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Involvement of heparanase in the pathogenesis of acute kidney injury: nephroprotective effect of PG545

Despite the high prevalence of acute kidney injury (AKI) and its association with increased morbidity and mortality, therapeutic approaches for AKI are disappointing. This is largely attributed to poor understanding of the pathogenesis of AKI. Heparanase, an endoglycosidase that cleaves heparan sulfate, is involved in extracellular matrix turnover, inflammation, kidney dysfunction, diabetes, fibrosis, angiogenesis and cancer progression. The current study examined the involvement of heparanase in the pathogenesis of ischemic reperfusion (I/R) AKI in a mouse model and the protective effect of PG545, a potent heparanase inhibitor. I/R induced tubular damage and elevation in serum creatinine and blood urea nitrogen to a higher extent in heparanase over-expressing transgenic mice vs. wild type mice. Moreover, TGF-β, vimentin, fibronectin and α-smooth muscle actin, biomarkers of fibrosis, and TNFα, IL6 and endothelin-1, biomarkers of inflammation, were upregulated in I/R induced AKI, primarily in heparanase transgenic mice, suggesting an adverse role of heparanase in the pathogenesis of AKI. Remarkably, pretreatment of mice with PG545 abolished kidney dysfunction and the up-regulation of heparanase, pro-inflammatory (i.e., IL-6) and pro-fibrotic (i.e., TGF-β) genes induced by I/R. The present study provides new insights into the involvement of heparanase in the pathogenesis of ischemic AKI. Our results demonstrate that heparanase plays a deleterious role in the development of renal injury and kidney dysfunction, attesting heparanase inhibition as a promising therapeutic approach for AKI.


INTRODUCTION
Acute kidney injury (AKI) is a common clinical disorder affecting 2-7% of hospitalized patients and some 20-50% of critically ill subjects [1,2]. Renal ischemia and hypoxia, toxic insult and sepsis are the leading causes of clinical AKI [1,2]. Specifically, AKI is characterized by decreased oxygen delivery, depletion of cellular energy stores and accumulation of toxic metabolites. The cellular depletion of ATP, a hallmark of ischemic injury, leads to a series of morphological, biochemical and physiological derangements [2][3][4]. Reperfusion of ischemic tissue, although necessary for repair, has been shown to exacerbate AKI owing to the generation of reactive oxygen and nitrogen species [2][3][4][5][6]. Despite the advances in critical care medicine, AKI is still associated with high morbidity and mortality assumedly due to delayed detection of the disease and lack of effective treatment [7].
Several strategies were proposed to deal with AKI [2,3,8]. Among these are antioxidants including acetylcysteine, antioxidant enzyme mimetics, erythropoietin, peroxisome-proliferator-activated receptor agonists, inhibitors of poly(ADP-ribose) polymerase, carbon monoxide-releasing molecules and other measures to enhance hypoxia inducible factors, statins, and adenosine [2,3,6]. Unfortunately, the clinical efficacy of these therapeutic approaches is inconsistent and disappointing. Therefore, there is a great need to improve our understanding of the pathogenesis of AKI and design new strategies to prevent kidney damage during AKI.
Heparan sulfate (HS) proteoglycans (HSPGs) are ubiquitous macromolecules associated with the cell surface and extracellular matrix (ECM) of a wide range of tissues including the kidney [9]. The HS chains bind to and assemble ECM proteins, thus playing important roles in ECM integrity, barrier function, and cell-ECM interactions [9]. HSPGs not only provide a storage depot for heparin binding molecules (i.e., growth factors, chemokines, enzymes) in the tissue microenvironment, but also regulate their accessibility, function and mode of action. It is therefore not surprising that a HS degrading enzyme, heparanase, is critically involved in pathological processes, including tumor growth, metastasis, angiogenesis, thrombosis, fibrosis, inflammation, autoimmunity and kidney dysfunction [10][11][12][13][14][15][16][17][18].
Although heparanase is recognized for its important role in diabetic nephropathy and other progressive CKD [34], its direct involvement and mode of action in the pathogenesis of AKI has not been studied thoroughly. We have previously reported that heparanase is involved in the onset and development of I/R-induced epithelial to mesenchymal transition (EMT) both in vitro and in vivo [35] and Lygizos et al [36] found that glomerular heparanase is activated during sepsis and contributes to septic AKI. Elucidation of the mechanism underlying the function of heparanase in AKI is critically important for the proper design of novel therapeutics approaches, including heparanase inhibitors such as PG545, Roneparstat and other compounds currently under intensive development and clinical testing [17,37]. PG545, a synthetic fully sulfated HS mimetic with potent, long acting heparanase inhibiting capacity [38,39], ended phase I clinical trial in cancer patients. The availability of PG545 and of heparanase over-expressing transgenic mice (Hpa-tg) [40] provides a most appropriate experimental platform to elucidate the involvement of heparanase in the pathogenesis of AKI. We report that PG545 abolished kidney dysfunction and the up-regulation of heparanase, pro-inflammatory (i.e., IL-6) and pro-fibrotic (i.e., TGF-β) mediators induced by I/R AKI.

