Biochem. J. (1993) 295, 599-605 (Printed in Great Britain) 599

lnositol lipid signalling occurs in brush-border membranes during initiationof compensatory renal growth in the ratHrvoje BANFIC,* Milena VUICA,* Mladen KNOTEK,* Sasa MOSLAVAC* and Nullin DIVECHAt*Department of Physiology, School of Medicine, University of Zagreb, 41000 Zagreb, Croatia,and tDeparment of Biochemistry, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, U.K.

Using highly specific mass assays, concentrations of inositollipids and 1,2-diacylglycerol (DAG) were determined in plasmamembranes isolated from rat kidney cortex. Significantly higherconcentrations of inositol lipids were determined in brush-border(BBM) than in basal-lateral (BLM) plasma membranes, althoughDAG concentrations were similar in both. After unilateralnephrectomy, a decrease in PtdIns(4,5)PJ and PtdIns4P, with aconcomitant increase in DAG and translocation of proteinkinase C (PKC), were observed in BBM but not in BLM isolatedfrom the remaining kidney. On the other hand, stimulation ofrenal cortical slices with insulin-like growth factor II (IGF-II) orphenylephrine caused similar effects in BLM but not in BBM.

INTRODUCTIONAfter unilateral nephrectomy, a variety offunctional, biochemicaland morphological changes develop in the remaining kidney,compensating to a great extent the loss of one kidney (Fine,1986; Wesson, 1989). Although the biochemical nature and theorigin of the stimulus (or stimuli) that triggers compensatoryrenal growth is still unclear, recent studies performed in vitrodemonstrate that plasma from uninephrectomized rats producescomplex signalling in renal cortical slices based on phospholipaseC activation (Banfic, 1990; Banfic et al., 1992). Furthermore, theincrease in activity of the Na+/H+ exchange in the brush-borderplasma membranes (BBM) could already be observed 15 minafter unilateral nephrectomy (Salihagic et al., 1988), whereas noincrease in activity of other transport processes in kidney plasmamembranes, e.g. (Na+ + K+)-ATPase, Ca2+-ATPase, Na+/Ca2+exchanger, Na+/HCO3- cotransporter and H+-ATPase, could beseen in such a short time period (Hadzic et al., 1990; Jurcevic etal., 1992; Salihagic et al., 1988). This suggests a close importantrelationship between inositol lipid signalling pathways andactivation of Na+/H+ exchanger during initiation of compensa-tory renal growth, as has also been suggested for cell growth inother experimental models (Berridge and Irvine, 1989; Moole-naar et al., 1983).The following study was performed to determine whether: (1)

inositol lipid signalling occurs after unilateral nephrectomy invivo; (2) the signalling is localized in BBM or basal-lateralplasma membranes (BLM), or both; (3) it could be switched onand off by blood occlusion and reflow through contralateralkidney, as demonstrated for Na+/H+ exchanger activity (Sali-hagic et al., 1988).

Stimulation of phospholipase C activity with translocation ofPKC only to BBM in one kidney was also induced by occlusionof blood flow through the contralateral kidney for 15 min. At30 min after the occlusion was removed and reflow established,DAG concentration and the amount of PKC in BBM returnedto control values. These results suggest that an early signal afterunilateral nephrectomy is transmitted to cells through BBM andcan be switched on and off by blood occlusion and reflowthrough the contralateral kidney, while hormonal signals causedby IGF-II and phenylephrine are transmitted to cells throughBLM.

MATERIALS AND METHODS

MaterialsPercoll, DEAE-Sepharose and Blue-Sepharose were fromPharmacia. Phenylephrine, insulin growth factor II (IGF-II) andanti-rabbit IgG conjugated to horseradish peroxidase were fromSigma. Pyruvate kinase, lactate dehydrogenase and phospho-enolpyruvate were from Boehringer. Silica gel plates were fromMerck. [32P]ATP (3000 Ci/mmol) was from either Amersham orICN. All other chemicals used were of p.a. grade purchasedcommercially.

