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UNC5B Receptor Deletion Exacerbates Tissue Injury in Response to AKI

† † Punithavathi Ranganathan,* Calpurnia Jayakumar,* Sutip Navankasattusas, Dean Y. Li, Il-man Kim,* and Ganesan Ramesh*

*Department of Medicine and Vascular Biology Center, Georgia Regents University, Augusta, Georgia; and †Program in Molecular Medicine, University of Utah, Salt Lake City, Utah

ABSTRACT Netrin-1 regulates cell survival and by activation of its receptors, including UNC5B. However, the in vivo role of UNC5B in cell survival during cellular stress and tissue injury is unknown. We investigated the role of UNC5B in cell survival in response to stress using mice heterozygously expressing the UNC5B 2 fl gene (UNC5B / ox) and mice with targeted homozygous deletion of UNC5B in kidney epithelial cells 2 fl (UNC5B / ox/GGT-cre). Mice were subjected to two different models of organ injury: ischemia reperfusion injury of the kidney and cisplatin-induced nephrotoxicity. Both mouse models of UNC5B depletion had 2 fl normal organ function and histology under basal conditions. After AKI, however, UNC5B / ox/GGT-cre mice BASIC RESEARCH exhibited significantly worse renal function and damage, increased tubular apoptosis, enhanced ac- 2 fl tivation, and exacerbated inflammation compared with UNC5B / ox and wild-type mice. shRNA-mediated suppression of UNC5B expression in cultured tubular epithelial cells exacerbated cisplatin-induced cell death in a p53-dependent manner and blunted Akt phosphorylation. Inhibition of PI3 kinase similarly exacerbated cisplatin-induced apoptosis; in contrast, overexpression of UNC5B reduced cisplatin-in- duced apoptosis in these cells. Taken together, these results show that the netrin-1 receptor UNC5B plays a critical role in cell survival and kidney injury through Akt-mediated inactivation of p53 in response to stress.

J Am Soc Nephrol 25: 239–249, 2014. doi: 10.1681/ASN.2013040418

AKI is a life-threatening disorder that has been Netrin-1 is a -related secreted molecule increasing in incidence. About 6% of hospitalized identified as a neuronal guidance cue that directs patients and more than 30% of patients in the neurons and their to targets during develop- intensive care unit develop AKI, which can lead to ment of the nervous system.14 Although netrin-1 is organ failure and death.1–3 Currently, there are effec- primarily thought of as an guidance cue, this tive therapies available to treat AKI. Several in vivo function is unlikely to be its only function, because studies suggest that administration of netrin-1 sup- expression studies have shown that netrins are presses inflammation and apoptosis in the kidney widely expressed outside the nervous system, in- and other organs caused by different insults.4–10 cluding in vascular endothelial cells as well as the 4,15 Moreover, netrin-1 is highly induced in tubular epi- liver, lung, colon, heart, and kidney. In fact, the thelial cells and secreted into the tubular lumen within hours after ischemia reperfusion.11 Overex- pression of netrin-1, specifically in proximal tubular Received April 25, 2013. Accepted July 3, 2013. epithelial cells, suppressed ischemia reperfusion and Published online ahead of print. Publication date available at nephrotoxin-induced kidney injury and dextran sul- www.jasn.org. fate sodium-induced colon injury by suppressing ap- Correspondence: Prof. Ganesan Ramesh, Vascular Biology Center, optosis.12,13 However, the receptor that mediates the CB-3702, Georgia Regents University, 1459 Laney-Walker Boulevard, protective effects of netrin-1 and the mechanism of Augusta, GA 30912. Email: [email protected] protection against cell death are unknown. Copyright © 2014 by the American Society of Nephrology

