EP1 receptor antagonism mitigates early and late stage renal fibrosis

Abstract Aim Renal fibrosis is a major driver of chronic kidney disease, yet current treatment strategies are ineffective in attenuating fibrogenesis. The cyclooxygenase/prostaglandin system plays a key role in renal injury and holds great promise as a therapeutic target. Here, we used a translational approach to evaluate the role of the PGE2‐EP1 receptor in the pathogenesis of renal fibrosis in several models of kidney injury, including human (fibrotic) kidney slices. Methods The anti‐fibrotic efficacy of a selective EP1 receptor antagonist (SC‐19220) was studied in mice subjected to unilateral ureteral obstruction (UUO), healthy and fibrotic human precision‐cut kidney slices (PCKS), Madin‐Darby Canine Kidney (MDCK) cells and primary human renal fibroblasts (HRFs). Fibrosis was evaluated on gene and protein level using qPCR, western blot and immunostaining. Results EP1 receptor inhibition diminished fibrosis in UUO mice, illustrated by a decreased protein expression of fibronectin (FN) and α‐smooth muscle actin (αSMA) and a reduction in collagen deposition. Moreover, treatment of healthy human PCKS with SC‐19220 reduced TGF‐β‐induced fibrosis as shown by decreased expression of collagen 1A1, FN and αSMA as well as reduced collagen deposition. Similar observations were made using fibrotic human PCKS. In addition, SC‐19220 reduced TGF‐β‐induced FN expression in MDCK cells and HRFs. Conclusion This study highlights the EP1 receptor as a promising target for preventing both the onset and late stage of renal fibrosis. Moreover, we provide strong evidence that the effect of SC‐19220 may translate to clinical care since its effects were observed in UUO mice, cells and human kidney slices.


| INTRODUCTION
Chronic kidney disease (CKD) is a leading cause of death and the global prevalence is currently estimated to be about 10%-15%. 1 Regardless of the underlying cause, CKD is characterized by fibrotic changes in the kidney and progressive loss of renal function, which can ultimately lead to end-stage renal disease (ESRD). 2 Persistent fibrogenesis is regarded as the most important pathologic process underlying the progression of CKD. Despite overwhelming efforts to find therapeutics that arrest the fibrotic process, current treatment strategies have proven ineffective in attenuating renal fibrogenesis. Thus, the identification of novel therapeutic targets is of the utmost importance.
Inflammatory processes mediated by the cyclooxygenase/prostaglandin (COX/PG) system play fundamental roles in the progression of renal injury. [3][4][5][6][7][8][9][10] Prostaglandin E 2 (PGE 2 ) is the major prostaglandin in the kidney, and it is involved in the regulation of several physiological processes such as renal haemodynamics and water and salt homeostasis. 11,12 The physiological effects of PGE 2 are mediated via the G-protein-coupled receptors EP 1-4 , 13 and thus, the most likely target for PGE 2 effects during renal injury. We have recently reported that activation of the EP 2 receptor with butaprost mitigates renal fibrosis in mice subjected to unilateral ureteral obstruction (UUO), MDCK cells and human precision-cut kidney slices. 14 In addition, EP 4 agonism has been shown to have anti-fibrotic effects in UUO mice and cultured renal fibroblasts. 15 Furthermore, it has been reported that antagonism or deletion of the EP 1 receptor has therapeutic potential by reducing renal fibrosis in diabetic mice 16 and hypertensive rats. 17 In contrast, EP 1 deletion caused severe renal impairment in glomerulonephritic mice. 18 Thus, the role of the EP 1 receptor in kidney diseases remains unclear. Based on previous work, we hypothesize that EP 1 receptor antagonism will reduce renal fibrosis.
In this study, we used a translational approach to investigate the impact of SC-19220, an EP 1 receptor antagonist, on renal fibrosis. To this end, we used a combination of well-known in vitro and in vivo fibrosis models as well as a novel ex vivo fibrosis model, namely human precision-cut kidney slices (PCKS). This unique model is suitable for studying multicellular (pathological) processes, including fibrosis, directly in human kidney tissue as cellular heterogeneity and organ architecture is preserved in the slices. 19,20 Moreover, we studied the impact of SC-19220 on the late stage of fibrogenesis using PCKS prepared from patients with established renal fibrosis.

