Fluid flow shear stress upregulates prostanoid receptor EP2 but not EP4 in murine podocytes

Prostaglandins & other Lipid Mediators 104–105 (2013) 49–57

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Prostaglandins and Other Lipid Mediators

Fluid flow shear stress upregulates prostanoid receptor EP2 but not EP4 in murine podocytes Tarak Srivastava a,∗ , Ellen T. McCarthy b , Ram Sharma c , Alexander Kats d , Carol G. Carlton b , Uri S. Alon a , Patricia A. Cudmore a , Ashraf El-Meanawy e , Mukut Sharma c a

Section of Nephrology, Children’s Mercy Hospital and University of Missouri at Kansas City, Kansas City, MO, United States Kidney Institute, University of Kansas Medical Center, Kansas City, KS, United States c Renal Research Laboratory, Research and Development, Kansas City VA Medical Center, Kansas City, MO, United States d Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital and University of Missouri at Kansas City, MO, United States e Division of Nephrology, Medical College of Wisconsin, Milwaukee, WI, United States b

a r t i c l e

i n f o

Article history: Received 4 May 2012 Received in revised form 30 July 2012 Accepted 15 November 2012 Available online 20 December 2012 Keywords: Podocytes Prostanoid receptors EP2 Prostaglandin E2 Fluid flow shear stress Cyclooxygenase Hyperfiltration

a b s t r a c t Podocytes in the glomerular filtration barrier regulate the passage of plasma proteins into urine. Capillary pressure and ultrafiltration impact the structure and function of podocytes. The mechanism of podocyte injury by fluid flow shear stress (FFSS) from hyperfiltration in chronic kidney disease (CKD) is not completely understood. Recently, we demonstrated increased synthesis of prostaglandin E2 in podocytes exposed to FFSS. Here, we determine the effect of FFSS on prostanoid receptors EP1–EP4 in cultured podocytes and in Os/+ mouse kidney, a model of hyperfiltration. Results of RT-PCR, qRT-PCR, immunoblotting and immunofluorescence studies indicate that cultured podocytes express EP1, EP2 and EP4 but not EP3. FFSS resulted in upregulated expression of only EP2 in podocytes. Kidney immunostaining showed significantly increased expression of EP2 in Os/+ mice compared with littermate controls. These novel results suggest that EP2 may be responsible for mediating podocyte injury from hyperfiltration-induced augmented FFSS in CKD. © 2012 Elsevier Inc. All rights reserved.

1. Introduction The glomerular filtration barrier is a three layer structure constituted by fenestrated endothelial cells, glomerular basement membrane (GBM), and podocytes (visceral epithelial cells) through which plasma ultrafiltrate passes from capillary into the urinary space [1]. Podocytes localized in the Bowman’s space restrict the passage of plasma proteins through their interdigitating foot processes surrounding the capillary. A number of receptors, ion channels and an extensive cytoskeletal complex are responsible for maintaining the unique structure and function of podocytes [2,3]. Podocytes respond to physiological mechanical stress caused by glomerular capillary pressure (PGC ) and ultrafiltrate flow [4,5] through changes in the actin cytoskeleton and signaling pathways that are modulated by mediators including angiotensin II and eicosanoids [5–9].

∗ Corresponding author at: Section of Nephrology, Children’s Mercy Hospital, 2401 Gillham Road, Kansas City, MO 64108, United States. Tel.: +1 816 2343010; fax: +1 816 2343494. E-mail address: [email protected] (T. Srivastava). 1098-8823/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.prostaglandins.2012.11.001

Increased single nephron glomerular filtration rate (SNGFR) observed with hyperfiltration elevates fluid flow shear stress (FFSS) on podocytes in chronic kidney disease (CKD) associated with diabetic nephropathy, reflux nephropathy, congenital anomalies of the kidney and urinary tract (CAKUT). While hyperfiltration is closely related to CKD, the mechanism of its contribution to progressive loss of glomerular function is not clear. Normal mice after surgical ablation of renal mass or heterozygous oligosyndactyly mice (OS/+) with about 50% nephron number are used to study the effect of hyperfiltration [10–17] in vivo. Cultured cells are routinely used as an in vitro model to study the effect of capillary pressure-induced mechanical stretch or hyperfiltration-induced FFSS. Recently, we showed that in vitro application of FFSS results in increased secretion of prostaglandin E2 (PGE2 ) and upregulation of cyclooxygenase (COX)-2 in podocyte-monolayers [8]. In contrast, in vitro application of mechanical stretch results in upregulation of COX-2 without a change in PGE2 secretion [9]. PGE2 is a major prostaglandin product of arachidonic acid metabolism by the COX activities and exerts its physiologic effects in an autocrine and paracrine manner [18,19]. Four G-protein coupled E-prostanoid (EP) receptors namely, EP1, EP2, EP3 and EP4 bind PGE2 in both human and mouse tissues [20]. Subtle differences in the ligandbinding capacity, tissue distribution and intracellular signaling

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elements of these receptors may be responsible for the diverse biological effects of PGE2 [20–23]. EP1 and EP4 are expressed in glomeruli and in other segments of the nephron. EP2, when present, is a low abundance receptor and is believed to be primarily expressed as a result of cell stimulation [20,23–27]. Furthermore, glomerular expression of EP2 is uncertain and remains to be confirmed in podocytes [9,28,29]. Other investigators have shown that stretch-induced signaling is mediated by EP4 but the mechanism of FFSS-induced signaling in podocytes is not clear [9,30–32]. In this research, we plan to determine the expression of key prostanoid receptors EP2 and EP4 in cultured podocytes and evaluate the effect of FFSS on their expression.