Animals and treatmentStudies were performed on Wistar rats of both sexes aged 3-4months. The rats were bred at the Department of Physiology,School of Medicine, University of Zagreb, Croatia. During theexperiments, the rats were offered pelleted food (Sljeme, Zagreb,Croatia) and tap water ad libitum.

Unilateral nephrectomy was performed under light ethernarcosis by the dorsolateral approach as described previously(Banfic et al., 1985). If not stated otherwise, the right kidney wasremoved. Control animals were sham-operated.

Renal cortical slices from intact animals were prepared afterthe animals had been killed by a blow on the neck. The kidneyswere removed, washed with ice-cold isotonic saline and strippedof capsule and fat. Sagittal cortical slices weighing 30-50 mgwere cut from each side of the kidney with a Stadie-Riggsmicrotome. Two slices were placed into a 25 ml Erlenmeyer flaskwith 2 ml of Krebs-Ringer bicarbonate medium (pH 7.4) con-

Abbreviations used: DAG, 1,2-diacylglycerol; PtdCho, phosphatidylcholine; BBM, brush-border plasma membranes; BLM, basal-lateral plasmamembranes; PKC, protein kinase C; IGF-Il, insulin-like growth factor 11; EGF, epidermal growth factor.

599Biochem. J. (1 993) 295, 599405 (Printed in Great Britain)

600 H. Banfic and others

taining 10 mM glucose. The flasks were gassed with 95 % 02/5%CO2, capped and incubated at 37 °C in a shaking water-bath forvarious time periods (Kukolja et al., 1986).

Preparation of BBM and BLMRat renal cortical BBM were isolated by the Mg/EGTA pre-cipitation method (Biber et al., 1981). Compared with thehomogenate, the isolated membranes were enriched in brush-border marker enzymes [leucine arylamidase (13.7 + 0.5, n = 25,homogenate = 1) and alkaline phosphatase (7.8 + 0.3, n= 12)]similar to published values (Salihagic et al., 1988), and theenrichment factor for (Na+ + K+)-ATPase activity, a marker forcontraluminal membranes, was 0.7 + 0.1 (n = 10). Unilateralnephrectomy did not affect any of the enrichment factors for theabove-mentioned marker enzymes (results not shown).The BLM were isolated from the kidney cortex using a Percoll

gradient method as described by Scalera et al. (1981). Theenrichment factor for (Na++ K+)-ATPase was 10.6 + 0.4(n = 19), whereas the enrichment factors for the BBM markerenzymes (leucine arylamidase and alkaline phosphatase) were1.2+0.2 (n = 17) and 1.1 +0.1 (n = 9) respectively, similar tothose published previously (Hadzic et al., 1990). Unilateralnephrectomy did not affect any of the enrichment factors for theabove-mentioned marker enzymes (results not shown).

Mass assay of lipid extraction and quantfficatlonDiacylglycerol (DAG) from the plasma membranes was extractedusing 0.75 ml of chloroform/methanol (1: 2, v/v). When inositollipids were to be measured, membranes were extracted using0.75 ml of chloroform/methanol/I M HCl (80:160: 1, by vol.).Further extraction was performed as described by Folch et al.(1957). Measurements of inositol lipid concentration were per-formed as described below. For measurement of DAG con-centration, lipid extract was dissolved in 0.5 ml of chloroform,loaded on a silicic acid column (0.5 ml made in a Pasteurpipette), eluted with 1 ml of chloroform and dried again; DAGwas then determined as described below.

