Role of meprin metalloproteinases in cytokine processing and inflammation.

Meprin metalloendopeptidases, comprising α and ß isoforms, are widely expressed in mammalian cells and organs including kidney, intestines, lungs, skin, and bladder, and in a variety of immune cells and cancer cells. Meprins proteolytically process many inflammatory mediators, including cytokines, chemokines, and other bioactive proteins and peptides that control the function of immune cells. The knowledge of meprin-mediated processing of inflammatory mediators and other target substrates provides a pathophysiologic link for the involvement of meprins in the pathogenesis of many inflammatory disorders. Meprins are now known to play important roles in inflammatory diseases including acute kidney injury, sepsis, urinary tract infections, bladder inflammation, and inflammatory bowel disease. The proteolysis of epithelial and endothelial barriers including cell junctional proteins by meprins promotes leukocyte influx into areas of tissue damage to result in inflammation. Meprins degrade extracellular matrix proteins; this ability of meprins is implicated in the cell migration of leukocytes and the invasion of tumor cells that express meprins. Proteolytic processing and maturation of procollagens provides evidence that meprins are involved in collagen maturation and deposition in the fibrotic processes involved in the formation of keloids and hypertrophic scars and lung fibrosis. This review highlights recent progress in understanding the role of meprins in inflammatory disorders in both human and mouse models.

Molecular Interactions Between Reactive Oxygen Species and Autophagy in Kidney Disease.

Reactive oxygen species (ROS) are highly reactive signaling molecules that maintain redox homeostasis in mammalian cells. Dysregulation of redox homeostasis under pathological conditions results in excessive generation of ROS, culminating in oxidative stress and the associated oxidative damage of cellular components. ROS and oxidative stress play a vital role in the pathogenesis of acute kidney injury and chronic kidney disease, and it is well documented that increased oxidative stress in patients enhances the progression of renal diseases. Oxidative stress activates autophagy, which facilitates cellular adaptation and diminishes oxidative damage by degrading and recycling intracellular oxidized and damaged macromolecules and dysfunctional organelles. In this review, we report the current understanding of the molecular regulation of autophagy in response to oxidative stress in general and in the pathogenesis of kidney diseases. We summarize how the molecular interactions between ROS and autophagy involve ROS-mediated activation of autophagy and autophagy-mediated reduction of oxidative stress. In particular, we describe how ROS impact various signaling pathways of autophagy, including mTORC1-ULK1, AMPK-mTORC1-ULK1, and Keap1-Nrf2-p62, as well as selective autophagy including mitophagy and pexophagy. Precise elucidation of the molecular mechanisms of interactions between ROS and autophagy in the pathogenesis of renal diseases may identify novel targets for development of drugs for preventing renal injury.

Autophagy in acute kidney injury.

Autophagy is a conserved multistep pathway that degrades and recycles damaged organelles and macromolecules to maintain intracellular homeostasis. The autophagy pathway is upregulated under stress conditions including cell starvation, hypoxia, nutrient and growth-factor deprivation, endoplasmic reticulum stress, and oxidant injury, most of which are involved in the pathogenesis of acute kidney injury (AKI). Recent studies demonstrate that basal autophagy in the kidney is vital for the normal homeostasis of the proximal tubules. Deletion of key autophagy proteins impaired renal function and increased p62 levels and oxidative stress. In models of AKI, autophagy deletion in proximal tubules worsened tubular injury and renal function, highlighting that autophagy is renoprotective in models of AKI. In addition to nonselective sequestration of autophagic cargo, autophagy can facilitate selective degradation of damaged organelles, particularly mitochondrial degradation through the process of mitophagy. Damaged mitochondria accumulate in autophagy-deficient kidneys of mice subjected to ischemia-reperfusion injury, but the precise mechanisms of regulation of mitophagy in AKI are not yet elucidated. Recent progress in identifying the interplay of autophagy, apoptosis, and regulated necrosis has revived interest in examining shared pathways/molecules in this crosstalk during the pathogenesis of AKI. Autophagy and its associated pathways pose potentially unique targets for therapeutic interventions in AKI.

Epigenetic regulation of KLK7 gene expression in pancreatic and cervical cancer cells.