Acute ischemic injury up-regulates renal heparanase expression and enzymatic activity
Acute ischemic injury was induced by clamping both renal arteries for 30 minutes. As previously described [35], real-time PCR analyses ( Figure 1A) confirmed an increment of heparanase expression in renal tissue of wt mice 48 h after ischemia and a more pronounced effect was noted in Hpa-tg mice at 72 h. Immunofluorescence Oncotarget 34193 www.impactjournals.com/oncotarget staining of renal tissue of wt animals confirmed that acute ischemic renal injury up-regulated heparanase in glomeruli, tubular cells and interstitial cells ( Figure 1B). As demonstrated in Figure 1C, heparanase enzymatic activity was markedly enhanced following AKI in wt mice. Thus, when incubated with sulfate-labeled ECM, extracts from kidney of wt mice that were subjected to AKI released high amounts of HS degradation fragments (peak at fraction 23) as compared with sham control mice. As expected, kidney tissue from untreated Hpa-tg mice exhibited high basal heparanase activity ( Figure 1D). These results are supported by qPCR analysis ( Figure 1A) and immunostaining ( Figure 1B) showing that heparanase expression and immunoreactivity were increased following AKI induction in both wt and Hpa-tg mice. Importantly, when wt and Hpa-tg mice were pretreated with PG545, heparanase gene expression ( Figure 1A), immunoreactivity ( Figure 1B) and enzymatic activity ( Figure 1C, 1D) were profoundly suppressed, signifying the remarkable protective effect of PG545 against I/R.

Heparanase induces acute ischemic injury Histology
The structural changes in the kidney tissue of control and I/R wt and Hpa-tg mice were evaluated by PAS staining. We confirmed that at 48 h after I/R, wt mice  Oncotarget 34195 www.impactjournals.com/oncotarget showed acute tubular necrosis which included tubular lysis, loss of brush border and sloughed debris in the tubular lumen spaces (Figure 2A). While in wt mice the damage was partially attenuated after 72 h, the injury in Hpa-tg mice was more profound and persistent also after 72 h (not shown). In Hpa-tg mice there was a significant alteration in glomeruli and tubular structures. In particular, in Hpa-tg mice I/R produced a severe tubular damage with tubular dilatation, cell detachment from basement membrane, cast formation and loss of brush border ( Figure  2A). Notably, these effects were partially prevented in response to pretreatment with PG545.

Ultrastructure alterations
Electron microscopy analyses of the renal tissue from the various experimental groups are presented in Figure 2B. As expected, induction of AKI resulted in remarkable mitochondrial alterations. Specifically, the mitochondria in the tubular cells of control wt and Hpatg mice exhibited elongated cylindrical shape, whereas induction of AKI in both wt and Hpa-tg mice resulted in fragmented mitochondria and transformation from filamentous shape into short rods ( Figure 2B). These deleterious alterations were more profound in Hpa-tg mice as was evident by spherical shape and matrix vacuoles (cristolysis). Pretreatment with PG545 of either wt or Hpa-tg mice partially restored the tubular mitochondrial morphological changes induced by AKI ( Figure 2B) as was evident by nearly normal ultrastructural appearance of the mitochondrial cristae ( Figure 2B).

