Regulation of renal aquaporin water channels in acute pyelonephritis.

Acute pyelonephritis (APN) is most frequently caused by uropathogenic Escherichia coli (UPEC), which ascends from the bladder to the kidneys during a urinary tract infection. Patients with APN have been reported to have reduced renal concentration capacity under challenged conditions, polyuria, and increased aquaporin-2 (AQP2) excretion in the urine. We have recently shown increased AQP2 accumulation in the plasma membrane in cell cultures exposed to E. coli lysates and in the apical plasma membrane of inner medullary collecting ducts in a 5-day APN mouse model. This study aimed to investigate if AQP2 expression in host cells increases UPEC infection efficiency and to identify specific bacterial components that mediate AQP2 plasma membrane insertion. As the transepithelial water permeability in the collecting duct is codetermined by AQP3 and AQP4, we also investigated whether AQP3 and AQP4 localization is altered in the APN mouse model. We show that AQP2 expression does not increase UPEC infection efficiency and that AQP2 was targeted to the plasma membrane in AQP2-expressing cells in response to the two pathogen-associated molecular patterns (PAMPs), lipopolysaccharide and peptidoglycan. In contrast to AQP2, the subcellular localizations of AQP1, AQP3, and AQP4 were unaffected both in lysate-incubated cell cultures and in the APN mouse model. Our finding demonstrated that cellular exposure to lipopolysaccharide and peptidoglycan can trigger the insertion of AQP2 in the plasma membrane revealing a new regulatory pathway for AQP2 plasma membrane translocation, which may potentially be exploited in intervention strategies.NEW & NOTEWORTHY Acute pyelonephritis (APN) is associated with reduced renal concentration capacity and increased aquaporin-2 (AQP2) excretion. Uropathogenic Escherichia coli (UPEC) mediates changes in the subcellular localization of AQP2 and we show that in vitro, these changes could be elicited by two pathogen-associated molecular patterns (PAMPs), namely, lipopolysaccharide and peptidoglycan. UPEC infection was unaltered by AQP2 expression and the other renal AQPs (AQP1, AQP3, and AQP4) were unaltered in APN.

Recovery of Water Homeostasis in Adenine-Induced Kidney Disease Is Mediated by Increased AQP2 Membrane Targeting.

Chronic kidney disease (CKD) represents a major public health burden with increasing prevalence. Current therapies focus on delaying CKD progression, underscoring the need for innovative treatments. This necessitates animal models that accurately reflect human kidney pathologies, particularly for studying potential reversibility and regenerative mechanisms, which are often hindered by the progressive and irreversible nature of most CKD models. In this study, CKD was induced in mice using a 0.2% adenine-enriched diet for 4 weeks, followed by a recovery period of 1 or 2 weeks. The aim was to characterize the impact of adenine feeding on kidney function and injury as well as water and salt homeostasis throughout disease progression and recovery. The adenine diet induced CKD is characterized by impaired renal function, tubular injury, inflammation, and fibrosis. A significant decrease in urine osmolality, coupled with diminished aquaporin-2 (AQP2) expression and membrane targeting, was observed after adenine treatment. Intriguingly, these parameters exhibited a substantial increase after a two-week recovery period. Despite these functional improvements, only partial reversal of inflammation, tubular damage, and fibrosis were observed after the recovery period, indicating that the inclusion of the molecular and structural parameters is needed for a more complete monitoring of kidney status.

P2X7 accelerate tissue fibrosis via metalloproteinase 8-dependent macrophage infiltration in a murine model of unilateral ureteral obstruction.

Renal fibrosis is tightly associated with chronic kidney disease, irrespective of the underlying pathogenesis. We previously demonstrated mild antifibrotic effects of targeting the P2X7 receptor in a pyelonephritis model. Reduced P2X7 R-activation elevated the neutrophil-to-macrophage ratio, resulting in less matrix accumulation without affecting the initial tissue healing. Here, we test if this P2X7 R-dependent modification of matrix accumulation also applies to a noninfectious fibrosis model of unilateral ureteral obstruction (7dUUO) and whether the response is gender-dependent. We found that P2X7 -/- mice show reduced fibrosis compared to wild type after 7dUUO: the effect was most pronounced in females, with a 55% decrease in collagen deposition after 7dUUO (p < 0.0068). P2X7 R deficiency did not affect early fibrosis markers (TGF-ß, α-SMA) or the renal infiltration of neutrophils. However, a UUO-induced increase in macrophages was observed in wildtypes only (p < 0.001), leaving the P2X7 -/- mice with ≈50% fewer CD68+ cells in the renal cortex (p = 0.018). In males, 7dUUO triggered an increase in diffusely interstitial scattering of the profibrotic, macrophage-attracting metalloproteinase MMP8 and showed significantly lower MMP8 tissue expression in both male and female P2X7 -/- mice (p < 0.0008). Thus, the P2X7 R is advocated as a late-stage fibrosis moderator by reducing neutrophil-dependent interstitial MMP8 release, resulting in less macrophage infiltration and reduced matrix accumulation.

