J Am Soc Nephrol 10: 2084–2093, 1999 Characterization of Prostanoid Receptors in Podocytes MARTIN BEK,* ROLF NUSING,¨ † PASCAL KOWARK,* ANNA HENGER,* PETER MUNDEL,‡ and HERMANN PAVENSTADT*¨ *Department of Medicine, Division of Nephrology, University of Freiburg, Germany; †Department of Medicine, University of Marburg, Germany; and ‡Division of Nephrology, Albert Einstein College of Medicine, Bronx, New York. Abstract. Prostaglandins participate in the regulation of impor- extracellular solution with a reduced Cl2 concentration (from tant glomerular functions and are involved in the pathogenesis 145 to 32 mM). PGE2 and the prostaglandin agonists, but not of glomerular diseases. This study investigates the influence of the IP receptor agonist iloprost and the EP2 receptor agonist prostaglandins on membrane voltage, ion conductances, cAMP butaprost, induced a time- and concentration-dependent cAMP 21 accumulation, and cytosolic calcium activity ([Ca ]i) in dif- accumulation in podocytes. In fura-2 fluorescence experi- ferentiated podocytes. Prostaglandin E2 (PGE2) caused a con- ments, PGE2, sulprostone, PGF2a, fluprostenol (a potent FP centration-dependent depolarization and an increase of the agonist), and U-46619 (a selective thromboxane A2 agonist) ' 21 whole cell conductance in podocytes (EC50 50 nM). Com- induced a biphasic increase of [Ca ]i in 60 to 80% of podo- pared with PGE2, the EP2/EP3/EP4 receptor agonist 11-deoxy- cytes. In reverse transcription-PCR studies, podocyte mRNA PGE1 caused an equipotent depolarization, whereas the DP for the EP1,EP4, FP, and TP receptor could be amplified. receptor agonist BW 245 C, the EP1/EP3 receptor agonist These data indicate that in podocytes, PGE2 regulates distinct sulprostone, and the IP receptor agonist iloprost were at least cellular functions via the EP1 and EP4 receptor, thereby in- 21 100 to 1000 times less potent than PGE2. The EP2 receptor creasing [Ca ]i and cAMP, respectively. Furthermore, PGF2a 21 agonist butaprost did not change membrane voltage of podo- and U-46619 increase [Ca ]i via their specific receptors. cytes. The depolarizing effect of PGE2 was increased in an Within the kidney, prostaglandins have been implicated to focal segmental glomerulosclerosis (7). Podocytes are also regulate important cellular functions in a variety of different believed to initiate and maintain the progression of glomeru- cell types through specific prostanoid receptors. They influ- losclerosis after subtotal nephrectomy, a model of chronic ence glomerular hemodynamics by increasing GFR and renal renal failure (8). blood flow, especially under conditions with an increased An imbalance of prostaglandin action seems to play a role in vascular tone (1,2). Their effects on GFR may partly be me- glomerular diseases, which are at least in part induced by diated by a change of the ultrafiltration coefficient (Kf). In this podocyte injury. Changes of prostaglandin action have been regard, it has been assumed that prostaglandins antagonize reported in several glomerular diseases such as membranous vasoactive hormone-induced mesangial cell contraction, which nephropathy (9), toxin-induced glomerular injury (10), and might lead to a decrease of Kf (3,4). development of glomerulosclerosis after subtotal renal ablation Knowledge about the influence of prostaglandins on cellular (11,12). functions of the cells that form the glomerular filtration barrier, Because prostaglandin-mediated changes of podocyte func- i.e., the glomerular endothelial cell and the podocyte, is lim- tion might be involved in the pathogenesis of these glomerular ited. Podocytes play a crucial role as a filter restricting the diseases, we attempted to investigate whether prostaglandins passage of macromolecules, and they maintain a large filtration influence cellular functions of differentiated cultured podo- surface through the slit membranes (5). It has been speculated cytes, and we characterized the prostanoid receptors involved. that they might modify Kf by contraction or dilation of their foot processes (6). Under pathologic conditions, podocytes are the target cell of injury in several forms of glomerular injury, Materials and Methods such as minimal change and membranous nephropathy, and Cell Culture In the present study, we used conditionally immortalized mouse podocyte clone cells harboring a temperature-sensitive mutant of immortalizing SV-40 large T-antigen coupled with a promoter whose Received July 1, 1998. Accepted April 30, 1999. g Correspondence to Dr. Hermann Pavensta¨dt, Medizinische Universita¨tsklinik, activity is enhanced by -interferon (13). Podocytes were maintained Abteilung IV, Labor C4, Hugstetter Strasse 55, 79106 Freiburg, Germany. in RPMI 1640 (Life Technologies, Eggenstein, Germany) supple- Phone: 149 76 1270 3270; Fax: 149 76 1270 3245; E-mail: paven@ mented with 10% fetal calf serum (Boehringer Mannheim, Mann- mm41.ukl.uni-freiburg.de heim, Germany), 100 U/ml penicillin, and 100 mg/ml streptomycin 1046-6673/1010-2084 (Life Technologies) in a humid atmosphere with 