Kidney function
Besides the histological changes, we further determined kidney function by measuring blood urea after I/R insult in wt and Hpa-tg mice. Note that AKI induced a more profound increase in both SCr and BUN in Hpa-tg mice as compared with wt mice. Additional two groups of wt and Hpa-tg mice were pretreated one day prior to AKI induction with PG545 (0.4 mg/mouse, ip) and sacrificed 48 h after renal injury A., C. *p < 0.05, **p < 0.01 vs. sham; #p < 0.01 vs. untreated mice; $ p < 0.05 vs. wt.
Oncotarget 34196 www.impactjournals.com/oncotarget nitrogen (BUN) and serum creatinine (SCr) in the studied experimental groups (Figure 3). Induction of AKI in both wt and Hpa-tg mice was characterized by significant elevation of serum creatinine (SCr) ( Figure 3A, 3B) and blood urea nitrogen (BUN) ( Figure 3C, 3D), which was evident after 48 h and 72 h in both the wt and Hpa-tg mice ( Figure 3), but was more prominent in the Hpa-tg mice after 72 h ( Figure 3B, 3D). Pretreatment with PG545 attenuated the elevation of SCr and BUN in the Hpa-tg mice ( Figure 3A, 3C).

Heparanase inhibition attenuates epithelial mesenchymal transition
Gene expression analysis of total kidney lysates and immunofluorescence staining for α-SMA ( Figure

Heparanase affects TGF-β, ET-1, IL6, TNFα and cathepsin L expression
In wt mice, 48 and 72 h after ischemia, TGF-β mRNA levels were similar to sham operated animals. In contrast, in Hpa-tg mice, acute I/R injury induced significant up-regulation of the TGF-β gene at both 48 h and 72 h ( Figure 5A). A similar pattern of ET-1 ( Figure  5B) and IL6 ( Figure 5C) upregulation was observed in Hpa-tg, but not wt mice that were subject to AKI, yet the upregulation of these genes was significant only at 72 h. Induction of AKI in wt and Hpa-tg mice was also associated with enhanced expression of TNFα ( Figure  5D) and cathepsin L ( Figure 5E), a key enzyme in the processing and activation of latent heparanase [41]. Again, the increased expression of these two genes was more profound in Hpa-tg mice. Remarkably, pretreatment with PG545 abolished the elevation in TGFβ, and to a lesser extent the upregulation of ET-1, IL-6, cathepsin L and TNFα. Altogether, it appears that biomarkers of fibrosis, EMT and inflammation were upregulated in I/R induced AKI, primarily in Hpa-tg mice, and this elevation was prevented to a large extent following pretreatment with PG545.

