Targeting pH Regulation in Cancer: Combined Mild Alkaline treatment and NHE1 Inhibition as a Potential Therapy for Clear Cell Renal Cell Carcinoma

Targeting pH Regulation in Cancer: Combined Mild Alkaline treatment and NHE1 Inhibition as a Potential Therapy for Clear Cell Renal Cell Carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Targeting pH Regulation in Cancer: Combined Mild Alkaline treatment and NHE1 Inhibition as a Potential Therapy for Clear Cell Renal Cell Carcinoma Ana Beatriz Celi, Ana Mechali, Natalia Beltramone, Juan Jose Casal, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5875263/v2 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Mar, 2026 Read the published version in Molecular and Cellular Biochemistry → Version 2 posted 11 You are reading this latest preprint version Show more versions Abstract Acid-base homeostasis is critical for maintaining physiological functions. An acidic tumor microenvironment, driven by altered cellular metabolism, plays a pivotal role in tumor progression by fostering aggressive phenotypes, immune evasion, and resistance to therapy, often at the detriment of surrounding normal tissues. The Na⁺/H⁺ exchanger isoform 1 (NHE1) is a key regulator of intracellular pH and a critical factor in cancer cell survival and proliferation. This study aimed to evaluate the effect of mild alkaline treatment, combined with NHE1 inhibition, on cell viability in normal renal cells and clear cell renal cell carcinoma (ccRCC) cells. Our findings reveal that this therapeutic combination selectively induces cell death in ccRCC cells while sparing normal renal cells. Mechanistically, we demonstrate that NHE1 activity is higher in ccRCC cells than in normal cells. In our experimental model, mild alkaline treatment differentially affected NHE1 activity, stimulating it in normal cells but suppressing it in cancer cells. Furthermore, prolonged alkaline exposure alters the subcellular localization of NHE1 in the plasma membrane, with distinct patterns observed between normal and cancer cells. These results suggest that targeting NHE1 activity in conjunction with alkaline treatment represents a promising strategy for ccRCC treatment, providing a potential therapeutic avenue to exploit the differential pH regulation between cancerous and normal cells. Figures Figure 3 Figure 4 Figure 5 Figure 6 Introduction One of the hallmarks of cancer cells is their acidic microenvironment. Cancer cells develop adaptive mechanisms that allow them to evade acid-mediated toxicity, promoting their growth while compromising the viability of surrounding normal cells. Localized acidosis further enhances tumor progression by facilitating extracellular matrix degradation, which promotes invasion and suppresses immune responses to tumor antigens [ 1 ]. Among the key regulators of intracellular pH (pHi) in cancer cells is the isoform 1 of the Na⁺/H⁺ exchanger (NHE1) [ 2 ]. NHE1 plays a vital role in physiological processes such as epithelial salt transport, cell volume regulation, proliferation, and apoptosis [ 3 ]. However, its influence on apoptosis is context-dependent, as it can either promote or inhibit cell death depending on the specific cell type and conditions studied [ 4 , 5 ]. In recent years, researchers have proposed neutralizing the acidic microenvironment of tumors to slow the progression of certain cancers. In silico simulations, which model changes in microenvironmental pH, suggest that increasing pH can reduce cancer progression rate [ 6 ]. Experimental studies in breast, prostate, and melanoma cancers further support this approach [ 7 – 10 ]. However, maintaining stable intracellular pH is critical for proper cell proliferation and death mechanisms under physiological conditions [ 11 , 12 ]. Therefore, to establish optimal conditions for safely applying this strategy, the potential adverse effects of pH neutralization on healthy tissues must also be evaluated. Renal cell carcinoma (RCC) includes a diverse group of cancers originating