Expression of peroxisomal proliferator-activated receptors and retinoid X receptors in the kidney.

The discovery that 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) is a ligand for the gamma-isoform of peroxisome proliferator-activated receptor (PPAR) suggests nuclear signaling by prostaglandins. Studies were undertaken to determine the nephron localization of PPAR isoforms and their heterodimer partners, retinoid X receptors (RXR), and to evaluate the function of this system in the kidney. PPARalpha mRNA, determined by RT-PCR, was found predominately in cortex and further localized to proximal convoluted tubule (PCT); PPARgamma was abundant in renal inner medulla, localized to inner medullary collecting duct (IMCD) and renal medullary interstitial cells (RMIC); PPARbeta, the ubiquitous form of PPAR, was abundant in all nephron segments examined. RXRalpha was localized to PCT and IMCD, whereas RXRbeta was expressed in almost all nephron segments examined. mRNA expression of acyl-CoA synthase (ACS), a known PPAR target gene, was stimulated in renal cortex of rats fed with fenofibrate, but the expression was not significantly altered in either cortex or inner medulla of rats fed with troglitazone. In cultured RMIC cells, both troglitazone and 15d-PGJ2 significantly inhibited cell proliferation and dramatically altered cell shape by induction of cell process formation. We conclude that PPAR and RXR isoforms are expressed in a nephron segment-specific manner, suggesting distinct functions, with PPARalpha being involved in energy metabolism through regulating ACS in PCT and with PPARgamma being involved in modulating RMIC growth and differentiation.

Cyclooxygenase-2 is expressed in bladder during fetal development and stimulated by outlet obstruction.

Studies were undertaken to assess expression of inducible cyclooxygenase (COX)-2 in bladder during fetal development and COX-1 and COX-2 expression after outlet obstruction. Bladder tissue or bladder progenitor tissue was harvested from CD-1 murine embryos at embryonic days 11.5 (E11.5), E14.5, E17.5, E20.5 (newborn), and from adult. Bladder obstruction was created in adult female mice by ligating the urethra, and bladders were harvested after 3-24 h of obstruction. Gene expression was assessed by semiquantitative reverse transcription-polymerase chain reaction and Western blotting. COX-2 was highly expressed at the early stages of bladder development and declined progressively throughout gestation. In adult bladder, both COX-1 and COX-2 were detectable at low levels under basal conditions. An approximately 30-fold increase in COX-2 mRNA was seen after 24 h of obstruction. In contrast, COX-1 did not change with obstruction. COX-2 mRNA levels peaked at 6 h of obstruction. In regional bladder-distention models, COX-2 induction was confined to the area of distention. Bladder outlet obstruction stimulates COX-2 expression dramatically, reactivating a gene that is highly expressed during fetal development.

Expression of PTHrP, PTH/PTHrP receptor, and Ca(2+)-sensing receptor mRNAs along the rat nephron.

To provide a frame of reference for studies of renal divalent cation and phosphate metabolism, we assessed the cellular localization of kidney calcium receptor (RaKCaR), parathyroid hormone-related protein (PTHrP), and parathyroid hormone/ parathyroid hormone-related protein (PTH/PTHrP) receptor mRNA. The studies used using reverse transcription-polymerase chain reaction (RT-PCR) applied to cDNA prepared from dissected rat nephron segments and from primary cultures of mouse juxtaglomerular granular cells. With species-specific primers, PCR products of expected size were obtained for RaKCaR (967 bp), PTHrP (420 bp), and PTH/PTHrP receptor (817 bp), with product identity being confirmed by restriction digestion. RaKCaR mRNA was found in medullary and cortical thick ascending limbs (MTAL and CTAL, respectively), the macula densa-containing segment, distal convoluted tubules (DCT), and, to a lesser extent, in cortical collecting ducts (CCD). It was not found in glomeruli, proximal convoluted and straight tubules (PCT and PST, respectively), outer and inner medullary collecting ducts (OMCD and IMCD, respectively), or in juxtaglomerular granular cell isolates. PTHrP mRNA was predominantly expressed in glomeruli and at lower levels in PCT and the macula densacontaining segment but was not detectable in CTAL, MTAL, DCT, and CD segments. Presence of PTH/PTHrP receptor mRNA was demonstrated in glomeruli, PCT, PST, CTAL, MTAL, and DCT but not in CD segments. These results suggest that the function of TAL and DCT cells, in addition to being affected by PTH, may be directly altered by extracellular divalent cations through RaKCaR and that PTHrP may act in the glomerulus and proximal tubule as an autocrine or paracrine regulator of hemodynamics and phosphate transport.

Cyclic AMP selectively increases renin mRNA stability in cultured juxtaglomerular granular cells.

This study was undertaken to examine the regulation of renin release and gene expression in primary cultures of juxtaglomerular granular (JGG) cells. JGG cells, isolated from mouse kidney, demonstrated high purity and showed regulated renin release in vitro. Changes in steady-state renin mRNA levels were assessed by quantitative polymerase chain reaction techniques, with polymerase chain reaction amplification efficiency monitored by co-amplification of experimental samples with a dilution series of cDNA for a mutant template. When the cells were incubated in the presence or absence of forskolin, isoproterenol, or 8-bromo-cAMP plus 3-isobutyl-1-methylxanthine for 24 h or cholera toxin for 12 h, renin mRNA levels were increased 3.9-, 4.4-, 5.1-, and 3.3-fold, respectively (all, p < 0.05). A significant increase in renin mRNA levels was observed 8 h after treatment with forskolin, but no change was detectable at 4 h. Cycloheximide did not prevent the increase in renin mRNA by isoproterenol. When RNA synthesis was inhibited by incubation with actinomycin D (5 micrograms/ml), renin mRNA levels declined with a half-life of 3.0 +/- 0.8 h. Treatment with forskolin increased renin mRNA half-life to 10.8 +/- 2.7 h (p < 0.025). The half-life of beta-actin, endothelin-1, or the facilitative glucose transporter-1 (GLUT-1) mRNA expressed in the same cells was not altered, although the steady-state levels of GLUT-1 mRNA increased 2.2-fold after treatment with forskolin. These data demonstrate that cAMP increases renin release and mRNA levels in JGG cells in vitro, that the stimulatory effect of cAMP on renin mRNA is delayed but does not require new protein synthesis, and that the increased renin mRNA levels induced by cAMP are due in part to a selective increase in renin mRNA stability.