DISCUSSION
Despite the high prevalence of AKI and its association with an alarming increase in morbidity and mortality, the therapeutic approaches for AKI are still disappointing and rely mainly on supportive measures. This deficiency stems mainly from the poor understanding of the pathogenesis of AKI. The current research is therefore of special significance since it provides new insights into the mechanism underlying ischemic AKI, a major etiology of AKI. Specifically, we demonstrated that I/R of the kidney induced renal injury in both wt and Hpatg mice as was evident by typical tubular damage including cell lysis, loss of brush border and sloughed debris in tubular lumen, which was more profound in Hpa-tg mice ( Figure 2). The latter also displayed exaggerated elevation in SCr and BUN. Moreover, TGF-β, fibronectin, vimentin and α-smooth muscle actin, biomarkers of epithelial mesenchymal transition [35,42], were upregulated in I/R induced AKI, especially in Hpa-tg mice, indicating an adverse role of heparanase in the pathogenesis AKI. This notion is further supported by the fact that pretreatment with PG545, a potent long acting inhibitor of heparanase [38], abolished the adverse renal changes induced by I/R and improved kidney function. Collectively, these findings clearly implicate heparanase in the pathophysiology of AKI, and suggest heparanase inhibitors as preventive/ therapeutic option for this common clinical setting. Given that transgenic mice may differ from their wt counterparts in various aspects, each experiment was performed with both wt and Hpa-tg mice, yielding the same results (i.e., upregulation of biomarkers of fibrosis, inflammation and EMT and down regulation/protection by the heparanase inhibitor PG545), albeit to a lower extent in wt mice. Overall it appears that the disease model is operating in wt mice in a manner similar to that seen in Hpa-tg mice.
Despite the remarkable advances in nephrology, the pathophysiology of AKI remains largely obscured. Thus, research involving genetically modified animal models is a critical step in providing a new perspective into the pathophysiology of AKI. Indeed, the Hpa-tg mouse model [40] provides an appropriate experimental platform to elucidate the involvement of heparanase in the pathogenesis of AKI. Of note, Hpa-tg mice revealed increased levels of urinary creatinine at baseline, indicating a slight effect of heparanase on renal function even without induction of AKI (40), although this increase did not reach statistical significance as compared with wt mice (Figure 3). This result further supports the involvement of heparanase in kidney dysfunction, www.impactjournals.com/oncotarget showing that the heparanase level in Hpa-tg mice is by itself not enough to induce acute damage, whereas its combination with I/R results in AKI to a higher extent in Hpa-tg than in wild type mice. Similar to other heparanase-inhibiting heparin-like compounds PG545 displays some anti-coagulant properties, but this side effect was not detected in the AKI model nor in numerous tumor models in which mice are treated with PG545 for weeks [38]. Also, PG545 was found safe in phase I clinical trial in cancer patients [17,37]. Our preliminary studies indicate that administration of PG545 post I/R failed to restore kidney damage and function. Yet, our pre-treatment approach maybe of clinical relevance in cases where AKI may occur (i.e., patients who undergo imaging involving i.v radiocontrast administration; patients undergoing major vascular surgery and cardiopulmonary bypass). So far there is no effective preventive treatment to minimize the incidence of AKI under these and other clinical settings, suggesting that PG545 may constitute a novel prophylactic therapeutic approach.
Unlike the Hpa-tg mice, results obtained with heparanase knockout mice [43] were not straightforward. AKI was induced by I/R also in the Hpa-KO mice, albeit to a lower extent than in wt and Hpa-tg mice, as revealed by the respective BUN and creatinine values. We have previously reported that the Hpa-KO mice reveal compensations for the lack of heparanase. These include up-regulation of matrix degrading enzymes and pro- representing relative gene expression of TGF-β A., ET-1B., IL6 C., TNFα D. and Cathepsin L E., evaluated by real-time PCR in renal tissue extracts from wt and Hpa-tg mice that underwent I/R kidney injury with or without pre-treatment with PG545. Results were normalized to GAPDH expression. *p < 0.05, **p < 0.001 vs. wt sham; #p < 0.05, ##p < 0.01 vs. corresponding group w/o I/R ; $p < 0.05, $$p < 0.01 vs. corresponding wt; +p < 0.05, ++p < 0.01 vs. untreated I/R corresponding group.
Oncotarget 34199 www.impactjournals.com/oncotarget inflammatory mediators [43], resulting, paradoxically, in several phenotypes that closely resemble phenotypes seen in the Hpa-tg mice, making it difficult to interpretate data obtained with the Hpa-KO mice.
The availability of PG545 allows exploring potential therapeutic effects of heparanase inhibition in AKI. The latter is of special interest since in contrast to the well-established association between heparanase and glomerular diseases [20,28], the role of heparanase in renal EMT associated with I/R injury has only recently been addressed [35]. Notably, a role for heparanase was identified in several proteinuric nephropathies [20,23,28], mainly in the pathogenesis of diabetic nephropathy [26,33] and in a model of septic AKI [36]. The latter is not surprising in light of the emerging data linking heparanase and inflammatory responses, suggesting a role of this enzyme in AKI. Indeed, Lygizos et al [36] found that glomerular heparanase is activated during sepsis and contributes to septic AKI. Specifically, the authors induced polymicrobial sepsis in mice using cecal ligation and puncture (CLP) in the presence or absence of competitive heparanase inhibitors (heparin or nonanticoagulant N-desulfated re-N-acetylated heparin). CLP-treated mice demonstrated early activation of glomerular heparanase with coincident loss of glomerular function, as indicated by an increase in blood urea nitrogen (BUN) and decrease in