Angiotensin II (Ang II) was obtained from Alomone (Jerusalem, Israel), lanthanum (La³⁺) chloride, penicillin (10,000 U) and streptomycin (10 mg/mL), carbenoxolone (CBX), the monoclonal anti-α-tubulin antibody, probenecid (PBC) and malondialdehyde (MDA) were obtained from Sigma‒Aldrich (St. Louis, MO, United States); ¹⁰panx1 (WRQAAFVDSY, first extracellular loop domain of Panx1) was obtained from GenScript (New Jersey, United States); and A740003, a specific P2X7 receptor blocker, was obtained from Tocris Biosciences (Bristol, United Kingdom). The monoclonal anti-unphosphorylated Panx1 antibody was obtained from Invitrogen (Carlsbad, CA, United States); and anti-mouse and anti-rabbit secondary antibodies conjugated to horseradish peroxidase were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, United States). HEPES, ATP, NaCl, KCl, CaCl₂, MgCl₂ and glucose were obtained from MERCK (Darmstadt, Germany). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT, United States), whereas trypsin 10X, Hank’s solution, ATP determination kit, FURA-2 AM Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS) and ethidium (Etd) bromide (10 mg/mL) were purchased from Thermo Fisher Scientific (Waltham, MA, United States). 4′,6-Diamidino-2-phenylindole (DAPI) was obtained from Abcam (Cambridge, United Kingdom).

C57BL/6 (U. Chile) mice of 6–8 weeks of age were housed in cages in a temperature-controlled (24°C) and humidity-controlled vivarium under a 12 h light/dark cycle (lights on 8:00 a.m.) with ad libitum access to food and water. All procedures were in accordance with institutional and international standards for the humane care and use of laboratory animals (Animal Welfare Assurance Publication A5427-01, Office for Protection from Research Risks, Division of Animal Welfare, NIH (National Institutes of Health), Bethesda, MD, United States). The Bioethical and Biosafety Committee of the Faculty of Biomedical Sciences at Universidad Autónoma de Chile (BE07-20; 26 October 2020) approved the described experimental procedures.

We used a well-known model of chronic administration of Ang II that causes hypertension (Giachelli et al., 1994). Briefly, animals were anesthetized with a dose of ketamine/xylazine (25:2.5 mg/kg). A small (1 cm) cut was performed in the mid-scapular region, and an osmotic pump was implanted; subsequently, the wound was sutured. After their recovery, the mice were kept with water and food ad libitum. Mice were infused with Ang II (1,000 ng/min) or saline for 6 weeks. Weekly measures were performed to examine various physiological parameters, including blood pressure (see below) (Gómez et al., 2019).

Systolic and diastolic blood pressure and mean arterial pressure (PAM) were determined once a week in the morning in conscious prewarmed restrained mice by noninvasive plethysmography (NIBP machine, IITC Inc., Woodland Hills, CA, United States) by the tail-cuff method. At least four determinations were made in every session, and the mean of the ten measurements was taken as the blood pressure value.

Plasma and urinary creatinine levels were measured by the Jaffé alkaline picrate assay (VALTEK Diagnostica, Santiago, Chile). The urinary protein concentration was determined by Bradford’s method (Bio-Rad protein assay, Kidlington, United Kingdom) (Salas et al., 2003). To determine the U prot/U Crea ratio, the value for urinary protein was divided by the value for urinary creatinine. The levels of blood urea nitrogen (BUN) in serum were determined by appropriate detection kits following the manufacturer’s instructions.

Primary mesangial cells were isolated from mouse glomeruli treated with collagenase (Menè and Stoppacciaro, 2009; Wilson and Stewart, 2012; Salva et al., 2017; Wang et al., 2019). In brief, kidney fragments were minced with a razor blade, and a 100 m nylon sieve was used to collect the glomeruli from the cortex homogenates of mouse kidneys under aseptic conditions. This glomeruli-enriched fraction was collected from underneath the sieve with HBSS. The diluted suspension was poured onto a second 70 m filter and washed with the same solution. The glomeruli and other fragments retained on the filter were transferred into a sterile tube. The glomerular suspension was incubated for digestion with sterile type IV collagenase in DMEM for 1 h at 37°C in an incubator. Then, it was triturated through a 21-gauge needle. Glomerular remnants were washed, and mesangial and endothelial cells were plated onto a six-well plate in a complete medium and incubated at 37°C in a humidified 5% CO₂ incubator (Menè and Stoppacciaro, 2009; Wilson and Stewart, 2012; Salva et al., 2017; Wang et al., 2019).

