Kidney International, Vol. 55 (1999), pp. 1251–1258

HORMONES – CYTOKINES – SIGNALING

Endothelial cells regulate proximal tubule epithelial cellsodium transport

STUART L. LINAS and JOHN E. REPINE

Denver Health Medical Center and the Webb-Waring Antioxidant Research Institute, University of ColoradoHealth Sciences Center, Denver, Colorado, USA

Endothelial cells regulate proximal tubule epithelial cell so- Burns demonstrated that cGMP inhibited apical Na/Hdium transport. exchange in rabbit proximal tubules [6]. Proximal tubule

Background. Proximal tubule epithelial cells are in close cGMP can be produced from membrane or soluble guan-contact with the renal microvasculature, but the effect of endo-ylate cyclase [7]. Soluble guanylate cyclase is regulatedthelial cells (ECs) on proximal tubule epithelial cell (PTEC)by nitric oxide (NO), and NO is an important determi-function is not known.

Methods. To determine if ECs regulate PTECs, we coincu- nant of proximal tubule function. For example, decreasesbated ECs with PTECs in a system that permitted cross-talk in NO are associated with decreases in lithium clearancebetween the two cell types and the vectorial transport of sodium. [8]. In addition to indirect evidence suggesting that NOResults. In the presence (but not absence) of ECs, adding

regulates proximal tubule Na reabsorption, adding Nabradykinin or acetylcholine increased cGMP and decreasedsodium transport, as well as Na,K-ATPase in PTECs. Interleukin nitroprusside (SNP) or other NO donors to perfusates(IL)1B preconditioning of ECs also increased cGMP and de- of perfused proximal tubules resulted in concentration-creased sodium transport and Na,K-ATPase in PTECs. Brady- dependent increases (high concentration) or decreaseskinin, acetylcholine, and IL1B EC-dependent effects were re-

(low concentrations) in proton flux [9]. Moreover, addingversed with the nitric oxide (NO) synthase inhibitor L-NNA.NO donors to proximal tubule cells inhibited key trans-In the absence of ECs, the addition of NO donors to PTECs

increased cGMP and decreased sodium transport and Na,K- porters that regulate proximal tubule Na reabsorptionATPase. 8Br-cGMP also decreased PTEC sodium transport [5–10].and Na,K-ATPase. Nitric oxide is made from arginine by several isoformsConclusion. Endothelial cells regulate PTEC function. This

of NO synthase (NOS), including type I, II, and III NOSeffect is mediated by NO synthase-dependent up-regulation of[11]. Type II NOS has been identified in proximal tubulecGMP in PTECs.epithelial cells (PTECs) [12]. Exposure of PTECs in cul-ture to agonists that induce NOS activity increased NO

Sixty percent of sodium (Na) filtered at the glomerulus and resulted in autocrine-mediated decreases in proxi-is reabsorbed by the proximal tubule. Proximal tubule mal tubule Na,K-ATPase activity [5]. Proximal tubulesNa reabsorption is regulated by the activity of renal nerves are apposed to vascular endothelial cells (ECs) that con-and by hormones, including angiotensin II (Ang II) and tain NOS. Paracrine regulation of proximal tubules bynorepinephrine. The major signaling pathways that ECs has not been considered but is possible because: (a)transduce Ang II and catecholamine-mediated Na trans- there is close contact between PTECs and ECs; (b) ECsport are the phospholipase C, protein kinase C, adenyl- make NO; (c) other renal tubule cells, including corticalate cyclase, and phospholipase A2 systems [1–4]. collecting tubule cell lines, are regulated by ECs [13];

The cGMP signaling pathway also contributes to the (d) the addition of agents eliciting EC NO productionregulation of proximal tubule Na transport. Guzman et to peritubular capillary perfusate increased proton fluxal found that cGMP analogues decreased Na,K-ATPase in perfused proximal tubule preparations [14].in mouse proximal tubules [5], whereas Roczniak and The purpose of this study, therefore, was to determine

if and how ECs regulate PTEC Na transport. To test forthis possibility, we used a coincubation system in whichKey words: nitric oxide, nitric oxide synthase, cGMP, polarity, sodium

transport, cell-cell interaction. PTECs were grown on semipermeable filters in apposi-tion to ECs. This approach enabled us to determineReceived for publication July 6, 1998cell–cell interactions while avoiding the confounding ef-and in revised form October 30, 1998

Accepted for publication October 30, 1998 fects resulting from changes in systemic and/or renalhemodynamics. 1999 by the International Society of Nephrology

1251

Linas and Repine: EC regulate PTEC Na transport1252

METHODS then centrifuged at 3000 3 g for 20 minutes at 48C.Aliquots of supernatant were extracted three times withRat proximal tubule epithelial cell isolationfour volumes of water-saturated ether, brought to a pHand cultureof 7.0 with Tris, and assayed for cGMP using a commer-

