p2y receptor localizes in the renal collecting duct and...

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P2Y12 Receptor Localizes in the Renal Collecting Ductand Its Blockade Augments Arginine VasopressinAction and Alleviates Nephrogenic Diabetes Insipidus

Yue Zhang,*† Janos Peti-Peterdi,‡§ Christa E. Müller,| Noel G. Carlson,¶**†† Younis Baqi,|‡‡

David L. Strasburg,*† Kristina M. Heiney,*† Karie Villanueva,‡§ Donald E. Kohan,*† andBellamkonda K. Kishore*†††

*Nephrology Research and ¶Geriatric Research, Education and Clinical Center, Department of Veterans Affairs SaltLake City Health Care System, Salt Lake City, Utah; †Department of Internal Medicine, **Department of Neurobiologyand Anatomy, and ††Center on Aging, University of Utah Health Sciences Center, Salt Lake City, Utah; ‡ZilkhaNeurogenetic Institute and §Department of Physiology and Biophysics, University of Southern California, Los Angeles,California; |PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Bonn,Germany; and ‡‡Department of Chemistry, Faculty of Science, Sultan Qaboos University, Muscat, Oman

ABSTRACTP2Y12 receptor (P2Y12-R) signaling ismediated throughGi, ultimately reducing cellular cAMP levels. BecausecAMP is a central modulator of arginine vasopressin (AVP)-induced water transport in the renal collectingduct (CD), we hypothesized that if expressed in the CD, P2Y12-R may play a role in renal handling of water inhealth and in nephrogenic diabetes insipidus. We found P2Y12-R mRNA expression in rat kidney, and immu-nolocalized its protein and aquaporin-2 (AQP2) in CD principal cells. Administration of clopidogrel bisulfate,an irreversible inhibitor of P2Y12-R, significantly increased urine concentration and AQP2 protein in thekidneys of Sprague–Dawley rats. Notably, clopidogrel did not alter urine concentration in Brattleboro ratsthat lack AVP. Clopidogrel administration also significantly ameliorated lithium-induced polyuria, improvedurine concentrating ability and AQP2 protein abundance, and reversed the lithium-induced increase in free-water excretion, without decreasing blood or kidney tissue lithium levels. Clopidogrel administration alsoaugmented the lithium-induced increase in urinary AVP excretion and suppressed the lithium-induced in-crease in urinary nitrates/nitrites (nitric oxide production) and 8-isoprostane (oxidative stress). Furthermore,selectiveblockadeofP2Y12-Rby the reversible antagonist PSB-0739 inprimary culturesof rat innermedullaryCD cells potentiated the expression of AQP2 and AQP3 mRNA, and cAMP production induced by dDAVP(desmopressin). In conclusion, pharmacologic blockade of renal P2Y12-R increases urinary concentratingability by augmenting the effect of AVP on the kidney and ameliorates lithium-induced NDI by potentiatingthe action of AVP on the CD. This strategy may offer a novel and effective therapy for lithium-induced NDI.

J Am Soc Nephrol 26: ccc–ccc, 2015. doi: 10.1681/ASN.2014010118

Water absorption in renal collecting duct (CD) isregulated by arginine vasopressin (AVP), through itsV2 receptor (R) and cyclic AMP signaling, resulting inincreased expression of aquaporin-2 (AQP2) and itsapicalmembrane targeting in theCDcells.1AVPactionon CD can be modulated by the autocrine/paracrineagents, such as extracellular nucleotides.2,3 TheAVP-V2-R-AQP2 pathway is disrupted in acquirednephrogenic diabetes insipidus (NDI), resulting inthe development of resistance of CD to AVP.4 Themost common acquired NDI is caused by chronic

usage of lithium (Li) for the treatment of bipolardisorder, which affects 2% of the general populationof the United States. The exact mechanism(s) by

Received January 30, 2014. Accepted January 27, 2015.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Bellamkonda K. Kishore, Nephrology Re-search (151M), VA SLC Health Care System, 500 Foothill Drive,Salt Lake City, UT 84148. Email: [email protected]

Copyright © 2015 by the American Society of Nephrology

J Am Soc Nephrol 26: ccc–ccc, 2015 ISSN : 1046-6673/2612-ccc 1

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which CD develops AVP resistance in Li-induced NDI is notestablished.5–8 Current therapies for Li-inducedNDI are proneto adverse effects.7,9–11 Understanding the mechanism(s) of Li-induced NDI or developing modalities to reverse the AVP re-sistance of CD will result in new and safer therapies.