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2 fl kidney was shown to have the highest level of netrin-1 mRNA and littermates without cre (UNC5B / ox)orWTmicewere compared with any other tissues studied so far.15 Netrin-1 subjected to 26 minutes of ischemia followed by reperfusion. 2 fl mediates its biologic activity by binding to two vertebrate fam- Both UNC5B / ox/GGT-cre mice and littermates without cre ilies of receptors: the Deleted in Colorectal Cancer (DCC) died within 24 hours after ischemia, but the WT mice sur- family comprising DCC and neogenin receptors and the un- vived, suggesting that deletion of a single allele of UNC5B alone coordinated 5 (UNC5) family UNC5A-D receptors.16 Both increases susceptibility to ischemia reperfusion of the kidney. DCC and UNC5 family members are called dependence re- To determine the contribution of UNC5B in the proximal 2 fl 2 fl ceptors because of the dependence of cell survival on the pres- tubular epithelial cells, UNC5B / ox/GGT-cre and UNC5B / ox ence of the netrin-1 ligand, and the absence of these receptors mice were subjected to 22 minutes of ischemia followed by is known to induce apoptosis.17–19 However, the in vivo rele- 48 hours of reperfusion. Kidney function was assessed by mea- vance of this dependence receptor hypothesis is not clear. For suring the levels of serum creatinine at different times after example, very little netrin-1 protein is expressed in normal reperfusion. WT mice did not show any increase in serum cre- kidney tubular epithelial cells, but UNC5B expression was atinine levels with a mild form of ischemia, whereas mice with found in the same cells. If dependence receptor hypothesis is proximal tubular epithelial cell-specificUNC5Bdeletion functioning, then we would expect to see a large number of showed a large increase in serum creatinine levels and BUN apoptotic cells in the normal kidney, which was not the case. (Figures 1 and 2, A and B). Most of the animals died by 72 hours, Therefore, we hypothesized that UNC5B receptor signaling is which suggests UNC5B has a critical protective role against critical for cell survival in response to injurious stimulus. In ischemic tubular injury. Consistent with kidney dysfunction, the absence of the UNC5B receptor, cells and tissues are highly histology showed more necrotic tubules, cast formation, and 2 fl prone to injury. Previous studies from our laboratory showed dilated tubules in UNC5B / ox/GGT-cre mice compared with 2 fl that the UNC5B receptor is highly expressed in proximal tu- UNC5B / ox mice kidneys (Figure 2C). Netrin-1 is known to bular epithelial cells of the kidney in vivo and in vitro, whereas regulate immune cell infiltration10,15 in the kidney. To determine DCC expression is very low or negligible. Moreover, UNC5C whether UNC5B deletion in proximal tubular epithelial cells in- expression is restricted to distal tubules where UNC5A and creased infiltration of neutrophils, immunolocalization was per- 2 fl UNC5D are not expressed in the kidney.20 In addition, netrin- formed. As shown in Figure 2, C and D, UNC5B / ox/GGT-cre 1–induced epithelial cell proliferation and migration in vitro mouse kidneys showed a large number of neutrophils compared 2 fl were mediated through UNC5B, and invivo antibody-based neu- with UNC5B / ox mice kidneys. tralization of UNC5B function abolished netrin-1 protective effects against ischemia reperfusion injury of the kidney.10,21 These data led us to investigate the critical role of UNC5B in renal epithelial survival in response to AKI. In this study, we investigated our hypothesis using tissue- specific UNC5B knockout animals as well as animal with partial deficiency of UNC5B receptors. We have generated kidney proximal tubular epithelial (TKPTS) cell-specificUNC5B knockout mice using Cre-lox technology. These mice were subjected to ischemia reperfusion injury and cisplatin-induced kidney injury. Here, we report that deletion of the netrin-1 receptor UNC5B rendered the mice highly susceptible to ischemic and toxic kidney injury. The exacerbated kidney injury is caused by increased apoptosis, p53 activation, and inflammatory response in the epithelial cells of the kidney compared with wild-type (WT) or heterozygous knockout mice. Moreover, this activation of p53 was caused by defective activation of protein kinase B (Akt) pathways in UNC5B Figure 1. Characterization of mice with proximal tubular epithelial knockout epithelial cells. cell specific deletion of UNC5B receptor. Characterization of TKPTS- specific deletion of UNC5B. (A) Genomic PCR showing successful fl fl 2 fl generation of three different genotypes: UNC5B ox/ ox,UNC5B / ox, 2/flox/GGT-cre RESULTS and UNC5B . The band marked deleted represents whole-body deletion of a single allele for UNC5B. (B and C) Immu- 2 fl nohistochemical localization of the UNC5B receptor in UNC5B / ox fi 2 fl Proximal Tubular Epithelial Cell-Speci c Deletion of and UNC5B / ox/GGT-cre mice. Heterzygous knockout mice without UNC5B in Mice Exacerbates Ischemia Reperfusion cre showed UNC5B staining in the proximal tubular epithelial cells, Injury of the Kidney whereas GGT-cre–positive mice showed no staining for UNC5B, To determine the role of tubular epithelial UNC5B receptor in suggesting successful deletion of the floxed UNC5B gene. Scale bar, 2 fl ischemic injury of the kidney, mice with UNC5B / ox/GGT-cre 100 mM.

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2 fl increased in UNC5B / ox/GGT-cre mice (Fig- ure 3). This result suggested that deletion of UNC5B exacerbates the inflammatory re- sponse of the kidney.

UNC5B Deletion in Proximal Tubular Epithelial Cells Increases Tubular Epithelial Cell Apoptosis Netrin-1 was shown to be a survival factor for intestinal epithelial cells, and its adminis- trationoroverexpressionsuppressedischemia- induced tubular epithelial cell apoptosis. Netrin-1 is also highly induced after ischemia and may act to limit the apoptosis. To de- terminewhetherUNC5Bdeletioninproximal tubular epithelial cells alters ischemia reper- fusion-induced apoptosis, apoptoticcells were quantified using the terminal deoxynucleo- tidyl transferase-mediated digoxigenin- deoxyuridine nick-end labeling (TUNEL) 2 fl assay. Sham-operated UNC5B / ox (Figure 2 fl 4A) and UNC5B / ox/GGT-cre (Figure 4C) mouse kidneys did not show any apoptotic cells. Ischemia reperfusion induced a small 2 fl increase in apoptosis in UNC5B / ox mice (Figure 4B). However, ischemia reperfu- sion-induced apoptosis was drastically 2 fl increased in UNC5B / ox/GGT-cre mice fi Figure 2. Kidney injury and histopathological changes in kidney-specificUNC5B (Figure 4D). Quanti cation of apoptotic 2 fl 2 fl fi knockout mice. (A and B) UNC5B / ox and UNC5B / ox/GGT-cre mice were subjected to cells showed a signi cant increase in 2/flox/GGT-cre 22 minutes of ischemia followed by 48 hours of reperfusion. Kidney function was UNC5B mice compared with 2 /flox measured by quantifying serum creatinine. Mice with kidney-specific UNC5B deletion UNC5B mice (Figure 4E). In- developed severe renal injury, which was seen by increased (A) serum creatinine and creased apoptosis was associated with in- (B) BUN, whereas heterozygous knockout mice did not develop any injury. *P,0.001 creased p53 activation and expression in 2/flox 2 fl 2 fl versus UNC5B . (C) Photomicrograph of periodic acid–Schiff (PAS)-stained UNC5B / ox and UNC5B / ox/GGT-cre mice and neutrophil-stained kidney tissue sections 6 hours after ischemia reperfusion. 2 fl compared with WT mice. To determine UNC5B / ox/GGT-cre mice kidney shows cast formation and brush border damage, 2 fl 2 fl whether the increased netrin-1 expression whereas UNC5B / ox mice did not develop any injury. In addition, UNC5B / ox/GGT-cre seen in proximal tubular epithelial cells after mouse kidney showed a large number of infiltrated neutrophils compared with 2 fl 15 UNC5B / ox mouse kidney. (D) Quantification of tubular damage and neutrophil in- reperfusion is associated with UNC5B re- 2 fl 2 fl filtration in UNC5B / ox and UNC5B / ox/GGT-cre mice subjected to ischemia reperfusion. ceptor expression, colocalization of netrin-1 n=8. Scale bar, 100 mM. and UNC5B was carried out in kidneys from WT mice that were subjected to 26 minutes of ischemia followed by 24 hours of reperfu- sion. As shown in Supplemental Figure 1, UNC5B Deletion in Proximal Tubular Epithelial Cells both netrin-1 and UNC5B expressions are colocalized in the Increases Inflammatory Response in the Kidney cortical tubular epithelial cells, suggesting that netrin-1 mediates Because UNC5B is known to regulate inflammatory and its activity through UNC5B in proximal tubular epithelial cells. apoptotic responses,10 we profiled inflammatory mediator genes and genes that regulate apoptosis expression in the kidney at 6 Partial or Proximal Tubular Epithelial Cell-Specific hours after ischemia reperfusion. Consistent with increased neu- Deletion of UNC5B in Mice Exacerbates Cisplatin- 2 fl trophil infiltration in the UNC5B / ox/GGT-cre mouse kidney Induced AKI (Figure 2, C and D), expression of several cytokines, chemo- To determine whether UNC5B has the protective role in other kines, and their receptors and genes known to regulate apoptosis modelsof tissueinjury, we treated WT,heterozygousknockout, was significantly upregulated after ischemia in heterozygous and proximal tubular epithelial cell-specific knockout mice UNC5B knockout mice without cre, and it was also dramatically with cisplatin. Kidney function was assessed by measuring