Kallikrein-related peptidase 7 (KLK7) is a serine protease encoded within the kallikrein gene cluster located on human chromosome region 19q13.3-13.4. KLK7 is overexpressed in human pancreatic ductal adenocarcinomas (PDACs), but not in normal pancreas. Examination of KLK7 mRNA levels in pancreatic cancer cell lines revealed that it is readily detected in MIA PaCa-2 and PK-1 cells, but not in Panc-1 cells. Treatment of Panc-1 cells with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) significantly induced KLK7 mRNA expression. Similarly, KLK7 is highly expressed in cervical cancer cells, but its expression in the human cervical cancer cell line HeLa is only detected following TSA treatment. Promoter deletion analysis revealed that the proximal -238 promoter region, containing a putative Sp1-binding site, was sufficient for TSA activation of luciferase reporter activity, which was abrogated by the disruption of the Sp1-binding sequence. Consistent with the notion that TSA induced KLK7 expression via Sp1, co-expression of Sp1 with the KLK7-promoter/luciferase construct produced a significant increase in reporter activity. Chromatin immunoprecipitation (ChIP) analysis revealed enriched Sp1 occupancy on the KLK7 promoter following TSA treatment. Similarly, ChIP analysis showed the histone active mark, H3K4Me3, in the KLK7 promoter region was significantly increased after exposure to TSA.

ADAM10 is the major sheddase responsible for the release of membrane-associated meprin A.

Meprin A, composed of α and ß subunits, is a membrane-bound metalloproteinase in renal proximal tubules. Meprin A plays an important role in tubular epithelial cell injury during acute kidney injury (AKI). The present study demonstrated that during ischemia-reperfusion-induced AKI, meprin A was shed from proximal tubule membranes, as evident from its redistribution toward the basolateral side, proteolytic processing in the membranes, and excretion in the urine. To identify the proteolytic enzyme responsible for shedding of meprin A, we generated stable HEK cell lines expressing meprin ß alone and both meprin α and meprin ß for the expression of meprin A. Phorbol 12-myristate 13-acetate and ionomycin stimulated ectodomain shedding of meprin ß and meprin A. Among the inhibitors of various proteases, the broad spectrum inhibitor of the ADAM family of proteases, tumor necrosis factor-α protease inhibitor (TAPI-1), was most effective in preventing constitutive, phorbol 12-myristate 13-acetate-, and ionomycin-stimulated shedding of meprin ß and meprin A in the medium of both transfectants. The use of differential inhibitors for ADAM10 and ADAM17 indicated that ADAM10 inhibition is sufficient to block shedding. In agreement with these results, small interfering RNA to ADAM10 but not to ADAM9 or ADAM17 inhibited meprin ß and meprin A shedding. Furthermore, overexpression of ADAM10 resulted in enhanced shedding of meprin ß from both transfectants. Our studies demonstrate that ADAM10 is the major ADAM metalloproteinase responsible for the constitutive and stimulated shedding of meprin ß and meprin A. These studies further suggest that inhibiting ADAM 10 activity could be of therapeutic benefit in AKI.

Challenges and advances in the treatment of AKI.

Treating or preventing AKI requires treating or preventing a rise in serum creatinine as well as the immediate and remote clinical consequences associated with AKI. Because a substantial number of patients with AKI progress to ESRD, identifying patients likely to progress and halting progression are important goals for treating AKI. Many therapies for AKI are being developed, including RenalGuard Therapy, which aims to maintain high urine output; α-melanocyte-stimulating hormone, with anti-inflammatory and antiapoptotic activities; alkaline phosphatase, which detoxifies proinflammatory substances; novel, small interfering RNA, directed at p53 activation; THR-184, a peptide agonist of bone morphogenetic proteins; removal of catalytic iron, important in free-radical formation; and cell-based therapies, including mesenchymal stem cells in vivo and renal cell therapy in situ. In this review, we explore what treatment of AKI really means, discuss the emerging therapies, and examine the windows of opportunity for treating AKI. Finally, we provide suggestions for accelerating the pathways toward preventing and treating AKI, such as establishing an AKI network, implementing models of catalytic philanthropy, and directing a small percentage of the Medicare ESRD budget for developing therapies to prevent and treat AKI and halt progression of CKD.

Meprin A metalloproteinase and its role in acute kidney injury.