Previously we reported that purinergic signaling mediated byATP/UTP-activated P2Y2 receptor (P2Y2-R) plays a significantrole in the genesis ofNDI. First, using a ratmodel, we identified apotential role for P2Y2-R in acquired NDI induced by Li treat-ment or postobstructive uropathy.12,13 P2Y2-R is known to exerttonic inhibitory effect on the AVP-V2-R-AQP2 pathway.14,15

Therefore, to further elucidate the role of P2Y2-R, we usedmice with genetic deletion of P2Y2-R and found that thesemice are significantly resistant to Li-inducedNDI.16,17 Althoughpromising, currently there are no Food and Drug Administra-tion (FDA)-approved or experimental drugs that can selectivelyblock P2Y2-R in vivo. Hence, we examined the ADP-activatedP2Y12-R as another potential P2Y receptor subtype that mayoppose AVP-induced water flow in the CD. PhylogeneticallyP2Y12-R is distinct from P2Y2-R and related receptors.18

P2Y12-R is expressed predominantly in blood platelets and mi-croglia and astrocytes in the brain. P2Y12-R signaling in plateletsis mediated through Gi, inhibiting adenylyl cyclase (AC) andactivation of class I phosphoinositol 3-kinase. P2Y12-R is alsocoupled to Gq, which through phospholipase C activation in-creases intracellular Ca2+ needed for platelet activation.19 Bothinhibition of AC activity and activation of phospholipase C are

known to decrease the sensitivity of CD to AVP.2 Hence, wehypothesized that if expressed in the kidney, P2Y12-R mayplay a role in renal handling of water and in pathophysiologyof NDI. The availability of an FDA-approved and time-testeddrug, clopidogrel bisulfate, to selectively block P2Y12-R allowedus to test our hypothesis in rat models. Clopidogrel is an oralthienopyridine class of drug that irreversibly inhibits P2Y12-R. Itis a prodrug activated in the liver by cytochrome P450 enzymes(e.g., CYP2C19) generating its active metabolite, which consti-tutes approximately 15% of the ingested drug molecule. In thisstudy we document that P2Y12-R is expressed in the rat kidney,and its pharmacologic blockade increases urine concentratingability and significantly ameliorates Li-induced NDI in rats.

RESULTS

Expression of P2Y12-R in the KidneyTo immunolocalize P2Y12-R in the kidney, we designed, gen-erated, and characterized a peptide-derived antibody specificfor rat or mouse P2Y12-R (Supplemental Table 1). We evalu-ated the specificity of our antibody by immunoblotting andimmunofluorescence (IF) microscopy in two different celllines, namely inner medullary collecting duct-3 (IMCD3)(mouse renal CDs) and HEK293T (human embryonic kid-ney), and by using two different approaches: P2Y12-R geneknockdown and overexpression (Figure 1). For proper

Figure 1. Characterization of specificity of P2Y12-R antibody. (A) Western analysis of knockdown (KD) of P2Y12-R showing receptorprotein abundance in IMCD3 cells transfected with scrambled (CT) or P2Y12-R specific shRNA (KD), relative to the respective proteinabundances of b-actin; the table below the blots shows the percent P2Y12-R protein remaining after KD. (B) IF detection of GFP (green)and P2Y12-R (red) in the IMCD3 cells transfected with shRNA plasmids specific for P2Y12-R. Cell nuclei were stained with propidiumiodide (blue). Upper panel shows P2Y12-R labeling in a profile of cells, whereas the lower panel is overlay of GFP labeling on the sameprofile of cells. Note a near complete KD of P2Y12-R expression (arrows in the upper panel) in transfected cells (arrows in the lowerpanel). Asterisks indicate nontransfected cells showing P2Y12-R labeling (red). (C) P2Y12-R protein abundance in control (CT) or humanP2Y12-R overexpressing (OE) HEK293T cells relative to the respective protein abundances of b-actin; the table below the blots showsthe percent overexpression of P2Y12-R protein. Act., actin.

2 Journal of the American Society of Nephrology J Am Soc Nephrol 26: ccc–ccc, 2015


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interpretation of the data presented in Figure 1, one needs totake into account that P2Y12-R exists in multiple forms(monomer, dimer, trimer, or oligomer), with each form inturn having different glycosylated subspecies, spanning from35 to .220 kD in immunoblots.20 Furthermore, the expres-sion of these multiple forms and glycosylated subspecies maydiffer depending on the nature of the cells under consider-ation. As shown in Figure 1A, the control IMCD3 cellsshowed a major band at approximately 80 kD and a faint bandat approximately 60 kD, which match with the same size bandsseen in rat kidney and brain (Figure 2B). In IMCD3 cells sub-jected to knockdown of P2Y12-R by shRNA (KD), there was anapproximate 55%decrease in relative intensity of the 80 kDbandand disappearance of 60 kD band. In parallel, IF imaging ofIMCD3 cells showed labeling for P2Y12-R (red) only in greenfluorescent protein (GFP)-negative untransfected cells (aster-isks), but not in GFP-positive transfected cells (arrows) (Figure1B). As shown in Figure 1C, in control HEK293T cells, ourantibody recognized a clear band at approximately 60 kD, whichmatches with the same size band in IMCD3 cells (Figure 1A). Inaddition,we could see a faint band at approximately 38 kD,whichmatches with the same size band in the native rat kidney (Figure2B). In cells over expressing P2Y12-R, both bands showed amarked increase in intensity, associated by the appearance of anew band at approximately 55 kD, which matches with the sim-ilar size band in human platelet lysates when probed with ourP2Y12-R antibody (Supplemental Figure 1). Therefore, taken to-gether, the data presented in Figure 1 validated the specificity ofour P2Y12-R antibody. Becausemanipulation of P2Y12-R proteinlevels in the cells by knockdown or overexpression at the genelevel matched with corresponding changes in the intensity of allof the detected bands, we can conclude that all detected bands arespecific to P2Y12-R. Further validation of our P2Y12-R antibody

was achieved by IF labeling of mouse micro-glial cells (Supplemental Figure 2).