J Am Soc Nephrol 25: 239–249, 2014 UNC5B and Cell Survival 241 BASIC RESEARCH www.jasn.org

in proximal tubular epithelial cell-specific UNC5B knockout 2 fl mice (UNC5B / ox/GGT-cre) (Figure 5). Consistent with kidney dysfunction, histology showed more necrotic tubules, cast forma- 2 fl 2 fl tion, and dilated tubules in UNC5B / ox and UNC5B / ox/GGT-cre fl fl mice kidneys compared with UNC5B ox/ ox mice kidneys (Figure 5, B–F). 2 fl Consistent with histologic damage, both UNC5B / ox and 2 fl UNC5B / ox/GGT-cre mouse kidneys treated with cisplatin showed increased expression of inflammatory cytokines and chemokines (Figure 5G). Because netrin-1 binding to UNC5B receptor is known to suppress apoptosis, the extent of apo- ptosis in the absence of UNC5B was quantified by TUNEL assay and proapoptotic gene expression analysis using PCR array. As shown in Figure 6, either partial deletion (Figure 6C) or proximal tubule–specific deletion of the UNC5B re- ceptor (Figure 6D) exacerbated apoptosis and proapoptotic gene expression (Figure 6F) compared with littermate con- trols (Figure 6B). No apoptosis was seen in untreated control mice (Figure 6A). Quantitative data of apoptosis are shown in Figure 6E. Our previous studies showed that netrin-1 over- expression suppressed p53 activation in response to cisplatin. Moreover, UNC5B signaling is known to suppress p53 expres- sion and activation. Therefore, we determined p53 activation in UNC5B knockout mice. As shown in Figure 6, G–J, cisplatin increased phopho-p53 levels in WT kidneys (Figure 6H), which was further increased in partial (Figure 6I) and proxi- mal tubule-specific (Figure 6J) UNC5B knockout mice. This finding suggests that UNC5B may regulate cisplatin-induced apoptosis through suppression of p53 activation; p53-positive nuclei were counted, and quantitative data are shown in Figure 6K.

Short Hairpin RNA-Mediated Knockdown of UNC5B Receptor Expression in Kidney Epithelial Cells Increased Sensitivity to Cisplatin-Induced Cell Death To determine whether UNC5B knockdown in epithelial cells Figure 3. Ischemia reperfusion–induced expressions of proin- flammatory and proapoptotic genes were enhanced in UNC5B increases sensitivity to cell stress (such as cisplatin), WT and knockout mouse kidney as compared to WT kidney. Regulation of UNC5B knockout cells were treated with cisplatin for 24 hours. genes that regulate inflammatory and apoptotic responses in Knockdown of UNC5B was confirmed with quantitative RT- 2 fl 2 fl UNC5B / ox and UNC5B / ox/GGT-cre mouse kidney. Gene ex- PCR (Figure 7A) and Western blot analyses (Figure 7B). The pression was analyzed by real-time PCR. (A) Kidney-specificde- expression of UNC5B is reduced over 80% compared with WT letion of UNC5B induced a large number of cytokines, chemokines, cells (Figure 7, A and B). Western blot analysis of p53 showed and their receptor expression compared with sham-operated increased accumulation of both total and phospho-p53 in control and heterozygous knockout mice subjected to ischemia UNC5B knockout cells compared with WT cells, suggesting , 2/flox reperfusion (IR). *P 0.05 versus sham operated UNC5B ; enhanced activation of p53 in the absence of the UNC5B re- + , fi P 0.001 versus all other groups. n=8. (B) Kidney-speci c deletion ceptor (Figure 7C). To determine whether enhanced activa- of UNC5B induced many genes that regulated apoptosis com- tion of p53 may contribute to increased cell death in UNC5B pared with sham-operated control and heterozygous knockout fi mice subjected to ischemia reperfusion (I/R). n=5. knockout cells, cell death was quanti ed as described in Con- cise Methods. As shown in Figure 7D, cisplatin treatment in- duced cell death in WTmice, which was significantly higher in UNC5B knockout cells. The addition of p53 inhibitors sup- serum creatinine levels at different times after cisplatin admin- pressed cisplatin-induced exacerbation of cell death in istration. WTmice developed AKI as expected by 72 hours after UNC5B knockout cells, suggesting that p53 activation may 2 fl cisplatin administration. However, UNC5B / ox developed play a critical role in cell death. Consistent with exacerbated much more severe renal injury, which was further exacerbated apoptosis in the absence of UNC5B in kidney epithelial cells,

