Role of meprin A in renal tubular epithelial cell injury

Meprins are zinc-dependent metalloproteinases that are highly expressed in the brush-border membranes of both the kidney and the intestines. Meprins are capable of proteolytically degrading extracellular matrix proteins, proteolytically processing bioactive proteins, and play a role in inflammatory processes. In this study, the function of meprin A in the acute kidney injury (AKI) model of cisplatin nephrotoxicity was examined. Normal linear localization of meprin A in the brush border membranes of proximal tubules was altered in AKI. The meprin A α-subunit was detected in the urine of both control and cisplatin-treated mice. A cleaved product of the meprin A β-subunit, undetected in the urine of control mice, was found to be significantly increased in the urine during the progression of cisplatin nephrotoxicity. The excretion of this β-fragment was found to be before the rise in serum creatinine and blood urea nitrogen (BUN) suggesting usefulness as a biomarker for AKI. Pretreatment of mice with a meprin A inhibitor afforded protection from cisplatin nephrotoxicity as reflected by significant decreases in serum creatinine, BUN, and the excretion of kidney injury molecule-1. These decreases in serum and urine biomarkers were accompanied by significant decreases in histologic markers such as leukocyte infiltration and apoptosis. Meprin A appears to be an important therapeutic target and urinary excretion appears to be a potential biomarker of AKI. Meprins are zinc-dependent metalloproteinases that are highly expressed in the brush-border membranes of both the kidney and the intestines. Meprins are capable of proteolytically degrading extracellular matrix proteins, proteolytically processing bioactive proteins, and play a role in inflammatory processes. In this study, the function of meprin A in the acute kidney injury (AKI) model of cisplatin nephrotoxicity was examined. Normal linear localization of meprin A in the brush border membranes of proximal tubules was altered in AKI. The meprin A α-subunit was detected in the urine of both control and cisplatin-treated mice. A cleaved product of the meprin A β-subunit, undetected in the urine of control mice, was found to be significantly increased in the urine during the progression of cisplatin nephrotoxicity. The excretion of this β-fragment was found to be before the rise in serum creatinine and blood urea nitrogen (BUN) suggesting usefulness as a biomarker for AKI. Pretreatment of mice with a meprin A inhibitor afforded protection from cisplatin nephrotoxicity as reflected by significant decreases in serum creatinine, BUN, and the excretion of kidney injury molecule-1. These decreases in serum and urine biomarkers were accompanied by significant decreases in histologic markers such as leukocyte infiltration and apoptosis. Meprin A appears to be an important therapeutic target and urinary excretion appears to be a potential biomarker of AKI. Meprins are zinc-dependent oligomeric metalloproteinases of the 'astacin' family that are highly expressed at the brush-border membranes of the kidney and intestines.1.Bertenshaw G.P. Bond J.S. Meprin A. Meprin B. Barrett A.J. Woessner F. Rawlings N. Handbook of Proteolytic Enzymes. Academic Press, London2004: 599-605Crossref Scopus (6) Google Scholar, 2.Bond J.S. Beynon R.J. The astacin family of metalloendopeptidases.Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (340) Google Scholar, 3.Bond J.S. Matters G.L. Banerjee S. Dusheck R.E. Meprin metalloprotease expression and regulation in kidney, intestine, urinary tract infections and cancer.FEBS Lett. 2005; 579: 3317-3322Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 4.Lottaz D. Hahn D. Müller S. et al.Secretion of human meprin from intestinal epithelial cells depends on differential expression of the α and β subunits.Eur J Biochem. 