Proximal tubule cell isolation techniques were derivedcially available kit (Amersham, Arlington Heights, IL,

from modification of methods of Vinay et al and wereUSA). cAMP and inositol phosphates were measured

previously described in detail by our laboratory [15, 16]. as described in earlier studies [16].Briefly, kidneys were removed from male Sprague-Daw-ley rats and were digested in collagenase (272 U/ml) for Assay of Na,K-ATPase activity30 minutes at 378C. Proximal tubules were isolated by Na,K-ATPase activity was determined by measuringPercoll gradient centrifugation and were mixed with Dul- ouabain-inhibitable 86Rb uptake from the basolateralbecco’s modified Eagle’s medium and Ham’s nutrient surface of PTECs as previously described [18]. Briefly,mixture (DMEM-F12) containing 1.7 nm epidermal at the time of the study, medium was aspirated fromgrowth factor, 6.5 mm transferrin, 10 mm dexamethasone, apical and basolateral sides and replaced with RPMI87 mm insulin, 14 mm NaHCO3, 10% bovine calf serum, buffer [in mm: 102 NaCl, 5.6 Na2HPO4, 5.4 KCl, 0.4 CaCl2,100 U/ml penicillin G, 100 mg/ml streptomycin sulfate, 0.4 MgSO4, 24 N-2-hydroxyethylpiperazine-N9-2-ethane-and 5 mm nonessential amino acids, pH 7.40. Proximal sulfonic acid (HEPES), 3 lactate, and 5 glutamine, pHtubules were plated on type I collagen-coated, 6.5 mm 7.45]. 86Rb (1 mCi/ml) was added to the basolateral sidediameter, 1.45 mm pore, permeable supports and were in the presence or absence of 2 mm ouabain. At the endsuspended in 24-well, 16 mm diameter culture dishes of study, supports were removed and washed with ice-(for cGMP and 86Rb uptake studies) and on 12 mm in cold MgCl2 (100 mm) to stop flux. The supports werediameter, 0.4 mm pore, permeable supports suspended counted by gamma scintillation (Packard Auto-Gammain six-well culture dishes (for 22Na transport studies). Scintillation Spectrometer model 5330). Ouabain-sensi-Both apical and basolateral surfaces were bathed with tive 86Rb uptake was calculated as the difference betweenDMEM-F12 containing 10% bovine calf serum. All stud- total counts per minute of 86Rb and counts per minute ofies were done after cells reached confluence between six 86Rb in the presence of ouabain. Because 86Rb uptake var-and nine days of culture. Confluence was considered to ied from plate to plate, each experiment had its own con-have occurred when 14C-inulin added to the upper cham- trol. In additional studies, 86Rb uptake was measured inber was prevented from appearing in the lower chamber. vesicles prepared as described by Molitoris and Simon [19].

Endothelial cell isolation and culture 3H adenine releaseBovine pulmonary artery ECs were harvested by Proximal tubule epithelial cell injury was assessed

methods described by our laboratory [17]. ECs were by measuring (3H) adenine release as previously de-grown in DMEM/F12 containing D-Val with 20% fetal scribed [20].calf serum. ECs were cultured in a 5% CO2/95% air

Measurement of sodium fluxincubator. Media were changed after 24 hours and twiceweekly thereafter, and ECs were passaged every four to Unidirectional apical-to-basolateral Na flux was deter-seven days. ECs from passages 3 through 6 were studied. mined by a modification of methods previously described

by our laboratory [21, 22]. Na (1 mCi/ml) was added toCoincubation the apical chamber in solution A (transport buffer), which

Confluent PTECs on permeable supports were coincu- contained (in mm) 128 NaCl, 5 NaHCO3, 5 KCl, 4 NaHPO4,bated with ECs grown in culture dishes for 30 minutes 1 CaCl2, 5 urea, 1 MgSO4, 3 lactate, 2 glutamine, andat room temperature prior to the addition of agonists. 20 HEPES, 305 mOsm/kg H2O, pH 7.40. BasolateralPTECs were oriented so that only the basolateral surface surfaces were exposed to the same buffer without 22Na.was apposed to ECs. The initiation of transport was defined as the time when

apical buffer was added. The final volume on both apicalAssay of cGMP, cAMP, and inositol phosphates and basolateral sides was 4 ml. Aliquots of 100 ml were

Confluent monolayers of PTECs grown on permeable removed from the basolateral buffer 15 minutes aftersupports were incubated for 15 minutes at room temper- the initiation of transport. In preliminary studies, weature in DMEM-F12 with 0.5 mm 3-isobutyl-1-methyl- found that 22Na transport was linear for up to 30 minutesxanthine (IBMX). Agonists were then added for the after the addition of agonists [21, 22]. 22Na was quanti-times and at the concentrations indicated. Reactions tated by liquid scintillation (Packard Tri-Carb 460), andwere stopped by removing ECs, aspiration of media, 22Na mass was calculated from simultaneously deter-and addition of ice-cold 10% trichloroacetic acid (TCA). mined standard curves. 22Na flux was defined as total

quantity of basolateral transport divided by permeableSuspensions were kept on ice for 30 minutes and were

