Extracellular vesicles contribute to early cyst development in autosomal dominant polycystic kidney disease by cell-to-cell communication.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid-filled cysts within the kidney due to mutations in PKD1 or PKD2. Although the disease remains incompletely understood, one of the factors associated with ADPKD progression is the release of nucleotides (including ATP), which can initiate autocrine or paracrine purinergic signaling by binding to their receptors. Recently, we and others have shown that increased extracellular vesicle (EVs) release from PKD1 knockout cells can stimulate cyst growth through effects on recipient cells. Given that EVs are an important communicator between different nephron segments, we hypothesize that EVs released from PKD1 knockout distal convoluted tubule (DCT) cells can stimulate cyst growth in the downstream collecting duct (CD). Here, we show that administration of EVs derived from Pkd1-/- mouse distal convoluted tubule (mDCT15) cells result in a significant increase in extracellular ATP release from Pkd1-/- mouse inner medullary collecting duct (iMCD3) cells. In addition, exposure of Pkd1-/- iMCD3 cells to EVs derived from Pkd1-/- mDCT15 cells led to an increase in the phosphorylation of the serine/threonine-specific protein Akt, suggesting activation of proliferative pathways. Finally, the exposure of iMCD3 Pkd1-/- cells to mDCT15 Pkd1-/- EVs increased cyst size in Matrigel. These findings indicate that EVs could be involved in intersegmental communication between the distal convoluted tubule and the collecting duct and potentially stimulate cyst growth.

Involvement of ceramide biosynthesis in increased extracellular vesicle release in Pkd1 knock out cells.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inherited disorder characterized by the development of renal cysts, which frequently leads to renal failure. Hypertension and other cardiovascular symptoms contribute to the high morbidity and mortality of the disease. ADPKD is caused by mutations in the PKD1 gene or, less frequently, in the PKD2 gene. The disease onset and progression are highly variable between patients, whereby the underlying mechanisms are not fully elucidated. Recently, a role of extracellular vesicles (EVs) in the progression of ADPKD has been postulated. However, the mechanisms stimulating EV release in ADPKD have not been addressed and the participation of the distal nephron segments is still uninvestigated. Here, we studied the effect of Pkd1 deficiency on EV release in wild type and Pkd1-/- mDCT15 and mIMCD3 cells as models of the distal convoluted tubule (DCT) and inner medullary collecting duct (IMCD), respectively. By using nanoparticle tracking analysis, we observed a significant increase in EV release in Pkd1-/- mDCT15 and mIMCD3 cells, with respect to the wild type cells. The molecular mechanisms leading to the changes in EV release were further investigated in mDCT15 cells through RNA sequencing and qPCR studies. Specifically, we assessed the relevance of purinergic signaling and ceramide biosynthesis enzymes. Pkd1-/- mDCT15 cells showed a clear upregulation of P2rx7 expression compared to wild type cells. Depletion of extracellular ATP by apyrase (ecto-nucleotidase) inhibited EV release only in wild type cells, suggesting an exacerbated signaling of the extracellular ATP/P2X7 pathway in Pkd1-/- cells. In addition, we identified a significant up-regulation of the ceramide biosynthesis enzymes CerS6 and Smpd3 in Pkd1-/- cells. Altogether, our findings suggest the involvement of the DCT in the EV-mediated ADPKD progression and points to the induction of ceramide biosynthesis as an underlying molecular mechanism. Further studies should be performed to investigate whether CerS6 and Smpd3 can be used as biomarkers of ADPKD onset, progression or severity.

Interplay between purinergic signalling and extracellular vesicles in health and disease.

Purinergic signalling is a receptor-mediated process characterized by the binding of extracellular nucleotides and nucleosides to purinergic receptors, which results in the activation intracellular signalling pathways, and, ultimately, leads to changes in cell physiology. Purinergic signalling has been related to the regulation of important physiological processes (e.g., renal electrolyte reabsorption; platelet aggregation; immune response). In addition, it has been associated with pathophysiological situations such as cancer and inflammation. Extracellular vesicles (EVs) are nanoparticles released by all cells of the organism, which play a key role in cell-cell communication. In this regard, EVs can mediate effects on target cells located at distant locations. Within their cargo, EVs contain molecules with the potential to affect purinergic signalling at the target cells and tissues. Here, we review the studies addressing the regulation of purinergic signalling by EVs based on the cell type or tissue where the regulation takes place. In this regard, EVs are found to play a major role in modulating the extracellular ATP levels and, specially, adenosine. This has a clear impact on, for instance, the inflammatory and immune response against cancer cells. Furthermore, we discuss the data available on the regulation of EV secretion and its cargo by purinergic signalling. Here, a major role of the purinergic receptor P2X7 and again, an impact on processes such as inflammation, immune response and cancer pathogenesis has been established. Finally, we highlight uninvestigated aspects of these two regulatory networks and address their potential as therapeutic targets.