DAGDAG kinase purification was achieved in a single step from ratbrain by using a DEAE-Sepharose column as described byDivecha and Irvine (1990). DAG concentration was determinedas follows. The dried lipid was dissolved in 20,u1 of CHAPS(9.2 mg/ml) and sonicated at room temperature for 15 s. Afterthe addition of 80 ,u of buffer (50 mM Tris/acetate, 80 mM KCl,10 mM magnesium acetate, 2 mM EGTA, pH 7.4), the reactionwas started by the addition of 20 ,1 of DAG kinase followed by80 #1 of buffer containing 5 #uM ATP and 1 ,uCi of [32P]ATP.After 1 h at room temperature, the reaction was stopped by theaddition of 750 ,u of chloroform/methanol/HCl (80:160: 1, byvol.). Phosphatidic acid was extracted as described by Folch etal. (1957) and chromatographed on oxalate (1 %)-sprayed t.l.c.plates using the following solvent system: chloroform/methanol/conc. ammonia/water (45:35:2:8, by vol.). After autoradio-graphy, the spots corresponding to phosphatidic acid werescraped off and their 32p content was determined by liquid-scintillation counting. The DAG content in each sample wasadjusted to between 5 and 50 pmol, as the sensitivity of the massassay is highest in this range (Divecha et al., 1991).

PtdinsP2To the dried PtdInsP2 was added 150,ul of monomethylaminereagent, prepared as described by Clarke and Dawson (1981),

and the sample was incubated at 53 °C for 60 min. The samplewas dried and 100 ,u1 of 50 mM sodium periodate was added. After30 min at room temperature, 20 ,1 of ethylene glycol (10 %, v/v)was added to stop the reaction. After 15 min, 50 4ul of 1 % (v/v)1,1'-dimethylhydrazine/formic acid (pH 4) was added, and themixture was allowed to stand for a further 1 h. The pH of thesample was adjusted to 7 with 1 M Tris/HCl and the sample wasevaporated to dryness. Preparation of binding protein and theprocedure for the binding assay for Ins(1,4,5)PJ was exactly asdescribed by Palmer and Wakelam (1990). The PtdInsP2 contentin each sample was adjusted to between 0.5 and 5 pmol, as thesensitivity of the mass assay is highest in this range (Divecha etal., 1991).

PtdinsPThis assay was performed using a highly purified bovine brainPtdInsP kinase as described by Divecha et al. (1991). Massmeasurement was performed as follows. Standard quantities ofPtdlnsP or lipid extract from cell membranes were dried byvacuum centrifugation and resuspended in 10O,l of buffer(50 mM Tris, 10 mM MgSO4, 80 mM KCl, 2 mM EGTA,pH 7.4), followed by sonication in a water-bath. The reactionwas started by the addition of 100 ,ul of the above-mentionedbuffer containing 20 #1 of enzyme, 10,M ATP and 1 ,tCi of[32P]ATP (3000 Ci/mmol)). After 1 h at room temperature, thereaction was terminated by the addition of 750 ,ul of chloroform/methanol/conc. HCl (80:160:1, by vol), and the lipids wereextracted as in Folch et al. (1957). After separation of lipids byt.l.c. (as for the DAG assay), PtdInsP2 spots were scraped off,after identification by autoradiography, and quantified by liquid-scintillation counting. The PtdlnsP content in each sample wasadjusted to between 2 and 16 pmol, as the sensitivity of the massassay is highest in this range (Divecha et al., 1991).

PtdinsPtdlns mass measurement was performed using a coupled assayemploying a mixture of PtdIns-specific phospholipase C and theDAG kinase as described previously (Divecha et al., 1991). Thedried lipid was dissolved by the addition of 50 1l of buffer(80 mM Tris/acetate, 10 mM magnesium acetate, 2 mM EGTA,10mM KCl, 70 mM NaCl, pH 7.4) and sonicated for 15 s.Phosphatidylethanolamine (50 nmol) in 80 ,1 of the above bufferwas then added. The reaction was started by the addition of 20 ,tlof enzyme mixture followed by 50 ,ul of buffer containing 5 ,tMATP and 1 etCi [32P[ATP. The reaction was continued for 1 h atroom temperature and phosphatidic acid was extracted andquantified as for the DAG assay (above). The PtdIns content ineach sample was adjusted to between 5 and 50 pmol, as thesensitivity of the mass assay is highest in this range (Divecha etal., 1991).

