T␤4 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 T␤4. 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 T␤4 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 (T␤4) 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, T␤4 is considered to be the precursor of 39). Our group has shown p...

Meprin metalloproteases are abundantly expressed in the brush-border membranes of kidney proximal tubules. Meprins are implicated in ischemia-reperfusion (IR)-induced renal injury and diabetic nephropathy. The protein kinase A (PKA) signaling pathway modulates extracellular matrix metabolism in diabetic kidneys. The present study evaluated isoform-specific interactions between the catalytic subunit of PKA (PKA C) and meprins. To this end, cytosolic-enriched kidney proteins from meprin αβ double knockout mice, and purified forms of recombinant mouse PKA Cα, Cβ1, and Cβ2, were incubated with activated forms of either homomeric meprin A or meprin B. The cleaved protein products were subjected to SDS-PAGE and analyzed by Coomassie staining and Western blot analysis. While meprin A only cleaved PKA Cβ1, meprin B cleaved all three PKA C isoforms. Analysis of the proteolytic fragments by mass spectrometry revealed that meprin A and B cleave the PKA C isoforms at defined sites, resulting in unique cleavage products. Michaelis-Menten enzyme kinetics demonstrated that meprin B-mediated cleavage of PKA Cα occurs at a rate consistent with that of other physiologically relevant meprin substrates. Meprin cleavage decreased the kinase activity of PKA Cα, Cβ1, and Cβ2. PKA C levels were higher in diabetic kidneys, with evidence of in vivo fragmentation in wild-type diabetic kidneys. Confocal microscopy showed localization of meprin A in the glomeruli of diabetic kidneys. At 3 h post-IR, PKA C levels in proximal tubules decreased compared with distal tubules, which lack meprins. These data suggest that meprins may impact kidney injury, in part, via modulation of PKA signaling pathways.

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