Fidelity (Invitrogen) and specific sets of primers for each of the EP receptors and ␤-actin. The sequences (5 –3 ) of primers used for RT-PCR are shown in Table 1. After an initialization step at 95 ◦ C for 3 min, cDNA was amplified using 30 cycles of denaturation (94 ◦ C, 30 s), annealing (55 ◦ C, 30 s) and elongation (72 ◦ C, 35 s). The final extension step was carried out at 72 ◦ C for 7 min. After PCR, a 10 ␮l aliquot from each amplified product was separated on a 2% Nu Sieve 3:1 agarose gel (Lonza, Rockland, MD), stained with SYBR Safe DNA gel stain (Invitrogen), and visualized using a UV light source. Product size was determined by comparing with the DNA ladder (Invitrogen) on the gel. Relative intensity of the bands was semi quantified using Alpha Imager Software (San Leandro, CA). PCR products were then sequenced to further confirm the EP receptors and ␤-actin at the KUMC Core Facilities (Kansas City, KS).

2. Methods These studies were carried out using protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the Medical College of Wisconsin, Milwaukee, WI and by the IACUC, Safety Subcommittee and the R&D Committee at the VA Medical Center, Kansas City, MO. 2.1. Podocyte cell culture The conditionally immortalized mouse podocyte line with thermosensitive tsA58 mutant T-antigen (a kind gift from Dr. Peter Mundel) was used as previously described [33,2]. Briefly, podocytes were maintained in RPMI 1640 medium containing l-glutamine supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 0.1 mg/ml streptomycin (Invitrogen, Carlsbad, CA). To propagate, podocytes were grown on collagen coated (BD Biosciences, San Diego, CA) tissue culture flasks or glass slides under permissive conditions, i.e. at 33 ◦ C with mouse ␥-interferon (Cell Sciences, Norwood, MA). To induce differentiation, cells were grown on 25 mm × 75 mm × 1 mm glass slides (Fisher Scientific, Pittsburgh, PA) under non-permissive conditions, i.e. at 37 ◦ C without mouse ␥-interferon. Differentiated podocytes were used for FFSS experiments on the 14th day [8]. 2.2. Fluid flow shear stress (FFSS) application to cultured podocytes Methodology for application of FFSS on differentiated podocytes has been described in detail [8]. Glass slides with podocyte monolayers were placed in the flow device chamber of the FlexCell Streamer Gold apparatus (FlexCell International, Hillsborough, NC). FFSS (2 dynes/cm2 ) was applied for 2 h at 37 ◦ C with 5% CO2 using a computer controlled program [8]. Control podocytes were incubated under identical conditions without FFSS. Following FFSS treatment, slides were transferred to fresh medium and allowed to recover for up to 24 h at 37 ◦ C at 5% CO2 in humidified atmosphere of the incubator. Pre-FFSS and post-FFSS 2 h/24 h cells were harvested for analysis. 2.3. RT-PCR and qRT-PCR to determine gene expression of EP1, EP2, EP3 and EP4 receptors in cultured podocytes 2.3.1. Reverse transcriptase-polymerase chain reaction (RT-PCR) Podocyte total RNA was extracted using the Micro-Midi Total RNA Purification System (Invitrogen) and analyzed for quality and quantity by absorbance at 260 and 280 nm using DNA/RNA calculator (Pharmacia Biotech/GE Healthcare, Uppsala). The OD260 :OD280 absorbance ratio was 1.8–2.0 indicating clean RNA preparations. One microgram of total RNA was reverse transcribed using SuperScript III First strand synthesis system for RT-PCR (Invitrogen) and cDNA synthesis and amplified using Platinum PCR SuperMix High