Western blotting with anti-PKC antibodyCell membranes were resuspended in 300 ,ul offinal buffer [10 mMTris/HCl (pH 8.0), 5 mM MgCl2, 0.32 M sucrose, 5 mM 2-mercaptoethanol and 1 mM EGTA]. Protein (50 ,ug) was sub-jected to SDS/PAGE (10% gel). Transfer to nitrocellulose wasachieved using 10 mM Na2CO3/3 mM NaHCO3 buffer (pH 9.9)for 1.5 h. The blot was blocked in buffer (20mM Tris/HCl(pH 7.4), 0.5 M NaCl, 5 % BSA, 0.2% Tween 20), followed byincubation ofprimary antibody (1: 1000) overnight at 4 °C in theabove buffer. After incubation with anti-rabbit IgG conjugatedto horseradish peroxidase, 1: 2000 at 4°C, visualization wasachieved using 3,3'-diaminobenzidine as substrate.

Inositol lipids in compensatory renal growth 601

Measurements of marker enzymes and proteinsLeucine arylamidase (EC 3.4.11.2) and alkaline phosphatase(EC 3.1.3.1) activities were determined colorimetrically at roomtemperature as described in the commercial kits (Merckotest no.3359 and 3344 respectively). The activity of (Na+ + K+)-ATPase(EC 3.6.1.3) was measured by the coupled optical assay at roomtemperature as described by Berner and Kinne (1976). Proteinswere determined by the method of Bradford (1976).

Statistical evaluationThe data are shown as means+S.E.M. For statistical analysesStudent's t test for unpaired samples at the level of significanceof 0.05 was used.

Table 1 Concentrations of DAG and inositol lipids in kidney cortex, BBMand BLMEach value is from five independent experiments and each measurement of lipid concentrationwas performed in duplicate.

Content (pmol/mg of protein)

DAG PtdlnsP2 PtdlnsP Ptdlns

Kidney cortexBBMBLM

207 +14327 + 35312 + 21

637 + 621875 +102911 + 73

1150 + 392728 + 2051822 + 201

15.7+ 0.245.1 + 2.330.2 + 0.9

RESULTSAs shown in Table 1, there is no significant difference betweenDAG concentrations in BBM and BLM, whereas concentrationsof inositol lipids are significantly higher (P < 0.01) in BBM thanin BLM. The values for Ptdlns concentration in BLM are almostidentical with those measured by Bode et al. (1976) and Hise etal. (1984), although we employed enzymic measurement of lipidconcentration, whereas their measurements were based on de-termination of total phospholipid phosphorus. On the otherhand, somewhat higher concentration of Ptdlns were observed inBBM than were reported by the above-mentioned authors,which can be explained by the different methods of BBMisolation; the Mg/EGTA precipitation method yields very pure

membrane preparations (Biber et al., 1981).Previous observations that plasma from uninephrectomized

rats stimulates phospholipase C in intact renal cortical slices(Kukolja et al., 1986; Banfic and Kukolja, 1988; Banfic, 1989)are in agreement with the present data shown in Figure 1.Increases in DAG concentration in BBM could be observed10 min to 3 h after unilateral nephrectomy, with a significantdecrease only in PtdInsP, 10 min after the operation. The increasein DAG is greater than the loss of inositol lipids, and we assume

that, as occurs in the plasma membrane in other tissues (Downeset al., 1989), some resynthesis of PtdlnsP and PtdInsP2 has takenplace; however, this would not produce an experimentallydetectable decrease in Ptdlns. There might also be a contributionto DAG from other lipids, such as phosphatidylcholine (PtdCho)(Exton, 1990), although we were not able to demonstrate any

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Blood flow through the left kidney was stopped by ligating the renal artery at zero time. The rats were killed either 15 min (U) or 45 min (-) later or the reflow was established by removingthe ligature 15 min after the blood flow occlusion and the animals were killed 30 min thereafter (*). Control animals (time 0) were sham-operated. Each point represents the mean + S.E.M. fromfour separate membrane preparations in each group. *Significantly different from control values; tsignificantly different from value obtained by ligating the renal artery.