Linas and Repine: EC regulate PTEC Na transport 1253

support surface area. Because basal 22Na transport varied pmol/mg, P , 0.001). Ach effects were blocked followingtreatment with L-NNA.from plate to plate (but not within individual plates), one

To determine whether ECs regulated PTEC Na trans-well per plate was used for the determination of 22Naport, we measured the effect of ECs on PTEC Na,K-transport in the absence of ECs or other antagonists. Re-ATPase activity and on transcellular Na transport. Coin-sults are expressed as femtomoles per square millimeter.cubation of ECs with PTECs did not affect the basal

Materials values of either of these parameters. In addition, addingBK did not decrease PTEC Na,K-ATPase activity or NaMaterials were obtained from the following sources:transport in the absence of ECs. However, adding BKDMEM-F12, type I collagen, penicillin G, streptomycinto coincubations of ECs and PTECs decreased PTECsulfate, transferrin, insulin, dexamethasone, epidermalNa,K-ATPase activity (Fig. 2) and Na transport (Fig. 3).growth factor, modified Eagle’s medium (MEM) nones-Figure 2A demonstrates that BK reduced Na,K-ATPasesential amino acids, dimethylamiloride, monensin, meta-activity from 6.1 6 0.2 to 4.8 6 0.3 pmol K · mg21 · min21

proterenol, and ouabain were from Cambridge Research(P , 0.01). Adding the NOS inhibitor L-NNA reversedBiochemicals (Wilmington, DE, USA). Sprague-DawleyEC-dependent decreases in Na,K-ATPase activity inrats were from Sasco (Omaha, NE, USA). CollagenasePTECs. Similar effects were found with Ach (Fig. 2B).was from Worthington Biochemicals (Freehold, NJ, USA).The BK addition to the coincubations also reduced trans-Percoll was from Pharmacia-LKB (Uppsala, Sweden).cellular Na transport. Figure 3A indicates that BK re-Permeable supports, cell culture dishes, were from Co-duced transcellular Na transport from 66.4 6 3.2 in thestar (Cambridge, MA, USA). Bovine calf serum wasabsence of ECs to 40.7 6 4 fmol/mm2 in the presencefrom Hyclone Laboratories (Logan, UT, USA). cAMPof ECs (P , 0.001). This effect was reversed withand cGMP assay kits and 22Na were from Amersham.L-NNA. In additional studies, we found that Ach also86Rb was from NEN Research Products (Boston, MA,decreased transcellular Na transport by 25% in the pres-USA), and Optifluor scintillation fluid was from Worldence but not absence of EC, and that this effect wasPrecision Instruments (Sarasota, FL, USA).reversed with L-NNA (Fig. 3B).

To further determine whether EC-dependent regula-Statisticstion of PTEC function was mediated by NO, ECs aloneResults are expressed as means 6 sem. Experimentswere conditioned with interleukin (IL)1B for 24 hours un-were repeated three times, and the mean was used toder conditions that up-regulate NOS and NO production

generate one data point. The results from each study[23]. Figure 4A indicates that coincubation of IL1B condi-

were repeated three to six times. Comparisons between tioned ECs with PTECs (which had not been exposedtwo groups was made by the unpaired, two-tailed Stu- to IL1B) resulted in increases in PTEC cGMP (1.0 6 0.1dent’s t-test. Comparisons between more than two groups to 2.7 6 0.2 pmol/mg, P , 0.001). These effects werewere made by one-way analysis of variance with the blocked with L-NNA. Figure 4B indicates that coincuba-Tukey test used for post hoc analysis. Statistical signifi- tion of IL1B-conditioned EC resulted in decreases incance is defined as a P of less than 0.05. PTEC transcellular Na transport (70.1 6 5.2 to 32.6 6 4.7

fmol/mm2, P , 0.001). These effects were also abrogatedwith L-NNA.RESULTS

Effect of endothelial cells on proximal tubule Role of nitric oxide and cGMP on proximal tubuleepithelial cell function epithelial cell function