2.3.2. Quantitative real time RT-PCR (qPCR) Briefly, total RNA isolated and tested for primer specificity as above was used for qRT-PCR in SYBR Supermix (Bio-Rad) using Bio-Rad iCycler (Bio-Rad, Hercules, CA). ␤-Actin was used as the housekeeping gene. The sequences (5 –3 ) of primers used for qRTPCR are shown in Table 2. Expression of EP2 receptor was further confirmed by qRT-PCR using a second set of primers with GAPDH as the housekeeping gene. Primer sequences used in the second series are also shown in Table 2. 2.4. Immunofluorescence microscopy to document EP receptor proteins in cultured podocytes Podocytes were fixed in 4% paraformaldehyde in PBS (15 min, room temperature) and permeabilized with 0.05% Triton X-100 (10 min). Triton X-100 was used as a permeabilizing agent to increase the fluorescence intensity of weakly expressed membrane bound proteins. After blocking with 5% goat serum (2 h, room temperature) cells were incubated with rabbit anti-mouse EP2 (1:100 dilution, Santa Cruz # sc-20675, Santa Cruz, CA) or rabbit anti-mouse EP4 (1:100 dilution, Abcam # ab93486, Cambridge, MA) antibodies in blocking solution (overnight, 4 ◦ C). Fluorescencetagged goat anti-rabbit (1:500 dilution, AlexaFluor 488, Invitrogen) was used as secondary antibody (1 h, room temperature) and 9:1 glycerol:PBS + 5% N-propyl gallate as the mounting medium. Negative control consisted of immunostaining using the same protocol in the absence of primary antibody for EP2 and EP4. Images were obtained using an Olympus BX60 fluorescence microscope (Hamburg, Germany) and analyzed in a masked manner by a pathologist (A.K.). 2.5. Immunofluorescence microscopy to document EP receptor proteins in mouse glomeruli Kidneys were harvested from CD-1 background mice and 4–10 ␮m frozen sections were cut and held on clean glass slides. Thawed sections were fixed in 1:1 mix of cold acetone and alcohol for 10 min at 4 ◦ C and blocked using 5% goat serum as described. Rabbit anti-mouse EP2 or EP4 antibodies were used at 1:150 dilution each. Fluorescent-tagged secondary (goat anti-rabbit AlexaFluor 488 and/or goat anti-rabbit AlexaFluor 594, Invitrogen) antibody was used at 1:500 dilution (1 h, room temperature). Images were obtained and analyzed as described. 2.6. Electrophoresis and Western blotting to document expression of EP receptor proteins in cultured podocytes Podocytes were lysed with RIPA buffer (50 mM Tris–HCl, 1% NP-40, 0.25% Na-deoxycholate and 150 mM NaCl) containing protease and phosphatase inhibitors (1 mM PMSF, 5 ␮l/ml Sigma

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Table 1 The primer sequences (5 –3 ) for prostanoid receptors EP1, EP2, EP3, EP4 and ␤-actin used for RT-PCR in this study. Target

PCR product size

EP1

228 bp

Forward Reverse

5 -GCTGTACGCCTCGCATCGTGG-3 5 -CCTTGAGCCCAGCCCAGAATG-3

EP2

246 bp

Forward Reverse

5 -CCTGCCGCTGCTCAACTACG-3 5 -GTCTCCTCTGCCATCGAAGTCCTC-3

EP3

266 bp

Forward Reverse

5 -AAGCCAGGCGAACTGCAATTAGAA-3 5 -AAGCCAGGCGAACTGCAATTAGAA-3

EP4

306 bp

Forward Reverse

5 -CTGCTGATCTCCTTTAACTCCC-3 5 -CTGCTGATCTCCTTTAACTCCC-3

␤-Actin

380 bp

Forward Reverse

5 -ACCAACTGGGACGACATGGAG-3 5 -GTCAGGATCTTCATGAGGTAGTC-3

Sequence

Protease inhibitor, 1 mM Na-vanadate and 10 ␮l/ml Sigma Phosphatase Cocktail 1, Sigma–Aldrich, St Louis, MO). Total protein was determined using a bicinchoninic acid protein assay kit (BCA1, Sigma–Aldrich). Proteins were denatured in sample buffer containing beta-mercaptoethanol at 94 ◦ C for 5 min. Ten micrograms total protein was electrophoresed using a 10% Bis–Tris gel (100 V, 90 min, 4 ◦ C). Proteins were transferred to a PVDF membrane (22 V, 4 ◦ C, overnight), washed with PBST (0.1% Tween-20) and blocked using 5% non-fat milk (4–6 h, room temperature). Rabbit polyclonal antimouse EP2 antibody (Santacruz # sc-20675) at 1:500 dilution or rabbit polyclonal EP4 antibody (Abcam # ab93486) at 1:2000 dilution in 5% milk for detection (4 ◦ C, overnight). After washing with PBST (0.1% Tween 20) the membrane was incubated with goat antirabbit HRP-conjugate (1 h, room temperature) as the secondary antibody (Santacruz # sc-2054). Membranes were incubated with enhanced chemiluminescence (ECL) reagent and exposed to Xray film. The developed X-ray films were imaged and analyzed on FluorChemTM using built in AlphaEaseFC software (Alpha Innotech Corporation, San Leandro, CA). 2.7. Immunohistochemistry to determine EP2 receptor expression in Os/+ mouse kidneys The Os/+ oligosyndactyl mouse is a radiation-induced mutant characterized by paws with ≤4 digits, kidneys with ∼50% fewer nephrons and by glomerular and tubular hypertrophy [10–12]. The reduced number of nephrons in the Os/+ mice results in hyperfiltration mediated augmentation of FFSS. We determined

EP2 expression by immunohistochemical examination of the kidneys from 26-week old Os/+ and littermate controls bred in ROP background. Immunohistochemistry was performed using anti-EP2 monoclonal primary antibody at 1:200 dilution and HRP-tagged rabbit anti-mouse secondary antibody at 1:1000 dilution (1 h at room temperature). Tissue sections were mounted in 9:1 glycerol:PBS + 5% N-propyl gallate and photomicrographed as described. We determined the 3,3-Diaminobenzidine (DAB) stained area as percent of total area using Metamorph software (Molecular Devices, LLC, Sunnyvale, CA).