Meprin A, composed of α- and ß-subunits, is a membrane-associated neutral metalloendoprotease that belongs to the astacin family of zinc endopeptidases. It was first discovered as an azocasein and benzoyl-l-tyrosyl-p-aminobenzoic acid hydrolase in the brush-border membranes of proximal tubules and intestines. Meprin isoforms are now found to be widely distributed in various organs (kidney, intestines, leukocytes, skin, bladder, and a variety of cancer cells) and are capable of hydrolyzing and processing a large number of substrates, including extracellular matrix proteins, cytokines, adherens junction proteins, hormones, bioactive peptides, and cell surface proteins. The ability of meprin A to cleave various substrates sheds new light on the functional properties of this enzyme, including matrix remodeling, inflammation, and cell-cell and cell-matrix processes. Following ischemia-reperfusion (IR)- and cisplatin-induced acute kidney injury (AKI), meprin A is redistributed toward the basolateral plasma membrane, and the cleaved form of meprin A is excreted in the urine. These studies suggest that altered localization and shedding of meprin A in places other than the apical membranes may be deleterious in vivo in acute tubular injury. These studies also provide new insight into the importance of a sheddase involved in the release of membrane-associated meprin A under pathological conditions. Meprin A is injurious to the kidney during AKI, as meprin A-knockout mice and meprin inhibition provide protective roles and improve renal function. Meprin A, therefore, plays an important role in AKI and potentially is a unique target for therapeutic intervention during AKI.

Proteolytic action of kallikrein-related peptidase 7 produces unique active matrix metalloproteinase-9 lacking the C-terminal hemopexin domains.

The gelatinases, matrix metalloproteinase (MMP)-9 and -2, are produced as latent, inactive enzymes that can be proteolytically activated by a number of proteases. In many normal and pathological conditions, where the expression of MMPs is deregulated, changes in the expression of other proteases have also been reported. Human kallikrein-related peptidase 7 (KLK7), a chymotryptic-like serine protease, is overexpressed in many different types of neoplastic conditions, which have also been shown to express high levels of both MMP-9 and -2. Since the activation of MMPs by KLK7 has never been examined, we sought to determine whether KLK7 can activate these MMPs. To test this hypothesis KLK7 was incubated with the recombinant MMPs and the products of the reaction were analyzed for their activity. Incubation of proMMP-9 with KLK7 resulted in the production of a novel truncated, active MMP-9 lacking the C-terminal hemopexin domains. In contrast, KLK7 degraded, but did not activate, proMMP-2. The novel activation of proMMP-9 by KLK7 was further confirmed using conditioned medium prepared from an MMP-9-expressing cell line, MDA-MMP-9. Our results clearly establish that KLK7 activates proMMP-9 to produce a novel truncated, active MMP-9 product not generated by other proteases. These findings suggest that KLK7 may play an important role in the activation of MMP-9 in tumors that express high levels of both these proteases and the resulting truncated MMP may possess altered substrate specificities compared with full-length MMP-9 activated by other proteases.

zVAD-fmk prevents cisplatin-induced cleavage of autophagy proteins but impairs autophagic flux and worsens renal function.

Cisplatin injury to renal tubular epithelial cells (RTEC) is accompanied by autophagy and caspase activation. However, autophagy gradually decreases during the course of cisplatin injury. The role of autophagy and the mechanism of its decrease during cisplatin injury are not well understood. This study demonstrated that autophagy proteins beclin-1, Atg5, and Atg12 were cleaved and degraded during the course of cisplatin injury in RTEC and the kidney. zVAD-fmk, a widely used pancaspase inhibitor, blocked cleavage of autophagy proteins suggesting that zVAD-fmk would promote the autophagy pathway. Unexpectedly, zVAD-fmk blocked clearance of the autophagosomal cargo, indicating lysosomal dysfunction. zVAD-fmk markedly inhibited cisplatin-induced lysosomal cathepsin B and calpain activities and therefore impaired autophagic flux. In a mouse model of cisplatin nephrotoxicity, zVAD-fmk impaired autophagic flux by blocking autophagosomal clearance as revealed by accumulation of key autophagic substrates p62 and LC3-II. Furthermore, zVAD-fmk worsened cisplatin-induced renal dysfunction. Chloroquine, a lysomotropic agent that is known to impair autophagic flux, also exacerbated cisplatin-induced decline in renal function. These findings demonstrate that impaired autophagic flux induced by zVAD-fmk or a lysomotropic agent worsened renal function in cisplatin acute kidney injury (AKI) and support a protective role of autophagy in AKI. These studies also highlight that the widely used antiapoptotic agent zVAD-fmk may be contraindicated as a therapeutic agent for preserving renal function in AKI.