| EP 1 receptor expression is not altered in response to UUO in mice
First, we set out to evaluate the potential anti-fibrotic effect of SC-19220 in vivo. To this end, we first investigated whether UUO affected the gene expression of the EP receptors using qPCR and protein expression of the EP 1 receptor using WB and IHC. After 7 days of UUO, mRNA levels of both EP 2 and EP 4 markedly increased in UUO mice ( Figure 1A), in line with the previous observations. 9 Conversely, both mRNA and protein levels of the EP 1 receptor were unchanged as compared to sham mice ( Figure 1A,B). Immunofluorescence staining of the EP 1 receptor showed labelling in the collecting ducts (CDs) and thick ascending limbs (TALs; Figure 1D). Additionally, the EP 1 receptor was detected in the glomeruli ( Figure 1D). No obvious differences in staining intensity of the EP 1 receptor between sham and UUO mice were observed ( Figure 1C, visual inspection).

| EP 1 receptor antagonism ameliorates UUO-induced fibrosis
To determine the effect of the EP 1 receptor antagonist SC-19220 on extracellular matrix (ECM) deposition, we determined the expression of FN, αSMA, Collagen 1a1 and Collagen 3a1. As shown in Figure 2, gene expression of all four markers increased after UUO. However, we did not observe a statistically significant decline in transcript levels following SC-19220 treatment. SC-19220 markedly reduced UUO-induced FN and αSMA protein expression, as shown by both WB (Figure 3A,B) and IHC ( Figure 3C). In addition, UUO resulted in tubular dilation, which was partially blocked by EP 1 receptor antagonism ( Figure 4A). Moreover, we observed a clear increase in interstitial collagen deposition in UUO mice as shown by both Sirius red ( Figure 4B) and Masson's trichrome ( Figure 4C) staining, which was significantly reduced by treatment with SC-19220 ( Figure 4B-E). Taken together, these results indicate that inhibition of the EP 1 receptor can attenuate UUOinduced fibrogenesis on the protein level in UUO mice.

| Effect of EP 1 inhibition on renal functional parameters in mice subjected to 7dUUO
To determine whether inhibition of the EP 1 receptor would affect renal function in UUO mice, we evaluated kidney F I G U R E 1 Renal EP 1 receptor expression and localization does not change following UUO. (A) EP receptor gene expression was studied by qPCR. Relative expression was calculated using the reference gene 18S (n = 10). (B) EP 1 protein expression was studied using western blotting. Protein levels were calculated in relation to total protein (n = 10). Data are presented as means ± SEM. (C) Representative immunofluorescent images of EP 1 stainings of sham and UUO mice (n = 3). Scale bar is 50 µm. (D) Representative images of renal autofluorescence captured with the FITC imaging setting as well as immunofluorescent staining of EP 1 , Tamm Horsfall protein (THP) and aquaporin-2 (AQP2). Double stainings were EP 1 and THP (panel 2) and EP 1 and AQP2 (panels 1, 3 and 4). Stainings are shown in inverted contrast, whereas in the merged images, cell nuclei are shown in blue, EP 1 in red and autofluorescence as well as THP and AQP2 are shown in green. Scale bar is 20 μm weight, creatinine, blood urea nitrogen (BUN) and major electrolytes. The results are summarized in Table 1. After 7 days, the bodyweight of the animals was unchanged, and the mice showed no signs of poor health. As expected, there was an increase in the weight of the left obstructed kidney of mice subjected to UUO when compared to sham-operated mice. This effect was similar in mice exposed to SC-19220. BUN was increased in mice subjected to UUO compared to sham-operated animals. However, in mice treated with SC-19220, BUN was significantly different from sham. Despite this finding, it must be noted that the BUN value after UUO was similar with and without SC-19220. Plasma creatinine, sodium and potassium did not differ among the four groups (Table 1). Collectively, these results show that renal function and overall health was not affected by inhibition of the EP 1 receptor.