242 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 239–249, 2014 www.jasn.org BASIC RESEARCH

signal-regulated kinase (ERK) pathways byWesternblotanalysisinWTand UNC5B knockout cells treated with either cisplatin or netrin-1. The addition of cisplatin or netrin-1 induced a signifi- cant increase in Akt phosphorylation in WT epithelial cells compared with vehicle- treated controls. However, both netrin- 1– and cisplatin-induced increases in Akt phosphorylation were completely blunted in UNC5B knockout cells (Figure 8, A and B) compared with WT epithelial cells. Moreover, the basal level of Akt phosphorylation was also blunted in the absence of UNC5B, suggesting the critical role of UNC5B in the activation of the PI3K-Akt pathway. Interestingly, a signif- icant increase in ERK phosphorylation was seen in WT epithelial cells at 3 hours but not 1 hour after the treatment of cis- platin and netrin-1. The increase in ERK phosphorylation with cisplatin and netrin-1 was not blunted in UNC5B knockout cells, suggesting that stress-induced activation Figure 4. UNC5B deletion exacerbates tubular epithelial cells apoptosis in response to of PI3K-Akt is specific for UNC5B (Figure ischemia reperfusion. Effect of UNC5B deletion in proximal tubular epithelial cells in 8, A and C). ischemia reperfusion (IR)-induced apoptosis. (A–D) Apoptotic cells were identified using 2 fl 2 fl To determine directly whether blunted TUNEL staining. Sham-operated (A) UNC5B / ox and (C) UNC5B / ox/GGT-cre mouse activation of the Akt pathway plays a kidneys did not show any apoptotic cells. Six hours after reperfusion, the number of 2 fl apoptotic cells (blue staining) was increased dramatically in (B) UNC5B / ox/GGT-cre mice critical role in the increased apoptosis 2 fl compared with (D) UNC5B / ox mice. Scale bar, 100 mM. (E) Quantification of apoptotic seen in UNC5B knockout cells, PI3K cells. Apoptotic-positive cells were counted in five 340 magnification fields. *P,0.05 activation in WT cells was inhibited with 2 fl versus sham-operated; #P,0.001 versus UNC5B / ox subjected to IR. n=4–6. (F) Western the specific pharmacological inhibitor blot analysis of p53 expression and phosphorylation in kidney from sham-operated mice LY294002 as described in Concise Meth- and mice that are subjected to IR. UNC5B deletion increased p53 expression and ods. As shown in Figure 8B, cisplatin ad- phosphorylation (P-p53) in response to IR compared with sham operation or flox/flox dition significantly increased cell death (WT) mice subjected to IR. over controls, which was further in- creased in PI3K inhibitor-treated cells, suggesting that PI3K-mediated Akt acti- the expression of several apoptotic genes was also signifi- vation plays a critical role in cell survival in response to cantly increased in UNC5B knockout cells compared with stresses and that UNC5B is a major activator of Akt pathways WT cells (Supplemental Table 1). Consistent with exacerba- under stresses. tion of cisplatin in UNC5B knockout renal epithelial cells, hydrogen peroxide-induced apoptosis and necrosis were Overexpression of UNC5B in TKPTS Cells Reduced also significantly higher in UNC5B knockout cells compared Cisplatin-Induced Apoptosis with WT cells (Supplemental Figure 2). To determine if forced overexpression of UNC5B in kidney epithelial cells reduced apoptosis in response to stress, Phosphatidylinositol 3 Kinase-Akt Pathway Is a Critical TKPTS cells were transfected with the rat UNC5B expression Mediator of UNC5B-Regulated Cell Survival in plasmid as described in Concise Methods. Expression was Response to Stresses. confirmed by quantitative RT-PCR and immunohistochem- Because our previous study showed that netrin-1 activates istry. Cisplatin induced a significant increase in apoptosis in Akt pathways through UNC5B21 and Akt is known to sup- mock-transfected cells, which was significantly reduced in press p53 activation, we hypothesized that UNC5B regu- UNC5B-transfected cells (Supplemental Figure 3). Consis- lates cell survival through phosphatidylinositol 3 kinase tent with reduced apoptosis in UNC5B-transfected cells, (PI3K) -mediated activation of Akt in response to stresses. caspase-3 activity was also reduced compared with mock- We determined the activation of Akt and extracellular transfected cells.