PtdCho breakdown by way of phospholipase C activation invitro (Banfic et al., 1992). It is important to note that, during theentire experimental period (5 min to 48 h), no changes in eitherDAG or inositol lipid concentrations were observed in BLMprepared from the remaining kidney (results not shown).

As classical hormonal signals in the kidneys are mediatedthrough BLM (Morel and Doucet, 1986), and as we failed toobserve activation of phospholipase C in BLM after unilateralnephrectomy, we decided to check inositol lipid signalling by wayof BLM using IGF-II, a well-known agonist which stimulates

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Inositol lipids in compensatory renal growth 603

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Figure 4 Translocation of PKC to BLM and BBM

Plasma membranes were isolated as described in the Materials and methods section. Protein(50 ul) was subjected to SDS/PAGE, transferred to nitrocellulose and probed with an anti-PKCantibody. Lane 1, BLM isolated 15 min after blood flow occlusion through the contralateralkidney; lane 2, control BLM; lane 3, BLM isolated 45 min after blood flow occlusion throughthe contralateral kidney; lane 4, BLM isolated 15 min after blood flow occlusion, which wasfollowed by re-established blood flow for 30 min through the contralateral kidney; lane 5, BBMisolated 15 min after blood flow occlusion through the contralateral kidney; lane 6, controlBBM; lane 7, BBM isolated 45 min after blood flow occlusion through the contralateral kidney;lane 8, BBM isolated 15 min after blood flow occlusion, which was followed by re-establishedblood flow for 30 min through the contralateral kidney; lane 9, BLM isolated after stimulationof renal cortical slices for 5 min with IGF-11 (10 nM); lane 10, BBM isolated after stimulationof renal cortical slices for 5 min with IGF-11 (10 nM).

inositol lipid signalling in canine BLM (Rogers and Hammerman,1988). As shown in Figure 2, IGF-II stimulates an increase inDAG concentration, with a concomitant decrease in PtdInsP2and PtdlnsP concentrations in BLM. In contrast, no changes ineither DAG or inositol lipid concentrations were observed inBBM (results not shown). Identical results were obtained whenwe used the al-agonist phenylephrine (0.1 mM) (results notshown), which has also been demonstrated to stimulate phospho-lipid metabolism in rat kidney cortical tubules (Wirthensohn etal., 1984).

In a previous paper (Salihagic et al., 1988), we have shown thatstimulation of Na+/H+ exchange in BBM in one kidney was alsoinduced by the occlusion of blood flow through the contralateralkidney and that Na+/H+ exchange returned to the controlpreoperative value when the reflow was established. Results fromidentically designed experiments are shown in Figure 3. At15 min after the occlusion of blood flow through the left kidneyby ligation of the left renal artery, the concentration of DAG inBBM from the right kidney significantly increased, but no

increase in DAG concentration could be observed in BLM. If theligature was kept in place for an additional 30 min, the DAGconcentration remained significantly elevated. Consequently, we

were able to mimic the increase in BBM DAG concentration seen

after unilateral nephrectomy (Figure 1). However, if blood flowthrough the left kidney was occluded for 15 min and then re-

established by removal of the ligature, 30 min later the DAGconcentration in the renal luminal membrane from the contra-lateral kidney did not differ from that in the control BBM. Theseresults indicate that the stimulation of phospholipase C in BBMis caused by a rapid and reversible mechanism.The simplest interpretation of the data described above is that

unilateral nephrectomy and blood occlusion through the contra-lateral kidney both induce phospholipase C-catalysed hydrolysisof inositol lipids in BBM, whereas 'classical hormones' do thesame thing in BLM. Phospholipase C activation is followed by