Autophagy protects proximal tubular cells from injury and apoptosis.

Autophagy is upregulated during ischemia-reperfusion (IR)-induced and cisplatin-induced acute kidney injury (AKI). Proximal tubule-specific Atg7 knockout mice exhibited increased renal injury compared with wild-type mice following cisplatin- and IR-induced AKI. Inhibition of autophagy by chloroquine aggravated AKI, whereas upregulation of autophagy by rapamycin recovered lost renal function and histology, further indicating a protective role of autophagy in AKI. These findings reported by Jiang et al. will provide stimulus to further examine the role and mechanism of the enhancement of autophagy in AKI.

Autophagy Function and Regulation in Kidney Disease.

Autophagy is a dynamic process by which intracellular damaged macromolecules and organelles are degraded and recycled for the synthesis of new cellular components. Basal autophagy in the kidney acts as a quality control system and is vital for cellular metabolic and organelle homeostasis. Under pathological conditions, autophagy facilitates cellular adaptation; however, activation of autophagy in response to renal injury may be insufficient to provide protection, especially under dysregulated conditions. Kidney-specific deletion of Atg genes in mice has consistently demonstrated worsened acute kidney injury (AKI) outcomes supporting the notion of a pro-survival role of autophagy. Recent studies have also begun to unfold the role of autophagy in progressive renal disease and subsequent fibrosis. Autophagy also influences tubular cell death in renal injury. In this review, we reported the current understanding of autophagy regulation and its role in the pathogenesis of renal injury. In particular, the classic mammalian target of rapamycin (mTOR)-dependent signaling pathway and other mTOR-independent alternative signaling pathways of autophagy regulation were described. Finally, we summarized the impact of autophagy activation on different forms of cell death, including apoptosis and regulated necrosis, associated with the pathophysiology of renal injury. Understanding the regulatory mechanisms of autophagy would identify important targets for therapeutic approaches.

Deletion of LOX-1 attenuates renal injury following angiotensin II infusion.

Angiotensin II upregulates the expression of LOX-1, a recently identified oxidized low-density lipoprotein receptor controlled by redox state which in turn upregulates angiotensin II activity on its activation. To test whether interruption of this positive feedback loop might reduce angiotensin II-induced hypertension and subsequent renal injury, we studied LOX-1 knockout mice. After infusion with angiotensin II for 4 weeks systolic blood pressure gradually increased in the wild-type mice; this rise was significantly attenuated in the LOX-1 knockout mice. Along with the rise in systolic blood pressure, renal function (blood urea nitrogen and creatinine) decreased in the wild-type mice, but the deterioration of function was significantly less in the LOX-1 knockout mice. Glomerulosclerosis, arteriolar sclerosis, tubulointerstitial damage, and renal collagen accumulation were all significantly less in the LOX-1 knockout mice. The reduction in collagen formation was accompanied by a decrease in connective tissue growth factor mRNA, angiotensin type 1 receptor expression, and phosphorylation of p38 and p44/42 mitogen-activated protein kinases. Expression of endothelial nitric oxide synthase was increased in the kidneys of the LOX-1 knockout mice compared to the wild-type mice. Overall, our study suggests that LOX-1 is a key modulator in the development of angiotensin II-induced hypertension and subsequent renal damage.

Meprin A and meprin alpha generate biologically functional IL-1beta from pro-IL-1beta.

The present study demonstrates that both oligomeric metalloendopeptidase meprin A purified from kidney cortex and recombinant meprin alpha are capable of generating biologically active IL-1beta from its precursor pro-IL-1beta. Amino-acid sequencing analysis reveals that meprin A and meprin alpha cleave pro-IL-1beta at the His(115)-Asp(116) bond, which is one amino acid N-terminal to the caspase-1 cleavage site and five amino acids C-terminal to the meprin beta site. The biological activity of the pro-IL-1beta cleaved product produced by meprin A, determined by proliferative response of helper T-cells, was 3-fold higher to that of the IL-1beta product produced by meprin beta or caspase-1. In a mouse model of sepsis induced by cecal ligation puncture that results in elevated levels of serum IL-1beta, meprin inhibitor actinonin significantly reduces levels of serum IL-1beta. Meprin A and meprin alpha may therefore play a critical role in the production of active IL-1beta during inflammation and tissue injury.

