Luminal NaCl delivery regulates basolateral PGE₂ release from macula densa cells

Macula densa (MD) cells express COX-2 and COX-2–derived PGs appear to signal the release of renin from the renal juxtaglomerular apparatus, especially during volume depletion. However, the synthetic machinery and identity of the specific prostanoid released from intact MD cells remains uncertain. In the present studies, a novel biosensor tool was engineered to directly determine whether MD cells release PGE₂ in response to low luminal NaCl concentration ([NaCl]_L). HEK293 cells were transfected with the Ca²⁺-coupled E-prostanoid receptor EP₁ (HEK/EP₁) and loaded with fura-2. HEK/EP₁ cells produced a significant elevation in intracellular [Ca²⁺] ([Ca²⁺]_i) by 29.6 ± 12.8 nM (n = 6) when positioned at the basolateral surface of isolated perfused MD cells and [NaCl]_L was reduced from 150 mM to zero. HEK/EP₁ [Ca²⁺]_i responses were observed mainly in preparations from rabbits on a low-salt diet and were completely inhibited by either a selective COX-2 inhibitor or an EP₁ antagonist, and also by 100 μM luminal furosemide. Also, 20-mM graduated reductions in [NaCl]_L between 80 and 0 mM caused step-by-step increases in HEK/EP₁ [Ca²⁺]_i. Low-salt diet greatly increased the expression of both COX-2 and microsome-associated PGE synthase (mPGES) in the MD. These studies provide the first direct evidence that intact MD cells synthesize and release PGE₂ during reduced luminal salt content and suggest that this response is important in the control of renin release and renal vascular resistance during salt deprivation.

PGE₂ is a major product of PGH₂ derived from COX metabolism and is an important paracrine regulator of salt and water homeostasis in the kidney. In the renal cortex, COX-1 expression predominates in collecting duct, vascular tissue, and glomerular mesangial cells (1). In contrast, COX-2 is expressed and presumably mediates PG production in the macula densa (MD) and surrounding cortical thick ascending limb (cTAL) cells (2–5). MD cells are in direct contact with the vascular pole of the same glomerulus from which the filtrate originates. These cells sense changes in tubular [NaCl] and send signals that control preglomerular vascular resistance and glomerular filtration rate in a process known as tubuloglomerular feedback. MD cells also control the release of renin from juxtaglomerular granular cells (3–6). COX-2–derived PGs may participate in MD-mediated control of juxtaglomerular function, particularly in high renin states such as low salt intake, loop diuretic treatment, and renovascular hypertension (4, 7). In particular, PGE₂ produced by MD cells has been suggested as the mediator of renin release induced by low luminal [NaCl] ([NaCl]_L) (3–10). In addition, PGE₂, as a potent vasodilator, may also modulate preglomerular vascular resistance (11–13) and tubuloglomerular feedback (14).

Although COX activity has been considered the key step in PG synthesis, metabolism of arachidonate by either COX-1 or COX-2 yields only the unstable intermediary PGH₂ (1). The subsequent fate of PGH₂ is dictated by coexpression of a PG synthase, which is the other key enzyme of PG synthesis. This enzyme is capable of converting PGH₂ to one of the prostanoid end products including PGE₂, PGF_2a, PGD₂, PGI₂, and thromboxane A₂ (11). Because of the limited number and inaccessibility of MD cells, as opposed to other nephron segments in the kidney (15), direct evidence for the expression of a PGE synthase in the MD and release of PGE₂ from intact MD cells is lacking. Investigating PGE₂ release from MD cells is important since a recent work actually questioned whether MD cells are equipped with PGE₂ synthetic machinery in the normal kidney (16).