Extracellular vesicles regulate purinergic signaling and epithelial sodium channel expression in renal collecting duct cells.

Purinergic signaling regulates several renal physiological and pathophysiological processes. Extracellular vesicles (EVs) are nanoparticles released by most cell types, which, in non-renal tissues, modulate purinergic signaling. The aim of this study was to investigate the effect of EVs from renal proximal tubule (HK2) and collecting duct cells (HCD) on intra- and intersegment modulation of extracellular ATP levels, the underlying molecular mechanisms, and the impact on the expression of the alpha subunit of the epithelial sodium channel (αENaC). HK2 cells were exposed to HK2 EVs, while HCD cells were exposed to HK2 and HCD EVs. Extracellular ATP levels and αENaC expression were measured by chemiluminescence and qRT-PCR, respectively. ATPases in EV populations were identified by mass spectrometry. The effect of aldosterone was assessed using EVs from aldosterone-treated cells and urinary EVs (uEVs) from primary aldosteronism (PA) patients. HK2 EVs downregulated ectonucleoside-triphosphate-diphosphohydrolase-1 (ENTPD1) expression, increased extracellular ATP and downregulated αENaC expression in HCD cells. ENTPD1 downregulation could be attributed to increased miR-205-3p and miR-505 levels. Conversely, HCD EVs decreased extracellular ATP levels and upregulated αENaC expression in HCD cells, probably due to enrichment of 14-3-3 isoforms with ATPase activity. Pretreatment of donor cells with aldosterone or exposure to uEVs from PA patients enhanced the effects on extracellular ATP and αENaC expression. We demonstrated inter- and intrasegment modulation of renal purinergic signaling by EVs. Our findings postulate EVs as carriers of information along the renal tubules, whereby processes affecting EV release and/or cargo may impact on purinergically regulated processes.

Intraluminal nutrients acutely strengthen rat intestinal MRP2 barrier function by a glucagon-like peptide-2-mediated mechanism.

AIM: MRP2 is an intestinal ABC transporter that prevents the absorption of dietary xenobiotics. The aims of this work were: (1) to evaluate whether a short-term regulation of intestinal MRP2 barrier function takes place in vivo after luminal incorporation of nutrients and (2) to explore the underlying mechanism. METHODS: MRP2 activity and localization were assessed in an in vivo rat model with preserved irrigation and innervation. Nutrients were administered into distal jejunum. After 30-minutes treatments, MRP2 activity was assessed in proximal jejunum by quantifying the transport of the model substrate 2,4-dinitrophenyl-S-glutathione. MRP2 localization was determined by quantitative confocal microscopy. Participation of extracellular mediators was evaluated using selective inhibitors and by immunoneutralization. Intracellular pathways were explored in differentiated Caco-2 cells. RESULTS: Oleic acid, administered intraluminally at dietary levels, acutely stimulated MRP2 insertion into brush border membrane. This was associated with increased efflux activity and, consequently, enhanced barrier function. Immunoneutralization of the gut hormone glucagon-like peptide-2 (GLP-2) prevented oleic acid effect on MRP2, demonstrating the participation of this trophic factor as a main mediator. Further experiments using selective inhibitors demonstrated that extracellular adenosine synthesis and its subsequent binding to enterocytic A2B adenosine receptor (A2BAR) take place downstream GLP-2. Finally, studies in intestinal Caco-2 cells revealed the participation of A2BAR/cAMP/PKA intracellular pathway, ultimately leading to increased MRP2 localization in apical domains. CONCLUSION: These findings reveal an on-demand, acute regulation of MRP2-associated barrier function, constituting a novel physiological mechanism of protection against the absorption of dietary xenobiotics in response to food intake.

Pannexin-1 mediates fluid shear stress-sensitive purinergic signaling and cyst growth in polycystic kidney disease.