Figure 2B shows identical P2Y12-R pro-tein bands in rat kidney and brain, whichare ablated by the blocking peptide andcould not be visualized when the blot wasprobed with preimmune IgG fraction. Inthe kidney samples our antibody identifiedtwo bands (one weak band at 38–40 kD,another strong band at 80 kD) in the cortexand outer medulla and one weak band (ap-proximately 80 kD) in the innermedulla. Onthe basis of the predicted molecular weightof the P2Y12-R protein (38 kD; 347 aminoacid residues), it appears that the weak bandat 38–40 kD is the monomeric form of thereceptor protein, whereas the strong band at80 kD represents a dimeric form. Real-timeRT-PCR detected P2Y12-R mRNA in the re-nal cortex and outer and inner medullas ofSprague–Dawley rats (Figure 2A).

Cellular Localization of P2Y12-R in the Rat KidneyImmunoperoxidase reaction revealed strong labeling forP2Y12-R on the brush border of the proximal tubule in thecortex (Figure 3A), which was ablated by peptide block andwas not seen when probed with preimmune IgG (Figure 3, Band C). Weak labeling was found at the apical domain of theCD in the medulla and cortex (Figure 3, D and E). Confocal IFimaging confirmed expression of P2Y12-R over the brush bor-der of proximal tubules, revealed labeling in arterioles (Figure3F), and showed P2Y12-R protein in AQP2 positive cells in thecortex (Figure 3, G) and medulla (Figure 3, H–K), with anapparent localization of both proteins on the apical membraneof the CD cells (Figure 3, G, H, and K). Similar observationswere made in IF when P2Y12-R antibody from Alomone Labswas used (Supplemental Figure 3).

Administration of Clopidogrel Ameliorated Li-InducedNDIAdministration of clopidogrel significantly ameliorated poly-dipsia, polyuria, and decrease in urine osmolality (Figure 4,A–C) and reversed the increase in electrolyte-free water excre-tion (Figure 4D) induced by Li feeding to rats for 2 weeks. Thesewere associated with significant improvement in Li-induceddecrease in AQP2 protein in the inner medulla (Figure 4, Eand F). IF labeling for AQP2 protein in the inner medullas ofcontrol rats was predominantly on the apical aspect of themed-ullary CD cells (Figure 5A). In Li-treated rat kidneys the AQP2labeling in the medullary CD was scanty (Figure 5B). Afterclopidogrel administration, AQP2 labeling was seen all overthe cells, with relatively higher intensity on the apical domain(Figure 5C). In rats coadministered with Li and clopidogrel, theintensity of AQP2 labeling improved with predominant apicallabeling (Figure 5D).

Figure 2. P2Y12-R expression in rat kidney. (A) P2Y12-R mRNA expression relative to thatof b-actin in the three major regions of the kidney and other organs of rat. CTX, cortex;OM, outer medulla; IM, inner medulla. Bars show mean6SEM of triplicate real-time PCRassays, plotted relative to the expression levels in the brain. (B) P2Y12-R protein in the ratkidney and brain. Identical immunoblots prepared by applying equal amounts of protein(30 mg) from solubilized homogenates of rat brain (BR), renal cortex (CTX), or outermedulla (OM) or inner medulla (IM) were incubated with the affinity purified P2Y12-Rantibody (Ab.) or the antibody blocked by the immunizing peptide or with proteinA-purified preimmune (Imm.) IgG as marked on the top of the panels.

J Am Soc Nephrol 26: ccc–ccc, 2015 P2Y12-R and Li-Induced NDI 3

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Clopidogrel administration augmented Li-induced in-crease in urinary AVP excretion (Figure 6A). Administrationof clopidogrel had no significant effect on Li-induced natri-uresis (Figure 6B), but effectively reversed the significantincreases in urinary total nitrates/nitrites, an index of produc-tion of nitric oxide (Figure 6C), and 8-isoprostane, a measureof oxidative stress (Figure 6D). Finally, clopidogrel did notdecreased lithium levels in the blood (Figure 7A) or in theinner medulla (Figure 7B).