Mesangial cells were treated with 1 µM Ang II for different periods (0, 24, 48 and 72 h) in a 2:1 mixture of DMEM and F-12 tissue culture media supplemented with 100 U/mL penicillin and 100 g/mL streptomycin. Cells were kept at 37°C in 5% CO₂/95% air at nearly 100% relative humidity. CBX (10 μM), PBC (500 μM) and ¹⁰panx1 (50 μM) were added 24 h before the end of a 3-day experiment to cell cultures treated with Ang II at time zero.

The activity of Panx1 channels was evaluated by using the Etd uptake method, as previously described (Orellana et al., 2012). In brief, cells at 70% confluence were plated onto glass coverslips and bathed with Locke’s saline solution (in mM: 154 NaCl, 5.4 KCl, 2.3 CaCl₂, 1.5 MgCl₂, 5 HEPES, 5 glucose, and pH 7.4) containing 5 μM Etd. Time-lapse images were measured (at regions of interest in different cells) every 30 s for 13 min using a Nikon Eclipse Ti inverted microscope (Tokyo, Japan) and NIS-Elements software. The fluorescence intensity recorded from 25 regions of interest (representing 25 cells per coverslip) was defined as the subtraction (F–F0) between the fluorescence (F) from the respective cell (25 cells per field) and the background fluorescence (F0) measured where no labeled cells were detected. The mean slope of the relationship F-F0 over a given time interval (ΔF/ΔT; F0 remained constant along the recording time) represents the Etd uptake rate. To determine changes in slope measurements, regression lines were fitted to points before and after the various experimental conditions using Excel software, and mean values of slopes were compared using GraphPad Prism software and expressed as AU/min. At least four replicates (four sister coverslips) were measured in each independent experiment (Sáez et al., 2018).

Mesangial cells plated on glass coverslips were loaded with 5 µM Fura-2-AM in DMEM without serum at 37°C for 45 min and then washed three times in recording solution (in mM): 140 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 5 glucose, and 10 HEPES, pH = 7.4, followed by de-esterification at 37°C for 15 min. The experimental protocol for Ca²⁺ signal imaging involved data acquisition every 10 s for 5 min (emission at 510 and 515 nm, respectively) at 340/380-nm excitation wavelengths (Xenon lamp) using an inverted microscope (Eclipse Ti-U, Nikon). NIS element advanced research software (version 4.0, Nikon) was used for data acquisition and image analysis. The fluorescence intensity recorded in 15 cells involved the determination of pixels assigned to each cell. The average pixel value allocated to each cell was obtained with excitation at each wavelength and corrected for background. The FURA-2 ratio was obtained after dividing the 340-nm by the 380-nm fluorescence image on a pixel-by-pixel basis (R = F_340 nm/F_380 nm).

The extracellular amount of ATP was measured using the ATP determination kit according to the protocol provided by its supplier Invitrogen (ATP Determination Kit, A22066). Using the conditioned medium of mesangial cells and applying the kit that includes D-Luciferin and recombinant firefly luciferase, the luminescence produced by luciferin upon binding with ATP was measured using a Tecan Infinite^® M200 PRO plate reader spectrometer (Männedorf, Switzerland) and i-control™ software.

Mesangial cells grown on coverslips were fixed at room temperature (RT) with 2% paraformaldehyde for 30 min and then washed three times with PBS. They were incubated three times for 5 min in 0.1 M PBS glycine and then in 0.1% PBS-Triton X-100 containing 10% NGS for 30 min. Cells were incubated with anti-Panx1 polyclonal antibody (1:100) diluted in 0.1% PBS-Triton X-100 with 2% NGS at 4°C overnight. After five rinses in 0.1% PBS-Triton X-100, cells were incubated with goat anti-rabbit IgG Alexa Fluor 555 (1:1,000) at RT for 60 min. After several rinses, coverslips were mounted in Paramount-DAPI fluorescent mounting medium and examined with high-resolution fluorescence microscopy (Leica, Wetzlar, Germany) with a 63X objective.