The present studies used a cloned PG receptor (E-prostanoid receptor-1, or EP₁) to engineer a novel biosensor tool that could be used to detect local PGE₂ production. PGE₂ interacts with four different G protein–coupled E-prostanoid receptors designated EP₁, EP₂, EP₃, and EP₄ (11, 12). EP₂, EP₃, and EP₄ couple to adenylyl cyclase–associated G proteins to increase or decrease cAMP generation. In contrast, EP₁ is coupled to a Ca²⁺-signaling mechanism that is thought to involve inositol 1,4,5-triphosphate and diacylglycerol formation (11, 12). Therefore, EP₁-mediated effects of PGE₂ can be detected using fluorescence microscopy and a biosensor cell expressing EP₁ and loaded with a calcium fluorophore. The present studies were undertaken to determine, for the first time, whether the enzymatic machinery necessary for PGE₂ synthesis is expressed in MD cells from rabbits on a standard or low-salt diet, and to provide direct functional evidence for the control of MD PGE₂ release in response to low [NaCl]_L.

Effects of exogenous PGE2. Initial studies were performed to assess the sensitivity of individual HEK/EP1 cells to exogenous PGE2. Representative recordings (Figure 2a) demonstrate that individual HEK/EP1 biosensor cells produced very similar, reversible, and dose-dependent (Figure 2b) elevations in [Ca²⁺]i in response to exogenous PGE2. Calcium signals were completely abolished (Figure 2a) when HEK/EP1 cells were preincubated with the EP1 blocker SC51322 (10 μM).

Figure 2

Effects of exogenous PGE2 (added to the bath) on [Ca²⁺]i of an HEK/EP1 biosensor cell. (a) Recordings of five different HEK/EP1 cells demonstrate almost identical sensitivities of cells to 1 μM PGE2. HEK/EP1 [Ca²⁺]i responses were prevented by the coadministration of an EP1 blocker (SC51322). (b) Dose-response curve. Each data point represents the average of four cells studied.

Effects of luminal NaCl removal. Since decreasing [NaCl]L produces MD signals that stimulate renin release, initial studies were performed in which [NaCl]L was decreased from 150 mM to 0 mM. Removing luminal NaCl resulted in a significant increase in [Ca²⁺]i of single HEK/EP1 cells positioned next to the MD basolateral membrane (Δ = 29.6 ± 12.8 nM, n = 6; Figure 3a and Figure 4b). This response was observed in preparations from animals kept on a low-salt diet for 1 week. In contrast, we were unable to detect significant elevations in HEK/EP1 cell [Ca²⁺]i (i.e., PGE2 release) from MD plaques obtained from rabbits kept on a standard salt diet (Δ = 2.8 ± 2.8 nM, n = 6) (Figure 3a and Figure 4b). Also, this response was MD cell–specific, since the same maneuver caused no detectable change in [Ca²⁺]i of HEK/EP1 cells placed next to distant cTAL cells (Figure 3a). Luminal NaCl removal–induced HEK/EP1 calcium responses in the low-salt group were completely inhibited by luminal addition of a selective COX-2 inhibitor (SC58236) or bath SC51322, a blocker of the receptor EP1 (Figure 3b and Figure 4b). It should also be noted that in low-salt diet studies there was a significant elevation in biosensor cell baseline [Ca²⁺]i (Figure 4a).

Figure 3

Representative recordings of changes in HEK/EP1 [Ca²⁺]i/MD PGE2 release. (a) Luminal NaCl removal in the low-salt group (in contrast to normal diet) induced significant PGE2 release from MD, but not from distant cTAL cells, as evidenced by increases in HEK/EP1 [Ca²⁺]i. (b) HEK/EP1 calcium responses (low-salt group) were inhibited by either luminal addition of furosemide, an Na:2Cl:K cotransport blocker, or by a selective COX-2 inhibitor (SC58236, 100 nM) or bath SC51322 (10 μM), an EP1 blocker.

Figure 4

Biosensor HEK/EP1 cell baseline [Ca²⁺]i (a) and tubular NaCl removal–induced increases in [Ca²⁺]i (Δ[Ca²⁺]i) (b). A low-salt diet significantly elevated biosensor cell baseline [Ca²⁺]i and Δ[Ca²⁺]i upon luminal NaCl removal, compared with a normal diet. Biosensor cell calcium responses were inhibited by luminal addition of a selective COX-2 inhibitor (SC58236, 100 nM) or bath SC51322 (10 μM), an EP1 blocker. (n = 6 in each group, *P < 0.01 compared with normal diet).