Blockade of P2Y12-R Enhanced the Effect ofDesmopressin in Rat IMCDInterestingly, clopidogrel administration per se significantly in-creased urinary excretion of AVP (0.5460.09 in controls versus1.2360.18 in clopidogrel treated, in mmol/24 h per 200 g bodywt, n=4/group, P,0.01). Preliminary studies revealed expres-sion of P2Y12-R mRNA in rat hypothalamus and significantincrease in AVP mRNA expression by selective blockade of

P2Y12-R by PSB-0739 in cultured rat hypo-thalamic cells (Supplemental Figure 4).These findings suggest that the observed in-crease inurinaryAVPexcretion in clopidogrel-treated rats was apparently mediated throughthe blockade of P2Y12-R in hypothalamus.In line with this, in the clopidogrel alonetreated group, there was a significant in-crease in AQP2 protein abundances in therenal cortex and innermedulla (Supplemen-tal Figure 5). These effects were apparentlymediated by an increase in AVP levels and bypotentiation of AVP effects on the CD by theblockadeof P2Y12-R (subsequentlydiscussed).However, the effects of clopidogrel werenot mediated through UTP/ATP-activatedP2Y2-R, because when administered toP2Y2-R knockout and wild-type mice,clopidogrel had identical effects on urinaryconcentration (Supplemental Figure 6).

To investigatewhetherblockadeofP2Y12-Rhad a direct effect on the CD, we performedexperiments onprimary cultures of rat IMCDcells, where we clamped the concentration ofdesmopressin (dDAVP; a V2 receptor specificanalog ofAVP) andusedPSB-0739, a selectiveand reversible antagonist of P2Y12-R. Unlikeclopidogrel, PSB-0739 is not a prodrug andtherefore is suitable for cell cultures. Blockadeof P2Y12-R with PSB-0739 significantly in-creased dDAVP-induced AQP2 expressionby 3.4-fold, but PSB-0739 alone had no effecton AQP2 expression, and enhanced thedDAVP-induced AQP3 expression (Figure8). PSB-0739 did not augment the increasein dDAVP-induced expression of V2 receptor.However, combination of dDAVP and PSB-

0739 significantly increased V2 receptor expression. In parallelcultures, we observed that PSB-0739 significantly enhanced thedDAVP-induced cAMP production (Figure 8). Taken together,these data suggest that blockade of P2Y12-R in IMCD in the ab-sence of dDAVP had little or no effect, but itmarkedly potentiatedthe effect of dDAVPonAQP2 andAQP3 expression andon cAMPproduction. Interestingly, the lack of effect of PSB-0739 alone inCD cells is further supported by our observation that clopidogrelper se had no effect on the urinary concentration in Brattlebororats that lack AVP (Supplemental Figure 7). Therefore, the ob-served effect of blockade of P2Y12-R on urinary concentration inSprague–Dawley rats is dependent on AVP.

DISCUSSION

We demonstrated that (1) P2Y12-R is expressed in rat kidneyand is localized in the CD cells; (2) administration of

Figure 3. Immunolocalization of P2Y12-R protein in rat kidney. Upper row: Immu-noperoxidase labeling for P2Y12-R: (A) labeling of brush border membrane of proximaltubules (PT); (B) peptide-block ablated the labeling in PT; (C) substitution of antibodywith preimmune (Imm.) IgG showed no labeling in the PT (arrows); weak labeling on theapical aspect of the CD in the medulla (D), and cortex (E) (arrows). Middle and lowerrows: IF labeling of AQP2 (green) and P2Y12-R (red): (F) and (G) are low magnificationprofiles of cortical region, showing labeling of arterioles (arrowhead in F), proximal tu-bules (arrows in F), and cortical CDs showing merging of labeling of AQP2 and P2Y12-R(arrows in G). (H) Low magnification profile of inner medullary region showing labeling ofCDs for AQP2 and P2Y12-R. (I)–(K) High magnification images of inner medullary regionshowing labeling of medullary CDs for AQP2 or P2Y12-R or merging of both. Cell nucleiwere labeled with DAPI. Bars are 20 mm in length.








clopidogrel, a selective and irreversible antagonist of P2Y12-Rameliorated Li-induced NDI in rats, without decreasing Li levelsin the blood or in the renal inner medulla; and (3) selectiveblockade of P2Y12-R by PSB-0739 in primary cultures of ratIMCD cells potentiated the expression of AQP2 and AQP3and cAMP production induced by dDAVP. In parallel, we docu-mented that administration of clopidogrel alone increased uri-nary concentrating ability and AQP2 protein abundance in ratkidney in an AVP-dependentmanner. Our observations define anew role of P2Y12-R in the kidney and suggest that pharmaco-logic blockade of P2Y12-R can be a new strategy for the devel-opment of a new class of drugs for the treatment of conditionsassociated with AVP resistance.