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proximal tubular epithelial cells in heterozy- gous knockout animal kidneys. In the con- ditional knockout mice, UNC5B deletion was complete and specifictotheproximal tubular epithelial cells in the renal cortex (Figure 1). Whole-body knockout of UNC5B or endothelial cell-specific condi- tional knockout of UNC5B in mice is lethal to , and mice die in utero because of defective .22 g-Glutamyl trans- ferase (GGT)-cre expression is known to oc- cur in late kidney development (beginning around postpartum day 14) when nephro- genesis is virtually complete.23 Moreover, bone marrow cells and other organs did not express GGT-cre.23 However, it is possi- ble that GGT-cre expression may start dur- ing early stages of development. Our studies show that the proximal tubular epithelial cell-specific knockout is not lethal and that the kidney shows normal histology. There- fore, UNC5B is not required for tubular epithelial cell development. During the obser- vations for the past 3 years and the period of Figure 5. UNC5B deletion exacerbates cisplatin-induced AKI and inflammatory 6–8 months of age, these mice do not de- response of the kidney. (A) Cisplatin was administered intraperitoneally at a dose of 25 mg/kg body wt. Kidney function was assessed by measuring serum creatinine. velop any abnormal histopathology in the fl fl Cisplatin-induced kidney dysfunction in WT (UNC5B ox/ ox) was exacerbated with partial kidney. The absence of developmental de- deletion of UNC5B. The cisplatin-induced kidney dysfunction was worse in UNC5B fects in these mice enables us to study the whole-body partial deletion with proximal tubular epithelial-specificdeletion.*P,0.05 role of UNC5B during adult life and in re- versus corresponding time in other groups; **P,0.05 versus corresponding time point sponse to ischemia reperfusion. Mice with 2 fl in WT and UNC5B / ox/GGT-cre mice; ***P,0.001 versus saline treatment (0 hours). cre expression in the tubular epithelial cells n=6. (B) Cisplatin administration induced the expression inflammatory cytokines and do not show any defect in kidney morphol- chemokines in WT, which was increased further in mice with both partial (heterozy- ogy and function (data not shown). More- fi , gous) and kidney-speci cdeletionofUNC5B.*P 0.05 versus saline treatment; over, they respond to ischemia or cisplatin # , P 0.05 versus all other groups. n=4. ICAM-1, intercellular adhesion molecule 1; KC; just like WT mice (data not shown). MCP-1; TLR4. (C–F) Periodic acid–Schiff-stained section showing tubular injury, In this study, we showed a remarkable including dilated tubules, cast formation, and necrotic tubules. (C) Saline-treated 2/flox/GGT-cre flox/flox flox/flox sensitivity of UNC5B mice to UNC5B mice kidneys. (D) Cisplatin-treated UNC5B mice kidneys. (E) 2 fl 2 fl 2 fl / ox Cisplatin-treated UNC5B / ox mice kidneys. (F) Cisplatin-treated UNC5B / ox/GGT-cre ischemic AKI compared with UNC5B mice kidneys. (G) Quantification of tubular injury score. *P,0.001 versus saline mice. Our initial results showed that deletion +/2 treatment; #P,0.05 versus all other groups. n=6. ofoneallele(UNC5B )mademicemore sensitive to ischemia reperfusion injury com- pared with WT littermates. All mice with whole-body deletion of one allele and dele- DISCUSSION tion of both alleles in proximal tubules died with longer ischemic time. With shorter ischemic time, both WT and heterozygous The pathophysiological role of UNC5B receptors in kidney UNC5B knockout mice did not develop kidney injury, but diseaseisunknown. Inthiswork,wedescribe for thefirst time the heterozygous mice showed an increased number of apoptotic critical role of the UNC5B receptor in renal tubular epithelial cell cells in the cortex. In contrast, deletion of both UNC5B alleles survivalusingaconditionalknockoutmousemodelwithtargeted in proximal tubular epithelial cells exacerbated tissue damage UNC5B deletion from renal proximal tubules. The conditional and increased the number of apoptotic cells, resulting in sig- knockoutmicedidnotshowovertdefectsinkidneydevelopment, nificantly worse renal function than their heterozygous litter- histology, or function. Interestingly, these animals are highly mates. Our PCR array analysis further revealed exacerbated sensitive to ischemic AKI. We show for the first time the role of expression of inflammatory mediators. We show for the first tubular epithelial cell UNC5B in ischemic AKI. Consistent with time that tubular epithelial cell-specific deletion of specific re- our earlier studies,20 UNC5B expression was localized only in the ceptors enhanced the inflammatory response. Taken together,