translocation of PKC from the cytosol to membranes, a processcaused primarily by the generation of DAG, but probably alsorequiring increased intracellular Ca2+ (Nishizuka, 1992). It isintriguing that phospholipase C-mediated cell stimulation mightbe exerted through BBM or BLM and could involve translocationof PKC to the opposite side of the plasma membrane. We haveinvestigated this using an anti-PKC antibody (one recognizing aconsensus sequence common to a, y and a PKCs). As shown inFigure 4, blood occlusion and reflow through the left kidney doesnot induce any translocation ofPKC to BLM in the right kidney(lanes 1-4), whereas blood occlusion through the left kidney foreither 15 or 45 min (lanes 5 and 7) stimulates translocation ofPKC (which has an Mr of about 80000) to BBM in the rightkidney, compared with the control preoperative amount (lane 6).Translocation of PKC to BBM but not BLM of the remainingkidney was also observed when unilateral nephrectomy wasperformed 15, 30, 60 and 180 min before membrane isolation(Western blots not shown). Moreover, if the blood flow throughthe left kidney was occluded for 15 min and then re-establishedby removal of the ligature, 30 min later the amount of PKC inBBM in the right kidney returned to the control preoperativevalue (lane 8), showing that translocation of PKC is caused by arapid and reversible mechanism. Finally, IGF-II induced trans-location of PKC to BLM (lane 9) but not BBM (lane 10),demonstrating once again the difference between a classicalhormonal signal and the signal produced by unilateral neph-rectomy.

DISCUSSIONThe results presented here show that in renal cells there is aspatial separation of the inositol lipid signalling system betweenBLM and BBM. Although specific binding of 1251-labelled IGF-TI was demonstrable in isolated BLM and BBM with equalbinding capacities for each membrane (Hammerman and Rogers,1987), our results corroborate the previous observation (Rogersand Hammerman, 1988) that IGF-II stimulates inositol lipidsignalling only in BLM. It is important to note that Rogers andHammerman (1988) measured phospholipase C activation innative membranes incubated with IGF-II, whereas we isolatedBLM after we had incubated intact renal cortical slices with anagonist. In preliminary experiments (H. Banfic, unpublishedwork), we were unable to observe any phospholipase C activationon stimulation of isolated membranes with IGF-II, althoughprocedures for isolation of BLM and BBM were almost identicalwith those described by Rogers and Hammerman (1988). Thedifference probably lies in the different animal species used in theexperiments (canine versus rat kidney). It can be argued that,during isolation of rat renal cell membranes, the delicate inter-relationship between receptor, G-protein and phospholipase C isbroken. Consequently, these proteins are not functionallycoupled in isolated membranes. On the other hand, our ob-servation extended the results ofRogers and Hammerman (1988),as we were able to demonstrate PKC translocation, a processcaused primarily by the generation ofDAG (Nishizuka, 1992), toBLM but not to BBM.The stimulus (or stimuli) that causes compensatory renal

growth is poorly understood, but, in principle, two hypothesesexist. The premise of the 'work hypothesis' is that the increasedwork load after uninephrectomy may be the triggering signal forrenal hypertrophy, whereas according to the 'renotropic hy-pothesis' a humoral factor is responsible for it (Fine, 1986;Wesson, 1989). Our recent study performed in vitro favours therenotropic hypothesis by demonstrating that plasma from uni-

604 H. Banfic and others

nephrectomized rats stimulates phospholipase C activation inrenal cortical slices (Banfic and Kukolja, 1988). In the presentstudy, in vivo activation of phospholipase C was demonstrated,which, together with the perfect temporal overlapping of in-creased phospholipase C activity in vitro and in vivo (10 min to3 h after unilateral nephrectomy), further suggests the existenceofa humoral factor responsible for the initiation ofcompensatoryrenal growth.