To determine whether ECs regulate PTEC functions, Because the data suggested that NO produced by EC-ECs were coincubated with PTECs. Under basal condi- mediated, EC-dependent regulation of PTECs, we per-tions, coincubating ECs with PTECs did not alter cGMP, formed additional studies in which we added an NONa,K-ATPase, or Na transport in PTECs. In contrast, donor directly to PTECs. Table 1 shows that addingalthough the addition of bradykinin (BK) did not change increasing concentrations of SNP progressively increasedcGMP in PTECs in the absence of ECs (Fig. 1), in the cGMP generation in PTECs. Compared with no SNPpresence of ECs, adding BK increased PTEC cGMP (0.9 6 0.1 pmol/mg), the threshold for stimulating cGMPproduction from 0.7 6 0.1 to 2.1 6 0.2 pmol/mg (P , was 1025 m SNP (1.9 6 0.2, P , 0.05), and the maximum0.001). Endothelial cell-dependent increases in cGMP stimulation was observed at 1023 m SNP (2.8 6 0.2).were abrogated following treatment with the NOS inhib- Table 1 also indicates that adding SNP decreaseditor L-NNA. Similar results were found with acetylcho- Na,K-ATPase activity in PTECs. Compared with no SNPline (Ach; Fig. 1B). Whereas adding Ach did not alter (5.3 6 0.3 pmol K · mg21 · min21), concentrations ofcGMP in PTECs in the absence of ECs, adding Ach in SNP of 1025 m or greater reduced Na,K-ATPase. To

determine whether NO inhibited Na,K-ATPase in thethe presence of ECs increased cGMP levels (2.5 6 0.2

Linas and Repine: EC regulate PTEC Na transport1254

Fig. 1. Effect of bradykinin (A) or acetylcholine (B) on proximal tubule epithelial cells (PTEC) cGMP in the absence or presence of epithelialcells (ECs). PTECs were incubated with bradykinin or acetylcholine (1027 m, 15 min) in the absence (PTECs) or presence (PTECs and ECs) ofECs or ECs and L-NNA (1025 m, added 15 min before bradykinin or acetylcholine; PTECs and ECs plus L-NNA). ECs and PTECs were coincubatedfor 30 minutes prior to the addition of agonists or antagonist. cGMP was determined as per the Methods section (N 5 5 in each group).

Fig. 2. Effect of bradykinin (A) or acetylcholine (B) on PTEC Na,K-ATPase activity in the presence or absence of ECs. PTECs were incubatedwith bradykinin or acetylcholine (1027 m, 15 min) in the absence (PTECs) or presence (PTECs and ECs) of ECs or ECs and L-NNA (1025 m,added 15 min before bradykinin; PTECs and ECs plus L-NNA). Na,K-ATPase activity was determined as per the Methods section (N 5 5 ineach group).

absence of activating guanylate cyclase, we exposed ba- To determine if NO decreased transcellular Na trans-port, Na transport was determined in PTECs exposedsolateral membranes of PTECs to SNP (1025 m). Under

these conditions, SNP did not decrease Na,K-ATPase to SNP. Figure 6 indicates that adding SNP decreased22Na transport in a concentration-dependent manner. Naactivity (9.6 6 1.1 pmol K · mg 21 · min21, control; 9.1 6

1.3 SNP; N 5 3, P 5 NS). transport was reduced from 50.5 6 2.6 with 1027 m SNPto 22.4 6 2.4 fmol/mm2 with 1023 m SNP (P , 0.001).Because NO decreases apical Na uptake [6], we con-

sidered the possibility that decreases in Na,K-ATPase Because NO has been shown to be toxic to renal epi-thelial cells, we questioned whether NO- dependent de-were mediated by SNP-induced decreases in apical Na

uptake. To determined whether NO had direct effects on creases in Na,K-ATPase and transcellular Na transportcould be caused by PTEC injury. Concentrations of SNPNa,K-ATPase, Na,K-ATPase was determined in PTECs

exposed to concentrations of dimethylamiloride (DMA), used for our studies (1027 to 1023 m for 30 min) did notincrease adenine (3H) adenine release from PTECs.which blocks apical Na/H exchange [24]. Figure 5 indi-

cates that DMA decreased Na,K-ATPase. However, the Because coincubation of ECs with PTECs and addingthe NO donor to PTECs increased in cGMP, we examinedaddition of SNP further decreased Na,K-ATPase from

3.8 6 0.2 to 2.8 6 0.2 pmol K · mg21 · min21 (P , 0.05). whether the inhibitory effects of NO on Na,K-ATPase

Linas and Repine: EC regulate PTEC Na transport 1255

Fig. 3. Effect of bradykinin (A) or acetylcholine (B) on PTEC 22Na transport in the absence or presence of ECs. PTECs were incubated withbradykinin or acetylcholine (1027 m, 15 min) in the absence (PTECs) or presence (PTECs and ECs) of ECs or ECs and L-NNA (1025 m added15 min before bradykinin; PTECs and ECs plus L-NNA). 22Na transport was determined as in the Methods section (N 5 4 or 5 in each group).