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these observations support a critical role for UNC5B in protecting tubular epithelial cells against ischemic AKI. Consistent with ischemia reperfusion injury, we see similar effects in cisplatin- induced acute injury with UNC5B deletion. Cisplatin-induced tissue injury and inflam- matory response exacerbated with either partial or proximal tubular epithelial cell- specific deletion of the UNC5B receptor. Moreover, there is increased cell death and accumulation of phospho-p53 in mice with partial and kidney-specific deletion of UNC5B receptors. UNC5B is a target for p53.24 UNC5B signaling was shown to ac- tively suppress p53 expression and activation through an Akt-dependent manner.24,25 Moreover, p53 can be stably expressed in cancer cells but does not induce apoptosis in the presence of netrin-1. It was shown that netrin-1 binding to UNC5B activates Akt pathways to inhibit p53 activation, which accumulates in an inactive form.24,26,27 It was proposed that netrin-1 inhibits post- translational modification of p53, thereby suppressing p53-dependent apoptosis. Consistent with this view, our previous data suggest that cisplatin-induced p53 phosphorylation is inhibited in netrin-1 transgenic mice9 and netrin-1–activated Akt in a time-dependent manner in renal epithelial cells in vitro, which was mediated through UNC5B receptor.21 Therefore, it is possible that the absence of UNC5B inhib- Figure 6. UNC5B deletion in proximal tubular epithelial cells increased epithelial itory signaling on the p53 may lead to excess cells apoptosis and p53 activation in response to cisplatin administration. (A–D) Ap- fl fl activation by phosphorylation and increase optotic cells were identified using TUNEL staining. Saline-treated (A) UNC5B ox/ ox 2/flox 2/flox/GGT-cre of p53-dependent apoptosis. This idea was (UNC5B and UNC5B are not shown) mouse kidney did not show any in vitro apoptotic cells; 72 hours after cisplatin administration, the number of apoptotic cells further supported by our studies, 2 fl (blue staining) was increased dramatically in (D) UNC5B / ox/GGT-cre mice compared where UNC5B knockdown increased the 2 fl fl fl with (C) UNC5B / ox or (B) UNC5B ox/ ox mice. Scale bar, 100 mM. (E) Quantification of accumulation of both total and phospho- apoptotic cells. Apoptotic-positive cells were counted in five 340 magnification fields. p53 in response to cisplatin and inhibition *P,0.05 versus saline treatment; #P,0.001 versus all other groups. n=4–6. (F) Reg- of p53 suppressed cisplatin-induced exacer- 2 fl ulation of genes that regulate inflammatory and apoptotic responses in UNC5B / ox bation of apoptosis in kidney epithelial 2 fl and UNC5B / ox/GGT-cre mouse kidney. Gene expression was analyzed by real-time cells. Moreover, Akt activation by cisplatin PCR. Kidney-specific deletion of UNC5B induced a large number of proapoptotic or netrin-1 was completely blunted in flox/flox genes and their receptor expression compared with cisplatin-treated UNC5B UNC5B knockout cells, suggesting the im- , flox/flox and heterozygous knockout mice. *P 0.05 versus cisplatin-treated UNC5B ; portance of UNC5B in activating cell survival **P,0.001 versus all other groups. n=4. (G–J) Immunohistochemical localization of fl fl fl fl pathway. The critical role of UNC5B-activated phospho-p53 in (G) saline-treated UNC5B ox/ ox, (H) cisplatin-treated UNC5B ox/ ox,(I) 2/flox 2/flox/GGT-cre Akt in cell survival was further supported by cisplatin-treated UNC5B , and (J) cisplatin-treated UNC5B kidney tis- in vitro sue sections. (K) Quantification of p53-positive cells in five 340 magnification fields. our studies (Figure 8), where addi- fi *P,0.05 versus saline treatment; #P,0.001 versus all other groups. n=4–6. Scale bar, tion of a speci c PI3K inhibitor exacerbated 100 mM. cisplatin-induced cell death. Necrosis is a common pathologic obser- vation in many forms of AKI.28–31 Although inhibition of p53 activation suppresses

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contributor of organ dysfunction in these diseases. Currently, there are no effective treatments available to treat ischemia reper- fusion injury or chemical-induced organ in- jury. Our studies shed light on the protective mechanisms and possible new ways to treat AKI based on increasing UNC5B receptor- mediated survival signaling.

CONCISE METHODS

Mouse Strains Generation of the UNC5B alleles has been de- scribed previously.22,35 GGT-cremicewere generously provided by Eric Nelson at Vander- bilt University School of Medicine. Genotypes were determined by PCR analysis of genomic DNA isolated from tail clips; amplification pri- mersandconditionsusedforUNC5B mice were 59-TAGCCTCAGGGTCTACTGTCTG, 59- CTCTCAGACTTCTCAAAGAGATTC, and 59- Figure 7. Deletion of UNC5B in TKPTS exacerbates cisplatin-induced apoptosis through increased p53 activation. Cisplatin-induced cell death in WT and UNC5B CCACTGTATGCCAGACGACATG under the knockout mouse proximal tubular epithelial cells was analyzed by flow cytometry after conditions of 94°C for 30 seconds, 62°C for 20 staining with annexin V–FITC and propidium iodide. (A) UNC5B knockout was con- seconds, and 72°C for 40 seconds. GGT-cre mice firmed by real-time RT-PCR. Over 80% knockdown was observed with short hairpin were genotyped using the primers 59-AGGTGTA- RNA (shRNA) to UNC5B in transfected cells compared with WT cells. *P,0.0001 GAGAAGGCACTTAGC and 59- CTAATCGC- versus WT. n=4. (B) Western blot analysis of UNC5B receptor expression in WT and CATCTTCCAGCAGG-39 under the conditions UNC5B shRNA-transfected tubular epithelial cells. UNC5B expression reduced to of 94°C for 30 seconds, 58°C for 20 seconds, a large extent with UNC5B shRNA transfection. (C) Western blot analysis of p53 and 72°C for 40 seconds. The Institutional Animal proteins in WT and UNC5B knockout (KO) cells. Cisplatin induced accumulation of Care and Use Committee of the Georgia Health both total and phospho-p53 (P-p53) in WT cells, which was highly enhanced in UNC5B Sciences University approved all of the protocols KO cells. The basal level of p53 was also higher in UNC5B KO cells compared with WT and procedures for using animals (approval num- cells. Bands are from the same gel but rearranged for better presentation. (D) A 10 mM – – cisplatin addition induced a significant increase cell death, which was exacerbated in ber BR10 10 369). UNC5B KO cells. The addition of p53 inhibitors suppressed cisplatin-induced cell death exacerbation in UNC5B KO cells. *P,0.001 versus all other groups; #P,0.05 Proximal Tubular Epithelial Cell- versus WT cisplatin treatment. n=4. PI, propidium iodide. Specific Deletion of UNC5B Shows Normal Phenotype in Mice flox Creation and characterization of the UNC5B apoptosis, it may have no effect on primary necrosis. It should, allele were described previously.22,35 To systematically inactivate however, reduce secondary necrosis, which is known to occur UNC5B in a tissue-specific fashion, animals homozygous for the con- flox/flox after apoptosis. Our data show that both apoptosis and necrosis ditional allele (UNC5B ) were mated with animals heterozygous +/2 flox/2 increased in the UNC5B knockout kidney. Although the extent of for the UNC5B null allele (UNC5B ). The UNC5B progeny were primary necrosis is not clear in our studies, our in vitro studies mated with mice carrying the Cre gene under the control of GGT pro- with hydrogen peroxide (Supplemental Figure 2) show that moter, with activity that was restricted to proximal tubular epithelial UNC5B deletion could increase both apoptosis and necrosis. cells. The tubular-restricted GGT promoter was shown to be active These data suggest that UNC5B may regulate both apoptosis only in adulthood but not during development.23 Deletion and necrosis in renal epithelial cells. However, the underlying was confirmed by PCR and immunohistochemical staining of UNC5B mechanisms remain to be determined. in kidney sections (Figure 1). Deletion of UNC5B in proximal tubular In conclusion, organ injury caused by toxic chemical and epithelial cells resulted in mice that survived to adulthood and seemed ischemia reperfusion is widespread in disease, which to be normal. includes AKI caused by vascular surgery, cardiac bypass surgery, sickle cell disease, anticancer drug-induced kidney Renal Ischemia Reperfusion 2 fl 2 fl injury, colon injury in Crohn’s disease, and inflammatory Eight- to nine-week-old UNC5B / ox/GGT-cre,UNC5B / ox,and fl fl bowel disease.9,12,13,32–34 Cell death by apoptosis is a major UNC5B ox/ ox mice were anesthetized with sodium pentobarbital

