Role of p38 in the regulation of renal cortical cyclooxygenase-2 expression by extracellular chloride

Abstract

We have previously shown that in renal cortex, COX-2 expression is localized to macula densa and surrounding cortical thick ascending limb of Henle (cTALH). Dietary salt restriction increases local expression of COX-2, which mediates renin production and secretion. Given that decreased luminal chloride [Cl(-)] at the level of the macula densa increases renin production and secretion, we investigated the role of extracellular ion concentration on COX-2 expression. Quiescent rabbit cTALH cells were incubated in a physiological salt solution containing high or low levels of NaCl. Immunoreactive COX-2 expression increased significantly in the low NaCl solution. COX-2 expression also increased after administration of the Na(+)/K(+)/2Cl(-) cotransport inhibitor, bumetanide. Selective substitution of chloride led to increased COX-2 expression, whereas selective substitution of sodium had no effect. The p38 MAP kinase inhibitor PD169316 decreased low NaCl-induced COX-2 expression. Low-salt or low-chloride medium induced cultured cTALH to accumulate >/= 3-fold higher levels of pp38, the activated (phosphorylated) form of p38; low-salt medium also increased pJNK and pERK levels. Feeding rats a low-salt diet for 14 days induced a significant increase in renal cortical pp38 expression, predominantly in the macula densa and cTALH. These results suggest that reduced extracellular chloride leads to increased COX-2 expression, which may be mediated by activation of a p38-dependent signaling pathway.

Figure 1

Decreased extracellular [Cl^–] increases immunoreactive COX-2 (irCOX-2) expression in cultured cTAL cells. Quiescent primary cultured rabbit cTAL cells were incubated with various media (described in Table 1) for 6 hours: control, low salt, NaCl, Na gluconate, Na isethionate, and choline chloride. ^AP < 0.01 compared with control. Inset: representative experiment.

Figure 2

Time course of COX-2 expression in cTAL induced by low-salt medium. Primary cultured cTAL cells were incubated in low-salt medium for the indicated time. (n = 4–13). ^AP < 0.05, ^BP < 0.01 compared with base line. Inset: representative experiment.

Figure 3

Effects of mannitol addition to low-salt medium. Lane 1, control; lane 2, low salt; lane 3, Low salt + NaCl; lane 4, low salt + 200 mM mannitol. ^AP < 0.01 compared with control. Inset: representative experiment.

Figure 4

Bumetanide increases COX-2 expression in cultured cTAL. Lane 1, control; lane 2, bumetanide (50 μM) in NaCl solution; lane 3, low salt. ^AP < 0.01 compared with control. Inset: representative experiment.

Figure 5

The p38 specific inhibitor, PD169316, prevents increases in the COX-2 expression in cTAL cells exposed to low-salt medium. Quiescent cTAL cells were maintained in DME/F12 medium (lane 1), incubated for 6 hours in low-salt medium alone (lane 2), or in low-salt medium in the presence of MEK1 inhibitor, PD98059 (1 μM) (lane 3); p38 MAP kinase inhibitor, PD169316 (1 μM) (lane 4); PKA kinase inhibitor, H8 (1 μM) (lane 5); PKC kinase inhibitor, H7 (1 μM) (lane 6); or adenylate cyclase inhibitor, dideoxyadenosine (100 μM) (lane 7) (n = 5). ^AP < 0.01 compared with control. Inset: representative experiment.

Figure 6

(a) Decreased extracellular [Cl^–] increases pp38 expression in cultured cTAL cells. Cultured cTAL cells were incubated in the indicated solution for 6 hours. (lane 1) control; (lane 2) low salt; (lane 3) Na isethionate; (lane 4) choline chloride; (lane 5) NaCl. ^AP < 0.01 compared with control; n = 5. Inset: representative experiment. (b) Decreased extracellular [Cl^-] increases p38 kinase activity in cTAL cells. cTAL cells were incubated in different media for 6 hours, and p38 kinase activity was measured as described in Methods, using ATF2 as substrate. (lane 1) control; (lane 2) low salt; (lane 3) NaCl; (lane 4) Na isethionate; and (lane 5) choline chloride. (c) Time course of pp38 expression in cTAL incubated in low-salt medium. Cells were incubated in low-salt medium 0–16 hours (n = 3). ^AP < 0.01 compared with base line. (d) Time course of pJNK and pERK expression in cTAL incubated in low-salt medium.

Figure 7

Expression of pp38 in rat renal cortex in response to dietary salt restriction. Male Sprague-Dawley rats (250 g) were maintained on normal chow or a salt-deficient diet for 14 days. Renal cortical pp38 increased significantly in low-salt rats. ^AP < 0.05 compared with control; n = 3. Inset: representative experiment.

Figure 8

Localization of pp38 in rat renal cortex in response to dietary salt. Immunoreactive pp38 expression was detected at low (×10) (a and b) and higher (×32) power (c and d) in rats maintained on normal chow (a, c, and e) or a salt-deficient diet for 14 days (b, d, and f). Note restriction of pp38 expression to cTALH and tubular expression in the animals on the salt-deficient diet (arrowheads in a and b). Low salt increased pp38 expression in macula densa and surrounding cTALH (arrows in c and d). For comparison, increased macula densa/cTALH COX-2 expression is presented (e and f). (Figure widths: a and b: 700 μm; c–f: 212 μm).