Fig. 4. Effect of interleukin (IL)1B conditioned ECs on cGMP (A) and 22Na transport (B) in PTECs. ECs were conditioned with no additions(ECs) or IL1B (10 ng/ml, 24 hr; ECs and IL1B). After 24 hours, IL1B was removed, and ECs were coincubated with PTECs for 30 minutes in DMEM-F12. In some studies, L-NNA (1025 m) was added to ECs 15 minutes before coincubation (ECs and IL1B plus L-NNA). cGMP and 22Na transportwere determined as per the Methods section (N 5 5 or 6 in each group).

Table 1. Effect of adding sodium nitroprusside (SNP) on cGMP and Na,K-ATPase in PTEC Concentration SNP

0 1027 m 1026 m 1025 m 1024 m 1023 m

PTEC cGMP pmol/mg 0.960.1 (6) 0.97 60.16 (6) 1.20 60.19 (6) 1.85 60.18a (6) 2.53 60.17a (6) 2.82 60.22a (6)PTEC Na, K-ATPase pmol K·lg21 min21 5.360.3 (6) 5.4 60.2 (4) 4.9 60.2 (6) 4.1 60.1a (6) 3.8 60.2a (5) 3.5 60.1a (5)

Data are mean 6 sem; the number of determinations is in parentheses.a Value significantly different (P , 0.05) than no additions

and Na transport were mediated by cGMP. Figure 7 2.1 6 0.2 (P , 0.01 vs. DMA). Exposure to 8Br-cGMPdecreased Na transport from 42.8 6 3.2 fmol/mm2 withindicates that adding the cGMP analogue 8Br-cGMP

to PTECs caused concentration-dependent decreases in 1027 m 8Br-cGMP to 35.2 6 2.6 with 1025 m 8Br-cGMPand to 27.7 6 1.8 with 1023 m 8Br-cGMP (Fig. 7).Na,K-ATPase. The maximum inhibition was 1024 m 8Br-

cGMP. The inhibitory effects of 8Br-cGMP on Na,K- Because the adenylate cyclase and phospholipase Csignaling systems regulate Na transport in PTECs, weATPase activity were observed when apical Na entry

was blocked with DMA: control 5.7 6 0.3 pmol K · mg21 · measured cAMP and IP3 generation in PTECs after theaddition of 8Br-cGMP (1024 m). We did not detect activa-min21, DMA 3.9 6 0.3; DMA plus 8Br cGMP (1024 m)

Linas and Repine: EC regulate PTEC Na transport1256

Fig. 5. Effect of sodium nitroprusside (SNP) on Na,K-ATPase. PTECswere exposed to no additions (N 5 5) or dimethylamiloride (1023 m,N 5 5) or dimethylamiloride plus SNP (1024 m, N 5 5) for 15 minutes.Na,K-ATPase was determined as described in the Methods section.

Fig. 6. Effect of SNP on 22Na transport across PTEC monolayers.PTECs were exposed to SNP in the concentrations indicated for 15minutes. 22Na transport was determined as described in the Methodssection (N 5 4 in each group).

tion of these signaling pathways with 8Br-cGMP [cAMP,control 100%, 8Br-cGMP 94 6 3% (P 5 NS, N 5 3 ineach group); IP3, control 100%; 8Br-cGMP 101.6 6 4%

addition of NO to proximal tubules has been reported.(P 5 NS, N 5 3 in each group)].Cantiello and Ausiello showed that SNP decreased Nauptake in LLC-PK cells [25]. Chevalier reported that

DISCUSSION SNP inhibited transepithelial Na transport in LLC-PK1

cells, whereas Roczniak and Burns showed that NO do-The main findings of our studies were that (a) ECshave the potential to regulate PTEC function, and (b) nors inhibited apical Na/H exchange in rabbit proximal

tubules [6]. In addition, Guzman et al reported that SNPNO mediates EC-dependent control of PTECs. The ad-dition of BK or Ach to EC/PTEC coincubations, but not and SIN-1, as well as interferon conditioning of mouse

proximal tubule cells, resulted in decreased Na,K-to PTEC alone, resulted in increases in PTEC cGMP,as well as decreases in Na,K-ATPase and transcellular ATPase [5]. Interferon-induced decreases in Na,K-

ATPase were reversed with an NOS inhibitor and wereNa transport. Moreover, IL1B conditioning of ECs re-sulted in the same findings. All of the EC-dependent attenuated by removing arginine from the culture sys-

tem. Taken together, the results of these studies indicatechanges were blocked with the NOS antagonist L-NNA.Renal epithelial cells are in close apposition to ECs. that NO decreases Na transport in proximal tubules by

decreasing the activity of apical Na/H exchange andHowever, there are few studies of EC regulation of epi-thelial cells. Stoos et al coincubated ECs with a cortical Na,K-ATPase. Our data confirm these results (Figs. 5

and 6). In addition, our results extend these observationscollecting duct cell line and found that EC activationwith BK or Ach reduced short circuit current and that and indicate that NO-mediated inactivation of Na,K-

ATPase (1) is independent of apical Na, hydrogen ex-this effect was reversed with an NOS inhibitor [13]. ECregulation of proximal tubule function has recently been change (but not necessarily of other apical transporters)

because NO-induced inhibition of Na,K-ATPase oc-reported. Amorena and Castro perfused peritubular cap-illaries with agents causing EC NO production [14]. Both curred in the presence of DMA and (2) is not mediated

directly by NO because the NO donor did not decreaseBK and carbamylcholine increased proximal tubule pro-ton flux. Because GMP analogues increased proton flux Na pump activity in basolateral membrane vesicles.