The finding that PSB-0739 augmented the effect of dDAVPon gene expression of AQP2 and AQP3 and enhanced cAMPproduction provides direct evidence that blockade of P2Y12-Rper se has marked effect on CD and the observed in vivo effectof clopidogrel in rats is not just caused by increased levels ofAVP. It is possible that under basal conditions P2Y12-Rthrough Gi may exert tonic inhibitory effect on the AC inthe CD. When P2Y12-R is blocked by clopidogrel/PSB-0739,the tonic inhibition is relieved; therefore, the effect of AVP/

dDAVP is markedly enhanced. The subdued activity of phos-pholipase C during the blockade of P2Y12-R may also contrib-ute to the potentiation of AVP/dDAVP effect on CD. However,in the absence of AVP, PSB-0739 alone has no effect on AQP2gene expression in the cultured IMCD cells, which is consis-tent with our finding of lack of effect of clopidogrel in Brattle-boro mutant rats.

Clopidogrel not only prevented Li-induced polyuria butreversed the significant increase in electrolyte-free waterexcretion, which suggest that clopidogrel is acting on CD.The latter is in turn supported by the improved abundance andcellular disposition of AQP2 protein in the renal medulla andCD, respectively. It is also clear that the ameliorating effect ofclopidogrel was not caused by impaired absorption of Li in theintestines, resulting in lower blood Li levels or reducedaccumulation of Li in the renal medulla. Although adminis-tration of clopidogrel augmented Li induced increase inurinary AVP excretion, one cannot attribute this effect ofclopidogrel per se to its ability to ameliorate Li-induced NDIbecause Li-induced NDI is caused by the resistance of thekidney to circulating AVP levels, which are often high. Becausewe showed that in primary cultures of rat IMCD cells blockade

Figure 4. Effect of clopidogrel administration on Li-induced polyuria and decrease in AQP2 protein abundance in rats. (A) Water intake;(B) urine output; (C) urine osmolality; and (D) electrolyte-free water clearance on the last day (day 13). CNT, control group; CLPD,clopidogrel-treated group; LI, Li-treated group; LI+CLPD, combined treatment with Li and clopidogrel. n=7 rats/group for (A), (B), and(C); n=4–5 rats for (D). *P,0.05 versus Li groups by ANOVA followed by Tukey–Kramer multiple comparison test [(A)–(D)]. (E) Rep-resentative immunoblot of AQP2 and b-actin proteins run with inner medullary tissue samples. (F) Densitometry of AQP2 protein bandsin Li and Li+CLPD groups relative to b-actin protein bands; P value by Mann–Whitney nonparametric method.



of P2Y12-R potentiates dDAVP effect on AQP2 water channelexpression and cAMP production, it is reasonable to suggestthat blockade of P2Y12-R by clopidogrel might have sensitizedthe CD during Li treatment and therefore relieved the NDI.

In addition to its effect on the CD, the blockade of P2Y12-Rhad other beneficial effects on the kidney that might contrib-ute to amelioration of Li-induced NDI. These are the reversalof Li-induced increase in nitric oxide production and oxida-tive stress. Endogenously derived nitric oxide exerts tonic in-hibition on the expression of sodium transporters,21 which inturn may contribute to impaired urinary concentrating ability

in Li-induced NDI. Oxidative stress is known to repress ex-pression of several genes, including transcription factors22;however, less is known about its significance in relation torenal transport functions.23

The significance of predominant expression of P2Y12-R on thebrush border of proximal tubules is not known at this stage.How-ever, P2Y12-R does not seem to have an effect on the bulk absorp-tion of sodium because urinary sodium levels were not altered bythe administration of clopidogrel alone or in combinationwith Li.A parallel study conducted in a mouse model, where we analyzedthe sodium transporter/channel/exchanger profile, also did notsupport a major role for the proximal tubules.24

Li-induced NDI is associated with CD remodeling,25 with adecreased number of principal cells and an increased number ofintercalated cells.26,27Hence, there is a possibility that the observedeffect of clopidogrel administration on Li-induced decrease inAQP2 protein may be related to its ability to interfere in CD re-modeling. However, the IF data in clopidogrel- and/or Li-treatedrats do not suggest that possibility in our short-term model.

Finally, previous reports showed thatP2Y12-Rmaycontributeto early mesangial cell transformation and renal vessel hypertro-phy28 or altered renal autoregulation,29 both in the setting ofangiotensin II–induced hypertension. Recent studies by Morelet al.30,31 also demonstrated an impaired platelet P2Y12-R inhi-bition in CKD, which appears to be related to the effect of path-ophysiologic milieu of CKD on platelet activity. Furthermore,most clinical studies on clopidogrel bisulfate were designed withthe criteria of evaluation on the basis of cardio- or cerebrovas-cular events, but not renal effects, and therefore were prone tomiss the findings documented in our study.

CONCISE METHODS

Animal ExperimentsAnimalprocedureswereapprovedbytheInstitutionalAnimalCareandUse

Committeesof theVASaltLakeCityHealthCareSystemandtheUniversity

of Utah.Male Sprague–Dawley rats were purchased

from Charles River Laboratories (Wilmington,

MA). Brattleboro mutant rats that lack AVP

(HsdBlu:BRAT-Avpdi) were obtained from the Rat

Resource&ResearchCenter, University ofMissouri

(Columbia, MO). All animals had free access to

drinkingwater and standard food during the exper-

imental period. NDI was induced by feeding

Li-added food(40mmolLiCl/kg food;MPBiomed-

icals, Solon, OH) to rats for 13 days. Clopidogrel

bisulfate was administered orally by mixing

finely powdered clopidogrel bisulfate tablets

(Bristol-Myers Squibb, New York, NY) in drink-

ingwater.Theconcentrationof thedrug in thedrink-

ing water was adjusted daily on the basis of water

consumption of the animals on the previous day.