It is surprising that signalling is mediated through BBM;although the presence of phospholipase C in rat kidney BBM hasbeen known for some time (Schwertz et al., 1983), until recentlyit was not known whether it could be functionally activated byany receptor on the apical side of the cell (Reskhin et al., 1991).Sustained elevated levels of DAG in BBM of the remainingkidney after uninephrectomy suggests that there should be othersources of DAG besides inositol lipids. Although we were notable to demonstrate any PtdCho breakdown in vitro by way ofphospholipase C activation (Banfic et al., 1992), increasedphospholipase D activity was observed in the cortex of theremaining kidney (Gatalica et al., 1993), the time course ofactivation being a little behind that ofphospholipase C activationobserved in this paper. Several lines of evidence suggest thatPtdCho is hydrolysed by phospholipase D, resulting in theformation of phosphatidic acid, which is then converted intoDAG by the action of phosphatidic acid phosphomonoesterase(Nishizuka, 1992). The process that occurs after phospholipase Cactivation and generation of DAG is translocation ofPKC fromthe cytosol to membranes (Nishizuka, 1992), which also happensin BBM after unilateral nephrectomy or blood occlusion throughthe contralateral kidney. Increased PKC activity in BBM of theremaining kidney has also been observed 24 and 48 h afterunilateral nephrectomy (Hise and Mehta, 1989), and, althoughDAG concentration at these time points is not changed, it isknown that there are other activators of PKC besides DAG(Nishizuka, 1992). The metabolites of phospholipase A2 ac-tivation might be of particular interest, as it has been observedboth in vitro (Banfic and Gatalica, 1989) and in vivo (Logan et al.,1986) that they influence compensatory renal growth. Anotherimportant possibility that emerges from separate inositol lipidsignalling systems in BBM and BLM is that renal cells can easilydistinguish from which side they are stimulated, and, despite thesame general signalling system used, can probably responddifferently. This might be even more important when a dualmessenger system is used, as has been demonstrated for para-thyroid hormone (Reskhin et al., 1991), or during initiation ofcompensatory renal growth (Banfic, 1990).The experiments involving uninephrectomy and temporary

occlusion of blood flow through the renal artery followed byreflow clearly indicate the presence of a mechanism that regulatesfast and reversible changes in Na+/H+ antiporter activity in theluminal membrane of proximal tubular cells (Salihagic et al.,1988). In the light of the fast and reversible changes in phospho-lipase C activity and PKC translocation to BBM under theexperimental conditions shown here, this strongly favours thepossibility that activation of exchanger is caused by PKC-mediated protein phosphorylation, as has already been shown insolubilized brush-border membrane proteins (Weinman et al.,1990) and primary cultures of renal proximal tubular cells (Hiseand Mehta, 1989; Horie et al., 1992).

Recently, it was demonstrated that denervation inhibits theearly increase in Na+/H+ exchange after uninephrectomy butdoes not suppress hypertrophy. This suggests that a renorenalreflex arc mediates the increase in antiporter activity throughsympathetic nerves, but dissociates renal growth from the Na+/H+ antiport activation (Mackovic-Basic et al., 1992). This means

that Na+/H+ exchanger activation does not have a crucial role ingrowth of renal cells as has been reported for some other celltypes (Grinstein and Rothstein, 1986; Moolenaar et al., 1983;Schwertz et al., 1983; Seiffer and Aronson, 1986). Proximaltubules have adrenergic receptors on their plasma membrane andare richly innervated (Sundaresan et al., 1987). Although acti-vation of al-receptors stimulates phosphoinositide hydrolysis inrat cortical tubules (Wirthensohn et al., 1984) and in MDCKcells (Slivka and Insel, 1987), the present results clearly demon-strate that signalling during initiation of compensatory renalgrowth is not mediated by way of al-receptor activation, asphenylephrine activates phospholipase C in BLM, whereasunilateral nephrectomy and blood flow occlusion stimulatephospholipase C in BBM. Similar conclusions can be madeabout the functioning of renal dopaminergic nerves, as it isknown that dopamine-I receptors that are coupled to phospho-lipase C are only located in BLM (Felder et al., 1989a,b).Nevertheless, renal nerves may be involved in the signallingprocesses to the remaining kidney via the afferent arc ofneurocirculatory reflex; 30 min after unilateral nephrectomy, anincrease in peripheral plasma concentration of y-melanocyte-stimulating hormone-like peptide from the pituitary was observed(Lin et al., 1985, 1987). Thus this peptide can be added to thelong list of humoral factors that might be involved in the controlof transport processes and growth of kidney cells (Fine, 1986;Morel and Doucet, 1986; Segal and Fine, 1989; Wesson, 1989).An increase in plasma concentration of atrial natriuretic