Mesangial cell cultures were placed on ice, washed twice with ice-cold PBS (pH 7.4), and harvested by scraping in 80 μL of a solution containing a protease and phosphatase inhibitor cocktail (Thermo Scientific, Pierce, Rockford, IL, United States; cat # 78430). Lysates were centrifuged (25,200X g, Eppendorf Centrifuge 5415C, Hamburg, Germany), and supernatants were collected for Western blot analysis. Protein concentration was determined using Lowry’s method (Lowry et al., 1951). Samples of homogenized cell cultures (50 μg of proteins) under different conditions were resolved by electrophoresis in 10% SDS-polyacrylamide gel, and prestained molecular weight markers were resolved in one lane. Proteins were transferred to a PVDF membrane (pore size: 0.45 μm), which was blocked at RT with Tris pH 7.4, 5% skim milk (w/v) and 1% BSA (w/v). Then, the PVDF membrane was incubated overnight at 4 °C with anti-Panx1 (1:1,000) antibody, followed by incubation with rabbit (1:2,000) for 1 h at RT. Then, the PVDF membrane was stripped and reblotted with the anti-α-tubulin antibody (1:5,000) used as a loading control, following the same procedure described above. After repeated rinses, immunoreactive proteins were detected by using ECL reagents (Pierce Biotechnology, Rockford, IL, United States) according to the manufacturer’s instructions. The bands detected were digitized and subjected to densitometry analysis using ImageJ software (Version 1.50i, NIH, Washington, DC, United States).

The total RNA of cellular and kidney homogenates was obtained using TRIzol reagent (Ambion). The procedure was established according to the manufacture’s instructions. Aliquots of 2 μg of the whole RNA were transformed into cDNA by using MMLV reverse transcriptase (Fermentas), and the amplicon amount was assessed by PCR amplification (GoTaq flexi DNA Polymerase, Promega). The primers for GADPH were the following: sense ’-ACC​ACA​GTC​CAT​GCC​ATC​AC-’ and antisense ’-TCC​ACC​ACC​CTG​TTG​CTG​TA-’. The primers for Panx1 were the following: sense ’-GTG​GCT​GCA​CAA​GTT​CTT​C-’ and antisense ’-CTC​TGC​CCC​ACA​TTC​TCA​GT-’.

The number of viable cells was quantified using an MTT/PMS reagent-based Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay Kit (Promega) according to the manufacturer’s instructions (Xu et al., 2018). 10 μL of aqueous MTT solution (4 mg/mL) was then added to each well (100 μL), and the mixture was incubated at 37°C for 3 h. The MTT solution was carefully decanted off, and formazan was extracted from the cells with 100 μL of a 4:1 DMSO–EtOH mixture in each well. Color intensity was measured with a 96-well ELISA plate reader at 550 nm, with the reference filter set to 620 nm. All MTT assays were repeated three times.

IL-1β was determined in the extracellular medium under different conditions in mesangial cells. Samples were centrifuged at 14.000 g for 40 min. Supernatants were collected and protein content was assayed using the BCA method. IL-1β levels were determined by sandwich ELISA, according to the manufacturer’s protocol (IL-1β EIA kit, Enzo Life Science, Farmingdale, NY, United States). For the assay, 100 µL of samples were added per ELISA plate well and incubated at 4°C overnight. A calibration curve with recombinant cytokine was included. Detection antibody was incubated at room temperature for 1 h and the reaction developed with avidin–HRP and substrate solution. Absorbance was measured at 450 nm with reference to 570 nm with the microplate reader Synergy HT (Biotek Instruments).