3. Results 3.1. Cultured podocytes express mRNA for E-type prostaglandin receptors As shown in Fig. 1A, we detected mRNA for EP1, EP2 and EP4 in total RNA preparations from podocytes using RT-PCR. However, we did not detect mRNA for EP3. The PCR products were sequenced and analyzed using BLAST algorithm to confirm their specificity. Fig. 1A also shows that EP2 expression was increased at 24 h following FFSS. Yet, no similar increase was detected in EP1 or EP4 expression. Documentation of mRNA expression for EP2 receptor by podocytes under basal conditions is a novel finding that will be valuable in our studies on the role of cyclooxygenase 1 or 2 and eicosanoids in the response of podocyte to FFSS. These observations were corroborated by qRT-PCR as described below.

Table 2 The primer sequences (5 –3 ) for prostanoid receptors EP1, EP2, EP3, EP4 and ␤-actin used for qRT-PCR in this study. Target

PCR product size

EP1

100 bp

Forward Reverse

5 -GAGCCAGGGAGTAGCTGGA-3 5 -GCTCATATCAGTGGCCAAGAG-3

Sequence

EP2

73 bp

Forward Reverse

5 -TGCTCCTTGCCTTTCACAAT-3 5 -CTCGGAGGTCCCACTTTTC-3

EP3

74 bp

Forward Reverse

5 -AGCTCATGGGGATCATGTG-3 5 -GAAGATCATTTTCAACATCATTATCAA-3

EP4

92 bp

Forward Reverse

5 -CGGTTCCGAGACAGCAAA-3 5 -CGGTTCGATCTAGGAATGG-3

␤-Actin

104 bp

Forward Reverse

5 -CTAAGGCCAACCGTGAAAAG-3 5 -ACCAGAGGCATACAGGGACA-3

EP2

206 bp

Forward Reverse

5 -CTAATGGAGGACTGCAAGA-3 5 -GTGAGCACCAATTCCGTTA-3

EP4

159 bp

Forward Reverse

5 -GCTGAGGTTGGAGGTACCAT-3 5 -GATGAACATCACTGCGGGAA-3

GAPDH

102 bp

Forward Reverse

5 -GCCTTCCGTGTTCCTACCC-3 5 -TGCCTGCTTCACCACCTTC-3

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Fig. 1. (A) Podocytes express EP1-4 genes: RT-PCR for prostanoid receptors EP1-4 gene expression separated on a 2% agarose gel. Podocytes exposed to FFSS show an increase in inducible EP2 gene expression at post-24 h FFSS. Prostanoid receptor EP1 and EP4 expression remain unchanged with FFSS. EP3 expression in podocyte was not seen. Figure (A) represents results of 4 separate experiments. (B) FFSS upregulates EP2 gene expression: Real-time quantitative RT-PCR for prostanoid receptors EP1-4 gene expression. Podocytes exposed to FFSS show an increase in inducible EP2 gene expression at post-24 h FFSS. Prostanoid receptor EP1 and EP4 expression remain unchanged with FFSS (values are mean ± SEM, n = 5).

3.2. Fluid flow shear stress (FFSS) alters the mRNA for E-type prostaglandin receptors in cultured podocytes Fig. 1B shows the results of qRT-PCR gene expression studies at 2 and 24 h following FFSS. EP2 mRNA expression for normalized to ␤actin showed an increase over control at 2 h post-FFSS (1.72 ± 0.22, p = 0.40, NS) and at 24 h post-FFSS (2.67 ± 0.62, p < 0.02). There was no change in mRNA expression for EP4 at 2 h post-FFSS (0.91 ± 0.12, p = 0.86, NS) and at 24 h post-FFSS (0.88 ± 0.16, p = 0.76, NS). Similarly, EP1 at 2 h post-FFSS (1.10 ± 0.15, p = 0.77, NS) and at 24 h post-FFSS (0.85 ± 0.09, p = 0.57, NS) by qRT-PCR also remained unchanged. PCR experiments were repeated using a second set of primers and similar results were obtained (data not shown). Gene identification of the qRT-PCR PCR products as EP2 was confirmed by sequencing at the Core Facilities, KU Medical Center. 3.3. Cultured podocytes express E-type prostaglandin receptor proteins Fig. 2A shows the results of Western blots using total protein lysate from podocytes. Total protein at 10, 15 and 20 ␮g were analyzed to determine the expression of WT1, synaptopodin, podoplanin and podocin as well as the presence of EP2 and EP4. These results confirm that cultured podocytes express proteins that are routinely used to characterize these glomerular epithelial cells. EP2 and EP4 proteins are expressed in podocytes. Fig. 2B shows the results of immunofluorescence microscopy for EP2 and EP4 proteins in podocytes. Upper panel show that both EP2 and EP4 proteins are expressed in podocytes. These findings corroborated the Western blotting results presented in Fig. 2A. The negative control did not show fluorescence (results not presented). Thus, our results using both immunostaining by Western blotting

and immunofluorescence microscopy showed the expression of EP2 and EP4 at the podocyte plasma membrane.