from renal epithelial cells and ranks among the ten most common cancers worldwide. Clear cell renal cell carcinoma (ccRCC) is the most prevalent subtype, accounting for 70–80% of RCC cases [ 13 ]. When detected at advanced stages or following metastatic spread, ccRCC exhibits high mortality in both men and women, primarily owing to its resistance to chemotherapy and radiotherapy [ 14 , 15 ]. Understanding the role of the cancer microenvironment and its interaction with transporters like NHE1 could offer new therapeutic strategies for ccRCC. For instance, Koltai et al. describe a case where a patient’s ccRCC stabilized following treatment with repurposed drugs that neutralized the inverted pH gradient [ 16 ]. This study investigates the effects of mild alkaline treatment combined with NHE1 inhibition on cell death in normal and cancerous renal cells. We use three human renal cell models: HK-2, representing healthy proximal tubular epithelial cells; 786-O, derived from a primary clear cell renal cell carcinoma; and Caki-1, derived from a ccRCC metastasis. Our findings demonstrate that the combination of mild alkaline treatment and NHE1 inhibition selectively induces cell death in ccRCC cells while minimally affecting normal renal cells. Moreover, prolonged exposure to alkaline treatment downregulates NHE1 expression in ccRCC cells, suggesting a potential mechanism for this selective effect. These results provide insights into the interplay between extracellular pH modulation and NHE1 activity, offering a promising avenue for therapeutic strategies targeting ccRCC. Materials and Methods Cell culture We cultured HK-2, 786-O, and Caki-1 cells using standard periodic subcultures on plastic supports. The culture media used were DMEM-F12 for HK-2 cells, RPMI for 786-O cells, and McCoy's 5A for Caki-1 cells, all supplemented with fetal bovine serum and antibiotics. The non-tumorigenic human proximal tubular epithelial cell line HK-2 (ATCC Cat# CRL-2190, RRID: CVCL_0302) was cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin [ 17 ]. HK-2 cells were kindly provided by Dr. María Marta Amaral (Laboratorio de Fisiopatogenia, IFIBIO-Houssay, CONICET–UBA, Argentina), who acquired them directly from the American Type Culture Collection (ATCC). The human renal epithelial cell lines 786-O (ATCC Cat# CRL-1932, RRID: CVCL_1051) and Caki-1 (ATCC Cat# HTB-46, RRID: CVCL_0234) were maintained on plastic supports using standard subculturing protocols. Caki-1 cells were cultured in McCoy’s 5A medium, and 786-O cells in RPMI 1640 medium both supplemented with 10% FBS and 1% penicillin/streptomycin [ 18 , 19 ]. These cell lines were kindly provided by Dr. Mariana Callero (Instituto de Oncología “Dr. Ángel Roffo”, UBA, Argentina), who obtained them from the National Cancer Institute cell repository. All cell lines are not listed as misidentified or problematic in the ICLAC or Cellosaurus databases. All cell lines were handled under consistent culture conditions and were routinely tested and confirmed negative for mycoplasma contamination throughout the course of the experiments. To create alkaline conditions, we adjusted the culture media by adding NaOH. For NHE1 blockade experiments, we added 1 µM HOE 694 (Aventis Pharma Deutschland GmbH, Germany) or vehicle (water, diluted 1:1000) to the medium. The osmolarity of the culture media and solutions, measured with a Vapro 5520 vapor pressure osmometer (Wescor), was not affected by the addition of NaOH, the vehicles, or the inhibitors. Cell Death analysis using Acridine Orange and Ethidium Bromide staining To quantify cell death, we washed the cells with 1X PBS and stained them with the fluorescent DNA-binding dyes acridine orange (AO, 15 µg/ml) and ethidium bromide (EB, 500 µg/ml) for 1 minute at room temperature. AO, which emits green fluorescence, penetrates both viable and dead cells, while EB, emitting red fluorescence, selectively marks dead cells. After staining, we washed the cells twice with PBS and immediately captured