Tubular ATP release is regulated by mechanosensation of fluid shear stress (FSS). Polycystin-1/polycystin-2 (PC1/PC2) functions as a mechanosensory complex in the kidney. Extracellular ATP is implicated in polycystic kidney disease (PKD), where PC1/PC2 is dysfunctional. This study aims to provide new insights into the ATP signaling under physiological conditions and PKD. Microfluidics, pharmacologic inhibition, and loss-of-function approaches were combined to assess the ATP release in mouse distal convoluted tubule 15 (mDCT15) cells. Kidney-specific Pkd1 knockout mice (iKsp-Pkd1-/- ) and zebrafish pkd2 morphants (pkd2-MO) were as models for PKD. FSS-exposed mDCT15 cells displayed increased ATP release. Pannexin-1 inhibition and knockout decreased FSS-modulated ATP release. In iKsp-Pkd1-/- mice, elevated renal pannexin-1 mRNA expression and urinary ATP were observed. In Pkd1-/- mDCT15 cells, elevated ATP release was observed upon the FSS mechanosensation. In these cells, increased pannexin-1 mRNA expression was observed. Importantly, pannexin-1 inhibition in pkd2-MO decreased the renal cyst growth. Our results demonstrate that pannexin-1 channels mediate ATP release into the tubular lumen due to pro-urinary flow. We present pannexin-1 as novel therapeutic target to prevent the renal cyst growth in PKD.

Up-regulation of ATP-binding cassette transporters in the THP-1 human macrophage cell line by the antichagasic benznidazole.

The effect of benznidazole (BZL) on the expression and activity of P-glycoprotein (P-gp, ABCB1) and multidrug resistance-associated protein 2 (MRP2, ABCC2), the two major transporters of endogenous and exogenous compounds, was evaluated in differentiated THP-1 cells. BZL induced P-gp and MRP2 proteins in a concentration-dependent manner. The increase in mRNA levels of both transporters suggests transcriptional regulation. P-gp and MRP2 activities correlated with increased protein levels. BZL intracellular accumulation was significantly lower in BZL-pre-treated cells than in control cells. PSC833 (a P-gp inhibitor) increased the intracellular BZL concentration in both pre-treated and control cells, confirming P-gp participation in BZL efflux.

Physiological and pathophysiological factors affecting the expression and activity of the drug transporter MRP2 in intestine. Impact on its function as membrane barrier.

The gastrointestinal epithelium functions as a selective barrier to absorb nutrients, electrolytes and water, but at the same time restricts the passage into the systemic circulation of intraluminal potentially toxic compounds. This epithelium maintains its selective barrier function through the presence of very selective and complex intercellular junctions and the ability of the absorptive cells to reject those compounds. Accordingly, the enterocytes metabolize orally incorporated xenobiotics and secrete the hydrophilic metabolites back into the intestinal lumen through specific transporters localized apically. In the recent decades, there has been increasing recognition of the existence of the intestinal cellular barrier. In the present review we focus on the role of the multidrug resistance-associated protein 2 (MRP2, ABCC2) in the apical membrane of the enterocytes, as an important component of this intestinal barrier, as well as on its regulation. We provide a detailed compilation of significant contributions demonstrating that MRP2 expression and function vary under relevant physiological and pathophysiological conditions. Because MRP2 activity modulates the availability and pharmacokinetics of many therapeutic drugs administered orally, their therapeutic efficacy and safety may vary as well.

Modulation of biotransformation systems and ABC transporters by benznidazole in rats.

The effect of antichagasic benznidazole (BZL; 100 mg/kg body weight/day, 3 consecutive days, intraperitoneally) on biotransformation systems and ABC transporters was evaluated in rats. Expression of cytochrome P-450 (CYP3A), UDP-glucuronosyltransferase (UGT1A), glutathione S-transferases (alpha glutathione S-transferase [GST-α], GST-µ, and GST-π), multidrug-resistance-associated protein 2 (Mrp2), and P glycoprotein (P-gp) in liver, small intestine, and kidney was estimated by Western blotting. Increases in hepatic CYP3A (30%) and GST-µ (40%) and in intestinal GST-α (72% in jejunum and 136% in ileum) were detected. Significant increases in Mrp2 (300%) and P-gp (500%) proteins in liver from BZL-treated rats were observed without changes in kidney. P-gp and Mrp2 were also increased by BZL in jejunum (170% and 120%, respectively). In ileum, only P-gp was increased by BZL (50%). The activities of GST, P-gp, and Mrp2 correlated well with the upregulation of proteins in liver and jejunum. Plasma decay of a test dose of BZL (5 mg/kg body weight) administered intraduodenally was faster (295%) and the area under the concentration-time curve (AUC) was lower (41%) for BZL-pretreated rats than for controls. The biliary excretion of BZL was higher (60%) in the BZL group, and urinary excretion of BZL did not show differences between groups. The amount of absorbed BZL in intestinal sacs was lower (25%) in pretreated rats than in controls. In conclusion, induction of biotransformation enzymes and/or transporters by BZL could increase the clearance and/or decrease the intestinal absorption of coadministered drugs that are substrates of these systems, including BZL itself.