3.4. Mouse glomeruli express EP2 and EP4 receptor proteins Fig. 2C shows mouse glomeruli in kidney sections immunostained with anti-EP2 or anti-EP4 antibody and detected using fluorescence microscopy. Both EP2 and EP4 proteins were detected along the glomerular basement membrane as expected for a podocyte protein. The expression of EP2 and EP4 proteins was faint and patchy suggesting low expression in normal kidneys. EP2 expression in a medium-sized vessel indicated by an arrow in Fig. 2C serves as a convenient internal positive control. These results provide evidence for in situ expression of EP2 in glomeruli and corroborate our in vitro results using cultured podocytes.

3.5. Fluid flow shear stress (FFSS) increases EP2 receptor protein expression in cultured podocytes Fig. 3A and B shows results of Western blotting for EP2 and EP4 proteins in podocyte lysates after FFSS treatment. Representative immunoblots are presented with results of semi-quantitative analysis of image densities normalized using ␤-actin as the loading control. Fig. 3A shows immunoblots for EP2 at 2 and 24 h post-FFSS. The ratio of intensities of EP2 and ␤-actin bands (EP2/␤actin) showed a time-dependent increase between 2 h post-FFSS (1.34 ± 0.46, p = 0.56, NS) and 24 h post-FFSS (2.49 ± 1.07, p < 0.04) compared to the control. Fig. 3B shows immunoblots for EP4 at 2 and 24 h post-FFSS. There was no change in the EP4/␤-actin intensity ratio at 2 h postFFSS (0.79 ± 0.42, p = 0.22) or 24 h post-FFSS (0.99 ± 0.06, p = 0.97)

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Fig. 2. (A) Immunoblot analysis show that podocytes express EP2 and EP4 receptor proteins: Total cell protein lysates (10, 15 and 20 ␮g) were analyzed by Western blotting for podocyte specific proteins WT-1, synaptopodin, podoplanin and podocin, and for prostanoid receptors EP2 and EP4. (B) Immunofluorescence shows in vitro expression of EP2 and EP4. Prostanoid receptors EP2 and EP4 were detected by immunofluorescence microscopy using specific antibodies in vitro mouse podocyte. EP2 and EP4 were detected on the cytoplasmic membrane. (C) Immunofluorescence shows in situ expression of EP2 and EP4. In situ expression of EP2 and EP4 receptor proteins was further confirmed in glomeruli from untreated CD1mouse. EP2 specific staining in a medium sized vessel is indicated by an arrow that also serves as an internal positive control.

compared to the control. Thus, the intensity of EP2 protein at 24 h post-FFSS was significantly different from that of the control. 3.6. ROP Os/+ mice show increased expression of EP2 receptor protein Fig. 4 shows results of immunohistochemical analysis for EP2 protein in kidney tissue slices. The Os/+ mouse is characterized by elevated SNGFR consequent to a reduced number of nephrons in the kidney [10–12] and is used as a model to study the effect of hyperfiltration. Fig. 4 shows results of immunohistochemical detection of EP2 receptor protein in Os/+ mouse kidney. The Os/+ kidney showed a strong expression of EP2 in the glomerulus compared to a faint staining in the ROP++ animal. We determined the DAB stained area as percent of total area using Metamorph software (Molecular Devices, LLC, Sunnyvale, CA). The DAB stained area as percent of total area increased significantly in the Os/+ mouse kidneys (0.69 ± 0.05 in the control vs. 1.35 ± 0.18 in the Os/+, p < 0.02, n = 3 in each group). These results further validate the significance of EP2 in FFSS. Thus our results demonstrate expression of EP2 mRNA and protein in cultured podocytes and in glomeruli in situ. Flow induced FFSS caused increased levels of EP2 mRNA and protein in cultured podocytes. Finally, initial immunohistochemical findings suggest increased expression of EP2 protein in OS/+ mouse glomeruli compared to their littermate++ ROP controls. These findings suggest a role for EP2 in the intracellular signaling initiated by FFSS. 4. Discussion Albuminuria (proteinuria) in CKD is associated with glomerular dysfunction, sclerosis and ultrastructural changes in podocyte.