Does the route matter? A preclinical review of mesenchymal stromal cell delivery to the kidney.

Mesenchymal stromal/stem cell (MSC) therapy has been thoroughly tested in preclinical animal models and holds great promise for the treatment of kidney diseases. It is becoming increasingly evident that the efficacy of MSC therapy is dependent on several factors including dosage, the tissue source of MSCs, the route of delivery and timing of administration. In a time where MSC therapy is moving from preclinical research to clinically therapeutic use, the importance of choice of delivery method, modality, and administration route increases. In this review, we provide an overview of the different MSC delivery routes used in preclinical kidney disease models, highlight the recent advances in the field, and summarize studies comparing delivery routes of MSCs to the kidney.

Obstructive nephropathy and molecular pathophysiology of renal interstitial fibrosis.

The kidneys play a key role in maintaining total body homeostasis. The complexity of this task is reflected in the unique architecture of the organ. Ureteral obstruction greatly affects renal physiology by altering hemodynamics, changing glomerular filtration and renal metabolism, and inducing architectural malformations of the kidney parenchyma, most importantly renal fibrosis. Persisting pathological changes lead to chronic kidney disease, which currently affects ∼10% of the global population and is one of the major causes of death worldwide. Studies on the consequences of ureteral obstruction date back to the 1800s. Even today, experimental unilateral ureteral obstruction (UUO) remains the standard model for tubulointerstitial fibrosis. However, the model has certain limitations when it comes to studying tubular injury and repair, as well as a limited potential for human translation. Nevertheless, ureteral obstruction has provided the scientific community with a wealth of knowledge on renal (patho)physiology. With the introduction of advanced omics techniques, the classical UUO model has remained relevant to this day and has been instrumental in understanding renal fibrosis at the molecular, genomic, and cellular levels. This review details key concepts and recent advances in the understanding of obstructive nephropathy, highlighting the pathophysiological hallmarks responsible for the functional and architectural changes induced by ureteral obstruction, with a special emphasis on renal fibrosis.

Standardized Protocol for the Preparation of Precision-Cut Kidney Slices: A Translational Model of Renal Fibrosis.

Renal fibrosis is a hallmark of progressive renal diseases. To date, there is a lack of effective therapeutics for the treatment of renal fibrosis, in part due to the scarcity of clinically relevant translational disease models. Since the early 1920s, hand-cut tissue slices have been used as a means to better understand organ (patho)physiology in a variety of scientific fields. From that time, the equipment and methodology for the preparation of tissue slices has continuously improved, thereby expanding the applicability of the model. Nowadays, precision-cut kidney slices (PCKS) have been demonstrated to be an extremely valuable translation model for renal (patho)physiology, bridging the gap between preclinical and clinical research. A key feature of PCKS is that the slices contain all cell types and acellular components of the whole organ in the original configuration while preserving cell-cell and cell-matrix interactions. In this chapter, we describe how to prepare PCKS and how the model can be implemented in fibrosis research.

Stimulation of the Hydroxycarboxylic Acid Receptor 2 With the Ketone Body 3-Hydroxybutyrate and Niacin in Patients With Chronic Heart Failure: Hemodynamic and Metabolic Effects.