| Inhibition of the EP 1 receptor ameliorates early and late stage fibrosis in human renal tissue
Next, we evaluated whether inhibition of the EP 1 receptor could ameliorate fibrosis directly in human precision-cut kidney slices (PCKS). To this end, human PCKS were incubated with transforming growth factor-β (TGF-β, 10 ng/ mL) for 24 and 48 hours in the absence or presence of SC-19220 (225 µM).
We first investigated whether TGF-β treatment could change the gene expression of the EP 1 receptor in PCKS. As shown in Figure 5A, incubation of PCKS with TGF-β up to 48 hours did not significantly affect the expression of the EP 1 receptor. This finding was confirmed by visual inspections of immunofluorescent stainings where labelling F I G U R E 2 EP 1 receptor inhibition does not alter the gene expression of fibrotic markers in UUO mice. Mice were subject to 7 days of UUO and treated with SC-19220 (25 mg/kg) via IP injection. Afterwards, gene expression of (A) fibronectin (FN), (B) alpha-smooth muscle actin (αSMA), (C) collagen 1a1 (Col-1a1) and (D) collagen 3a1 (Col-3a1) was studied by qPCR. Relative expression was calculated using the reference gene 18S (n = 10). Data are presented as means ± SEM; ***P < .001 intensity of the EP 1 receptor in both TGF-β and control samples seemed identical ( Figure 5B).
Exposure to TGF-β induced fibrosis in human PCKS as illustrated by an increase in the gene expression of FN, αSMA and collagen 1a1 ( Figure 5C,D). Inhibition of the EP 1 receptor mitigated TGF-βinduced fibrogenesis, without affecting PCKS viability ( Figure 5C-E). We observed that the inhibitory effect of SC-19220 on TGF-βinduced fibrogenesis was similar both in the presence and absence of indomethacin. To further examine the effect of EP 1 receptor antagonism on collagen deposition in human PCKS, we performed Sirius red ( Figure 6A) and Masson's trichrome staining ( Figure 6B). Figure 6A-D shows increased collagen deposition upon TGF-β exposure, which could be attenuated by SC-19220 treatment.
Additionally, SC-19220 treatment reduced the expression of FN, αSMA and collagen 1a1 in PCKS prepared from patients with established renal fibrosis ( Figure 7). Taken together, these data indicate that antagonizing the EP 1 receptor with SC-19220 ameliorates early and late stage renal fibrosis in a translational model of human disease.

| TGF-βinduced epithelial de-differentiation is prevented by EP 1 receptor antagonism in MDCK cells
Epithelial plasticity is an essential part of renal fibrogenesis. 21 To mimic this process, we used TGF-β to induce F I G U R E 3 Inhibition of the EP 1 receptor reduces UUO-induced protein expression of fibrotic markers. Mice were subject to 7 days of UUO and treated with SC-19220 (25 mg/kg) via IP injection, followed by Western Blot analysis of fibrotic markers (A) fibronectin (FN) and (B) alpha-smooth muscle actin (αSMA) (n = 10). Data are presented as means ± SEM; *P < .05, **P < .01 and ***P < .001. (C) Representative immunohistochemistry images of FN (upper) and αSMA (lower) expression, 20× magnification, scale bar is 50 μm. n = 4 F I G U R E 4 EP 1 receptor antagonism mitigates UUO-induced renal injury. Mice were subject to 7 days of UUO and treated with SC-19220 (25 mg/kg) via IP injection. Afterwards, renal injury was assessed by IHC. Representative microscopy images of kidney tissue following (A) Mayers haematoxylin and eosin staining, (B) Sirius red staining, (C) Masson's trichrome staining; 20× magnification, scale bar is 50 μm. Quantification of (D) Sirius Red and (E) Masson's trichrome staining as a percentage of total area from eight pictures from each slice (n = 3-4). Data are presented as means ± SEM; *P < .05 compared to control, # P < .05 compared to UUO de-differentiation of MDCK cells. Subsequently, we evaluated the impact of SC-19220 on TGF-βinduced phenotypical changes. First, we confirmed the presence of the EP 1 receptor in MDCK cells by RT-PCR ( Figure 8A). Next, we wanted to ensure that the effects of SC-19220 did not interfere with endogenous PGE 2 production. Therefore, PGE 2 production was measured in MDCK cells in the presence or absence of the non-specific COX inhibitor indomethacin to eliminate endogenous PGE 2 production ( Figure 8B,C). In the absence of indomethacin, endogenous PGE 2 production was significantly increased when cells were exposed to TGF-β. Surprisingly, this response was completely abolished when cells were treated with the EP 1 receptor antagonist SC-19220, suggesting an EP 1 receptor-dependent feed-forward response of TGF-βinduced de novo prostaglandin synthesis. The addition of indomethacin markedly reduced endogenous PGE 2 production in all groups, short-circuiting both the effect of TGF-β and SC-19220 when using PGE 2 production as the read out.
As shown in Figure 8D, exposure of MDCK cells to TGF-β increased the protein expression of FN. This effect of TGF-β was concentration-dependently reduced by SC-19220. Additionally, indomethacin potentiated the effect of SC-19220, suggesting that other receptors than EP 1 are involved in the TGF-βdependent induction of FN expression. Moreover, in the presence of indomethacin, one can still observe a concentration-dependent reduction of FN expression, suggesting that there still is sufficient baseline PGE 2 production to support EP 1 receptor activation. In addition, light microscopy revealed that treatment with TGF-βinduced a phenotypic transition of MDCK cells, causing the cells to acquire a spindle-like morphology ( Figure 8E). Moreover, treatment of the cells with SC-19220 reduced TGF-βinduced epithelial de-differentiation ( Figure 8E). These findings demonstrate that EP 1 receptor antagonism can prevent the loss of epithelial characteristics.