Sprague–Dawley rats received 20 mg clopidogrel/kg

body wt per day, whereas the Brattleboro rats

Figure 5. Effect of clopidogrel treatment on Li-induced cellular ex-pression and disposition of AQP2 (green) and P2Y12-R (red) proteins inrat IMCD. Representative low magnification profiles of IF labeling forAQP2 and P2Y12-R in the renal medullas of rats treated with no drug(A), Li (B), clopidogrel (C), or a combination of Li and clopidogrel (D).Insets show corresponding higher magnification profiles. Bar 20 mm.

Figure 6. Effect of clopidogrel on Li-induced alterations in urinary parameters. (A) UrineAVP; (B) urine sodium; (C) urine total nitrates (NO3) and nitrites (NO2); and (D) urine 8-isoprostane. bw, body weight; CNT, control group; CLPD, clopidogral group; LI, Li alone;LI+CLPD, combination of Li and CLPD. n=4–6 rats per group. *Significantly different fromall other groups by ANOVA followed by Tukey–Kramer multiple comparison test.



received double this dose (40 mg/kg body wt per day). The rationale for

the doses of clopidogrel used is provided in the Supplemental Material.

Twenty-four hour urine samples were collected by placing the animals in

plastic metabolic cages (1 rat per cage). Blood and kidney tissues were

collected at necropsy. Cortical, outer, and inner medullary regions of the

kidneys were dissected out and processed for laboratory analysis. In some

untreated Sprague–Dawley rats, samples of cerebral cortex and whole

hypothalamic tissue were collected and processed.

Synthesis and Characterization of PSB-0739PSB-0739 (1-amino-4-[4-phenylamino-3-sulfophenylamino]-9,10-

dioxo-9,10-dihydroanthracene-2-sulfonate) is a highly potent, selective,

reversible non-nucleotide antagonist of P2Y12-R that is not toxic to the

cells.32,33 Unlike clopidogrel, PSB-0739 does not require bioactivation;

therefore, it is suitable for use in cell cultures. PSB-0739 was designed,

synthesized, purified, and characterized in the laboratory of Dr. Christa

Müller at the University of Bonn, Bonn, Germany.32,34

Blood, Urine, and Tissue AnalysisOsmolalities of clear urine samples were measured by vapor pressure

method(Wescor, Logan,UT). Sodium,potassium, andLi levels in serum

and/or urine were determined on EasyElectrolytes (Medica, Bedford,

MA). On the basis of serum and urine analysis, electrolyte-free water

clearance [CH2O(e)] was computed using the formula CH2O(e)=V(1–

UNa+UK)/PNa, where V=urine volume, UNa and UK are urine Na and

K concentrations, and PNa is plasma Na concentration, respectively. Li

levels in the inner medullary tissue homogenates were assayed by in-

ductively coupled plasma-mass spectrometry (Exova, Santa Fe Springs,

CA) and were normalized to their respective protein contents. Induc-

tively coupled plasma-mass spectrometry has a detection limit of 0.06

parts per million for Li, with recovery of 91% in spiked samples. Li was

not detectable in the homogenization medium (0.5% SDS and 0.02%

sodium azide in ultrapure water). Commercial enzyme immunoassay/

ELISAor colorimetric assay kits were used to quantify urinary excretion

ofAVP (Enzo Life Sciences, Farmingdale,NY), total nitrates and nitrites

(NO3/NO2) (Cayman Chemical, Ann Arbor, MI), and 8-isoprostane

(Oxford Biomedical Research, Oxford, MI) as per the manufacturers’

instructions and published in our previous study.35

Detection of mRNA by Real-Time RT-PCR AssayThis was performed essentially as described previously.16,35,36 Briefly,

total RNA was extracted from samples of kidney cortex, outer and

inner medullas, cerebral cortex, and whole hypothalamus. cDNAwas

synthesized by SuperScript Reverse transcription (Invitrogen, Carlsbad,

Figure 7. Effect of clopidogrel on Li disposition in the rat. (A)Terminal Li levels in serum (n=6 rats per group). (B) Li accumulationin the inner medulla (n=4–5 rats per group). Statistical analysis byunpaired t test. LI, Li alone; LI+CLPD, combination of Li and CLPD.