1999; 259: 496-504Crossref PubMed Scopus (65) Google Scholar Meprins are expressed as an oligomeric complex made of α- and/or β-subunits.1.Bertenshaw G.P. Bond J.S. Meprin A. Meprin B. Barrett A.J. Woessner F. Rawlings N. Handbook of Proteolytic Enzymes. Academic Press, London2004: 599-605Crossref Scopus (6) Google Scholar The distribution of meprin subunits in adult mice demonstrates that meprin A, the heterooligomeric form comprised of both α and β-subunits, is the predominant form of meprin expressed abundantly in the apical membranes of renal proximal tubules and comprises about 5% of the total brush-border membrane proteins in murine kidneys.5.Kaushal G.P. Walker P.D. Shah S.V. An old enzyme with a new function: purification and characterization of a distinct matrix-degrading metalloproteinase in rat kidney cortex and its identification as meprin.J Cell Biol. 1994; 126: 1319-1327Crossref PubMed Scopus (88) Google Scholar, 6.Kumar J.M. Bond J.S. Developmental expression of meprin metalloprotease subunits in ICR and C3H/He mouse kidney and intestine in the embryo, postnatally and after weaning.Bioche Biophys Acta. 2001; 1518: 106-114Crossref PubMed Scopus (25) Google Scholar, 7.Craig S.S. Reckelhoff J.F. Bond J.S. Distribution of meprin in kidneys from mice with high- and low-meprin activity.Am J Physiol. 1987; 253: C535-C540PubMed Google Scholar Both meprin α and meprin β-isoforms are initially synthesized as membrane-spanning proteins but, during biosynthesis, the transmembrane domain of the α-isoform is proteolytically cleaved. Meprin β with its transmembrane domain is anchored in the plasma membrane of the brush border and associates with transmembrane domain-deficient meprin α by disulfide linkages or noncovalent linkages.1.Bertenshaw G.P. Bond J.S. Meprin A. Meprin B. Barrett A.J. Woessner F. Rawlings N. Handbook of Proteolytic Enzymes. Academic Press, London2004: 599-605Crossref Scopus (6) Google Scholar,8.Villa J.P. Bertenshaw G.P. Bylander J.E. Bond J.S. Meprin proteolytic complexes at the cell surface and in extracellular spaces.Biochem Soc Symp. 2003; 70: 53-63Crossref PubMed Scopus (31) Google Scholar Thus meprin A, anchored to brush-border membranes only by β-subunit, is a membrane-associated metalloendopeptidase of renal proximal tubules. Our previous studies have demonstrated that meprin A purified from the rat kidney cortex is the major protease in the renal cortex capable of degrading components of the extracellular matrix proteins including collagen IV, fibronectin, laminin, and nidogen in vitro.5.Kaushal G.P. Walker P.D. Shah S.V. An old enzyme with a new function: purification and characterization of a distinct matrix-degrading metalloproteinase in rat kidney cortex and its identification as meprin.J Cell Biol. 1994; 126: 1319-1327Crossref PubMed Scopus (88) Google Scholar,9.Walker P.D. Kaushal G.P. Shah S.V. Meprin A. The major matrix degrading enzyme in renal tubules, produces a novel nidogen fragment in vitro and in vivo.Kidney Int. 1998; 53: 1673-1680Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar Consistent with the purified meprin from rat kidney, recombinant human meprin was recently shown to degrade extracellular matrix proteins in vitro.10.Kruse M.N. Becker C. Lottaz D. et al.Human meprin alpha and beta homo-oligomers: cleavage of basement membrane proteins and sensitivity to metalloprotease inhibitors.Biochem J. 2004; 378: 383-389Crossref PubMed Scopus (131) Google Scholar,11.Köhler D. Kruse M.-N. Stöcker W. Sterchi E.E. Heterologously overexpressed, affinity purified human meprin-alpha is functionally active and cleaves components of the basement membrane in vitro.FEBS Lett. 2000; 465: 2-7Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar Meprins are also capable of proteolytically processing bioactive peptides, cytokines, and peptide hormones.2.Bond J.S. Beynon R.J. The astacin family of metalloendopeptidases.Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (340) Google Scholar, 3.Bond J.S. Matters G.L. Banerjee S. Dusheck R.E. Meprin metalloprotease expression and regulation in kidney, intestine, urinary tract infections and cancer.FEBS Lett. 2005; 579: 3317-3322Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 12.Chestukhin A. Litovchick L. Muradov K. et al.Unveiling the substrate specificity of meprin β on the basis of the site in protein kinase A cleaved by the kinase splitting membranal proteinase.J Biol Chem. 1997; 272: 3153-3160Crossref PubMed Scopus (45) Google Scholar, 13.Bertenshaw G.P. Turk B.E. Hubbard S.J. et al.Marked differences between metalloproteases meprin A and B in substrate and peptide bond specificity.J Biol Chem. 2001; 276: 13248-13255Crossref PubMed Scopus (100) Google Scholar Our recent studies demonstrated that meprins can produce biologically active interleukin-1β from its inactive proform,14.Herzog C. Kaushal G.P. Haun R.S. Generation of biologically active interleukin-1beta by meprin B.Cytokine. 