The amount of TBARS was estimated using the method described by Ramanathan and collaborators (Ramanathan et al., 1994) with slight modifications. Culture medium was mixed with SDS (8% w/v), thiobarbituric acid (0.8% TBA w/v), and acetic acid (20% v/v) and heated for 60 min at 90°C. The material that precipitated was removed by centrifugation, and the absorbance of the supernatant was evaluated at 532 nm. The amount of TBARS was calculated using a calibration curve obtained with malondialdehyde (MDA) as a standard. MDA was obtained from Merck (Darmstadt, Germany).

The results are expressed as the mean ± standard error of the mean (SEM); n refers to the number of independent experiments performed. Statistical analysis was performed using GraphPrad Prism (version 9, GraphPad Software, La Jolla, CA, United States). Normality and equal variances were assessed by the Shapiro‒Wilk normality test and Brown-Forsythe test, respectively. Depending on the nature of the data, they were analyzed with parametric or nonparametric tests. When the data were normal, without unequal variance and nonheteroscedastic, a t-test was used to compare two groups, and in the case of multiple comparisons, one or two-way analysis of variance (ANOVA) was used, followed; in the case of significance, by Bonferroni’s post hoc test. When the data were heteroscedastic as well as nonnormal and had unequal variation, nonparametric tests were used, such as the Kruskal‒Wallis test, followed, in the case of significance, by Dunn’s post hoc test. Details of the statistical results, together with the n and number of replicates, are included in the figure legends. A probability of p < 0.05 was considered statistically significant.

Ang II-mediated activation of AT1 receptors is pivotal in the progressive deterioration of glomerular function, contributing to inflammatory and oxidative damage in renal diseases (Rupérez et al., 2005; Lucero et al., 2022b). Additionally, mesangial cells critically participate in glomerular hypertension and progressive renal disease (da Silva Novaes et al., 2018; Zhao, 2019). We recently found that in vitro treatment with Ang II boosts the activity of Panx1 channels in the mesangial cell line MES-13 (Gómez et al., 2018). However, whether in vivo treatment with Ang II could modulate the activity of these channels in mesangial cells remains unknown. To explore this question, mice were subjected to a 6-week subcutaneous infusion of either Ang II or saline via osmotic pumps. Previous studies have shown that constant delivery of Ang II under these conditions elicits hypertension in weeks (Menzies et al., 2015; Gómez et al., 2019). Consistently, after 1 week of Ang II infusion, mice showed a substantial rise in systolic blood pressure (Ang II: 163 ± 9 mmHg vs saline: 117 ± 1 mmHg), diastolic blood pressure (Ang II: 111 ± 8 mmHg vs saline: 78 ± 2 mmHg) and mean arterial pressure (Ang II: 120 ± 6 mmHg vs saline: 87 ± 2 mmHg) compared to the control saline group (Figures 1A–C). These responses steadily increased until reaching a plateau around 5–6 weeks of Ang II infusion (Figures 1A–C). The quantification of proteinuria serves as a pivotal diagnostic and treatment monitoring tool in CKD (Clermont et al., 2000). A single sample of urine and plasma, coupled with measurements of creatinine, protein, and blood urea nitrogen (BUN), can provide valuable insights into kidney health and function (Clermont et al., 2000; Methven et al., 2010; Gómez and Velarde, 2018; Gómez et al., 2019). As expected, after 6 weeks of Ang II infusion, mice showed increased levels of plasma creatinine (Ang II: 0.61 ± 0.05 mg/mL vs saline: 0.41 ± 0.04 mg/mL) (Figure 1D), BUN (Ang II: 38.71 ± 5.71 mg/dL vs saline: 19.46 ± 1.39 mg/dL) (Figure 1E) and the ratio of urine protein to urine creatinine (Ang II: 39.75 ± 3.26 AU vs saline: 12.88 ± 2.90 AU) (Figure 1F). These findings collectively indicate that our model of Ang II infusion exacerbates hypertensive conditions and induces renal damage in mice.