Loss of podocyte structure and function are used clinically to determine glomerular dysfunction in chronic disease. Podocytes are located in the Bowman’s capsule and their foot processes adhere to glomerular basement membrane (GBM) in an interdigitating manner around the outer aspect of the glomerular capillary. An extensive actin cytoskeleton and other structural features enable podocytes to maintain a flexible structure and function. While these unique structural features and spatial organization are largely responsible for regulating the passage of plasma proteins, they also render podocytes subject to physical stress resulting from the mechanical forces associated with plasma filtration. Under physiological conditions podocytes respond to tensile stress due to glomerular capillary pressure (PGC ) by permitting the flow of the plasma ultrafiltrate, changes in the cytoskeleton and signaling pathways [4–9]. Glomerular capillaries, unlike systemic capillaries, maintain much higher arteriolar pressure to achieve ultrafiltration that induces stretch (elongation) of podocytes that attach to the outer aspect of the capillary through the GBM. In vitro mechanical stretch is used as a model to study tensile stress from podocyte elongation [4,6,7]. In addition, a constant flow of the ultrafiltrate generates shear stress tangential to cell surface. Human kidneys generate approximately 180 L ultrafiltrate each day and podocytes must adjust to the constantly changing microenvironment [5]. Thus, efficient filtration in each nephron depends upon structural and functional integrity of podocytes. Pathophysiological changes associated with a decreased number of functional nephrons result in increased capillary pressure and greater ultrafiltrate flow per nephron – termed adaptive hyperfiltration. Relative effects of stretch and shear stress have been directly compared in osteocytes. Computation of changes in cytoskeleton, cytoplasm, nucleus and membrane components showed that FFSS causes greater cellular deformity and transmits

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Fig. 3. (A) Effect of FFSS on EP2 receptor protein. Aliquots of total protein from cell lysates were analyzed by immunoblotting using anti-EP2 antibody. Expression of EP2 was evident at 2 and 24 h post-FFSS compared to untreated control. The blot shown on the left are representative of three separate experiments. The blots were subjected to image analysis, and EP protein was normalized to ␤-actin levels. Bar graphs in the adjoining panel represent mean ± SEM of three experiments (n = 3). (B) Effect of FFSS on EP2 receptor protein. Aliquots of total protein from cell lysates were analyzed by immunoblotting using anti-EP4 antibody. Expression of EP4 did not change at 2 or 24 h post-FFSS compared to untreated control. The blot shown on the left is representative of three separate experiments. The blots were subjected to image analysis, and EP protein was normalized to ␤-actin levels. Bar graphs on the right present results as mean ± SEM (n = 3).

Fig. 4. Prostanoid receptor, EP2 expression was evaluated in ROP (+/+) kidney (left) and ROP (Os/+) kidney (right) at 26 weeks by immunohistochemistry using a monoclonal anti-mouse EP2 antibody. Glomeruli from the ROP (Os/+) mice show structural hypertrophy with an increase in EP2 expression (shown as brown staining). In contrast, glomeruli from ROP (+/+) kidney showed a faint EP2 expression.

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deformation more effectively from outer to inner compartments of the cell compared to stretch [34]. Since podocytes play an important role in glomerular hemodynamics and barrier function, a better understanding of the effect of increased FFSS on the podocyte is warranted. In vitro studies using cultured cells or isolated glomeruli as well as in vivo studies in animal models or in patients with CKD indicate a role for PGE2 in hyperfiltration-induced glomerular dysfunction. Recently, we showed that in vitro application of FFSS to podocyte monolayers results in increased synthesis and secretion of PGE2 , reduced transversal F-actin stress filaments and formation of a cortical actin network [8]. These changes in the actin cytoskeleton were reproduced by direct exposure of podocytes to PGE2 , an effect that was blocked by the cyclooxygenase inhibitor Indomethacin [8]. Since the podocyte cytoskeletal derangement changes may undermine the integrity of the filtration barrier, the observed cytoskeletal changes may alter glomerular filtration characteristics and lead to increased permeability to albumin. Our observations on cultured cells were corroborated by experiments using isolated glomeruli. We found that addition of exogenous PGE2 to glomerular suspensions or ex vivo application of FFSS to isolated glomeruli resulted in increased glomerular albumin permeability that was effectively blocked by Indomethacin [35,36]. In vivo studies also suggest a role for PGE2 in hyperfiltration-induced renal changes since the synthesis of PGE2 and expression of COX-2 are increased in the 5/6 nephrectomized and diabetic rats [37,38]. In both animal models and human subjects, proteinuria is reduced by COX inhibition using NSAIDs. NSAIDs that reduce renal PGE2 excretion decrease proteinuria whereas Sulindac, which does not influence PGE2 , shows no effect on proteinuria [39–42]. However, the nature of receptor(s) and signaling elements that mediate the effect of FFSS-induced increase in PGE2 are not clear. We hypothesized that mechano-transduction induced by FFSS involves the COX2–PGE2 system and a specific prostanoid-E receptor but the nature the PGE2 receptors in podocytes is not clear. Four E-type prostanoid receptors namely, EP1, EP2, EP3 and EP4 that mediate the biological effects of PGE2 . EP receptors are cell surface G protein-coupled receptors containing seven trans-membrane domains [43,44]. EP1 is coupled to Ca2+ mobilization; EP2 and EP4 are linked to Gs protein; and, EP3 is coupled to Gi protein [43–45]. EP2 is a low abundance but inducible protein with proven expression in the lung, uterus and spleen, and in medium sized vessels of the kidney [20,23,25,43,46]. Our results show that EP1, EP2 and EP4 are expressed in podocytes (see Fig. 1). Demonstration of low abundance prostanoid receptor EP2 in podocytes, a novel finding, was corroborated by performing RT-PCR and qRT-PCR using three separate sets of primers (see Fig. 1, Tables 1 and 2). In addition to gene expression, the presence of EP2 protein was demonstrated by Western blotting and immunofluorescence microscopy in podocytes in vitro (see Fig. 2A and B), and by immunofluorescence and immunohistochemistry in isolated glomeruli and tissue sections (see Figs. 2C and 4). Prostanoid receptor EP4 mediates the effect of mechanical stretch stimulus in podocytes [9,47,48]. In contrast, application of FF SS to podocytes resulted in upregulation of both gene and protein expression for EP2 and not EP4 (see Figs. 1 and 3). EP2, when present, is a low abundance receptor and is believed to be inducible [20,23,27]. Earlier studies using showed that EP2 is relevant in mediating the effect of FFSS in osteocytes [49–51]. The absence of a change in EP4 expression was equally interesting. Although EP2 and EP4 are both membrane localized GPCRs, share ∼30% homology and activate adenylate cyclase they, however, differ with regard to their responses to stimuli, nature of ligands and regulatory mechanisms. A complex interplay between EP2 and EP4 is illustrated by their response to lipopolysaccharide (LPS) in peritoneal macrophages and a macrophage cell line J774.1 [47,48] that