peptide 2 h after unilateral nephrectomy has also been reported,together with an increase in urinary cyclic GMP excretion(Valentin et al., 1990). Although it is known that unilateralnephrectomy increases cyclic GMP concentration in the renalcortex of the remaining kidney (Dicker, 1977; Schlondorff andWeber, 1976), there is little chance that atrial natriuretic peptidecauses inositol lipid signalling in BBM, as it is known that thispeptide mainly affects the distal portion of the nephron (DeWardener and Clarkson, 1985), whereas, using the above-mentioned methods for the preparation of BBM and BLM, themajority of the plasma membrane is found to originate from theproximal tubule. Also, to our knowledge, there has been noreport of inositol lipid signalling in the renal cells on stimulationwith atrial natriuretic peptide. Moreover, inositol lipid signallingprecedes any increase in cyclic GMP concentration both in vivo[present results compared with the data of Dicker (1977) andSchlondorffand Weber (1976)] and in vitro, where it was observedthat an increase in cyclic GMP depends on extracellular Ca2+(Banfic and Kukolja, 1988), suggesting that an increase in cyclicGMP concentration is a consequence of inositol lipid signalling.To summarize, the present results demonstrate that, in renal

cells, there is a spatial separation of the inositol lipid signallingsystem between BLM and BBM, and that uninephrectomy causesactivation of phospholipase C only in BBM. It seems that thestimulus factor for compensatory renal growth is present intubular fluid and might be excreted in urine. Indeed, it has beenobserved that reinfusion of half the urine output in rat stimulatesrenal growth, and this is not diminished by dialysis of the urinebefore its reinfusion, suggesting that the renal growth stimu-lator(s) in urine is not readily dialysable (Harris and Best, 1979).Unfortunately, one peptide of particular interest, epidermalgrowth factor (EGF), which can be isolated from urine in its low-and high-M, forms (Nexo et al., 1990) and is largely synthesizedin the kidney (Salido et al., 1991) and might act within it as aparacrine agent, is not a candidate to explain the effects observedin this paper, as it is known that its major site of action is thecollecting tubule, particularly the inner medullary region (Breyeret al., 1988; Harris, 1989), and only there can it stimulate

Inositol lipids in compensatory renal growth 605

phosphoinositide hydrolysis (Teitelbaum et al., 1990). Moreover,although increased distal nephron EGF content was observed inthe remaining kidney (Miller et al., 1992), there is no specificbinding ofEGF to rat kidney apical cortical membranes (Behrenset al., 1989) and, in renal cortical slices, EGF is not capable ofstimulating phospholipase C (Gatalica and Banfic, 1988). Finally,although the biochemical nature of the stimulus that initiatescompensatory renal growth is still unknown, experiments withblood flow occlusion and reflow through renal artery clearlyindicate the presence of a fast and reversible mechanism ofchanges in phospholipase C activity and PKC translocation toBBM.

We are most grateful to Stewart Gilmour for the anti-PKC antibody, to David Landerfor the preparation of the lipid standards and to Visnja Kabalin for editing themanuscript. We also thank Robin F. Irvine for helpful discussions. This work wassupported by the Ministry for Science of the Republic of Croatia and by the WellcomeTrust.

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Received 8 April 1993; accepted 4 June 1993