2005; 31: 394-403Crossref PubMed Scopus (55) Google Scholar suggesting that meprins may play an important role in inflammatory processes in addition to their ability to degrade extracellular matrix components. Active meprin A purified from rat kidney was found to be cytotoxic in renal tubular epithelial cells in culture,15.Carmago S. Shah S.V. Walker P.D. Meprin, a brush-border enzyme, plays an important role in hypoxic/ischemic acute renal tubular injury in rats.Kidney Int. 2002; 61: 959-966Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar further suggesting a detrimental role of meprin A during renal injury. Cisplatin nephrotoxicity is associated with damage in the S3 segment of proximal tubules in the corticomedullary junction that results in decline in renal function. Thus, the manifestation of cisplatin-induced acute kidney injury (AKI) is at the site where meprin A is predominantly expressed. In addition, recent studies provide evidence that cisplatin nephrotoxicity is associated with apoptosis, necrosis, and inflammatory mechanisms.16.Ramesh G. Reeves W.B. TNF-alpha mediates chemokine and cytokine expression and renal injury in cisplatin nephrotoxicity.J Clin Invest. 2002; 110: 835-842Crossref PubMed Scopus (651) Google Scholar, 17.Zager R.A. Johnson A.C. Hanson S.Y. Lund S. Acute nephrotoxic and obstructive injury primes the kidney to endotoxin-driven cytokine/chemokine production.Kidney Int. 2006; 69: 1181-1188Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 18.Kaushal G.P. Kaushal V. Hong X. Shah S.V. Role and regulation of activation of caspases in cisplatin-induced injury to renal tubular epithelial cells.Kidney Int. 2001; 60: 1726-1736Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar Therefore, this study was performed to determine if meprin A plays a role in cisplatin nephrotoxicity. We examined whether meprin A (i) undergoes altered distribution and (ii) is excreted in the urine in response to cisplatin-induced AKI injury, and whether meprin A inhibition affords (i) protection from cisplatin-induced cell death and inflammation; (ii) functional and histologic protection from AKI; and (iii) prevention in the shedding of kidney injury molecule (KIM), as the release of KIM is dependent on a metalloproteinase. Meprin A comprised of both α and β-subunits is expressed abundantly in the apical brush border of the renal proximal tubule epithelium. The distribution pattern of meprin A in kidney sections was first examined by immunostaining using meprin β polyclonal antibody. Tissue sections from normal kidneys revealed a specific linear staining of meprin A in the brush border or luminal surface of normal proximal tubules in the corticomedullary junction. No staining was observed in the luminal surface of distal tubules, glomeruli, and collecting duct. Following 2 days of cisplatin treatment of mice, staining for meprin A was altered and some staining was observed in the cytoplasm and toward the basolateral side in the proximal tubules but much of the staining remained in the apical region. This positive staining was scattered throughout the cytoplasm and basolateral plasma membrane in a severely injured kidney after 3 days of cisplatin treatment (Figure 1). These studies suggest that, owing to enormous degradative potential, meprin A redistribution may play a significant role in renal injury. Similar altered staining in cisplatin injury was observed when kidney sections were stained with meprin α antibody (data not shown). Expression of meprin α and meprin β were examined by Western blot using specific antibodies. The protein levels of meprin expression in the kidney cortex did not change significantly and remained more or less at the same level in the kidney cortex in response to cisplatin (data not shown). We also analyzed levels of meprin α and meprin β in urine