GFR. Administration of heparanase inhibitors two hours prior to CLP attenuated the deleterious consequences of sepsis, suggesting that glomerular heparanase is active during sepsis and contributes to septic renal dysfunction via uncharacterized mechanisms. In line with these findings, bilateral renal ischemia/reperfusion (I/R) in syndecan-1 deficient mice resulted in increased initial renal failure and tubular injury compared with wt mice [44]. In additional study, these authors have shown that within 24 h after renal I/R, HSPGs expressed at the abluminal side of peri-tubular capillaries are induced to bind L-selectin and the monocyte chemoattractant protein-1, and that HSPGs facilitate monocyte extravasation [45]. A vicious circuit of heparanase-driven molecular events promoting chronic inflammation and renal injury has recently been described [11,33]. This circuit is fueled by heterotypic interactions among glomerular, tubular, and immune cell compartments. It appears that latent heparanase, over-expressed by glomerular, epithelial and interstitial cells and post-translationally activated by cathepsin L, sustains continuous activation of kidneydamaging macrophages via degradation of HS and release of chemokines anchored within the ECM network and cell surfaces [11]. Moreover, active heparanase governs macrophage activation via HS degradation fragments which activate TLR2 and TLR4 [46], leading to increased levels of TNF-α thereby creating inflammatory conditions fostering macrophage-mediated renal injury and upregulation of heparanase expression. This scenario is further supported by our findings. First, Hpa-tg mice were more susceptible to I/R-induced renal damage and kidney dysfunction as compared with their wt counterparts subjected to the same surgical maneuver. Second, using gene silencing approaches we further revealed an essential involvement of heparanase in the development of I/R-induced epithelial damage and suggested that its inhibition may signify a therapeutic approach to minimize/ prevent ischemic-induced renal fibrosis [35,47]. Both, our previous in vitro studies applying cultured epithelial cells that were subjected to I/R [35,47] and the current in vivo ischemic AKI model in wt and Hpa-tg mice exhibited upregulation of heparanase along over-expression of proinflammatory and pro-fibrotic cytokines (i.e., TGF-β, IL-6, TNFα) as well as markers of EMT (i.e., VIM, FN, αSMA). Activated macrophages secrete TNFα and cathepsin L, which stimulate further production and activation of heparanase by these cells and by ischemia-induced renal injured cells.
Upregulation of the TGF-β gene suggests that I/R triggers renal fibrogenesis, most likely through epithelialmesenchimal transition (EMT) of tubular cells [48]. Hparanase seems to play a major role in these processes as was evident by the exaggerated overexpression of TGF-β in Hpa-tg mice exposed to I/R. While the critical causal involvement of TGF-β in EMT and tissue fibrosis is well documented [42,49], little is known about a crosstalk between heparanase and TGF-β [48]. Even more so, a crosstalk between heparanase and ET-1 [50,51] in AKI has not been reported. Evidence for the combined involvement of heparanase, TGF-β and ET-1 in the fibrosis/inflammatory response to AKI is provided by showing that treatment with PG545 prior to I/R maintained a normal epithelial phenotype and abolished the EMT transcriptional program exemplified by a marked decrease in the levels of VIM, FN, α-SMA, IL-6, ET-1 and TGF-β. PG545, a potent heparin-like inhibitor of heparanase enzymatic activity, markedly decreases the ability of heparanase to release and potentiate the activity of ECMbound growth factors [37], further supporting PG545 as potential anti-fibrotic and kidney protective drug. Downregulation of heparanase expression in response to PG545 was noted in 4T1 breast carcinoma model [52]. This effect was attributed in part to inhibition of growth factorstimulated ERK activation by PG545 and the resulting inhibitory effect on the transcription factor early growth response 1 (EGR1), known to induce heparanase gene expression [53]. Given the multi target genes regulated by EGR1 [54], the same mechanism may explain the effect of PG545 on the expression TGF-β, TNF-α, fibronectin and possibly other cytokines. Similarly, we have recently reported that heparanase activates Erk, p38 and JNK signaling, leading to increased c-Fos levels and induction of cytokine expression [55]. Suppression of this cascade by PG545 may affect the transcription of multiple genes including heparanase.
One of the studied parameters that might be of special relevance to kidney damage is ET-1, a vasoconstrictor released by endothelial cells [56]. ET-1 www.impactjournals.com/oncotarget acts via two receptors subtypes ETA and ETB to modulate vasoconstriction, proliferation, inflammation, ECM production, and fibrosis in various target organs including the kidney [57,58]. An evidence for the deleterious role of ET-1 in renal damage is derived from the nephroprotective effects of ET-1 antagonists in experimental and clinical models of AKI [59]. Previous studies have demonstrated a linkage between ET-1 and heparanase [60]. Such an association is essential for the development of DN, as was proven by the observation that heparanase-deficient mice were protected against diabetes-induced proteinuria and renal damage [33]. Addition of ET-1 increased heparanase mRNA expression and enzymatic activity in cultured podocytes [60]. Our results clearly show a positive correlation between renal heparanase and ET-1. Both were upregulated during AKI and inhibition of heparanase was associated with a decline in ET-1, suggesting a causal relationship between ET-1 and heparanase during ischemic AKI.
In summary, our results indicate that heparanase is involved in the pathogenesis of ischemic AKI. Moreover, we demonstrated that pretreatment with a potent heparanase inhibitor (PG545) exerts nephroprotective effect against this clinical setting. Thus, our study provides a better perspective on the pathogenesis of AKI and the potential development of therapeutic/preventive interventions.