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express only EP4 at resting conditions. However, change of the medium induces EP2 without affecting EP4 expression. And, LPS further augments EP2, but down regulates EP4 expression that is reversed by Indomethacin suggesting that the down-regulation of EP4 is mediated by LPS-induced prostaglandin synthesis [47]. Additionally, EP2 and EP4 differ in regard to their response to various ligands. In Chinese hamster ovary cells, PGE2 causes short-term desensitization of EP4 without affecting EP2. 15-keto-PGE2 causes a greater and gradual loss of EP2 but a lower and immediate reduction in EP4 activity [52]. Thus, EP2 and EP4 display differences in agonist-induced short term desensitization and susceptibility to prostaglandins. Our findings provide further evidence that EP2 and EP4 have diverse characteristics. Present results show that FFSS up-regulates the expression of EP2 but not of EP4 at both gene and protein levels (Fig. 3). Previously, we showed that FFSS up regulates COX-2 and increases PGE2 secretion [8]. These findings are consistent with our hypothesis that mechano-transduction induced by FFSS is mediated via the COX2–PGE2 system through specific receptors. Preliminary results suggest that FFSS induces increased cAMP production and translocation of ␤-catenin indicating the activation of EP2-PKA-cAMP and EP2-PI3K-Akt-GSK3␤-␤ catenin signaling pathways (unpublished observations). In contrast, other investigators found that mechanical stretch applied to podocytes upregulates EP4 but not EP2 [9,30–32]. Thus, podocytes appear to respond to FFSS and stretch through different mechanisms. Briefly, mechanical stretch: (i) induces a decrease in transverse stress fibers and causes formation of actin-rich centers and radial stress fibers, effects that are disrupted by post-stretch addition of exogenous PGE2 [9], (ii) up regulates COX-2 without an increase in PGE2 synthesis or secretion [9], and (iii) up regulates EP4, but not EP2, in podocytes cultures [9,30–32]. In contrast, FFSS: (i) disrupts the actin stress fibers with the formation of cortical actin [5,8], (ii) upregulates COX-2 with increased levels of PGE2 , and (iii) up regulates EP2 but not EP4, in cultured podocytes. Differences between cellular response to mechanical stretch and shear stress have been reported in other cells. For example, osteocytes in bone cavities form an anastomized network of processes and utilize different mechanisms to respond to stretch and shear stress [34]. FFSS applied to cultured osteocytes results in: (i) significant increase in PGE2 but not in collagen-I expression while mechanical stretch results in opposite effects [34] and (ii) up regulation of COX-2 and EP2, but not EP4 [49–51,53,54]. Thus, osteocytes and podocytes appear to respond to FFSS in a comparable manner. Although a detailed mechanism remains to be determined, differences in the response of EP2 and EP4 to prostaglandin ligands and to mechanical stretch and FFSS are corroborated by the results from experiments using diverse cell types including macrophages, CHO cells and osteocytes. Our attempts to delineate the differences between FFSS and stretch in cultured podocytes will lead to a better understanding of the mechanism of hyperfiltration-induced injury in CKD. Glomerular hyperfiltration is an important characteristic of CKD associated with diabetes, obesity, hypertension and congenital kidney abnormalities [55–58]. A gradual reduction in functional renal mass in CKD results in elevated renal blood flow and PGC in the remnant nephrons leading to an adaptive hyperfiltration and increase in SNGFR. Renal mass reduction by unilateral nephrectomy and congenital reduction in the number of nephrons provide two convenient models to study the effect of hyperfiltration-induced renal dysfunction in vivo. Ongoing studies in our laboratory show that unilateral nephrectomy results in increased FFSS over podocytes in rats (manuscript in preparation). Additionally, we used Os/+ ROP mice as a model to study the effect of hyperfiltration on glomerular structure and function. Os/+ ROP mice are born with ∼50% fewer nephrons, and have increased SNGFR [10]. Kidneys from these mice