To investigate whether in vivo Ang II infusion increases Panx1 channel activity in mesangial cells, we measured the uptake of Etd. This molecule crosses the plasma membrane in healthy cells through selective large-pore channels, including Panx1 channels (Orellana et al., 2012). Upon intercalation with base pairs of DNA and RNA, Etd becomes fluorescent, reflecting the activity of channels (Johnson et al., 2016). Interestingly, primary cultures of mesangial cells derived from animals treated with Ang II for 6 weeks did not exhibit significant differences in Etd uptake compared to mesangial cells from saline-treated mice (Figures 2A,B). Previous studies have shown that various pathological conditions can sensitize cells to exhibit Panx1 channel activity only when subjected to a subsequent pathological challenge (Orellana et al., 2014; Cibelli et al., 2022). With this in mind, we further evaluated whether in vivo Ang II infusion could sensitize mesangial cells on its response to a subsequent in vitro exposure to Ang II. For that purpose, we first examined whether in vitro Ang II exposure affects the expression and levels of Panx1 in mesangial cells derived from saline-treated mice. We observed that 1 µM Ang II induced a time-dependent increase in Panx1 expression and protein levels, with 72 h of treatment representing the period that elicited the maximum effect (Figures 2C–F). In this context, we opted for the treatment of 1 µM Ang II for 72 h in further experiments. Interestingly, this treatment led to a significant increase in Etd uptake in mesangial cells compared to control conditions (untreated cells) (Figure 3). Strikingly, this response was notably higher in cells derived from mice infused with Ang II for 6 weeks compared to those treated only with saline (Ang II: 3.5-fold vs saline: 1.5-fold) (Figure 3). Given that Panx1 channels represent one of the primary conduits for Etd influx in the mesangial cell line MES-13 (Gómez et al., 2018), we further explored the potential involvement of these channels in the Ang II-induced Etd uptake observed in our system. Remarkably, 10 μM CBX and 500 μM PBC, two well-known blockers of Panx1 channels (Crespo Yanguas et al., 2018; Caufriez et al., 2023), completely suppressed the Ang II-induced Etd uptake in mesangial cells derived from Ang II- or saline-treated mice (Figure 3). Supporting these data, a similar inhibitory effect was found with the mimetic peptide ¹⁰panx1 (50 µM), which specifically inhibit Panx1 channel activity by binding the first extracellular loop of Panx1 (Pelegrin and Surprenant, 2006; Caufriez et al., 2023). These findings highlight that in vivo Ang II infusion sensitizes mesangial cells to show increased Panx1 channel activity in response to a subsequent in vitro exposure to Ang II.

Over the past decade, a body of research has demonstrated that the persistent activation of Panx1 channels contributes to the pathogenesis and progression of various diseases (Penuela et al., 2014). For instance, Panx1 channel activation can trigger the release of molecules, such as ATP, which, when secreted at high concentrations, may exert toxicity on neighboring cells (Abudara et al., 2018). In this context, we examined whether in vivo and/or in vitro treatment with Ang II could alter the release of ATP in primary mesangial cell cultures. As observed in experiments measuring Etd uptake, the baseline release of ATP remains unchanged in mesangial cells from mice treated with Ang II for 6 weeks compared to those infused only with saline (Figure 4). However, treatment with 1 µM Ang II for 72 h resulted in a substantial elevation in the release of ATP compared to untreated cells, which was comparatively higher in mesangial cells derived from mice infused with Ang II than saline (Figure 4). Notably, these responses were totally attenuated by blocking Panx1 channels with 10 µM CBX, 500 µM PBC, or 50 µM ¹⁰panx1 (Figure 4).