Effects of various [NaCl]L’s and furosemide. Next we tested whether our model is sensitive in the physiological range of [NaCl]L, and if more subtle changes in [NaCl]L can cause HEK/EP1 calcium responses. Figure 5 demonstrates that graduated changes in [NaCl]L between 80 mM and 0 mM caused step-by-step increases in HEK/EP1 cell fura-2 ratio. The most sensitive range of [NaCl]L where the most significant changes occurred was between 40 mM and 20 mM (Figure 5a). The [NaCl]L producing the half-maximal effect was around 30 mM (Figure 5b). Additional experiments examined whether HEK/EP1 cell calcium signals are related to changes in MD NaCl transport. Furosemide (100 μM), a Na:2Cl:K cotransport blocker and loop diuretic, added to the luminal perfusate significantly inhibited the magnitude of HEK/EP1 [Ca²⁺]i increases (by 81.0% ± 7.6% compared with control; n = 5, P < 0.01) in response to 150 mM [NaCl]L removal, as illustrated by the representative recording in Figure 3b. In the presence of furosemide, HEK/EP1 cells were still sensitive to exogenous PGE2 added to the bathing solution (Figure 3b). Similar confirmation of HEK/EP1 biosensor cell reactivity was always performed when detecting no calcium response (such as in the normal salt group and with COX-2 inhibitor, not shown).

Figure 5

Luminal [NaCl] dependency of MD PGE2 release in cell preparations from rabbits fed a low-salt diet. (a) Representative recording shows step-by-step increases in HEK/EP1 cell fura-2 ratio in response to graduated reductions in luminal [NaCl] from 80 to 0 mM. (b) Relationship between biosensor cell responses and luminal [NaCl]. Data points represent mean ± SE, n = 4, all low-salt preparations.

Expression of COX-2 and mPGES in rabbit MD. As illustrated in Figure 6a, there was very little staining of the rabbit MD with an antibody directed toward a C-terminal COX-2 peptide in animals on a normal salt diet. In contrast, the low-salt diet greatly increased the number of COX-2–immunoreactive cells in the MD (Figure 6, b and c). As with COX-2, there was a low level of mPGES expression in the MD in the normal kidney, but mPGES expression greatly increased in response to the low-salt diet (Figure 6e). In addition to localizing the mPGES protein, in situ hybridization clearly demonstrated the expression of mPGES mRNA in rabbit MD cells (Figure 6f). These findings indicate that the complete enzymatic machinery necessary for PGH2 synthesis and its conversion to PGE2 is indeed present in MD cells, and more importantly, both COX-2 and mPGES expression are upregulated by low salt intake.

Figure 6

COX-2 (a–c) and mPGES (d and e) immunofluorescence of kidney cortex in control rabbits (a and d) and rabbits fed a low-salt diet (b and e). Number of COX-2–positive cells (c, n = 6 in each group, *P < 0.05 compared with normal salt diet) and mPGES expression in the MD significantly increased in response to the low-salt diet (LS) compared with the normal salt diet (NS). Nuclei are blue. In situ hybridization clearly demonstrated mPGES mRNA expression in MD cells (f).

