Renal Ischemia/Reperfusion Injury: Functional Tissue Preservation by Anti-Activated ␤1 Integrin Therapy Ana Molina,* Marı´a Ubeda,* Marı´a M. Escribese,* Laura Garcı´a-Bermejo,* David Sancho,† ʈ Guillermo Pe´rez de Lema,‡ Fernando Lian˜o,§ Carlos Caban˜as, Francisco Sa´nchez-Madrid,† and Francisco Mampaso* *Department of Pathology, Hospital Ramo´n y Cajal, Universidad de Alcala´, Madrid, Spain; †Department of Immunology, Hospital de la Princesa, Universidad Auto´noma de Madrid, Madrid, Spain; ‡Medizinische Poliklinic der Ludwig Maximillians-Universitat, Munich, Germany; §Department of Nephrology, Hospital Ramo´n y Cajal, ʈ Universidad de Alcala´, Madrid, Spain; and Institute of Pharmacology and Toxicology (Centro Mixto CSIC-UCM), Facultad de Medicina, Universidad Complutense, Madrid, Spain Renal ischemia/reperfusion injury (IRI) is an important cause of acute renal failure. Cellular and molecular responses of the kidney to IRI are complex and not fully understood. ␤1 integrins localize to the basal surface of tubular epithelium interacting with extracellular matrix components of the basal membrane, including collagen IV. Whether preservation of tubular epithelium integrity could be a therapeutic approach for IRI was assessed. The effects of HUTS-21 mAb administration, which recognizes an activation-dependent epitope of ␤1 integrins, in a rat model of IRI were investigated. Preischemic HUTS-21 administration resulted in the preservation of renal functional and histopathologic parameters. Analyses of activated ␤1 integrins expression and focal adhesion kinase phosphorylation suggest that its deactivation after IRI was prevented by HUTS-21 treatment. Moreover, HUTS-21 impaired the inflammatory response in vivo, as indicated by inhibition of proin- flammatory mediators and the absence of infiltrating cells. Ex vivo adhesion assays using reperfused kidneys revealed that HUTS-21 induced a significant increase of epithelial cell attachment to collagen IV. In conclusion, the data provide evidence that HUTS-21 has a protective effect in renal IRI, preventing tubular epithelial cell detachment by preserving activated ␤1 integrins functions. J Am Soc Nephrol 16: 374–382, 2005. doi: 10.1681/ASN.2004070528 schemia/reperfusion injury (IRI) is an important cause of the basement membrane (BM) (15). Recently, attention has fo- acute renal failure in native kidneys and allografts (1). cused on the role of ␤1 integrins in the loss of tubular cells and I Cellular and molecular responses of the kidney to acute tubular lumen occlusion by cellular debris during acute IRI (2). ischemic injury are complex and not fully understood (2–4). Using an in vitro model of renal epithelial cells that were Several studies have examined the role of leukocytes and their exposed to oxidative stress, it was demonstrated redistribution surface adhesion molecules in the pathogenesis of postischemic of the ␣3␤1 integrin-subunit from the basolateral to apical cell renal damage. Leukocyte adhesion molecules seem to facilitate membranes (16). Also, in an in vivo model of IRI, it has been polymorphonuclear neutrophils recruitment during reperfu- proposed that the decrease of ␤1 integrins on basal surfaces sion, being implicated as mediators of renal IRI (5,6). Comple- contributes to tubular epithelia detachment into the lumen. mentary studies using mAb, antisense oligonucleotides, or Such observations provide a potential mechanism to explain ␤ gene “knockout” indicate that blockade of 2 integrins and/or tubular obstruction and backleak of glomerular filtrate after intercellular adhesion molecule-1 attenuates IRI in some exper- acute IRI (17). Administration of synthetic arginine-glycine- imental models (7–13). Furthermore, it was proposed recently aspartic acid peptides that block integrins ameliorates renal IRI, that T cells might also mediate IRI through the interaction with suggesting that arginine-glycine-aspartic acid peptides inhibit renal tubular epithelial cells (14). cell–cell adhesion in the tubular lumen, thus reducing intratu- ␤ In adult kidney, members of the 1 integrin subfamily are the bular obstruction (18–20). most common and localize to basal surfaces of tubular epithe- Integrin activation and deactivation are not fully understood lia, interacting with extracellular matrix (ECM) components of in in vivo processes. It is conceivable that after IR, ␤1 integrins, which are basally localized in the stressed tubular cells, are ␤ Received July 6, 2004. Accepted October 15, 2004. deactivated. Conversely, as a new epithelium reforms, 1 inte- grins again become activated and return to an exclusively basal Published online ahead of print. Publication date available at www.jasn.org. location. The state of integrin activation can be assessed by a Address