samples. Urine samples collected at 0, 4, 8, 16 h, 1, 2, and 3 days after cisplatin administration were immunoblotted with specific anti-meprin α and anti-meprin β antibodies. Meprin α was detected in urine samples of both untreated control and cisplatin-treated mice. As meprin α is not directly associated with membranes but only bound through meprin β by disulfide bridges or by noncovalent interactions, it is quite conceivable that meprin α is detected in the urine of control mice. In contrast, there was no detectable meprin β in the urine samples of untreated control mice. A cleaved form of meprin β (∼55 kDa) was detected in the urine samples collected as early as 8 h after cisplatin treatment (Figure 2). This suggests that membrane-associated meprin β (∼80 kDa) is somehow cleaved by an unknown protease at initial stages of injury and the cleaved product released from the membranes is excreted in the urine. The amount of urinary meprin β cleaved fragment was increased markedly at 12 h and persisted at higher levels at 2 days of cisplatin treatment. We assessed the role of meprin A in the pathogenesis of cisplatin nephrotoxicity by measuring renal function and kidney histology in mice treated with cisplatin in the presence and absence of actinonin. We administered actinonin intraperitoneally 1 h before the cisplatin injection. The effect of actinonin on cisplatin-induced impairment of renal function was determined by blood urea nitrogen (BUN) and creatinine values at 1, 2, and 3 days. At 3 days after cisplatin treatment, the mice showed marked deterioration in renal function as reflected in increased levels of BUN and serum creatinine. Intraperitoneal administration of 15 mg/kg body weight (b.w.) actinonin significantly reduced cisplatin-induced elevated levels of both BUN and serum creatinine (Figure 3). As shown, actinonin alone did not effect either BUN or creatinine values. We have previously shown that 15 mg/kg b.w. actinonin administered intraperitoneally inhibited meprin A activity completely within 3 h in the kidney cortex in vivo.15.Carmago S. Shah S.V. Walker P.D. Meprin, a brush-border enzyme, plays an important role in hypoxic/ischemic acute renal tubular injury in rats.Kidney Int. 2002; 61: 959-966Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar We also examined the effect of actinonin on cisplatin-induced histological damage. Histological assessment following cisplatin treatment revealed severe tubular damage characterized by extensive loss of tubular epithelial cells, loss of brush-border membranes, tubular dilation, cellular desquamation, and proteinaceous casts of the tubular cells. The meprin inhibitor actinonin ameliorated most of the tubular injury (Figure 4). The glomeruli maintained their normal structure upon cisplatin treatment. Animals treated with cisplatin and actinonin resulted in considerable improvement in histology as reflected in significant protection from the severe tubular injury (Figure 4). Semiquantitative evaluation of kidney sections showed a tubular necrosis score of 0.05±0.02 in kidneys from mice treated with 20% ethanol in saline alone, 0.04±0.04 in kidneys from mice treated with actinonin alone, 3.4±0.5 in kidneys of 3 days cisplatin-treated mice, and 0.4±0.08 in mice treated with 3 days cisplatin and actinonin. The statistical differences were significant between kidneys of cisplatin-treated mice and either 20% ethanol in saline or cisplatin plus actinonin treated mice (n=6, P<0.005). These studies thus demonstrate that meprin inhibition provides significant protection from cisplatin-induced AKI.Figure 4Effect of actinonin on renal histology in cisplatin-induced AKI. PAS staining of kidney sections from mice treated with vehicle 20% ethanol/saline (Control), vehicle plus 15 mg/kg b.w. actinonin (control+actinonin), 20 mg/kg b.w. cisplatin (3 d (day) CP), and cisplatin plus actinonin (3 d CP+actinonin). Administration of actinonin alone has no effect on kidney histology.View Large Image Figure ViewerDownload (PPT) To ensure that actinonin did not block the uptake of cisplatin in the kidney, we isolated kidneys from mice (n=4 in each group) following administration with cisplatin (20 mg/kg b.w.) and cisplatin (20 mg/kg b.w.) plus