The increase in [Ca²⁺]_i has been linked with heightened Panx1 channel activity in various cell types (Zorzi et al., 2017; Grimmer et al., 2022; Jaque-Fernandez et al., 2023). This facilitates the release of ATP and further engagement of purinergic receptors, perpetuating the sustained opening of Panx1 channels (Sáez et al., 2013; Garré et al., 2016). Accordingly, we examined whether in vivo and/or in vitro treatment with Ang II could modulate basal [Ca²⁺]_i levels in primary mesangial cell cultures. FURA-2 ratio (340/380) time-lapse recordings showed that basal [Ca²⁺]_i in mesangial cells from Ang II-or saline-treated mice remained unchanged (Figures 5A–C). Nevertheless, upon treatment with 1 µM Ang II for 72 h, mesangial cells showed a rise in basal [Ca²⁺]_i levels compared to untreated cells (Figures 5A–C). As occurred with the release of ATP, the above effect was more robust in mesangial cells derived from mice treated with Ang II for 6 weeks than those infused with saline (Figures 5A–C). Because we found in our system that Ang II triggers the release of ATP via Panx1 channels, we investigated the effect of Ang II on [Ca²⁺]_i responses elicited by this paracrine messenger. As previously reported (Gutierrez et al., 1999), acute stimulation with 10 µM ATP in mesangial cells induced a rapid peak in [Ca²⁺]_i that rapidly returned to baseline levels (Figures 5A,B). Relevantly, in vitro treatment with Ang II for 72 h augmented the ATP-induced amplitude of the peak and the integrated [Ca²⁺]_i signal (Figures 5D,E). Notably, these responses were more prominent in mesangial cells derived from mice treated with Ang II for 6 weeks than those infused with saline (Figures 5D,E). Of note, the remaining difference between the final and initial basal [Ca²⁺]_i signal triggered by ATP was elevated by in vitro Ang II treatment only in mesangial cells from Ang II-treated mice (Figure 5F).

Mounting evidence indicates that Panx1 channels indirectly elevate [Ca²⁺]_i via the ATP-dependent activation of purinergic receptors (Pinheiro et al., 2013b; Murali and Nurse, 2016; Murali et al., 2017). Consistent with this notion, we observed that inhibition of Panx1 channels with 10 µM CBX, 500 µM PBC, or 50 µM ¹⁰panx1 strongly diminished Ang II-induced increase in both [Ca²⁺]_i basal levels and ATP-mediated [Ca²⁺]_i signal amplitude (Figures 5A–D). Likewise, the blockade of Panx1 channels prominently abolished the augment in the integrated and remaining basal ATP-dependent [Ca²⁺]_i signal responses induced by the in vitro treatment with Ang II (Figures 5E,F). Overall, these data point out that the opening of Panx1 channels contributes to the in vivo Ang II-induced sensitization of mesangial cells in showing increased basal and ATP-induced [Ca²⁺]_i dynamics.

In various renal pathologies, Ang II is recognized for its capacity to generate inflammatory mediators, including cytokines and oxygen radicals (Peng et al., 2008; Gómez et al., 2018). In this scenario, we scrutinized whether in vitro and/or in vivo treatment with Ang II affects the production of pro-inflammatory cytokines and oxidative stress in mesangial cells. For that purpose, we measured the production of IL-1β by ELISA and the generation of thiobarbituric acid reactive substances (TBARS), which are formed as a byproduct of lipid peroxidation. We found that in vitro treatment with 1 µM Ang II for 72 h elicited a marked increase in the extracellular levels of IL-1β and TBARS when contrasted with untreated cells (Figures 6A,B). These effects were significantly greater in mesangial cells from Ang II-treated mice than those infused with saline (Figures 6A,B). No changes in the production of IL-1β or TBARS were detected between control mesangial cells from mice infused with Ang II or saline (Figures 6A,B). Remarkably, the Ang II-induced augment in IL-1β or TBARS production was strongly mitigated upon blockade of Panx1 channels with 10 µM CBX, 500 µM PBC, or 50 µM ¹⁰panx1 (Figures 6A,B).

We further examined the potential impact of in vitro and/or in vivo treatment with Ang II on mesangial cell survival. To assess this, we measured cell viability by quantifying the reduction of MTT to formazan, a process directly correlated with the number of metabolically active cells in the culture. Mesangial cells stimulated with 1 µM Ang II for 72 h exhibited a substantial reduction in cell viability compared to untreated cells (Figure 7). Interestingly, no changes in MTT production were observed in mesangial cells from mice infused with either Ang II or saline, regardless of whether they were treated with Ang II in vitro or not (Figure 7). Relevantly, 50 µM ¹⁰panx1 completely prevented the Ang II-induced increase in MTT production in mesangial cells, whereas 10 μM CBX or 500 µM PBC only were effective in cells derived from Ang II-treated mice (Figure 7). These findings highlight the pivotal role of Panx1 channels in the Ang II-induced cell death and production of pro-inflammatory cytokines and lipid peroxidation in mesangial cells.