246 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 239–249, 2014 www.jasn.org BASIC RESEARCH

Renal Function Renal function was assessed by measurements of serum creatinine (DZ072B; Diazyme Laborato- ries, Poway, CA) and BUN (BioAssay Systems, Hayward, CA).10,15

Histology and Immunostaining Kidney tissue was fixed in buffered 10% formalin for 12 hours and then embedded in paraffinwax. For assessment of injury, 5-mmsectionswere stained with periodic acid–Schiff followed by hematoxylin. Tubular injury was assessed in pe- riodic acid–Schiff-stained sections using a semi- quantitative scale,12,15 in which the percentage of cortical tubules showing epithelial cell necro- sis, brush border loss, cast formation, and apo- ptotic bodies in the cortex was assigned a score: 0, normal; 1, ,10%; 2, 10%–25%; 3, 26%–75%; 4, .75%. Sections were scored independently by two investigators who were blinded to the treatment of the animal. Ten fields of 340 mag- nification were examined and averaged. The in- dividual scoring of the slides was blinded to the genotype of the animal. To quantify leukocyte infiltration, sections were stained with rat anti-mouse neutrophil antibody (Abcam, Cambridge, MA) (1:200 dilution) followed by goat Figure 8. Inhibition of PI3K exacerbates cisplatin-induced cell death in mouse prox- anti-rat biotin conjugate. Color was developed imal tubular epithelial cells. (A) Cisplatin- and netrin-1–induced Akt activation (phos- after incubation with ABC reagent (Vector Lab- phorylation [P-Akt]) was blunted, but ERK phosphorylation (P-ERK) in UNC5B knockout (KO) cells was analyzed by Western blot analysis. (B and C) Densitometric quantifi- oratories). Stained sections were photographed, fi 3 fi cation of Western blot analysis of (B) phospho-Akt (1 and 3 hours) and (C) ERK (3 and ve 40 elds of neutrophils were examined hours). *P,0.05 versus vehicle-treated and UNC5B KO cells. n=4. (D) Cell death was for quantification of leukocytes. To determine analyzed by flow cytometry after staining with annexin V-FITC and propidium iodide endogenous mouse netrin-1 and UNC5B pro- (PI). A 10 mM cisplatin addition induced a significant increase in cell death (red box), tein expression, sections were stained with goat which was exacerbated with specific inhibition of PI3K with 50 mM LY294002. antinetrin-1 polyclonal antibody (Santa Cruz LY294002 alone did not increase cell death significantly without cisplatin. *P,0.001 Biotechnology, Inc., Dallas, TX) and rabbit # , versus all other groups; P 0.05 versus WT cisplatin treatment. n=4. anti-UNC5B polyclonal antibody (MD Milli- pore Corporation, Billerica, MA) followed by secondary antibody conjugated with fluoro- (50 mg/kg body wt intraperitoneally) and placed on a heating pad to phors or goat anti-rabbit peroxidase polymer (Vector Laboratories). maintain body temperature at 37°C. Both renal pedicles were identified To determine phospho-p53–positive cells, sections were stained with through dorsal incisions and clamped for a period of 26 or 22 minutes. rabbit antiphospho-p53 (Ser15; Cell Signaling Technologies, Inc.). Reperfusion was confirmed visually on release of the clamps. As a control, Color was developed after incubation with ABC reagent (Vector Lab- sham-operated animals were subjected to the same surgical procedure, oratories). Stained sections were photographed using an Olympus except that the renal pedicles were not clamped. Surgical wounds were inverted microscope with a color charge-coupled device camera. closed, and mice were given 1 ml warm saline intraperitoneally. The mice were kept in a warm incubator until they regained consciousness. TACS TdT In Situ Apoptosis Detection Toidentify apoptotic cells, tissue sections were stained using the TACS Cisplatin-Induced AKI TdT in situ Apoptosis Detection Kit (R&D Systems, Inc.) according to Cisplatin was dissolved in saline at a concentration of 1 mg/ml. Mice the manufacturer’sinstruction.Briefly, tissue sections were deparaffi- were given a single intraperitoneal injection of either saline or cisplatin nized, hydrated, and washed with PBS. Sections were digested with (25 mg/kg body wt). Animals were euthanized 72 hours after cisplatin proteinase K for 15 minutes at 24°C. Slides were then washed, and injection, and blood and kidney tissues were collected. Kidney tissues endogenous peroxidase activity was quenched with 3% H202 in meth- were processed for histology, TUNEL assay, neutrophil and p53 anol. Slides were washed and incubated with TdT labeling reaction staining, and RNA isolation. mixat37°Cfor1hourandthenstreptavidin–horseradish