In addition to NO generated from NO donors, ourwhile an NOS inhibitor blocked the effects of carbamyl-choline, the authors concluded that ECs increase proxi- studies indicate that NO produced by ECs regulates

proximal tubule function. NO is produced from argininemal tubule proton flux by activating the Na/H exchange[14]. In contrast to Amorena and Castro, our data indi- by the enzyme NO synthase. There are at least three

isoforms of NOS [11]. PTECs contain type II [12] andcate that ECs down-regulate important proximal tubulefunctions, including signaling enzymes, transporters, and possibly type III [26] NOS, and cytokine activation of

PTEC NOS results in autocrine regulation of PTECtranscellular Na transport.Nitric oxide produced by ECs appears to be an impor- function. ECs contain type III and type II NOS [23, 27].

Endothelial cell NOS produces NO constitutively andtant mediator by which ECs regulate PTEC functionsbecause the NOS inhibitor blocked the EC regulation can be induced to form NO by cytokines [23, 28]. The

exposure of coincubations to BK or Ach or EC condi-of PTEC functions (Figs. 1, 2, 3, 4). The effect of direct

Linas and Repine: EC regulate PTEC Na transport 1257

these investigators demonstrated stimulatory effects ofECs, NO, and cGMP on proton flux (and, presumably,Na uptake), we found inhibitory effects of EC, NO, andcGMP on Na uptake and transport. These differencesin findings are especially confusing because a number ofinvestigators have reported that cGMP decreases proxi-mal tubule Na transport and Na/H exchange [6, 10, 25,33, 34], and others have reported inhibitory [5, 6, 10] orbiphasic effects of NO [9] on these parameters.

There are several caveats to our studies. In vivo, basalsurfaces of ECs are apposed to basolateral membranesof PTECs. In the coincubation system, apical surfacesof ECs are apposed to the basolateral surfaces of PTECs.Fig. 7. Effect of 8Br-cGMP on Na,K-ATPase and 22Na transport PTEC

were exposed to 8Br-cGMP in the concentrations indicated for 15 Although this difference in orientation could effect ourminutes. Na,K-ATPase (broken line) and 22Na transport (solid line) results, in preliminary studies, we oriented the basal sur-were determined as described in the Methods section (N 5 4 or 5 in

faces of ECs to the basolateral membranes of PTECseach group).and found that EC-dependent PTEC cGMP formationwas comparable to the results obtained when apical sur-faces of ECs were apposed to PTEC (data not shown).

tioning with IL1B resulted in changes in PTEC signaling Other caveats include our use of a coincubation systemand transport functions. Each of these effects was as well as our use of bovine macrovascular ECs and ratblocked by treating ECs with the NOS inhibitor. PTECs. Although there may be differences in function

Many effects of NO are mediated by cGMP formed between microvascular and macrovascular ECs, we wereby guanylate cyclase. However, NO is highly reactive, not able to culture sufficient quantities of rat microvascu-and some effects of NO are mediated by secondary oxi- lar ECs to perform these studies. Also, the short durationdants produced after the interaction with NO [29]. The of our studies would minimize the differences betweenresults of our studies suggest that in our coincubation cells of different origins. An additional concern is thesystem, all of the effects of NO were mediated by cGMP. use of a coincubation system to infer in vivo interactionsIn this regard, exogenous cGMP reproduced the effects between cells.of SNP on Na,K-ATPase and Na transport (Fig. 7). Ear- With these caveats, the results of our studies may belier studies indicate that cGMP inhibits proximal tubule relevant to proximal tubule function in vivo. ProximalNa reabsorption [6, 10]. There are limited reports on the tubule Na transport is up-regulated by the filtered loadtransporters regulated by cGMP. A specific target of of Na, as well as by a number of circulating hormonescGMP appears to be the apical Na/H exchanger because (angiotensin II, norepinephrine, etc.) and renal nerves.the addition of exogenous cGMP blocked apical Na/H In addition, our studies indicate that the endotheliumexchange [6]. The effect of cGMP on regulation of proxi- down-regulates proximal tubule Na reabsorption. In ear-mal tubule Na,K-ATPase activity is less clear. Guzman lier studies, NO was shown to contribute to proximalet al reported that cytokine treatment of mouse proximal tubule injury during ischemia/reperfusion in the absencetubules decreased Na,K-ATPase [5]. Although this effect of neutrophils and to protect proximal tubules from neu-was blocked by a NOS inhibitor, it was only partially trophil-mediated oxidant attack [35, 36]. Moreover, ECreproduced with an exogenous cGMP analogue. In our NO was shown to protect vascular smooth muscle cellssystem, exogenous cGMP was as effective as NO donors from oxidant attack [20]. These results indicate that ECin inhibiting Na,K-ATPase (Figs. 5 and 7). We considered dysfunction such as that which occurs in a number ofthe possibility that cGMP inhibition of Na,K-ATPase pathological states (for example, sepsis, acute renal fail-was mediated by inhibition of apical Na entry. However, ure) could result in dysregulation of proximal tubule Nathe inhibitory effects of the cGMP analogue on Na,K- reabsorption and abnormalities in Na balance.ATPase occurred when apical Na uptake was blockedwith amiloride. The effect of downstream kinases or ACKNOWLEDGMENTSphosphatases regulated by cGMP on Na,K-ATPase ac-