micrographs using an Olympus OPTIPHOT IMT-2 microscope equipped with an SPlan 10 PL 10X/0.30 NA objective. A Sony Alpha 3000 camera digitized the captures. We analyzed at least six fields from three independent coverslips for each condition. To evaluate the percentage of cells displaying morphological features of cell death, we performed each experiment in triplicate and scored between 1,000 and 5,000 cells for each condition. We identified dead cells using established criteria such as nuclear morphological changes, chromatin condensation, and staining [ 20 ], ImageJ software facilitated image analysis and counting. We designed a KNIME workflow to perform total cell counts (see Data and/or Code availability). Cell Death Analysis using Annexin V and Propidium iodide staining To assess cell death using an alternative method, we performed Annexin V/Propidium Iodide (PI) staining as described by Sánchez et al. [ 21 ]. We stained the cells with FITC-conjugated Annexin V (AV, BD Pharmingen) and Propidium Iodide, following the manufacturer's protocol. We captured micrographs with an Olympus OPTIPHOT IMT-2 microscope equipped with an SPlan 10 PL 10X/0.30 NA objective and digitized them using a Sony Alpha 3000 camera attached to the microscope. For each condition, we analyzed at least six fields from a minimum of three independent coverslips. We classified cells based on staining patterns: AV-/PI- cells were considered viable, AV+/PI- cells were in early apoptosis, AV+/PI + cells were in late apoptosis, and AV-/PI + cells were necrotic. Spheroid Formation and Image Analysis Caki-1 cells were pre-exposed to vehicle or experimental treatments for 72 hours before spheroid generation. We assembled spheroids using the hanging drop method (2,000 cells in 30 µL of McCoy’s medium supplemented with 10% FBS and antibiotics) maintaining the same treatment conditions throughout the process. After 4 days, we transferred the spheroids to 96-well plates containing fresh medium with either vehicle or treatments, which were maintained for the entire 11-day follow-up period. Images were acquired using a Sony Alpha 3000 camera and analyzed in ImageJ for area and compactness, with values normalized to day 0. For the quantification of areas of cell death, a custom Python script was developed in Google Colab, utilizing adaptive thresholding based on image intensity histograms (see Data and/or Code availability). Analyses were performed in three independent experiments, evaluating 20–26 spheroids per treatment. Immunofluorescence studies We conducted immunofluorescence studies on all cell lines to detect NHE1 expression. First, we seeded the cells onto coverslips and allowed them to grow in the presence or absence of 9.6 mM NaOH. In some experiments, we incubated the cells with 5 µg/ml wheat germ agglutinin Alexa 488 conjugate (WGA, Thermo Fisher Scientific, Cat# W11261). We then fixed the cells with 3% paraformaldehyde for 15 minutes and permeabilized them with 0.2% saponin at room temperature. Next, we incubated the samples overnight at 4ºC with monoclonal anti-NHE1 (dilution 1:200, Santa Cruz Biotechnology, Cat# sc-518041, RRID: AB_3481247) and followed with a 2-hour incubation at room temperature with secondary antibodies (anti-mouse Alexa 594 conjugate, dilution 1:200, Thermo Fisher Scientific, Cat# A-11032, RRID: AB_2534091). To stain the nuclei, we used Hoechst (5 µg/ml) for 3 minutes and then mounted the coverslips with Vectashield mounting medium (Vector Laboratories Inc., CA, USA) at room temperature. We captured and digitized the images using Fluoview FV1000 software on an Olympus FV1000 confocal microscope (objective UPLSAPO 40X/1.30 NA). We analyzed at least five fields from three independent coverslips per condition. Finally, we performed 3D reconstruction and colocalization analysis using Imaris software, calculating the amount of colocalization with the Mander’s coefficient. Intracellular pH Measurement and NHE1 Activity Assessment Using BCECF-AM and NH 4 Cl Pre-Pulse Technique