J Am Soc Nephrol 25: 239–249, 2014 UNC5B and Cell Survival 247 BASIC RESEARCH www.jasn.org peroxidase. Color was developed using TACS blue label substrate protein assay reagent (Pierce Biotechnology, Rockford, IL), and solution. Slides were washed, counterstained, and mounted with Per- 50 mg total protein was loaded onto 4%–12% polyacrylamide gels, mount. Sections were photographed, and labeled cells were counted separated, and then transferred onto a polyvinylidene difluoride and quantified. membrane. The membrane was probed with rabbit antiphospho-p53 (S15 and S392), antitotal-p53 (Cell Signaling Technologies), rabbit anti- Gene Expression Analysis by Real-Time RT-PCR phospho-Akt and total Akt antibodies, rabbit antiphospho- and Real-time RT-PCR was performed in an Applied Biosystems, Inc. 7700 total ERK antibodies, rabbit anti-p38 (Cell Signaling Technolo- Sequence Detection System (Foster City, CA); 1.5 mgtotalRNAwas gies), and rabbit anti-UNC5B antibodies (MD Millipore Corpora- reverse transcribed in a reaction volume of 20 ml using the Omni- tion,Billerica,MA).Proteinsweredetectedwithenhancedchem- script RT Kit and random primers. The product was diluted to a iluminescence detection reagents (Amersham Pharmacia Biotech). volume of 150 ml, and 6-ml aliquots were used as templates for am- Protein loading was normalized by probing the membrane with plification using the SYBR Green PCR Amplification Reagent (Qiagen) antiactin antibodies. and inflammatory cytokine and chemokine PCR array and apoptosis PCR array (SABiosciences). The amount of DNA was normalized to a Quantification of Apoptosis by Flow Cytometry housekeeping gene, such as b-actin, glyceraldehyde-3-phosphate To quantify the dead cells in WT and UNC5B knockout TKPTS cells, dehydrogenase, and hypoxanthine-guanine phosphoribosyl trans- cells were harvested at 24 hours after treatment of cisplatin (10 mM) ferase 1, amplifiedinthesameplate. or hydrogen peroxide (100 mM). To quantify apoptosis, cells were washed and stained for Annexin V–FITC and propidium iodide Apoptosis PCR Array (640914; Biolegend, San Diego, CA). Stained cells were immediately To determine apoptotic genes regulated in response to UNC5B analyzed by flow cytometry (BD FACSCalibur), and the data were deletion, PCR array (Realtimeprimers.com) was used to quantify analyzed using Cyflogic V.1.2.1 software. apoptotic pathway genes. UNC5B Overexpression in TKPTS Cells Creation of Stable TKPTS Cell Lines Expressing Short To determine the effects of UNC5B overexpression on renal epithelial cell Hairpin RNA for UNC5B apoptosis, TKPTS cells were transfected with 2 mg/well rat UNC5B ex- TKPTS cells stably expressing short hairpin RNA for UNC5B were pression construct (gift from Patrick Mehlen, Centre Leon Berard, Lyon, created by transfecting lentiviral vectors containing short hairpin France) in a six-well plate; 48 hours after transfection, cells were treated RNAs and the puromycin drug resistance selection marker (Sigma- with saline or 10 mM cisplatin, and 24 hours after cisplatin addition, cells Aldrich, St. Louis, MO). Colonies were selected based on drug resistance were harvested and used for Western blot and flow cytometry to deter- and propagated separately. Knockdown of UNC5B expression was mine apoptosis and caspase-3 activity. Caspase-3 activity was quantified determined by real-time RT-PCR. Fifteen clones for UNC5B were using an assay kit (BioAssay Systems, Hayward, CA). screened. Five clones for each gene showing $80% knockdown were selected and frozen in liquid nitrogen. All clones selected were shown to Statistical Methods have normal expression of the other receptors.21 All assays were performed in duplicate or triplicate. The data are reported as mean 6 SEM. Statistical significance was assessed by an Cell Culture unpaired, two-tailed t test for single comparison or ANOVA for mul- Mouse TKPTS cells were cultured in advanced DMEM/F12 medium tiple comparisons. P,0.05 was considered significant. with 5% serum. To determine whether cisplatin-induced cell death is exacerbated in the absence of UNC5B receptor expression, WT TKPTS cells and UNC5B knockout cells were treated with vehicle, ACKNOWLEDGMENTS 10 mM cisplatin, or 100 mM hydrogen peroxide; 24 hours after the addition of cisplatin or hydrogen peroxide, cells were harvested, This work was supported by National Institutes of Health Grant R01 stained with PI and annexin V, and analyzed by flow cytometry or 7R01DK083379-02 (to G.R.). used for caspase-3 assay or p53 expression by Western blot analysis. To determine the role of the PI3K pathway in UNC5B-mediated DISCLOSURES suppression of apoptosis and inactivation of p53, TKPTS cells were treated with 10 mM cisplatin with or without the PI3K inhibitor None. (10 mM LY294002; Cell Signaling Technologies) for 24 hours. Cells were harvested and used for Western blot analysis, PCR, and quan- REFERENCES tification of apoptosis by flow cytometry.

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