actinonin (15 mg/kg b.w.). The kidney tissue was analyzed for platinum content by Mayo Medical Lab (Rochester, MN, USA). The results of this study revealed that meprin inhibitor actinonin did not prevent uptake of cisplatin in the kidney (amount of platinum in kidney tissue: control kidney; 0.032±0.01 μg/g dry weight tissue, cisplatin; 34.6±5.6 μg/g dry weight tissue, cisplatin plus actinonin; 35.2±4.5 μg/g dry weight tissue). As apoptosis has been implicated in tubule cell death in response to cisplatin, we tested whether meprin inhibitor is able to reduce cisplatin-induced renal cell apoptosis. The TdT-mediated dUTP-biotin nick end labeling (TUNEL) method to localize DNA fragmentation was performed to detect apoptosis in situ. Cisplatin-treated mice displayed extensive incidence of apoptotic cell death in kidney sections compared with the saline-treated control mice. TUNEL-positive nuclei were restricted mainly to the tubular epithelial cells of the corticomedullary junction (Figure 5). Actinonin-treated mice significantly attenuated cisplatin-induced apoptotic response. Recent studies show that cisplatin-induced AKI is associated with infiltration of inflammatory cells into the kidney cortex.16.Ramesh G. Reeves W.B. TNF-alpha mediates chemokine and cytokine expression and renal injury in cisplatin nephrotoxicity.J Clin Invest. 2002; 110: 835-842Crossref PubMed Scopus (651) Google Scholar,17.Zager R.A. Johnson A.C. Hanson S.Y. Lund S. Acute nephrotoxic and obstructive injury primes the kidney to endotoxin-driven cytokine/chemokine production.Kidney Int. 2006; 69: 1181-1188Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar Therefore, we performed naphthol AS-D chloroacetate staining and counted the number of esterase-positive leukocytes in kidney tissue after treatment with cisplatin alone or in combination with the meprin A inhibitor actinonin. Cisplatin treatment dramatically increased the number of infiltrating leukocytes in the cortex and outer medulla (28±3, 10–12 fields/kidney). Following actinonin treatment, these infiltrating neutrophils were significantly reduced (4±1, 12 fields/kidney) (Figure 6). Recent studies have demonstrated that both kidney expression and urinary excretion of Kim-1 are considerably increased in renal injury and can serve as a useful biomarker for renal tubular injury.19.Vaidya V.S. Ramirez V. Ichimura T. et al.Urinary kidney injury molecule-1: a sensitive quantitative biomarker for early detection of kidney tubular injury.Am J Physiol Renal Physiol. 2006; 290: F517-F529Crossref PubMed Scopus (494) Google Scholar,20.Ichimura T. Hung C.C. Yang S.A. et al.Kidney injury molecule-1: a tissue and urinary biomarker for nephrotoxicant-induced renal injury.Am J Physiol-Renal Physiol. 2004; 286: F552-F563Crossref PubMed Scopus (494) Google Scholar Previous studies demonstrated that KIM is shed into the extracellular space by proteolytic cleavage mediated by a metalloproteinase.21.Bailly V. Zhang Z. Meier W. et al.Shedding of kidney injury molecule-1, a putative adhesion protein involved in renal regeneration.J Biol Chem. 2002; 277: 39739-39748Crossref PubMed Scopus (254) Google Scholar As actinonin inhibits the metalloendopeptidase meprin A, we explored whether actinonin is capable of reducing urinary excretion of Kim-1. As shown in Figure 7, Kim-1 excretion was markedly increased in the urine of cisplatin-treated mice as reported in a rat model of cisplatin nephrotoxicity.21.Bailly V. Zhang Z. Meier W. et al.Shedding of kidney injury molecule-1, a putative adhesion protein involved in renal regeneration.J Biol Chem. 2002; 277: 39739-39748Crossref PubMed Scopus (254) Google Scholar Actinonin was quite effective in significantly reducing the levels of cisplatin-induced Kim-1 excretion in the urine. Meprin A-deficient inbred strain of mice C3H/He lack more than 90% of meprin A.22.Trachtman H. Valderrama E. Dietrich J.M. Bond J.S. The role of meprin A in the pathogenesis of acute renal failure.Biochem Biophys Res Commun. 1995; 208: 498-505Crossref PubMed Scopus (58) Google Scholar,23.Craig S.S. Reckelhoff J.F. Bond J.S. Distribution of meprin in kidneys from mice with high- andlow-meprin activity.Am J Physiol. 