Proteolytic processing and inactivation of CCL2/MCP-1 by meprins.

Monocyte chemotactic protein 1 (CCL2/MCP-1) is a small chemokine involved in the recruitment and trafficking of mononuclear immune cells to inflammation sites. Our studies demonstrate that the metalloendopeptidases meprin A (purified from kidney cortex), recombinant meprin α, and recombinant meprin ß can all process CCL2/MCP-1. The cleavage sites were determined by amino acid sequencing and mass spectrometry analysis of the generated products, and the biological activity of the products was evaluated by chemotactic migration assay using THP-1 cells. The cleavage sites generated by the meprin isoforms revealed that meprin A and meprin α cleaved the N-terminal domain of mouse CCL2/MCP-1 at the Asn6 and Ala7 bond, resulting in significant reduction in the chemotactic activity of the cleaved CCL2/MCP-1. Meprin ß was unable to cleave the N-terminus of mouse CCL2/MCP-1 but cleaved the C-terminal region between Ser74 and Glu75. Human CCL2/MCP-1 that lacks the murine C-terminal region was also cleaved by meprin α at the N-terminus resulting in significant loss of CCL2/MCP-1 biological activity, whereas meprin ß did not affect the biological activity. These studies suggest that meprin α and meprin ß may play important roles in regulating the CCL2/MCP-1 chemokine activity during inflammation.

Carbamylated Low-Density Lipoprotein (cLDL)-Mediated Induction of Autophagy and Its Role in Endothelial Cell Injury.

Patients with chronic kidney disease (CKD) have high risk of cardiovascular complications. Plasma levels of carbamylated proteins produced by urea-derived isocyanate or thiocyanate are elevated in CKD patients and that they are significant predictors of cardiovascular events and all-cause mortality. Carbamylated LDL (cLDL) has pro-atherogenic properties and is known to affect major biological processes relevant to atherosclerosis including endothelial cell injury. The underlying mechanisms of cLDL-induced endothelial cell injury are not well understood. Although autophagy has been implicated in atherosclerosis, cLDL-mediated induction of autophagy and its role in endothelial cell injury is unknown. Our studies demonstrate that human coronary artery endothelial cells (HCAECs) respond to cLDL by specific induction of key autophagy proteins including LC3-I, beclin-1, Atg5, formation of lipid-conjugated LC3-II protein, and formation of punctate dots of autophagosome-associated LC3-II. We demonstrated that autophagy induction is an immediate response to cLDL and occurred in a dose and time-dependent manner. Inhibition of cLDL-induced autophagy by a specific siRNA to LC3 as well as by an autophagy inhibitor provided protection from cLDL-induced cell death and DNA fragmentation. Our studies demonstrate that autophagy plays an important role in cLDL-mediated endothelial cell injury and may provide one of the underlying mechanisms for the pathogenesis of cLDL-induced atherosclerosis in CKD patients.

Basement membrane protein nidogen-1 is a target of meprin ß in cisplatin nephrotoxicity.

Meprins are oligomeric metalloproteinases that are abundantly expressed in the brush-border membranes of renal proximal tubules. During acute kidney injury (AKI) induced by cisplatin or ischemia-reperfusion, membrane-bound meprins are shed and their localization is altered from the apical membranes toward the basolateral surface of the proximal tubules. Meprins are capable of cleaving basement membrane proteins in vitro, however, it is not known whether meprins are able to degrade extracellular matrix proteins under pathophysiological conditions in vivo. The present study demonstrates that a basement membrane protein, nidogen-1, is cleaved and excreted in the urine of mice subjected to cisplatin-induced nephrotoxicity, a model of AKI. Cleaved nidogen-1 was not detected in the urine of untreated mice, but during the progression of cisplatin nephrotoxicity, the excretion of cleaved nidogen-1 increased in a time-dependent manner. The meprin inhibitor actinonin markedly prevented urinary excretion of the cleaved nidogen-1. In addition, meprin ß-deficient mice, but not meprin α-deficient mice, subjected to cisplatin nephrotoxicity significantly suppressed excretion of cleaved nidogen-1, further suggesting that meprin ß is involved in the cleavage of nidogen-1. These studies provide strong evidence for a pathophysiological link between meprin ß and urinary excretion of cleaved nidogen-1 during cisplatin-induced AKI.