Figure 8. Effect of blockade of P2Y12-R on dDAVP effect in primary cultures of rat IMCD cells. Effect of PSB-0739 (100 nM), a selectiveantagonist of P2Y12-R on dDAVP (50 nM) induced expression of AQP2 and AQP3 water channels and vasopressin V2 receptor mRNAand cAMP production. PSB-0739 was added 24 hours before the addition of dDAVP, and the incubation was continued for another 24hours. Results of two independent experiments were pooled to obtain n=6 for each condition (mean6SEM). For gene expression,before pooling the data, the values were normalized as percent maximum response for each set of culture. Statistical significanceshown above the bars is derived by ANOVA followed by Tukey–Kramer multiple comparison test (AQP2, AQP3, and V2-R) or unpairedt test (cAMP). CNT, controls; dDAVP, desmopressin; PSB, PSB-0739.




CA). Using gene-specific primers (Supplemental Table 2), real-time

PCR amplifications were carried out on cDNA samples in Applied Bio-

systems 7500 Real-Time PCR System (Foster City, CA), with AmpliTaq

Gold and SYBR Green for detection. Expression of target genes was

normalized to that of expression of the housekeeping gene b-actin.

Generation and Characterization of P2Y12-R AntibodyWedesigned and generated apeptide-derived rabbit polyclonal antibody

for a 16-amino acidC-terminus sequence (KKKGQEGGEPSEETPM)of

mouse P2Y12-R (accession no. Q9CPV9), which differs from the cor-

responding rat sequence by one amino acid residue. This peptide has no

sequence homologywith related proteins, such as P2Y13, P2Y14, GPR34,

GPR82, GPR87, or GPR171 (Supplemental Table 1). Peptide synthesis

and purification, coupling to keyhole limpet hemocyanin, immuniza-

tion of the rabbits, and determination of antibody titers by ELISAwere

contracted to Thermo Fisher Scientific (Rockford, IL). Affinity purifi-

cation of the antibody and isolation of preimmune IgG fractions and

characterization of the specificity of the antibody were done in our

laboratories as described previously.36,37

P2Y12-R Knockdown and OverexpressionThe specificity of the antibody was evaluated using knockdown and

overexpression approaches in cells. IMCD3 cells, a mouse inner

medullary CD cell line (ATCC, Manassas, VA), were maintained in a

1:1 mixture of Dulbecco’s modified Eagle’s medium and F-12 (Life

Technologies, Grand Island, NY), supplemented with 10% FBS (Life

Technologies) at 37°C with 5% CO2. For transfection via electropora-

tion, IMCD3 cells were grown to 80%–90% confluence, trypsinized,

and resuspended in warm complete medium. Cells were pelleted, sus-

pended in Nucleofector solution (Lonza Cell Transfection System),

and transfected with mouse P2ry12 SureSilencing shRNA, GFP plas-

mids (Qiagen, Valencia, CA). DNA from the four individual P2Y12

shRNA plasmids (1.5 mg of each plasmid, totaling 6 mg) were mixed

with the suspended cells (0.53106 cells). Cells were transfected using

the program A24 on the transfection device (Lonza Amax Biosystems

Nucleofector I). Each transfection was plated in an individual well of a

12-well plate. Two days after transfection, the cells were processed for

either immunoblotting or IF microscopy. For immunoblotting, cells

were washed with PBS and collected into 13 blue loading buffer con-

taining DTT (Cell Signaling, Danvers, MA). Lysed cells were homog-

enized using TissueLyser (Qiagen) for 10 minutes and heated at 95°C

for 5minutes. The samples were processed forWestern blotting. For IF

microscopy, cells growing on chamber slides were washed briefly with

PBS and then fixed in 2% paraformaldehyde for 20 minutes and per-

meabilized in 0.2% Triton X-100. P2Y12-R antibody (which we gen-

erated) was used in combination with Cy5 anti-rabbit secondary

antibody (Thermo Fisher Scientific, Pittsburgh, PA) to detect the re-

ceptor protein in the transected IMCD3 cells. Propidium iodide

(Sigma-Aldrich, St. Louis, MO) was used to stain the nuclei. Cells

were visualized in Personal Confocal Microscope PCM-2000 (Nikon).

In another experiment, lysates of HEK293T cells overexpressing

human P2Y12-R and control lysates were purchased from the Novus

Biologicals. The overexpression cells were created using plasmid ID

RC205005 and on the basis of accession number NM_022788. The

cells were lysed in modified RIPA buffer containing protease cocktail

mixture by the Novus and were shipped to us. The control and over-

expressing cell lysates were processed for immunoblotting using our

P2Y12-R antibody.

ImmunoblottingProcessing of the kidney tissue samples and immunoblotting were

performed as described previously.13,16 Negative controls for P2Y12-R

antibody were generated by preadsorbing the affinity purified antibody

with a molar excess of the immunizing peptide or by substituting the

antibody with preimmune IgG fraction. Generation and characteriza-

tion of rabbit polyclonal antibody (GN-762) to AQP2 was described

previously.37 Rabbit polyclonal antibody to AQP1 was a kind gift from

Dr. Janet Klein (Emory University, Atlanta, GA), and b-actin rabbit

polyclonal antibody was purchased from Biolegend (San Diego, CA).

Where applicable, protein band densities were determined, and differ-

ences in protein loading were corrected by normalizing the band den-

sities of the target proteins to that of b-actin.