Background The ketone body 3-hydroxybutyrate (3-OHB) increases cardiac output (CO) in patients with heart failure through unknown mechanisms. 3-OHB activates the hydroxycarboxylic acid receptor 2 (HCA2), which increases prostaglandins and suppresses circulating free fatty acids. We investigated whether the cardiovascular effects of 3-OHB involved HCA2 activation and if the potent HCA2-stimulator niacin may increase CO. Methods and Results Twelve patients with heart failure with reduced ejection fraction were included in a randomized crossover study and examined by right heart catheterization, echocardiography, and blood sampling on 2 separate days. On study day 1, patients received aspirin to block the HCA2 downstream cyclooxygenase enzyme, followed by 3-OHB and placebo infusions in random order. We compared the results with those of a previous study in which patients received no aspirin. On study day 2, patients received niacin and placebo. The primary end point was CO. 3-OHB increased CO (2.3 L/min, P<0.01), stroke volume (19 mL, P<0.01), heart rate (10 bpm, P<0.01), and mixed venous saturation (5%, P<0.01) with preceding aspirin. 3-OHB did not change prostaglandin levels, neither in the ketone/placebo group receiving aspirin nor the previous study cohort. Aspirin did not block 3-OHB-induced changes in CO (P=0.43). 3-OHB decreased free fatty acids by 58% (P=0.01). Niacin increased prostaglandin D2 levels by 330% (P<0.02) and reduced free fatty acids by 75% (P<0.01) but did not affect CO. Conclusions The acute increase in CO during 3-OHB infusion was not modified by aspirin, and niacin had no hemodynamic effects. These findings show that HCA2 receptor-mediated effects were not involved in the hemodynamic response to 3-OHB. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT04703361.

Transcutaneous measurement of renal function in two rodent models of obstructive nephropathy.

OBJECTIVE: Glomerular filtration rate (GFR) is a key indicator of renal function. In both clinical practice and pre-clinical research, serum levels of endogenous filtration markers, such as creatinine, are often used to estimate GFR. However, these markers often do not reflect minor changes in renal function. In this study, we therefore set out to evaluate the applicability of transcutaneous GFR (tGFR) measurements to monitor the changes in renal function, as compared to plasma creatinine (pCreatinine), in two models of obstructive nephropathy, namely unilateral ureteral obstruction (UUO) or bilateral ureteral obstruction followed by release (BUO-R) in male Wistar rats. RESULTS: UUO animals showed a significant reduction in tGFR compared to baseline; whereas pCreatinine levels were not significantly changed. In BUO animals, tGFR drops 24 h post BUO and remains lower upon release of the obstruction until day 11. Concomitantly, pCreatinine levels were also increased 24 h after obstruction and 24 h post release, however after 4 days, pCreatinine returned to baseline levels. In conclusion, this study revealed that the tGFR method is superior at detecting minor changes in renal function as compared to pCreatinine measurements.

Tamoxifen Affects Aquaporin-3 Expression and Subcellular Localization in Rat and Human Renal Collecting Ducts.

Sex hormones play an important role in the regulation of water homeostasis, and we have previously shown that tamoxifen (TAM), a selective estrogen receptor modulator (SERM), affects the regulation of aquaporin (AQP)-2. In this study, we investigated the effect of TAM on the expression and localization of AQP3 in collecting ducts using various animal, tissue, and cell models. The impact of TAM on AQP3 regulation was studied in rats subjected to 7 days of unilateral ureteral obstruction (UUO), with the rats fed a lithium-containing diet to induce nephrogenic diabetes insipidus (NDI), as well as in human precision-cut kidney slices (PCKS). Moreover, intracellular trafficking of AQP3 after TAM treatment was investigated in Madin-Darby Canine Kidney (MDCK) cells stably expressing AQP3. In all models, the expression of AQP3 was evaluated by Western blotting, immunohistochemistry and qPCR. TAM administration attenuated UUO-induced downregulation of AQP3 and affected the localization of AQP3 in both the UUO model and the lithium-induced NDI model. In parallel, TAM also affected the expression profile of other basolateral proteins, including AQP4 and Na/K-ATPase. In addition, TGF-ß and TGF-ß+TAM treatment affected the localization of AQP3 in stably transfected MDCK cells, and TAM partly attenuated the reduced AQP3 expression in TGF-ß exposed human tissue slices. These findings suggest that TAM attenuates the downregulation of AQP3 in a UUO model and a lithium-induced NDI model and affects the intracellular localization in the collecting ducts.

Endotrophin Levels Are Associated with Allograft Outcomes in Kidney Transplant Recipients.