This work was supported by funds provided in part by Nationaltivity has not been characterized. Although there areInstitutes of Health Grant 2P50 HL-40784. We are indebted to Ms.

a number of potential phosphorylation sites on protein Jacqueline D. Smith for graphic production and Ms. Holly Poitra forsecretarial support.subunits of Na,K-ATPase, exogenous cGMP has not been

found to consistently inactivate the enzyme [5, 30–32].Reprint requests to Stuart L. Linas, M.D., Denver Health Medical

It is difficult to reconcile the differences between the Center, 777 Bannock Street, Denver, Colorado 80204, USA.E-mail: STUART.LINAS@UCHSC.EDUresults of Armorena and Castro and ours [14]. Whereas

Linas and Repine: EC regulate PTEC Na transport1258

cells from hydrogen peroxide attack. Am J Physiol (Renal FluidREFERENCESElectrolyte Physiol) 272:F767–F773, 1997

21. Schelling JR, Linas SL: Angiotensin II-dependent proximal tu-1. Liu FY, Cogan MG: Role of protein kinase C in proximal bicarbon-ate absorption and angiotensin signaling. Am J Physiol 258:F927– bule sodium transport requires receptor-mediated endocytosis. Am

J Physiol 266:C669–C675, 1994F933, 19902. Liu FY, Cogan MG: Angiotensin II stimulates early proximal 22. Schelling JR, Singh H, Marzec R, Linas SL: Angiotensin II-

dependent proximal tubule sodium transport is mediated by cAMPbicarbonate absorption in the rat by decreasing cyclic adenosinemonophosphate. J Clin Invest 84:83–91, 1989 modulation of phospholipase C. Am J Physiol 267:C1239–C1245,

19943. Li L, Wang YP, Capparelli AW, Jo OD, Yanagawa N: Effectof luminal angiotensin II on proximal tubule fluid transport: Role 23. Kanno K, Hirata Y, Imai T, Iwashina M, Marumo F: Regulation

of inducible nitric oxide synthase gene by interleukin-1 beta in ratof apical phospholipase A2. Am J Physiol 266:F202–F209, 19944. Andreatta-van Leyen S, Romero MF, Khosla MC, Douglas JG: vascular endothelial cells. Am J Physiol 267:H2318–H2324, 1994

24. Haggerty JG, Agarwal N, Reilly RF, Adelberg EA, SlaymanModulation of phospholipase A2 activity and sodium transport byangiotensin-(1-7). Kidney Int 44:932–936, 1993 CW: Pharmacologically different Na/H antiporters on the apical

and basolateral surfaces of cultured porcine kidney cells (LLC-5. Guzman NJ, Fang M-Z, Tang S-S, Ingelfinger JR, Garg LC:Autocrine inhibition of Na1/K1-ATPase by nitric oxide in mouse PK1). Proc Natl Acad Sci USA 85:6797–6801, 1988

25. Cantiello HF, Ausiello DA: Atrial natriuretic factor and cGMPproximal tubule epithelial cells. J Clin Invest 95:2083–2088, 19956. Roczniak A, Burns KD: Nitric oxide stimulates guanylate cyclase inhibit amiloride-sensitive Na1 transport in the cultured renal epi-

thelial cell line, LLC-PK1. Biochem Biophys Res Commun 134:852–and regulates sodium transport in rabbit proximal tubule. Am JPhysiol 270:F106–F115, 1996 860, 1986

26. Tracey WR, Pollock JS, Murad F, Nakane M, Forstermann U:7. Barroso-Vicens E, Ramirez G, Rabb H: Multiple primary malig-nancies in a renal transplant patient. Transplantation 61:1655–1656, Identification of an endothelial-like type III NO synthase in LLC-