| Influence of the EP 1 receptor on the intracellular Ca 2+ ([Ca 2+ ] i ) response in MDCK cells
It has been suggested that the EP 1 receptor can couple to both G q and G i . 22 It has previously been demonstrated that the EP 1 receptor contributes to fibrogenesis in cardiac fibroblasts via a Ca 2+ -dependent signalling pathway involving release from intracellular Ca 2+ stores. 23 Thus, one could speculate that inhibition of the EP 1 receptor mitigates TGF-βinduced fibrosis by interfering with intracellular Ca 2+ signalling. Therefore, we examined whether stimulating the EP 1 receptor with 17-phenyl trinor PGE 2 could trigger a [Ca 2+ ] i response in MDCK cells.
Our results demonstrated that the EP 1 receptor agonist 17-phenyl trinor PGE 2 potentiated TGF-βinduced fibrosis, as evaluated by FN protein levels, further supporting the notion that EP 1 receptor activity stimulates fibrogenesis. Interestingly, the pro-fibrotic effect of TGF-β in combination with 17-phenyl trinor PGE 2 was markedly reduced by SC-19220 treatment ( Figure 9A).
Next, we measured the effect of SC-19220 on the [Ca 2+ ] i response in MDCK cells in the absence or presence of TGFβ. To this end, cells were loaded with the fluorescent Ca 2+ probe Fluo-4 AM. As positive control for cell responsiveness, we used ATP (100 µM). ATP induced a brisk, transient increase in [Ca 2+ ] i , which is a trademark for G q coupled IP 3 -mediated Ca 2+ release from intracellular Ca 2+ stores mediated by the P2Y 2 receptor. Pre-incubation with TGF-β increased the ATP-induced [Ca 2+ ] i response ( Figure 9B). Our data showed that neither PGE 2 nor 17-phenyl trinor PGE 2 was able to elicit a [Ca 2+ ] i response resembling G q activation in MDCK cells, irrespective of whether the cells were incubated with or without TGF-β ( Figure 9C,D). Thus, we were unable to evaluate the effect of SC-19220 on EP 1 activation with [Ca 2+ ] i as readout. However, we did ascertain whether SC-19220 had any unexpected effects on [Ca 2+ ] i . Our data shows that SC-19220, at low, pharmacologically Abbreviations: BUN, blood urea nitrogen; K, potassium; Na, sodium.

ERK1/2 signalling in MDCK cells
In order to elucidate the mechanisms of action of SC-19220, we explored its impact on the TGF-β signalling pathway, which plays an important role in fibrogenesis. As shown in Figure 10A, the EP 1 receptor antagonist reduced TGF-β mRNA expression in MDCK cells. Next, we studied the expression of plasminogen activation inhibitor 1 (PAI-1) and the activation of Smad2, which are both major downstream elements in TGF-β signalling. TGF-β induced Smad2 phosphorylation and increased the expression of PAI-1, which was not impacted by SC-19220 treatment ( Figure 10B,C). Thus, it appears that the mechanism of action of SC-19220 in MDCK cells does not involve the Smad or PAI pathway ( Figure 10B,C). It has previously been shown that TGF-β can activate MAPK pathways in MCDK cells, which is an important intracellular signalling pathway in renal fibrosis. 24 So, we examined whether the anti-fibrotic effect of SC-19220 could be mediated via the MAPK signalling pathway. Our results revealed that inhibition of the EP 1 receptor suppressed TGF-βinduced phosphorylation of ERK1/2 whereas no effect was observed on p38 ( Figure 10B,C). Taken together, these results indicate that EP 1 receptor antagonism with SC-19220 mitigates fibrogenesis by reducing ERK1/2 signalling.

| Inhibition of the EP 1 receptor reduces matrix formation
Lastly, we set out to confirm our observations in human renal fibroblasts-key cellular players in the fibrotic process. As shown in Figure 11A, EP 1 , EP 2 and EP 4 are all expressed in HRFs. Moreover, exposure to TGF-β markedly increased the protein expression of FN, which was mitigated by SC-19220 treatment ( Figure 11B). In addition, the anti-fibrotic effect of EP 1 antagonism was not affected by PGE 2 .