Induction of hepatic multidrug resistance-associated protein 3 by ethynylestradiol is independent of cholestasis and mediated by estrogen receptor.

Multidrug resistance-associated protein 3 (Mrp3; Abcc3) expression and activity are up-regulated in rat liver after in vivo repeated administration of ethynylestradiol (EE), a cholestatic synthetic estrogen, whereas multidrug resistance-associated protein 2 (Mrp2) is down-regulated. This study was undertaken to determine whether Mrp3 induction results from a direct effect of EE, independent of accumulation of any endogenous common Mrp2/Mrp3 substrates resulting from cholestasis and the potential mediation of estrogen receptor (ER). In in vivo studies, male rats were given a single, noncholestatic dose of EE (5 mg/kg s.c.), and basal bile flow and the biliary excretion rate of bile salts and glutathione were measured 5 hours later. This treatment increased Mrp3 mRNA by 4-fold, detected by real-time polymerase chain reaction, despite the absence of cholestasis. Primary culture of rat hepatocytes incubated with EE (1-10 µM) for 5 hours exhibited a 3-fold increase in Mrp3 mRNA (10 µM), consistent with in vivo findings. The increase in Mrp3 mRNA by EE was prevented by actinomycin D, indicating transcriptional regulation. When hepatocytes were incubated with an ER antagonist [7α,17ß-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI182/780), 1 µM], in addition to EE, induction of Mrp3 mRNA was abolished, implicating ER as a key mediator. EE induced an increase in ER-α phosphorylation at 30 minutes and expression of c-Jun, a well-known ER target gene, at 60 minutes, as detected by Western blotting of nuclear extracts. These increases were prevented by ICI182/780. In summary, EE increased the expression of hepatic Mrp3 transcriptionally and independently of any cholestatic manifestation and required participation of an ER, most likely ER-α, through its phosphorylation.

Regulation of biotransformation systems and ABC transporters by benznidazole in HepG2 cells: involvement of pregnane X-receptor.

BACKGROUND: Benznidazole (BZL) is the only antichagasic drug available in most endemic countries. Its effect on the expression and activity of drug-metabolizing and transporter proteins has not been studied yet. METHODOLOGY/PRINCIPAL FINDINGS: Expression and activity of P-glycoprotein (P-gp), Multidrug resistance-associated protein 2 (MRP2), Cytochrome P450 3A4 (CYP3A4), and Glutathione S-transferase (GST) were evaluated in HepG2 cells after treatment with BZL. Expression was estimated by immunoblotting and real time PCR. P-gp and MRP2 activities were estimated using model substrates rhodamine 123 and dinitrophenyl-S-glutathione (DNP-SG), respectively. CYP3A4 and GST activities were evaluated through their abilities to convert proluciferin into luciferin and 1-chloro-2,4-dinitrobenzene into DNP-SG, respectively. BZL (200 µM) increased the expression (protein and mRNA) of P-gp, MRP2, CYP3A4, and GSTπ class. A concomitant enhancement of activity was observed for all these proteins, except for CYP3A4, which exhibited a decreased activity. To elucidate if pregnane X receptor (PXR) mediates BZL response, its expression was knocked down with a specific siRNA. In this condition, the effect of BZL on P-gp, MRP2, CYP3A4, and GSTπ protein up-regulation was completely abolished. Consistent with this, BZL was able to activate PXR, as detected by reporter gene assay. Additional studies, using transporter inhibitors and P-gp-knock down cells, demonstrated that P-gp is involved in BZL extrusion. Pre-treatment of HepG2 cells with BZL increased its own efflux, as a consequence of P-gp up-regulation. CONCLUSIONS/SIGNIFICANCE: Modifications in the activity of biotransformation and transport systems by BZL may alter the pharmacokinetics and efficiency of drugs that are substrates of these systems, including BZL itself.

Effect of glucagon-like peptide 2 on hepatic, renal, and intestinal disposition of 1-chloro-2,4-dinitrobenzene.