Early prediction of kidney graft function may assist clinical management, and for this, reliable non-invasive biomarkers are needed. We evaluated endotrophin (ETP), a novel non-invasive biomarker of collagen type VI formation, as a prognostic marker in kidney transplant recipients. ETP levels were measured with the PRO-C6 ELISA in the plasma (P-ETP) of 218 and urine (U-ETP/Cr) of 172 kidney transplant recipients, one (D1) and five (D5) days, as well as three (M3) and twelve (M12) months, after transplantation. P-ETP and U-ETP/Cr at D1 (P-ETP AUC = 0.86, p < 0.0001; U-ETP/Cr AUC = 0.70, p = 0.0002) were independent markers of delayed graft function (DGF) and P-ETP at D1 had an odds ratio of 6.3 (p < 0.0001) for DGF when adjusted for plasma creatinine. The results for P-ETP at D1 were confirmed in a validation cohort of 146 transplant recipients (AUC = 0.92, p < 0.0001). U-ETP/Cr at M3 was negatively associated with kidney graft function at M12 (p = 0.007). This study suggests that ETP at D1 can identify patients at risk of delayed graft function and that U-ETP/Cr at M3 can predict the future status of the allograft. Thus, measuring collagen type VI formation could aid in predicting graft function in kidney transplant recipients.

Multiplex immunofluorescence staining of coverslip-mounted paraffin-embedded tissue sections.

Animal and human tissues are used extensively in physiological and pathophysiological research. Due to both ethical considerations and low availability, it is essential to maximize the use of these tissues. Therefore, the aim was to develop a new method allowing for multiplex immunofluorescence (IF) staining of kidney sections in order to reuse the same tissue section multiple times. The paraffin-embedded kidney sections were placed onto coated coverslips and multiplex IF staining was performed. Five rounds of staining were performed where each round consisted of indirect antibody labelling, imaging on a widefield epifluorescence microscope, removal of the antibodies using a stripping buffer, and then re-staining. In the final round, the tissue was stained with hematoxylin/eosin. Using this method, tubular segments in the nephron, blood vessels, and interstitial cells were labeled. Furthermore, by placing the tissue on coverslips, confocal-like resolution was obtained using a conventional widefield epifluorescence microscope and a 60x oil objective. Thus, using standard reagents and equipment, paraffin-embedded tissue was used for multiplex IF staining with increased Z-resolution. In summary, this method offers time-saving multiplex IF staining and allows for the retrieval of both quantitative and spatial expressional information of multiple proteins and subsequently for an assessment of the tissue morphology. Due to the simplicity and integrated effectivity of this multiplex IF protocol, it holds the potential to supplement standard IF staining protocols and maximize use of tissue.

An animal-free preclinical drug screening platform based on human precision-cut kidney slices.

OBJECTIVE: Renal fibrosis is one of the main pathophysiological processes underlying the progression of chronic kidney disease and kidney allograft failure. In the past decades, overwhelming efforts have been undertaken to find druggable targets for the treatment of renal fibrosis, mainly using cell- and animal models. However, the latter often do not adequately reflect human pathogenesis, obtained results differ per strain within a given species, and the models are associated with considerable discomfort for the animals. Therefore, the objective of this study is to implement the 3Rs in renal fibrosis research by establishing an animal-free drug screening platform for renal fibrosis based on human precision-cut kidney slices (PCKS) and by limiting the use of reagents that are associated with significant animal welfare concerns. RESULTS: Using Western blotting and gene expression arrays, we show that transforming growth factor-ß (TGF-ß) induced fibrosis in human PCKS. In addition, our results demonstrated that butaprost, SC-19220 and tamoxifen - all putative anti-fibrotic compounds - altered TGF-ß-induced pro-fibrotic gene expression in human PCKS. Moreover, we observed that all compounds modulated fairly distinct sets of genes, however they all impacted TGF-ß/SMAD signaling. In conclusion, this study revealed that it is feasible to use an animal-free approach to test drug efficacy and elucidate mechanisms of action.

Gadolinium-enhanced MRI visualizing backflow at increasing intra-renal pressure in a porcine model.