| DISCUSSION
Preclinical studies have identified a number of approaches to either stop or reverse the progression of renal fibrosis. 25 However, none of these treatments have been successfully implemented in the clinic. 26 The availability of good translational models of human fibrosis, which can be used to directly validate preclinical findings, would improve drug development. In this study, we investigated the antifibrotic properties of a specific EP 1 receptor antagonist with a translational approach utilizing well-established in vitro and in vivo fibrosis models as well as an ex vivo human model of renal fibrosis, namely human (fibrotic) PCKS. Thus, we were able to validate our findings directly in a human model. By using these models, we show that antagonizing the EP 1 receptor has considerable antifibrotic effects.
In the current study, EP 1 antagonism alleviates kidney fibrogenesis in MDCK cells, HRFs, UUO mice as well as in human PCKS. To our knowledge, our work is the first to confirm that pharmacological inhibition of the EP 1 receptor using SC-19220 prevents the progression of fibrosis in a human renal fibrosis model. We and others have previously confirmed that other EP receptors, including EP 2 and EP 4 play significant roles in preventing kidney fibrosis. 15,27 Recently, Jensen et al showed that activation of the EP 2 receptor with butaprost mitigates fibrogenesis in human PCKS, 14 indicating that targeting the EP 2 receptor also directly prevents renal fibrosis in human kidney slices.
In the clinic, renal fibrosis is often diagnosed when the disease is in its final stages, making treatment even more difficult. Here, we show that EP 1 receptor antagonism might also be beneficial for the treatment of established fibrosis, further supporting the notion that the EP 1 receptor is a clinically relevant therapeutic target.
Several in vitro studies have demonstrated that EP 1 receptor activation can cause detrimental responses in different renal cell types, including mesangial cells, proximal tubule cells and podocytes. 16,28,29 In addition, in vivo studies have shown that a lack of the EP 1 receptor or EP 1 antagonism protects against hyperfiltration, albuminuria, and reduces injury/fibrotic markers in spontaneously hypertensive rats 17,30 and in diabetic mice models. 16 Conjointly, these data suggest that the EP 1 receptor mediates many pathologic effects in different kidney diseases, highlighting the importance of this receptor in renal diseases. Thus, the anti-fibrotic effect of EP 1 antagonism shown here corroborates previous work.
To eliminate the possibility that endogenous prostaglandins elicited the observed anti-fibrotic effects via the EP 2 or the EP 4 receptor, we performed both our in vitro and ex vivo experiments in the absence and presence of the non-specific COX inhibitor, indomethacin. MDCK cells exposed to TGF-β in the absence of indomethacin showed dramatically increased levels of PGE 2 , in line with previous studies showing autoamplification of PGE 2 through the EP 1 receptor. 31 Moreover, indomethacin lowered endogenous PGE 2 production considerably; however, there is still sufficient baseline PGE 2 production to support EP 1 receptor activation. Therefore, the anti-fibrotic effect of SC-19220 might be because of its impact on the EP 1 receptor, illustrating that the PGE 2 receptors are promising therapeutic targets for the treatment of fibrosis, in line with the study of Jensen and colleagues. 14 Based on our work, and that of others, it is clear that PGE 2 signalling can be pro-or anti-fibrotic depending on which receptor is activated. 16,29,32,33 Our results did not reveal how EP 1 receptor antagonism impacted signalling via EP 2 and EP 4 during fibrogenesis. In order to fully unravel the role of PGE 2 in human renal fibrosis, future studies are needed that can show which of the four PGE 2 receptors is dominant during the fibrotic process. It is highly likely that the net effect of PGE 2 on fibrosis will depend greatly on disease aetiology as well as cell-and tissue-specific expression of the PGE 2 receptors. Thus, even though our study provides another piece of the puzzle, the complex F I G U R E 1 0 Anti-fibrotic effect of SC-19220 in MDCK cells involves ERK1/2 signalling. (A) MDCK cells were exposed to TGF-β (10 ng/ mL) treatment in the presence or absence of the EP 1 antagonist SC-19220 (225 µM) for 48 hours. TGF-β mRNA expression was studied by qPCR. Relative expression was calculated using the reference gene GAPDH (n = 6). (B) Representative Western Blots of proteins related to SMAD and MAPK signalling pathways. (C) Quantification of protein expression relative to control (n = 6). Data are presented as means ± SEM *P < .05 compared to control, # P < .05 compared to TGF-β and dual role of PGE 2 in renal fibrosis requires further scrutiny.