The present studies used a modification of a recently established biosensor technique (26) to provide direct functional evidence, for the first time, that PGE2 is released from the basolateral membrane of intact, perfused MD cells in a setting of reduced luminal salt delivery. This bioassay was originally used to measure cellular ATP release from the MD (19) using a rat pheochromocytoma clonal cell line (PC12) that endogenously expresses purinergic P2X receptors. Instead of using a native cell line, we engineered a novel biosensor tool, HEK293 cells transfected with the mouse receptor EP1, to selectively detect PGE2. The fura-2 fluorescence ratio, and consequently [Ca²⁺]i of individual HEK/EP1 cells, increased in response to exogenous PGE2 added to the bathing solution (Figure 2). The calcium responses of several individual HEK/EP1 cells were almost identical, indicating similar EP1 density and PGE2 sensitivity (Figure 2a). Also, calcium responses were dose-dependent and were completely abolished by preincubation with the EP1 blocker SC51322, which is consistent with EP1-mediated calcium signaling (11, 12). It is important to note that nontransfected HEK293 cells did not increase [Ca²⁺]i in response to PGE2 but did respond to exogenous ATP (data not shown), presumably through endogenously expressed purinergic receptors. This latter effect was completely blocked by gadolinium, which not only blocks P2X receptors (21) but also inhibits ATP release (19, 20). The experiments measuring PGE2 release were therefore carried out in the presence of gadolinium to exclude ATP purinergic signaling and its effects on cell [Ca²⁺]i. Gadolinium did not affect PGE2 detection by HEK/EP1 cells (similar responses in Figure 2a and Figure 3b).

Because of the limited number and inaccessibility of MD cells (only 15–20 cells per nephron), it has not been possible to directly test for the release of prostanoids by MD cells. The HEK/EP1 biosensor technique provides a unique tool allowing the detection of PGE2 release from single MD plaques. As shown in Figure 1, a fura-2–loaded HEK/EP1 cell can be positioned at the MD basolateral membrane surface, allowing real-time determination of basolateral PGE2 release from an intact microperfused cTAL-MD segment.

Removing NaCl from the luminal perfusate significantly elevated [Ca²⁺]i in HEK/EP1 biosensor cells, indicating PGE2 release from MD cells. Importantly, not only a single large change in luminal NaCl delivery (Figure 3a), but also more subtle reductions in [NaCl]L in the physiological range (Figure 5a), caused detectable PGE2 release from MD cells. The largest change in biosensor cell responses were observed between 40 mM and 20 mM [NaCl]L, and the half-maximal effect was around 30 mM [NaCl]L (Figure 5b). These findings are not only consistent with earlier work (27), but the data are essentially identical to the previously established [Cl]1/2, the luminal chloride concentration producing a half-maximal renin secretory effect (27).

Additional studies using furosemide provided further evidence that biosensor cell calcium responses (and therefore MD PGE2 release) were related to MD NaCl transport rather than a nonspecific effect of luminal NaCl. Inhibition of MD Na:2Cl:K cotransport with furosemide prevented [NaCl]L-dependent increases in HEK/EP1 [Ca²⁺]i (Figure 3b), consistent with earlier findings that furosemide blocks MD NaCl transport dependency of renin release (27). Low distal tubular [NaCl] is associated with states of volume depletion and is a stimulus for MD signaling that increases synthesis and release of renin from juxtaglomerular granular cells (3–6). Basolateral MD PGE2 release in response to decreased luminal salt delivery represents the physiological direction of signal transmission from MD cells into the juxtaglomerular apparatus area. Recent evidence has accumulated that this signaling mechanism involves MD-derived PGs produced by COX-2 in these cells (3–12). Consistent with this, we found that HEK/EP1 calcium responses were inhibited by luminal addition of a selective COX-2 inhibitor (SC58236) or bath SC51322, a blocker of the PGE2 receptor EP1. These findings provide direct evidence that intact MD cells release PGE2, consistent with PGE2 synthesis reported in a recently developed putative MD cell line (25). In our experiments, PGE2 release appeared to be MD cell–specific, since applying this biosensor approach upstream using HEK/EP1 cells placed next to distant cTAL cells produced no detectable signals (Figure 3a). However, it is possible that cTAL cells adjacent to the MD can also synthesize and release PGE2 and that this contributed to the detected biosensor signal.