INTRODUCTION: Intrarenal backflow (IRB) is known to occur at increased intrarenal pressure (IRP). Irrigation during ureteroscopy increases IRP. Complications such as sepsis is more frequent after prolonged high-pressure ureteroscopy. We evaluated a new method to document and visualize intrarenal backflow as a function of IRP and time in a pig model. METHODS: Studies were performed on five female pigs. A ureteral catheter was placed in the renal pelvis and connected to a Gadolinium/ saline solution 3 ml/L for irrigation. An occlusion balloon-catheter was left inflated at the uretero-pelvic junction and connected to a pressure monitor. Irrigation was successively regulated to maintain steady IRP levels at 10, 20, 30, 40 and 50 mmHg. MRI of the kidneys was performed at 5-minute intervals. PCR and immunoassay analyses were executed on the harvested kidneys to detect potential changes in inflammatory markers. RESULTS: MRI showed backflow of Gadolinium into the kidney cortex in all cases. The mean time to first visual damage was 15 minutes and the mean registered pressure at first visual damage was 21 mmHg. On the final MRI the mean percentage of IRB affected kidney was 66% after irrigation with a mean maximum pressure of 43 mmHg for a mean duration of 70 minutes. Immunoassay analyses showed increased MCP-1 mRNA expression in the treated kidneys compared to contralateral control kidneys. CONCLUSIONS: Gadolinium enhanced MRI provided detailed information about IRB that has not previously been documented. IRB occurs at even very low pressures, and these findings are in conflict with the general consensus that keeping IRP below 30-35 mmHg eliminates the risk of post-operative infection and sepsis. Moreover, the level of IRB was documented to be a function of both IRP and time. The results of this study emphasize the importance of keeping IRP and OR time low during ureteroscopy.

The Release of 24 h Infravesical Obstruction in Mice: Changes in Molecular, Morphological, and Functional Parameters for 14-Day Observation.

Bladder outlet obstruction (BOO) induces bladder dysfunction and altered bladder architecture. Irrespective of the release of the obstruction, persistent bladder dysfunction severely affects the quality of life. A better understanding of the repair process offers an opportunity to enhance postintervention management. We subsequently evaluated the postobstructive repair process in mice subjected to 24 h BOO followed by release. Male and female mice bladders were obstructed for 24 h by placing a clip around the bladder neck. After the release of obstruction, the mice were studied for 3, 7, and 14 days to observe the bladder repair process over time. Voiding frequency and volume were recorded using the voiding spot assay, and the transcutaneous glomerular filtration rate (tGFR) was measured. Fibrogenesis and associated gene expressions and altered protein levels were evaluated in the bladder using histology, quantatative polymerase chain reaction (qPCR), and Western blot analyses. Bladder wall thickness was increased in both genders over time but occurred later in female mice. Moreover, collagen deposition in the smooth muscle layer increased over time in both genders. Male mice showed a decreased average voided volume at 3 days post release, while female mice showed no significant change during the time course. Fibrosis-related molecular events, including upregulation of fibronectin (FN) protein and Collagen-3 (Col-3) mRNA expression, were transient and normalized again at 14 days in both genders. Transforming growth factor-ß (TGF-ß) and bone morphogenic protein (BMP)-7 mRNA expressions were upregulated at 14 days post release in both genders. Transcutaneous GFR remained normal during the time course. Release of 24 h BOO initiated a bladder remodeling process. The animal model enables a wide range of experiments to study bladder remodeling, and gender differences offer potential targets for understanding bladder fibrosis and adaptation with BOO.

Increased COX-2 after ureter obstruction attenuates fibrosis and is associated with EP2 receptor upregulation in mouse and human kidney.

AIM: Cyclooxygenase-2 (COX-2) activity protects against oxidative stress and apoptosis early in experimental kidney injury. The present study was designed to test the hypothesis that COX-2 activity attenuates fibrosis and preserves microvasculature in injured kidney. The murine unilateral ureteral-obstruction (UUO) model of kidney fibrosis was employed and compared with human nephrectomy tissue with and without chronic hydronephrosis. METHODS: Fibrosis and angiogenic markers were quantified in kidney tissue from wild-type and COX-2-/- mice subjected to UUO for 7 days and in human kidney tissue. COX-enzymes, prostaglandin (PG) synthases, PG receptors, PGE2 , and thromboxane were determined in human tissue. RESULTS: COX-2 immunosignal was observed in interstitial fibroblasts at baseline and after UUO. Fibronectin, collagen I, III, alpha-smooth muscle actin, and fibroblast specific protein-1 mRNAs increased significantly more after UUO in COX-2-/- vs wild-type mice. In vitro, fibroblasts from COX-2-/- kidneys showed higher matrix synthesis. Compared to control, human hydronephrotic kidneys showed (i) fibrosis, (ii) no significant changes in COX-2, COX-1, PGE2 -, and prostacyclin synthases, and prostacyclin and thromboxane receptor mRNAs, (iii) increased mRNA and protein of PGE2 -EP2 receptor level but unchanged PGE2 tissue concentration, and (iv) two- to threefold increased thromboxane synthase mRNA and protein levels, and increased thromboxane B2 tissue concentration in cortex and outer medulla. CONCLUSION: COX-2 protects in the early phase against obstruction-induced fibrosis and maintains angiogenic factors. Increased PGE2 -EP2 receptor in obstructed human and murine kidneys could contribute to protection.