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showed increased glomerular expression of EP2 receptor compared to their littermate controls (see Fig. 4). These preliminary in vivo results corroborate our “proof of concept” that EP2 is important in mounting a podocyte response to increased FFSS under systemic conditions that result in hyperfiltration. In summary, we show that podocytes express the elusive prostanoid receptor EP2 and that EP2 is specifically upregulated by FFSS. Of greater interest, overlapping responses to mechanical forces in different cell types suggest generalizable fundamental cellular mechanisms for response to mechanical forces. A better understanding of EP2 receptor will likely provide novel molecules for attenuation of hyperfiltration-induced glomerular dysfunction in CKD. Conflict of interest None. Acknowledgments This work was supported in part by The Norman S. Coplon Extramural Research Grant and The Sam and Helen Kaplan Research Fund in Pediatric Nephrology awarded to Tarak Srivastava. Parts of the work were presented the Winter Eicosanoids Conference in Baltimore in March 2012. References [1] Rodewald R, Karnovsky MJ. Porous substructure of the glomerular slit diaphragm in the rat and mouse. J Cell Biol 1974;60:423–33. [2] Shankland SJ, Pippin JW, Reiser J, Mundel P. Podocytes in culture: past, present, and future. Kidney Int 2007;72:26–36. [3] Faul C, Asanuma K, Yanagida-Asanuma E, Kim K, Mundel P. Actin up: regulation of podocyte structure and function by components of the actin cytoskeleton. Trends Cell Biol 2007;17:428–37. [4] Endlich N, Endlich K. Stretch, tension and adhesion – adaptive mechanisms of the actin cytoskeleton in podocytes. Eur J Cell Biol 2006;85:229–34. [5] Friedrich C, Endlich N, Kriz W, Endlich K. Podocytes are sensitive to fluid shear stress in vitro. Am J Physiol Renal Physiol 2006;291:F856–65. [6] Endlich N, Kress KR, Reiser J, et al. Podocytes respond to mechanical stress in vitro. J Am Soc Nephrol 2001;12:413–22. [7] Durvasula RV, Petermann AT, Hiromura K, et al. Activation of a local tissue angiotensin system in podocytes by mechanical strain. Kidney Int 2004;65:30–9. [8] Srivastava T, McCarthy ET, Sharma R, et al. Prostaglandin E2 is crucial in the response of podocytes to fluid flow shear stress. J Cell Commun Signal 2010;4:79–90. [9] Martineau LC, McVeigh LI, Jasmin BJ, Kennedy CR. p38 MAP kinase mediates mechanically induced COX-2 and PG EP4 receptor expression in podocytes: implications for the actin cytoskeleton. Am J Physiol Renal Physiol 2004;286(4):F693–701. [10] Zalups RK. The Os/+ mouse: a genetic animal model of reduced renal mass. Am J Pathol 1993;264:F53–60. [11] He C, Zalups RK, Henderson DA, Striker GE, Striker LJ. Molecular analysis of spontaneous glomerulosclerosis in Os/+ mice, a model with reduced nephron mass. Am J Pathol 1995;269:F266–73. [12] Esposito C, He CJ, Striker GE, Zalups RK, Striker LJ. Nature and severity of the glomerular response to nephron reduction is strain-dependent in mice. Am J Pathol 1999;154:891–7. [13] Celsi G, Savin J, Henter JI, Sohtell M. The contribution of ultrafiltration pressure for glomerular hyperfiltration in young nephrectomized rats. Acta Physiol Scand 1991;141:483–7. [14] Celsi G, Larsson L, Seri I, Savin V, Aperia A. Glomerular adaptation in uninephrectomized young rats. Pediatr Nephrol 1989;3:280–5. [15] Brenner BM. Nephron adaptation to renal injury or ablation. Am J Pathol 1985;249:F324–37. [16] Brenner BM, Mackenzie HS. Nephron mass as a risk factor for progression of renal disease. Kidney Int Suppl 1997;63:S124–7. [17] Brenner BM, Lawler EV, Mackenzie HS. The hyperfiltration theory: a paradigm shift in nephrology. Kidney Int 1996;49:1774–7. [18] Scholz H. Prostaglandins. Am J Physiol Regul Integr Comp Physiol 2003;285:R512–4. [19] Vitzthum H, Abt I, Einhellig S, Kurtz A. Gene expression of prostanoid forming enzymes along the rat nephron. Kidney Int 2002;62:1570–81. [20] Narumiya S, Sugimoto Y, Ushikubi F. Prostanoid receptors: structures, properties, and functions. Physiol Rev 1999;79:1193–226.

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