The ability of the liver, small intestine, and kidney to synthesize and subsequently eliminate dinitrophenyl-S-glutathione (DNP-SG), a substrate for multidrug resistance-associated protein 2 (Mrp2), was assessed in rats treated with glucagon-like peptide 2 (GLP-2, 12 µg/100 g b.wt. s.c. every 12 h for 5 consecutive days). An in vivo perfused jejunum model with simultaneous bile and urine collection was used. A single intravenous dose of 30 µmol/kg b.wt. 1-chloro-2,4-dinitrobenzene (CDNB) was administered, and its conjugate, DNP-SG, and dinitrophenyl cysteinyl glycine (DNP-CG), resulting from the action of γ-glutamyltransferase on DNP-SG, were determined in bile, intestinal perfusate, and urine by high-performance liquid chromatography. Tissue content of DNP-SG was also assessed in liver, intestine, and kidneys. Biliary excretion of DNP-SG+DNP-CG was decreased in GLP-2 rats with respect to controls. In contrast, their intestinal excretion was substantially increased, whereas urinary elimination was not affected. Western blot and real-time polymerase chain reaction studies revealed preserved levels of Mrp2 protein and mRNA in liver and renal cortex and a significant increase in intestine in response to GLP-2 treatment. Tissue content of DNP-SG detected 5 min after CDNB administration was decreased in liver, increased in intestine, and unchanged in kidney in GLP-2 versus control group, consistent with GLP-2-induced down-regulation of expression of glutathione transferase (GST) Mu in liver and up-regulation of GST-Alpha in intestine at both protein and mRNA levels. In conclusion, GLP-2 induced selective changes in hepatic and intestinal disposition of a common GST and Mrp2 substrate administered systemically that could be of pharmacological or toxicological relevance under therapeutic treatment conditions.

Induction of intestinal multidrug resistance-associated protein 2 by glucagon-like Peptide 2 in the rat.

The effects of glucagon-like peptide 2 (GLP-2) on expression and activity of jejunal multidrug resistance-associated protein 2 (Mrp2; Abcc2) and glutathione transferase (GST) were evaluated. After GLP-2 treatment (12 µg/100 g b.wt. s.c., every 12 h, for 5 consecutive days), Mrp2 and the α class of GST proteins and their corresponding mRNAs were increased, suggesting a transcriptional regulation. Mrp2 was localized at the apical membrane of the enterocyte in control and GLP-2 groups, as detected by confocal immunofluorescence microscopy. As a functional assay, everted intestinal sacs were incubated in the presence of 1-chloro-2,4-dinitrobenzene in the mucosal compartment, and the glutathione-conjugated derivative, dinitrophenyl-S-glutathione (DNP-SG; model Mrp2 substrate), was detected in the same compartment by high-performance liquid chromatography. A significant increase in apical secretion of DNP-SG was detected in the GLP-2 group, consistent with simultaneous up-regulation of Mrp2 and GST. GLP-2 also promoted an increase in cAMP levels as detected in homogenates of intestinal mucosa. Treatment of rats with 2',3'-dideoxyadenosine (DDA), a specific inhibitor of adenylyl cyclase, abolished the increase in cAMP levels and Mrp2 protein promoted by GLP-2, suggesting cAMP as a mediator of Mrp2 modulation. Increased expression of Mrp2 and cAMP levels in response to GLP-2 occurred not only at the tip but also at the middle region of the villus, where constitutive expression of Mrp2 is normally low. In conclusion, our study suggests a role for GLP-2 in the prevention of cell toxicity of the intestinal mucosa by increasing Mrp2 chemical barrier function.

Induction of intestinal multidrug resistance-associated protein 2 (Mrp2) by spironolactone in rats.

The effect of spironolactone (SL) pretreatment (200micromol/kg b.w./day, 3 consecutive days) on intestinal multidrug resistance-associated protein 2 (Mrp2) was evaluated in rats. A significant increase in protein levels in upper regions of small intestine, where Mrp2 is mainly present, was detected by western blotting. Real time PCR studies suggest a transcriptional regulation. The administration of ketoconazole, a pregnane X receptor (PXR) antagonist, was able to prevent the increase in Mrp2 mRNA levels induced by SL. The serosal to mucosal transport of dinitrophenyl S-glutathione, a model substrate of Mrp2 was evaluated in jejunal sac model. The data indicate that SL increased Mrp2 activity, well correlating with its up-regulation. We conclude that SL is able to induce intestinal Mrp2 transcriptionally, PXR being a potential mediator. We propose that SL could be of potential therapeutic application particularly in situations of down-regulation of intestinal Mrp2.