Pretransplant endotrophin predicts delayed graft function after kidney transplantation.

Delayed graft function after kidney transplantation is common and increases morbidity and health care costs. There is evidence that endotrophin, a specific fragment of pro-collagen type VI, promotes the inflammatory response in kidney diseases. We tested the hypothesis that pretransplant endotrophin in kidney transplant recipients may be associated with the risk of delayed graft function. Pretransplant plasma endotrophin was assessed using an enzyme-linked immunosorbent assay in three independent cohorts with 806 kidney transplant recipients. The primary outcome was delayed graft function, i.e., the necessity of at least one dialysis session within one-week posttransplant. In the discovery cohort median pretransplant plasma endotrophin was higher in 32 recipients (12%) who showed delayed graft function when compared to 225 recipients without delayed graft function (58.4 ng/mL [IQR 33.4-69.0]; N = 32; vs. 39.5 ng/mL [IQR 30.6-54.5]; N = 225; P = 0.009). Multivariable logistic regression, fully adjusted for confounders showed, that pretransplant plasma endotrophin as a continuous variable was independently associated with delayed graft function in both validation cohorts, odds ratio 2.09 [95% CI 1.30-3.36] and 2.06 [95% CI 1.43-2.97]. Pretransplant plasma endotrophin, a potentially modifiable factor, was independently associated with increased risk of delayed graft function and may be a new avenue for therapeutic interventions.

Prostaglandin E2 receptors as therapeutic targets in renal fibrosis.

Prostaglandin E2 (PGE2), a lipid mediator produced by the cyclooxygenase enzyme system, is the main prostaglandin in the kidney. PGE2 is involved in various physiological and pathophysiological processes in the kidney, including renal hemodynamics, water and salt balance, and renal fibrosis-a key pathological feature of progressive kidney diseases. PGE2 functions by binding to four G-protein-coupled EP receptors (EP1 to EP4), which stimulate different intracellular signaling pathways. The intrarenal distribution of the four EP receptors as well as the different downstream signaling pathways associated with each receptor give rise to the distinct functional consequence of activating each receptor subtype. This review summarizes the current data on the renal expression of the four EP receptors and delineates the role of each receptor in renal fibrosis.

Integrative omics reveals subtle molecular perturbations following ischemic conditioning in a porcine kidney transplant model.

BACKGROUND: Remote Ischemic Conditioning (RIC) has been proposed as a therapeutic intervention to circumvent the ischemia/reperfusion injury (IRI) that is inherent to organ transplantation. Using a porcine kidney transplant model, we aimed to decipher the subclinical molecular effects of a RIC regime, compared to non-RIC controls. METHODS: Kidney pairs (n = 8 + 8) were extracted from brain dead donor pigs and transplanted in juvenile recipient pigs following a period of cold ischemia. One of the two kidney recipients in each pair was subjected to RIC prior to kidney graft reperfusion, while the other served as non-RIC control. We designed an integrative Omics strategy combining transcriptomics, proteomics, and phosphoproteomics to deduce molecular signatures in kidney tissue that could be attributed to RIC. RESULTS: In kidney grafts taken out 10 h after transplantation we detected minimal molecular perturbations following RIC compared to non-RIC at the transcriptome level, which was mirrored at the proteome level. In particular, we noted that RIC resulted in suppression of tissue inflammatory profiles. Furthermore, an accumulation of muscle extracellular matrix assembly proteins in kidney tissues was detected at the protein level, which may be in response to muscle tissue damage and/or fibrosis. However, the majority of these protein changes did not reach significance (p < 0.05). CONCLUSIONS: Our data identifies subtle molecular phenotypes in porcine kidneys following RIC, and this knowledge could potentially aid optimization of remote ischemic conditioning protocols in renal transplantation.

EP1 receptor antagonism mitigates early and late stage renal fibrosis.

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-ß-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-ß-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.