PK1 kidney epithelial cells. Am J Physiol 266:C22–C28, 199419968. Alberola A, Pinilla JM, Quesada T, Romero JC, Salazar FJ: 27. Lamas S, Marsden PA, Li GK, Tempst P, Michel T: Endothelial

nitric oxide synthase: Molecular cloning and characterization of aRole of nitric oxide in mediating renal response to volume expan-sion. Hypertension 19:780–784, 1992 distinct constitutive enzyme isoform. Proc Natl Acad Sci USA

89:6348–6352, 19929. Wang T: Nitric oxide regulates HCO32 and Na1 transport by a

cGMP-mediated mechanism in the kidney proximal tubule. Am J 28. Balligand JL, Ungureanu-Longrois D, Simmons WW, KobzikL, Lowenstein CJ, Lamas S, Kelly RA, Smith TW, Michel T:Physiol 272:F242–F248, 1997

10. Chevalier RL, Fang GD, Garmey M: Extracellular cGMP inhibits Induction of NO synthase in rat cardiac microvascular endothelialcells by IL-1 beta and IFN-gamma. Am J Physiol 268:H1293–1303,transepithelial sodium transport by LLC-PK1 renal tubular cells.

Am J Physiol 270:F283–F288, 1996 199529. Miles AM, Bohle DS, Glassbrenner PA, Hansert B, Wink DA,11. Moncada S, Higgs EA: Molecular mechanisms and therapeutic

strategies related to nitric oxide. FASEB J 9:1319–1330, 1995 Grisham MB: Modulation of superoxide-dependent oxidation andhydroxylation reactions by nitric oxide. J Biol Chem 271:40–47,12. Markewitz BA, Michael JR, Kohan DE: Cytokine-induced ex-

pression of a nitric oxide synthase in rat renal tubule cells. J Clin 199630. McKee M, Scavone C, Nathanson JA: Nitric oxide, cGMP, andInvest 91:2138–2143, 1993

13. Stoos BA, Carretero OA, Farhy RD, Scicli G, Garvin JL: Endo- hormone regulation of active sodium transport. Proc Natl AcadSci USA 91:12056–12060, 1994thelium-derived relaxing factor inhibits transport and increases

cGMP content in cultured mouse cortical collecting duct cells. J 31. Gupta S, McArthur C, Grady C, Ruderman NB: Stimulation ofvascular Na(1)-K(1)-ATPase activity by nitric oxide: A cGMP-Clin Invest 89:761–765, 1992

14. Amorena C, Castro AF: Control of proximal tubule acidification independent effect. Am J Physiol 266:H2146–H2151, 199432. Aperia A, Holtback U, Syren ML, Svensson LB, Fryckstedt J,by the endothelium of the peritubular capillaries. Am J Physiol

272:R691–R694, 1997 Greengard P: Activation/deactivation of renal Na1,K(1)-ATPase:A final common pathway for regulation of natriuresis. FASEB J15. Vinay P, Gougoux A, Lemieux G: Isolation of a pure suspension

of rat proximal tubules. Am J Physiol 241:F403–F411, 1981 8:436–439, 199433. Moran A, Montrose MH, Murer H: Regulation of Na1-H1 ex-16. Schelling JR, Hanson AS, Marzec R, Linas SL: Cytoskeleton-

dependent endocytosis is required for apical type 1 angiotensin change in cultured opossum kidney cells by parathyroid hormone,atrial natriuretic peptide and cyclic nucleotides. Biochim BiophysII receptor-mediated phospholipase C activation in cultured rat

proximal tubule cells. J Clin Invest 90:2472–2480, 1992 Acta 969:48–56, 198834. Eitle E, Hiranyachattada S, Wang H, Harris PJ: Inhibition17. Bowman CM, Butler EN, Vatter AE, Repine JE: Hyperoxia

injuries endothelial cells in culture and causes increased neutrophil of proximal tubular fluid absorption by nitric oxide and atrialnatriuretic peptide in rat kidney. Am J Physiol 274:C1075–C1080,adherence. Chest 83:33S–35S, 1983

18. Singh H, Linas SL: Beta 2-adrenergic function in cultured rat 199835. Yu L, Gengaro PE, Niederberger M, Burke TJ, Schrier RW:proximal tubule epithelial cells. Am J Physiol Renal (Fluid Electro-

lyte Physiol) 271:F71–F77, 1996 Nitric oxide: A mediator in rat tubular hypoxia/reoxygenation in-jury. Proc Natl Acad Sci USA 91:1691–1695, 199419. Molitoris BA, Simon FR: Renal cortical brush-border and basolat-

eral membranes: Cholesterol and phospholipid composition and 36. Linas SL, Whittenburg D, Repine JE: Nitric oxide prevents neu-trophil mediated acute renal failure. Am J Physiol 272:F48–F54,relative turnover. J Membr Biol 83:207–215, 1985

20. Linas SL, Repine JE: Endothelial cells protect vascular smooth 1996