Stimulation of the EP 1 receptor is known to increase intracellular Ca 2+ concentration by a G q dependent IP 3 mobilization. 34,35 As seen in Figure 9, we were unable to elicit abrupt G q dependent increases in [Ca 2+ ] i with neither PGE 2 nor 17-phenyl trinor PGE 2 . Together, our results indicated that the anti-fibrotic effect of SC-19220 is independent of [Ca 2+ ] i signalling, suggesting that other signalling pathways must be involved. Previous studies have demonstrated that TGF-β can induce phenotypical changes via Smad-2/3, PAI-1, ERK1/2 and p38 MAPK signalling pathways in MDCK cells. 24,36 In line with this, our data revealed that TGF-β stimulates phosphorylation of Smad2, ERK1/2 and p38 MAPK as well as induces PAI-1 expression. SC-19220 clearly inhibited the phosphorylation of ERK1/2 but had no effect on the phosphorylation of Smad2 and p38 MAPK nor on the expression of PAI-1.
This observation is in line with the study by Chen et al, showing that TGF-βinduced activation of the ERK1/2 MAPK pathway can be alleviated by EP 1 knockdown and stimulated by EP 1 expression in mesangial cells. 28 These findings support the notion that the EP 1 receptor might contribute to fibrogenesis via activation of the ERK1/2 signalling pathway.
In this study, we observed that SC-19220 mitigated TGF-βinduced phenotypical changes in MDCK cells. The loss of epithelial characteristics upon TGF-β exposure is well-documented and is linked to the progression of renal fibrosis. 37 For decades, it was believed that epithelial cells could convert to collagen-producing myofibroblasts via a process called epithelial-to-mesenchymal transition. However, lineage-tracing studies and singlecell RNA sequencing has revealed that only 1%-5% of dedifferentiated epithelial cells ultimately contribute to ECM production. 38,39 Nevertheless, the observed phenotypical changes might reflect a metabolic reprogramming, especially related to fatty acid oxidation, which can contribute to fibrogenesis. 37,39 Further studies are needed to reveal whether EP 1 receptor antagonism can indeed restore fatty acid metabolism in TGF-β exposed cells.
Gene and protein expression of the EP 1 receptor does not seem to be affected after 7 days of UUO. This is consistent with a previous study by Sun et al, showing that seven days of UUO in mice did not change the expression of the EP 1 receptor either at the mRNA or protein level. 40 However, we have previously demonstrated that UUO increased EP 1 receptor mRNA expression in wildtype and COX-2 KO mice. 9 In this study, we used another strain of mice, possibly altering the phenotype for this particular outcome. Consistent with previous studies, the EP 1 receptor was mainly found in cells of the collecting ducts 41 and TAL. 42 In addition, the EP 1 receptor was also detected in glomeruli. 43,44 A limitation of this study is that we administered SC-19220 via IP injection, whereas oral delivery of an EP 1 receptor antagonist would most likely be needed for treatment in humans. However, it is generally considered that the pharmacokinetics of small molecular drugs administered IP are fairly similar to those seen after oral administration, because the primary route of absorption is into the mesenteric vessels, which drain into the portal vein and pass through the liver, thus the compounds are subjected to first pass metabolism. 45,46 Moreover, from a technical point of view, IP delivery is easy, reproducible, ensures therapeutic bioavailability, allows for repeated treatment and is generally safer and less stressful for the animals as compared to other delivery methods. Thus, we believe that this route of administration is justifiable for preclinical drug efficacy studies. Moreover, most preclinical studies only use cell and animal models. By using human F I G U R E 1 1 Impact of SC-19220 treatment in human renal fibroblasts. (A) EP receptor mRNA expression was studied in HRFs using RT-PCR with (+) and without (−) reverse transcriptase (RT) enzyme. (B) HRFs were exposed to TGF-β (10 ng/mL) in the presence or absence of the EP 1 antagonist SC-19220 (225 µM) and PGE 2 (1 µM) for 48 hours. Fibronectin (FN) protein expression was studied using Western Blotting. Protein levels were calculated in relation to total protein (n = 6). Data are presented as means ± SEM; # P < .05 compared to TGF-β (fibrotic) kidney slices we have already greatly improved the clinical translatability of our findings.
In conclusion, this study indicates that the EP 1 receptor might be regarded as a potential target for the treatment of renal fibrosis. The present study provides strong evidence that the effect of the EP 1 antagonist SC-19220 may translate to clinical care since its effects on fibrosis is demonstrated in both UUO mice as well as human kidney slices.