Endoplasmic Reticulum Stress-Induced Autophagy Provides Cytoprotection from Chemical Hypoxia and Oxidant Injury and Ameliorates Renal Ischemia-Reperfusion Injury.

We examined whether endoplasmic reticulum (ER) stress-induced autophagy provides cytoprotection from renal tubular epithelial cell injury due to oxidants and chemical hypoxia in vitro, as well as from ischemia-reperfusion (IR) injury in vivo. We demonstrate that the ER stress inducer tunicamycin triggers an unfolded protein response, upregulates ER chaperone Grp78, and activates the autophagy pathway in renal tubular epithelial cells in culture. Inhibition of ER stress-induced autophagy accelerated caspase-3 activation and cell death suggesting a pro-survival role of ER stress-induced autophagy. Compared to wild-type cells, autophagy-deficient MEFs subjected to ER stress had enhanced caspase-3 activation and cell death, a finding that further supports the cytoprotective role of ER stress-induced autophagy. Induction of autophagy by ER stress markedly afforded cytoprotection from oxidants H2O2 and tert-Butyl hydroperoxide and from chemical hypoxia induced by antimycin A. In contrast, inhibition of ER stress-induced autophagy or autophagy-deficient cells markedly enhanced cell death in response to oxidant injury and chemical hypoxia. In mouse kidney, similarly to renal epithelial cells in culture, tunicamycin triggered ER stress, markedly upregulated Grp78, and activated autophagy without impairing the autophagic flux. In addition, ER stress-induced autophagy markedly ameliorated renal IR injury as evident from significant improvement in renal function and histology. Inhibition of autophagy by chloroquine markedly increased renal IR injury. These studies highlight beneficial impact of ER stress-induced autophagy in renal ischemia-reperfusion injury both in vitro and in vivo.

Impact of Hydroxychloroquine on Atherosclerosis and Vascular Stiffness in the Presence of Chronic Kidney Disease.

Cardiovascular disease is the largest cause of morbidity and mortality among patients with chronic kidney disease (CKD) and end-stage kidney disease, with nearly half of all deaths attributed to cardiovascular disease. Hydroxychloroquine (HCQ), an anti-inflammatory drug, has been shown to have multiple pleiotropic actions relevant to atherosclerosis. We conducted a proof-of-efficacy study to evaluate the effects of hydroxychloroquine in an animal model of atherosclerosis in ApoE knockout mice with and without chronic kidney disease. Forty male, 6-week-old mice were divided into four groups in a 2 x 2 design: sham placebo group; sham treatment group; CKD placebo group; and CKD treatment group. CKD was induced by a two-step surgical procedure. All mice received a high-fat diet through the study duration and were sacrificed after 16 weeks of therapy. Mice were monitored with ante-mortem ultrasonic echography (AUE) for atherosclerosis and vascular stiffness and with post-mortem histology studies for atherosclerosis. Therapy with HCQ significantly reduced the severity of atherosclerosis in CKD mice and sham treated mice. HCQ reduced the area of aortic atherosclerosis on en face examination by approximately 60% in HCQ treated groups compared to the non-treated groups. Additionally, therapy with HCQ resulted in significant reduction in vascular endothelial dysfunction with improvement in vascular elasticity and flow patterns and better-preserved vascular wall thickness across multiple vascular beds. More importantly, we found that presence of CKD had no mitigating effect on HCQ's anti-atherosclerotic and vasculoprotective effects. These beneficial effects were not due to any significant effect of HCQ on inflammation, renal function, or lipid profile at the end of 16 weeks of therapy. This study, which demonstrates structural and functional protection against atherosclerosis by HCQ, provides a rationale to evaluate its use in CKD patients. Further studies are needed to define the exact mechanisms through which HCQ confers these benefits.