Animal model
Studies were conducted on wt (Balb/c) and corresponding heparanase-overexpressing (Hpa-tg) mice weighing 23-30 gr and maintained on standard rat chow and water ad libitum. Mice were crossed for over 10 generations with Balb/c wt mice to produce pure genetic background. The Hpa-tg mice appeared normal and exhibited a normal life span and breeding behavior [40]. Western blot analysis revealed high expression of heparanase in the liver and kidney vs. low expression in the spleen of Hpa-tg vs. control mice [40,61]. The study was performed according to the guidelines of the Animal Use and Care Committee, Technion (Haifa), and according to the Guide for the Care and Use of Laboratory Animals (NIH Publication no. 1996) as approved by the local committee for supervision of animal experiments.

Induction of renal ischemia
Acute ischemia was induced in wt (Balb/c) and Hpa-tg mice as described by Mishra et al [62]. Wt and Hpa-tg mice were anesthetized with sodium pentobarbital (50 mg/kg, IP) and placed on a controlled heating table, keeping the body temperature at 37 o C. Both right and left renal arteries were exposed and clamped for 30 min during which time the kidney was kept warm and moist. The clamp was then removed, the kidney was observed for the return of blood flow, and the abdominal wall incision was sutured. Mice were allowed to recover in a warmed cage for 48 and 72 h and blood samples were obtained at each time point for measurement of creatinine and urea. At these time points, kidney dysfunction and renal damage peak, especially 48 h post I/R [63]. Additional groups of wt and Hpa-tg mice were pretreated one day prior to AKI induction with PG545 (0.4 mg/mouse, ip). The kidneys were harvested and weighted. One half of the left kidney was snap frozen in liquid nitrogen and stored at −70°C until further molecular processing; the other half was fixed in formalin, paraffin-embedded, and sectioned (4 μm). Paraffin sections were stained with hematoxylineosin and examined histologically. The right kidney was embedded in isopentane and frozen sections (4 μm) were obtained for immunohistochemistry [35]. Sham operated mice that underwent an identical procedure except renal artery clamping, served as controls. The number of mice per group (n) ranged from 6 to 12.

Quantitative real-time PCR
Total RNA was extracted with TRIzol (Sigma) and RNA (1 µg) was amplified using one step PCR amplification kit, according to the manufacturer's (ABgene, Epsom, UK) instructions, essentially as described [55]. The moue primer sets used are listed in Table 1.

Electron microscopy
Renal tissues from the various experimental groups were fixed in 3.5% glutaraldehyde and rinsed in 0.1 M sodium cacodylate buffer, pH 7.4. Tissue blocks (1 mm 3 ) were post-fixed with 2% OsO 4 in 0.2 M cacodylate buffer for 1 h, rinsed again in cacodylate buffer to remove excess osmium, immersed in saturated aqueous uranyl acetate, dehydrated in graded alcohol solutions, immersed in propylene oxide, and embedded in Epon 812. Ultrathin sections (80 nm) were mounted on 300-mesh, thinbar copper grid, counterstained with saturated uranyl acetate and lead citrate. Sections were examined with a transmission electron microscope (Jeol 1011 JEM) at 80 KV.

Chemical analyses
Blood urea nitrogen and serum creatinine levels were determined using commercially available colorimetric kits (Cayman, Ann Arbor, MI; Bioassay Systems, Hayward, CA, respectively).

Statistical analysis
Mean ± SEM of the real-time PCR data were calculated with Rest2009 software. Gene expression differences in mouse samples were analyzed by linear regression models with group (WT-sham, WT-I/R 48h, WT-I/R 72h, TG-sham, TG-I/R 48 h, TG-I/R 72 h) entered as a categorical variable. Bonferroni-corrected adjusted means and differences were computed using the WT-sham as the referent group. A Bonferroni-corrected p-value < 0.05 was considered as statistically significant.

ACKNOWLEDGMENTS
We are grateful to the Israeli Society of Nephrology for their financial support. This study was supported in part by a research grant awarded to I.V. by the Israel Science Foundation (grant 601/14); I. V. is a Research Professor of the ICRF. P.S. is supported by a postdoctoral fellowship awarded by the Israeli Council for Higher Education (PBC program). The authors are grateful for the professional help of Irena Minkov, Department of Pathology, Rambam Health Campus, Haifa, Israel.

CONFLICTS OF INTEREST
Edward Hammond is employed by Zucero Therapeutics, Darra, Queensland, Australia.