| Ethics statement
The procedures described below were performed in concordance with the Danish national guidelines for animal care and the published guidelines of the National Institutes

| Experimental animals and surgical procedures
Experiments were performed using male C57BL/6 mice, 7-8 weeks of age and weighing 20.1 ± 1.9 g (Janvier Labs, Le Genest-Saint-Isle, France). Animals were housed with a 12 h:12 h light-dark cycle, a temperature of 21 ± 2°C and a humidity of 55 ± 2%. Animals had ad libitum access to standard rodent chow (Altromin, Lage, Germany) and tap water. Animals were allowed to acclimatize 7 days before surgery. A preliminary dose-response study, using SC-19220 (5, 10 and 25 mg/kg, Cayman Chemical, Michigan, USA) was performed on 3-4 animals per group. Since 25 mg/kg was most effective in lowering the expression of αSMA and FN in the pilot study ( Figure S1), this dose was validated in a larger cohort. In the subsequent study, C57BL/6 male mice were allocated into the following experimental groups: SHAM operated mice (n = 14, 10 kidneys were used for qPCR and WB, and 4 kidneys were used for IHC), SHAM operated mice treated with 25 mg/kg of SC-19220 (n = 10, 10 kidneys were used for qPCR and WB), UUO (n = 14, 10 kidneys were used for qPCR and WB and 4 kidneys were used for IHC) and UUO operated mice treated with 25 mg/ kg once a day (n = 14, 10 kidneys were used for qPCR and WB and 4 kidneys were used for IHC).
On the day of surgery, mice were anaesthetized with 2% Sevoflurane (Abbott Scandinavia AB, Solna, Sweden) mixed with atmospheric air at 2 L/min, and injected with buprenorphine (Temgesic, Indivior UK Limited, Berkshire, UK). A midline incision was made and the left ureter was located and occluded with a 6-0 silk ligature. The EP 1 receptor antagonist SC-19220 was diluted in saline and administered once daily via intraperitoneal injection starting at the day of the surgery. Buprenorphine was added to the drinking water to maintain analgesia for 48 hours post-surgery. After 7 days of UUO, blood samples were collected by cardiac puncture, the kidneys were collected, and the mice were sacrificed by cervical dislocation. Biochemical analysis of blood samples was performed on a Roche Cobas 6000 analyzer (Roche Diagnostic, Rotkreuz, Switzerland) and creatinine levels were determined using a creatinine assay kit (Sigma-Aldrich, Missouri, USA), according to the manufacturer's instructions.

| Human precision-cut kidney slices
PCKS were prepared from functional (eGFR >60 mL/ min/1.73 cm 2 ) and macroscopically healthy renal cortical tissue obtained from both male and female patients following tumour nephrectomies as described previously. 14 In addition, PCKS were also prepared from human fibrotic kidneys (see Table 2 for patient demographics). In short, tissue samples were obtained using a 6 mm biopsy punch (Kai Medical, Japan), and slices were prepared in ice-cold Krebs-Henseleit buffer (25 mM D-glucose, 25 mM NaHCO 3 , 10 mM HEPES, saturated with 95% O 2 and 5% CO 2 ) using the Krumdieck Tissue slicer. Slices were cultured in William's medium E containing GlutaMAX, 10 mg/mL ciprofloxacin and 2.7 g/L D-(+)-Glucose in an 80% O 2 , 5% CO 2 atmosphere at 37°C. The slices were kept in constant moderate motion and the medium

| Western blotting
Total protein from mouse cortex was extracted using RIPA buffer including phosphatase inhibitor 2 and 3 (Sigma-Aldrich, St. Louis, MO, USA) and a protease inhibitor cocktail tablet (Roche Diagnostic, Rotkreuz, Switzerland) and total protein from cell experiments was extracted using M-PER including 2% SDS and DTT. Protein was then separated on a 12% Criterion TGX Stain-free gel and proteins were transferred to a nitrocellulose membrane, which was blocked with 5% skimmed milk in PBS-Tween. The membrane was washed in PBS-Tween and incubated with specific primary antibodies (see Table 3 for target and dilution). Next, the membrane was washed with PBS-Tween and incubated with the appropriate secondary antibody. Finally, the membrane was incubated with the detection reagent ECL-Prime (GE-healthcare, Chicago, IL, USA) and processed in the Western Blot Imager (ChemiDoc MP, Bio-Rad, CA, USA). Detected protein was normalized against total protein levels. 47 To confirm the expression of the EP receptors in MDCK cells and HRFs, RT-PCR was performed either in the presence (+) or absence (−) of reverse transcriptase. To visualize the PCR product, electrophoresis was performed using a 1% agarose gel including the Genruler DNA marker (Invitrogen, CA, USA) and images were obtained on an Azure c200 gel imaging workstation.