correspondence to: Dr. Francisco Mampaso, Hospital Ramo´n y Cajal, group of mAb (HUTS) that selectively recognize ␤1 integrins in Departamento de Patologı´a, Carretera de Colmenar, Km 9.1, 28034 Madrid, Spain. Phone: 34-91-3368052; Fax: 34-91-3369016; E-mail: fmampaso.hrc@salud. their active form (21). In fact, an activated epitope has been madrid.org defined in the rat using the anti-human HUTS-21 mAb, which Copyright © 2005 by the American Society of Nephrology ISSN: 1046-6673/1602-0374 J Am Soc Nephrol 16: 374–382, 2005 ␤1 Integrin in Ischemia/Reperfusion Injury 375 recognizes ␤1 integrins on rat lymphocytes after activation with concentration was quantified by Bradford colorimetric assay (24). Equal ϩ ϩ divalent cations Mn2 and Hg2 . This mAb confers protective amount of proteins (30 ␮g) were separated by 10% SDS-PAGE and effects in an in vivo autoimmune nephritis model (22). immunoblotted onto polyvinylidene difluoride membranes (Bio-Rad, ␤ ␤ We assessed herein the effects of HUTS-21 systemic admin- Hercules, CA). 1 integrin expression was detected using anti- 1 anti- body (M-106; Santa Cruz) and ECL Western blotting detection system istration, infused before the renal artery clamp in an IRI rat (Amersham Pharmacia Biotech., Buckinghamshire, UK). Results were model. A single administration of this anti-activated ␤1 mAb expressed as percentage of ␤1 integrin expression of sham-operated rat resulted in the preservation of renal function and prevention of kidneys. tissue damage after ischemic insult. Quantitative Reverse Transcriptase–PCR Materials and Methods Total RNA was obtained from snap-frozen kidney tissue using the Animals Ultraspec RNA isolation system (Biotecx, Houston, TX). Two micro- Male Sprague-Dawley rats (IFFA-Credo, Paris, France) that weighed grams of DNaseI-treated RNA were reverse-transcribed with MuLV 180 to 200 g were used throughout the experiments. Animals were reverse transcriptase (Roche, Indianapolis, IN). mRNA expression was treated according to the institutional guidelines that are in compliance quantified by real-time PCR following the manufacturer’s instructions with the National Institutes of Health Guide for the Care and Use of (Lightcycler rapid thermal cycler; Roche) using the primers specific for Laboratory Animals. exon sequences (5Ј33Ј): ATG CCA TCC TGC GTC TGG ACC TGG C (␤-actin, sense), AGC ATT TGC GGT GCA CGA TGG C (␤-actin, Surgery and Experimental Protocol antisense), CAC CTC TCA AGC AGA GCA CAG (IL-1␤, sense), GGG Rats were anesthetized with an inhaled anesthesia mixture of isoflu- TTC CAT GGT GAA GTC AAC (IL-1␤, antisense), CCA GGA GAA rane 2% (Abbott Laboratories Ltd., Queenborough, Kent, England) and AGT CAG CCT CCT (TNF-␣, sense), TCA TAC CAG GGC TTG AGC oxygen 1 L/min and placed on a temperature-regulated table (39°C) to TCA (TNF␣, antisense), CCC TTC CGA AGT TTC TGG CAG CAG maintain body temperature. Renal ischemia was induced by clamping (inducible nitric oxide synthase [iNOS], sense), GGG CTC CTC CAA both renal pedicles during 45 min. Vehicle group (n ϭ 36), received an GGT GTT GCC C (iNOS-antisense). intravenous injection of phosphate buffer (pH 7.4) 5 min before clamp- ϭ ing. The HUTS-treated group (n 36) received a single intravenous Isolation of Proximal Tubular Epithelial Cells ␤ injection of 0.28 g/100 g body wt anti-activated 1 integrin HUTS-21 Proximal tubular epithelial cells (PTEC) were obtained from rat ϭ mAb (19) 5 min before clamping. The sham-operated group (n 8) kidney cortices that were subjected to 45 min of ischemia and3hof underwent the same surgical procedure, except that the clamp was not reperfusion or sham operation, following the process described by applied. Blood samples and kidneys were collected at different reper- Vinay et al. (25) with some modifications. Briefly, renal cortices were fusion times and processed for different studies. incubated in gassed Krebs-Henseleit saline (pH 7.4) that contained 0.1 mg/ml collagenase type I (Sigma, St. Louis, MO) at 37°C for1hand Functional and Histopathologic Studies sieved throughout 0.250- to 0.035-mm mesh. Cell populations were Serum creatinine was determined using a modified Jaffe’s reaction, separated by gradient centrifugation in 50% Percoll (Sigma) solution and blood urea nitrogen (BUN) was measured on the AEROSET System (Krebs-Henseleit saline:Percoll, 1:1, gassed for1honice). PTEC that (Abbott Laboratories, Abbott Park, IL). Kidneys were processed for were obtained from the appropriate band were immediately used for light microscopy examination according to standard procedures, and cell adhesion experiments. sections were stained with hematoxylin and eosin and periodic acid- Schiff. Histopathologic changes were analyzed for tubular epithelial Cell Adhesion Assay cell necrosis, tubular