Localization of P2Y12-R Protein by ImmunoperoxidaseLabelingFormalin-fixed and paraffin-embedded kidney sections (5 mm) were

processed for immunoperoxidase labeling as described previously,36

except for the following modifications. Dako target retrieval solution

(Dako North America, Carpinteria, CA) was used for antigen retrieval.

Negative controls for labeling were generated by incubating the sections

with P2Y12-R antibody preadsorbed with a molar excess of the immu-

nizing peptide or preimmune IgG fraction in lieu of the affinity purified

P2Y12-R antibody. Sections were counterstained with methyl green.

Localization of P2Y12-R with AQP2 Protein by ConfocalIFThis was performed essentially as described previously.36 Sections were

probed with P2Y12-R rabbit polyclonal antibody overnight followed by

incubationwith secondaryAlexa Fluor 594-conjugated goat anti-rabbit

antibody (Invitrogen) for 1 hour. Some sections were double labeled

first by incubation with AQP2 mouse monoclonal antibody, followed

by incubation for 1 hour with goat anti-mouse secondary Alexa Fluor

488-conjugated antibody. DAPI (49,6-diamino-2-pheylindole) was

used to stain the nuclei.

Primary Cultures of Rat Inner Medullary CD CellsPrimary cultures of rat IMCD cells were prepared as described

previously38 with the followingmodifications. Freshly obtained inner

medullas were cut into small pieces and digested in PBS containing

1 mg/ml collagenase B (Roche Applied Sciences, Indianapolis, IN)

and 0.1 mg/ml DNase (Sigma-Aldrich) at 37°C for 1 hour. After that,

non-IMCD cells were eliminated by osmotic shock by adding two

volumes of sterile water. The suspensionwas filtered through 100-mm

cell strainer and centrifuged at 1000 rpm for 5minutes. The pellet was

washed with 10% BSA in PBS, suspended in modified defined me-

dium (DMEM/Ham’s F12; composition given in the Supplemental

Material) containing 10% FBS, and seeded into Transwell semiperme-

able support inserts (Corning Costar, Cambridge, MA). Cells were in-

cubated at 37°C in an atmosphere of 5%CO2. Themediumwas replaced

every 2 days, and when the cells became 70%–80% confluent, the







medium was changed to the starvation one (composition given in the

Supplemental Material). After 24 hours the test agents, namely dDAVP

(desmopressin; Sigma-Aldrich) and/or PSB-0739, were added, and the

incubation continued for another 48 hours. After removing themedium,

cells were collected into Trizol reagent for total RNA extraction. For

cAMP assay, the medium was gently aspirated an 200 ml of 0.1 M HCl

was added to each insert and incubated for 20 minutes at room temper-

ature. Then cells were scrapped off the semipermeable support and lysed

by pipetting up and down. The cell lysate was centrifuged at 1000 xg for

10 minutes. cAMP concentration in the supernatant was determined by

an EIA Kit (Cayman Chemical) and normalized to the protein content

(Pierce BCA Protein Assay Kit; Rockford, IL).

Statistical AnalysesAll quantitative datawere expressed asmean6SEM.Comparisons among

the means of multiple groups were made by ANOVA, followed by the

assessment of statistical significance by Tukey–Kramer multiple com-

parison test or Student–Newman–Keuls multiple comparison test of

Bonferroni test. Differences between the means of two groups were de-

termined by unpaired t test or where applicable, byMann–Whitney non-

parametric test. P values ,0.05 were considered significant. GraphPad

Instat (GraphPad Software, La Jolla, CA) was used for statistical analyses.

ACKNOWLEDGMENTS

We thank Dr. Janet D. Klein and Dr. Andrew Weyrich lab for gen-

erously providing AQP1 antibody and human platelet lysates, re-

spectively, and Linda Schmidt for expert technical assistance.

Dr. Beverly Koller kindly supplied breeders of P2Y2-R knockoutmice.

Supported by grants from the US Department of Veterans Affairs

Merit Review Program (to B.K.K.) and the National Kidney Foun-

dation ofUtah and Idaho (to B.K.K.) and resources and facilities at the

Veterans Affairs Salt Lake City Health Care System (Salt Lake City,

UT). J.P.-P. and D.E.K. were supported by the National Institute of

Diabetes and Digestive and Kidney Diseases (grant nos. DK-64324,

DK-100944 and DK-097007, respectively).

Parts of the data were presented at the American Society of

Nephrology’s Kidney Week 2011; November 8–13, 2011; Philadelphia,

Pennsylvania; and at the American Society of Nephrology’s Kidney

Week; November 5–10, 2013; Atlanta, Georgia.

DISCLOSURESThe use of P2Y12 receptor selective antagonists for the treatment of NDI is

proprietary to the US Department of Veterans Affairs and the University of

Utah Research Foundation and is protected by a pending patent application

with the US Patent and Trademark Office.

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This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2014010118/-/DCSupplemental.





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