5% CO2. To prop- Journal of the American Society of Nephrology agate podocytes, the culture medium was supplemented with 10 U/ml Copyright © 1999 by the American Society of Nephrology mouse recombinant g-interferon (Sigma, Deisenhofen, Germany) to J Am Soc Nephrol 10: 2084–2093, 1999 Characterization of Prostanoid Receptors 2085 enhance the expression of T-antigen, and cells were cultivated at 33°C ny). The autofluorescence signal of cells, which had not been loaded (permissive conditions). To induce differentiation, podocytes were with fura-2, was measured, and the results and the noise were sub- cultured on type I collagen (Biochrom, Berlin, Germany) at 37°C tracted from the results obtained in fura-2-loaded cells. This had no without g-interferon (nonpermissive conditions) and with 1% fetal effect on the bandwidth of the measurements. A calibration of the calf serum for 2 to 3 wk. Arborized cells were immunologically fura-2 fluorescence signal was attempted at the end of each experi- characterized and stained positive for the Wilms’ tumor antigen and ment using the Ca21 ionophore ionomycin (1 mM) and low and high 21 21 synaptopodin, a characteristic marker for podocyte foot processes in Ca buffers. [Ca ]i was calculated from the fluorescence ratio vivo and in vitro (13,14). Podocytes between passages 15 and 25 were 340/380 nm according to the equation described by Grynkiewicz et al. used in all experiments. (18). Patch-Clamp Experiments Expression of Prostanoid Receptor mRNA in Mouse The patch-clamp method used in these experiments has been de- Glomeruli and Podocytes scribed in detail (15,16). In brief, podocytes were placed in a bath The RNA preparation, the reverse transcription, and the PCR chamber on the stage of an inverted microscope, kept at 37°C, and amplification (PCR) were performed according to the method recently were superperfused with a phosphate-buffered Ringer’s-like solution. described (19). In brief, total RNA from cultured mouse podocytes 21 To vary free Ca activity, the solutions were prepared according to was isolated with guanidinium/acid phenol/chloroform extraction, and b established techniques with ethyleneglycol-bis( -aminoethyl ether)- the amount of RNA was measured by spectrophotometry. For first 9 9 21 N,N,N N -tetra-acetic acid and nitrilotriacetic acid as Ca buffers. strand synthesis, 2 mg of total RNA from podocytes was mixed in 53 21 The Ca activity was calculated from a standard equation and was reverse transcription buffer and incubated with5UofDNase I for 15 determined with a Ca21-selective electrode (Radiometer, Copenha- 1 min. After termination, the reaction was completed with 0.5 mM gen, Denmark). In ion replacement studies, the extracellular Na or m 2 1 dNTP, 0.5 M sequence-specific primers, 10 mM dithiothreitol, and Cl was replaced by N-methyl-D-glucamine (NMDG ) or gluconate, 200 U Superscript II transcriptase. (Reverse transcriptase was omitted respectively. The patch pipettes were filled with a solution containing in some experiments to control for amplification of contaminating (in mM): 95 K-gluconate, 30 KCl, 4.8 Na2HPO4, 1.2 NaH2PO4, 0.73 DNA.) The reverse transcription was performed at 42°C for 1 h, b 9 CaCl2, 1.03 MgCl2, 1 ethyleneglycol-bis( -aminoethyl ether)-N,N - followed by a denaturation at 95°C for 5 min. cDNA was purified 21 27 tetra-acetic acid, and 5 D-glucose, pH 7.2; Ca activity 10 M. The from amplification reaction and dissolved in 30 ml of 10 mM Tris V patch pipettes had an input resistance of 2 to 3 M . A flowing (10 buffer, pH 8. PCR was performed in duplicate in a total volume of 20 m l/h) KCl (2 M) electrode was used as a reference. The data were ml, each containing 2 ml of reverse transcription reaction and 18 mlof recorded using a patch-clamp amplifier (Fro¨be and Busche, Physiolo- PCR master mixture containing 10 pmol each of sense and antisense gisches Institut, Freiburg, Germany) and continuously displayed on a primer and 2.5 U of Taq DNA polymerase. The cycle profile included pen recorder. The access conductance (Ga) was monitored in most of denaturation for 30 s at 94°C, annealing for 30 s at 52°C, and the experiments by the method recently described (16). The mem- extension for 30 s at 72°C. The amplification products of 10 mlof brane voltage (Vm) of the cells was recorded continuously using the each PCR reaction were separated on a 1.5% agarose gel, stained with current clamp mode of the patch-clamp amplifier. To obtain the whole ethidium bromide, and visualized by ultraviolet irradiation. cell conductance (Gm), the voltage of the respective cell was clamped The primers were: in the voltage clamp mode (V )toV . Starting from this value, the 9 9 c m EP1 receptor [D16338]: f-5 -GCTGTACGCCTCGCATCGTGG-3 whole cell current was measured by depolarizing or hyperpolarizing r-59-CCTTGAGCCCAGCCCAGAATG-39 V in steps of 10 mV to 640 mV. G was calculated from the 9 9 c m EP2 receptor [D50589]: f-5 -CCTGCCGCTGCTCAACTACG-3 measured whole cell current (I), Ga and Vc using Kirchhoff’s and r-59-GTCTCCTCTGCCATCGAAGTCCTC-39 Ohm’s laws.