| Perfusion fixation and immunolabelling
For the preparation of tissue slices for histological analysis and IHC, we performed whole animal perfusion with subsequent immersion fixation (renal tissue) or immersion fixation (PCKS). Kidneys were fixed by perfusion via the left ventricle using 4% paraformaldehyde (PFA). Afterwards, the kidney was removed and immersed in 4% PFA for one hour, rinsed with PBS and dehydrated in a series of alcohol and embedded in paraffin. Tissue sections (2 and 5 μm) were deparaffinized, rehydrated and rinsed. Then, they were blocked with 1.5 mL 35% hydrogen peroxide (H 2 O 2 ) in methanol for 30 minutes. For epitope retrieval, sections were boiled in TEG buffer (1 mM Tris, 0.5 mM ETA, pH of 9.0) then left to cool and blocked with 50 mM NH 4 Cl in PBS. Sections were then incubated with primary antibodies (EP 1 receptor and FN for immunoperoxidase, see Table 3 for target and dilution) diluted in PBS containing 0.1% BSA and 0.3% Triton X100 for 1 hour at room temperature in a humidity chamber, followed by overnight incubation at 4°C. The sections were rinsed three times with PBS containing 0.1% BSA, 0.05% saponin and 0.2% gelatine followed by incubation with a secondary antibody (P448, diluted 1:300 in washing solution) for one hour at room temperature. Afterwards, sections were rinsed three times with washing solution and incubated with 3,3'diaminobenzidine tetrachloride (DAB) dissolved in water containing 0.1% H 2 O 2 to visualize the sites of antibody-antigen reactions. Light microscopy was carried out with an Olympus BX50 light microscope and images were processed using CellSens imaging software.
For immunofluorescence labelling of αSMA, sections were incubated with mouse-on-mouse blocking solution containing unconjugated AffiniPure Fab Fragment Donkey Anti-Mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) in PBS for one hour at room temperature and then post-fixed for 10 minutes in 4% PFA. Sections were incubated overnight at 4°C with the primary antibody αSMA diluted in PBS containing 0.1% BSA and 0.3% Triton X100. Hereafter, sections were washed for 30 minutes in PBS with 0.1% BSA, 0.2% gelatine and 0.05% saponin and then incubated with Alexa Flour 488-conjugated secondary antibody (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 30 minutes. Then, counterstaining with 4,6-diamindino-2-phenylindole (DAPI; diluted 1:200 in PBS, Thermo Fisher, D1306) was carried out and the sections were rinsed with PBS and mounted with SlowFade Gold Antifade Mountant (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence microscopy was carried out using an Olympus BX61 microscope and the images were processed in Xcellence Rt software.
For immunofluorescent labelling of paraffin sections for EP 1 and markers for TAL and CDs, labelling and imaging was carried our as previously described. 48 In brief, 2 µm paraffin sections were deparaffinized overnight in xylene and subsequently rehydrated. Antigens were retrieved by boiling in TEG buffer (10 mM Tris pH 9, 0.5 mM EGTA) and aldehyde groups quenched by incubation in 50 mM NH4Cl in PBS for 30 minutes. Sections were blocked and permeabilized in 1% BSA, 0.2% gelatin and 0.05% saponin in PBS and labelled with rabbit-anti EP 1 , followed by incubation with Alexa Fluor-647-conjugated antibody donkeyanti-rabbit and 2 μg/mL Hoechst33342. The sections were washed in PBS and mounted using glycergel mounting medium. For double stainings, goat-anti-AQP2 (sc-9880 Santa Cruz Biotechnology) and sheep-anti-TAM (AB733 Megapixel camera (Andor). The system was controlled by NIS Elements software from Nikon. The fluorescence illumination system was Cool LED-pE-300 white, and fluorescence filter sets were standard DAPI, GFP, TxRed and Cy5. Image analysis was performed using ImageJ Fiji software. 49 Background and contrast were adjusted liniary. Additionally, sections were stained with Mayers Hematoxylin for visualization of tubular dilation, and Picro Sirius Red and Masson's trichrome to visualize collagen deposition. The latter two stains were quantified in the mice study by capturing eight pictures from each slide at ×20 magnification and in the human slices by capturing five pictures from each slide at ×40 magnification. The fibrotic area was measured as percentage of total tissue area in ImageJ software. Light microscopy was carried out with the Olympus BX50 light microscope and the CellSens imaging software.

| Statistics
Data were analysed in GraphPad Prism. Data with two or more parameters were analysed by two-way ANOVA 5′-CTGTGAGCCTCTGCTCCTG-3′ 5′-GACAGCCAGCACCACTAACA-3′ T A B L E 4 Primers used for qPCR followed by Tukey's or Bonferroni's post-hoc comparison. When data comprised of one parameter, One-way ANOVA followed by Tukey's post hoc test or two-tailed Students t-test were used. Results were considered to be statistically significant when P < .05.