Interestingly, we were unable to detect [NaCl]L-dependent PGE2 release from MD cells obtained from animals kept on a standard salt diet. Only the low-salt diet caused consistent and significant [NaCl]L-dependent increases in HEK/EP1 cell [Ca²⁺]i. A low-salt diet produces an experimental model characterized by a high renin state and is also known to increase the expression of COX-2 in MD cells (2). Upregulation of COX-2 in kidneys obtained from rabbits on a low-salt diet was indeed confirmed in the present studies by immunohistochemistry (Figure 6, a–c). Thus, similar to findings observed in other species (2, 28), the number of COX-2–immunoreactive cells in the MD plaque was substantially increased (by about sixfold) when animals were subjected to a low-salt diet for 1 week. This number actually nicely correlates to findings of biosensor studies in that only one of six normal salt preparations produced a biosensor calcium signal. We believe that the failure to show MD PGE2 release in the majority of normal salt preparations is mainly technical: the chance that the biosensor cell is positioned next to a COX-2– and mPGES-expressing (and consequently PGE2-producing) MD cell is much higher in the low-salt group. In other words, the sensitivity of this biosensor technique is probably limited to those MD cells in direct contact with the biosensor cell. Still, average MD PGE2 release was much higher on the low-salt diet. If we compare Figure 4b with Figure 2b, the estimated amount of PGE2 released from MD cells is around 50–60 nM on the low-salt diet and only 1–2 nM on normal salt intake.

In addition to COX-2, the present studies also demonstrated that mPGES, an enzyme capable of converting COX-2–derived PGH2 to PGE2 (29), was also present in the rabbit MD (Figure 6, d and f), and that mPGES expression was greatly upregulated in response to low salt intake (Figure 6e). Previous studies performed in rats failed to detect mPGES in microdissected MD (16), but whether this represents a species difference or a difference in the physiologic status of the animals studied remains unclear.

Not only did MD cells obtained from animals on a low-salt diet exhibit increased PGE2 production in response to reduced [NaCl]L, but baseline [Ca²⁺]i was also increased in HEK/EP1 cells placed adjacent to the MD (Figure 5a). Increased baseline HEK/EP1 cell [Ca²⁺]i is consistent with increased COX-2 and mPGES expression in MD cells and higher baseline PGE2 production in these MD. NaClL removal caused an almost immediate elevation in biosensor cell [Ca²⁺]i (Figure 3), suggesting a rapid activation of COX-2 and mPGES, with synthesis and release of PGE2 from MD cells. The rapidity of PGE2 synthesis in response to low luminal Cl^– suggests rapid activation of an intracellular signaling cascade. At present the nature of this immediate signaling cascade, i.e., the link between reduced [NaCl]L and COX-2 activation, remains uncertain. Cl^– transport–dependent activation of the MAPKs ERK and p38 has recently been suggested in cultured cells (25). However, tyrosine phosphorylation (30) could also stimulate MD COX-2 as well as activation of phospholipase A2, and increased substrate availability could be the cause of acute PGE2 generation (31). The biosensor technique should provide a novel tool that will help to clarify in detail the intracellular signaling mechanisms involved in MD NaCl transport and related PGE2 production in response to low [NaCl]L during salt deprivation.

In summary, these studies provided direct evidence for the synthesis and release of PGE2 from intact, perfused MD cells. Our findings are consistent with the recent view (9) that renin release and renal vascular resistance are critically dependent on MD COX-2– and mPGES-derived PGE2, particularly during volume depletion.

This work was supported by an NIH grant (5RO1 DK-32032) to P.D. Bell; an American Heart Association Scientist Development Grant (0230074N); an American Society of Nephrology Carl W. Gottschalk Research Scholar grant to J. Peti-Peterdi; a Hungarian Ministry of Welfare (ETT) grant and NATO Science Fellowships to J. Peti-Peterdi and L. Rosivall; and a Veterans Merit Award and NIH grant (RO1 DK-37097) to M.D. Breyer.

Conflict of interest: The authors have declared that no conflict of interest exists.

Nonstandard abbreviations used: macula densa (MD); cortical thick ascending limb (cTAL); luminal NaCl concentration ([NaCl]_L); E-prostanoid receptor-1 (EP₁); EP₁-transfected HEK293 cells (HEK/EP₁ cells); intracellular [Ca²⁺] ([Ca²⁺]i); microsome-associated PGE synthase (mPGES).

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