1987; 253: C535-C540PubMed Google Scholar Previous studies demonstrated that in response to ischemia–reperfusion injury these mice exhibited significantly less tubular necrosis, serum creatinine, and BUN values compared with the normal C57 Bl/6 mice.23.Craig S.S. Reckelhoff J.F. Bond J.S. Distribution of meprin in kidneys from mice with high- andlow-meprin activity.Am J Physiol. 1987; 253: C535-C540PubMed Google Scholar We have examined whether these mice afford protection from cisplatin-induced AKI. C3H/He mice ameliorated toxic renal failure as reflected by improvement in histologic damage (Figure 8). Cisplatin-induced changes in normal C57Bl/6 mice such as loss of brush-border membranes, sloughing of tubular epithelial cells, dialation of tubules, and cast formation were almost absent in kidneys from C3H/He mice injected with cisplatin (Figure 8a). The amelioration of renal failure was also reflected in significant decreases in BUN and serum creatinine (Figure 8b). This study supports a role for meprin A in renal tubular epithelial cell injury in cisplatin nephrotoxicity. In normal renal tissue, meprin A is localized on the apical brush-border membranes of the proximal tubule.2.Bond J.S. Beynon R.J. The astacin family of metalloendopeptidases.Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (340) Google Scholar,9.Walker P.D. Kaushal G.P. Shah S.V. Meprin A. The major matrix degrading enzyme in renal tubules, produces a novel nidogen fragment in vitro and in vivo.Kidney Int. 1998; 53: 1673-1680Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar In response to cisplatin injury, we demonstrate that meprin A's linear localization in the brush-border membranes is changed and is distributed throughout the cytoplasm and some staining was observed toward the underlying basement membrane of the proximal tubules in the corticomedullary junction. Target substrates for meprin A in the cytosol have not yet been identified that may have detrimental effects within the cytosol. In a rat model of ischemia–reperfusion injury, we have previously demonstrated that meprin A undergoes redistribution from the apical brush-border membranes to the cytoplasm to reach the basolateral tubular basement membrane9.Walker P.D. Kaushal G.P. Shah S.V. Meprin A. The major matrix degrading enzyme in renal tubules, produces a novel nidogen fragment in vitro and in vivo.Kidney Int. 1998; 53: 1673-1680Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar suggesting that common mechanisms for meprin redistribution are involved in toxic and ischemic renal injury. Considering the enormous proteolytic potential of meprin A, the altered location to places other than the apical membranes during renal injury may be detrimental to the kidney. Meprins are capable of degrading a wide variety of substrates ranging from the major components of the basement membrane such as collagen, laminin, fibronectin, and nidogen5.Kaushal G.P. Walker P.D. Shah S.V. An old enzyme with a new function: purification and characterization of a distinct matrix-degrading metalloproteinase in rat kidney cortex and its identification as meprin.J Cell Biol. 1994; 126: 1319-1327Crossref PubMed Scopus (88) Google Scholar, 9.Walker P.D. Kaushal G.P. Shah S.V. Meprin A. The major matrix degrading enzyme in renal tubules, produces a novel nidogen fragment in vitro and in vivo.Kidney Int. 1998; 53: 1673-1680Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 10.Kruse M.N. Becker C. Lottaz D. et al.Human meprin alpha and beta homo-oligomers: cleavage of basement membrane proteins and sensitivity to metalloprotease inhibitors.Biochem J. 2004; 378: 383-389Crossref PubMed Scopus (131) Google Scholar, 11.Köhler D. Kruse M.-N. Stöcker W. Sterchi E.E. Heterologously overexpressed, affinity purified human meprin-alpha is functionally active and cleaves components of the basement membrane in vitro.FEBS Lett. 2000; 465: 2-7Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar as well as growth factors, protein kinases, and other bioactive peptides.1.Bertenshaw G.P. Bond J.S. Meprin A. Meprin B. Barrett A.J. Woessner F. Rawlings N. Handbook of Proteolytic Enzymes. Academic Press, London2004: 599-605Crossref Scopus (6) Google Scholar, 2.Bond J.S. Beynon R.J. The astacin family of metalloendopeptidases.Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (340) Google Scholar, 12.Chestukhin A. Litovchick L. Muradov K. et al.Unveiling the substrate specificity of meprin β on the basis of the site in protein kinase A cleaved by the kinase splitting membranal proteinase.J Biol Chem. 1997; 272: 3153-3160Crossref PubMed Scopus (45) Google Scholar, 14.Herzog C. Kaushal G.P. Haun R.S. Generation of biologically active interleukin-1beta by meprin B.Cytokine. 2005; 31: 394-403Crossref PubMed Scopus (55) Google Scholar Previous studies in different inbred strains of mice have demonstrated that meprin A is involved in renal injury. It was shown that mice strains with low levels of meprin A expression developed less acute renal failure compared with those that express high levels of meprin A when exposed to ischemia–reperfusion.22.Trachtman H. Valderrama E. Dietrich J.M. Bond J.S. The role of meprin A in the pathogenesis of acute renal failure.Biochem Biophys Res Commun. 1995; 208: 498-505Crossref PubMed Scopus (58) Google Scholar Also, when meprin A was added exogenously it was found to be cytotoxic in cultured renal cells.15.Carmago S. Shah S.V. Walker P.D. Meprin, a brush-border enzyme, plays an important role in hypoxic/ischemic acute renal tubular injury in rats.Kidney Int. 2002; 61: 959-966Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar Our studies demonstrate that a 55-kDa cleaved fragment meprin β isoform undetected in the urine of normal mice is significantly increased in cisplatin-treated mice. This increase occurred much earlier than rise in the BUN and creatinine values. Our results show that in response to cisplatin treatment, urinary meprin β fragments were excreted as early as 8 h after cisplatin administration and subsequently at later times early low levels were significantly increased and very high levels of meprin β were excreted in the urine after 2–3 days following cisplatin administration as shown in Figure 2. Thus cellular and histological changes appear at a time when very high levels of meprin β fragments are excreted in the urine. Compared to the excretion of meprin β fragments, maximum urinary excretion of a recently discovered potential urinary marker KIM19.Vaidya V.S. Ramirez V. Ichimura T. et al.Urinary kidney injury molecule-1: a sensitive quantitative biomarker for early detection of kidney tubular injury.Am J Physiol Renal Physiol. 2006; 290: F517-F529Crossref PubMed Scopus (494) Google Scholar,20.Ichimura T. Hung C.C. Yang S.A. et al.Kidney injury molecule-1: a tissue and urinary biomarker for nephrotoxicant-induced renal injury.Am J Physiol-Renal Physiol. 2004; 286: F552-F563Crossref PubMed Scopus (494) Google Scholar occurred after 1 day of cisplatin administration. Some excretion of KIM was also detected in the urine of normal mice, whereas no meprin β was detected in the urine of normal mice. As meprin β under normal conditions is anchored to the membranes, a proteolytic cleavage by unknown protease can produce the 55-kDa fragment. These studies suggest that meprin β can be a potential biomarker of AKI. The excretion of meprin α detected in the urine of normal mice increased marginally in cisplatin nephrotoxicity. Consistent to our studies, meprin α has been previously shown to be excreted in the urine of normal mice.24.Beynon R.J. Oliver S. Robertson D.H. Characterization of the soluble, secreted form of urinary meprin.Biochem J. 1996; 315: 461-465Crossref PubMed Scopus (31) Google Scholar A recent study on proteomics in a sepsis model has shown that meprin α is a potential biomarker of this injury.25.Holly M.K. Dear J.W. Hu X. et al.Biomarker and drug-target discovery using proteomics in a new rat model of sepsis-induced acute renal failure.Kidney Int. 2006; 70: 496-506Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar The excretion of meprin α observed in the normal mice was not changed significantly in the cisplatin-treated mice. Thus, more studies are needed to explore whether meprins can serve as an important biomarker in the list of newly discovered potential biomarkers.26.Hewitt S.M. Dear J. Star R.A. Discovery of protein biomarkers for renal diseases.J Am Soc Nephrol. 2004; 15: 1677-8916Crossref