For intracellular pH (pHi) measurements, we grew cells on glass coverslips and loaded them with 13 µM 2’,7’bis(2-carboxyethyl)-5-(and–6) carboxyfluorescein acetoxymethyl ester (BCECF-AM, Thermo Fisher Scientific, Cat# B1170) for 15 minutes at 37°C. Afterward, we washed away the excess dye. We then placed the chamber on a Nikon TE-200 epifluorescence inverted microscope (Nikon Plan Fluor 40X oil immersion objective lens) with a micro-incubator system and temperature controller (Harvard Apparatus Inc, MA, USA). Fluorescence data were collected every 10 seconds from a small circular region in the cells using a charge-coupled-device camera (Hamamatsu C4742-95) connected to a computer running the Metafluor acquisition program (Universal Imaging Corporation, PA, USA). We determined intracellular pH by calculating the ratio of BCECF emission intensity at 535 nm. The dye was excited at ~ 490 nm, and its isosbestic point was set at ~ 440 nm. We calibrated the 490/440 BCECF fluorescence ratio by incubating the cells in a high K + solution (140mM KCl, 4.6mM, NaCl, 1mM MgCl2, 2mM CaCl 2 , 10mM Hepes, 5mM glucose), followed by permeabilization with 5mM nigericin to equilibrate extracellular pH (pHo) with pHi. We then stepped the pH-bathing solution between 6.6 and 8.5. The 490/440 ratio remained linear within this pH range (r = 0.96, n = 6). Table 1 Composition of solutions used for determination of pH i recovery. Solution Control 0 Na + NH 4 Cl NaCl 145 ---- 127 NaHCO 3 ---- ---- ---- Hepes 30 30 30 KCl 3 3 3 CaCl 2 1.8 1.8 1.8 MgCl 2 ---- ---- ---- MgSO 4 1 1 1 KH 2 PO 4 1 1 1 K 2 SO 4 1 1 1 NH 4 Cl ---- ---- 20 TMA ---- 147 ---- glucose 5.6 5.6 5.6 All concentrations are given in mM; pH was adjusted to 7.4 at 37°C. In some experiments, we monitored NHE1 activity by evaluating the recovery of pHi after an acid load, using the NH 4 Cl pre-pulse technique as previously described [ 22 – 24 ]. We first exposed the cells to a control solution, followed by a 20 mM NH 4 Cl pulse (see Table 1 ) and then Na + removal (0 Na + in Table 1 ). Afterward, we reintroduced Na + to the bath and evaluated Na + -dependent pHi recovery mechanisms, see Online Resource 1. Online Resource 1 pH experiments A- Representative of pinhole placement. B- Representative intracellular pH (pHi) vs time in control 786-O cells We calculated acid fluxes using the following equation Eq. (A.1): \(\:JH=\beta\:i\times\:\frac{dpH}{dt}\) Eq. (A.1) H + represents the H + flux, dpH/dt is the recovery rate from the acid load, and βi is the intracellular buffering power. We estimated intracellular H + buffering power (βi) using the application of external permeant weak bases, as described by Roos, Boron, and Saleh [ 25 , 26 ]. In this study, we used NH 4 Cl, which permeates the cell membrane in its uncharged form but exists mainly in the ionized form once inside the cell. The ionization of this weak base absorbs H + ions. Thus, NH 4 Cl induces changes in pHi, which we used to estimate βi using the following equation Eq. (A.2): \(\:\beta\:i\left(mM\right)=\left(\right[NH\_4\:]\_o\cdot\:10^((pHo-pHip)\left)\right)/\varDelta\:pHi\) Eq. (A.2) Where ΔpHi is the change in pHi following the ammonium load, [NH 4 + ] 0 is the concentration of external NH4 + , pHo is the pH of the external solution, and pHip is the intracellular pH after the ammonium load. Statistics We presented the values as mean ± standard error of the mean (SEM), with "n" indicating the number of cells compared. To compare data between the two groups, we used an unpaired Student's t-test, considering differences significant at p < 0.05. For comparisons among multiple groups, we performed a one-way analysis of variance (ANOVA) followed by a Tukey post-hoc test, defining significance as p < 0.05. Results Cancer cell death induced by alkali and NHE1 inhibition with minimal impact on healthy cells. We used three human renal cell models: HK-2, representing healthy proximal tubular epithelial cells; 786-O, derived from primary clear cell renal cell carcinoma (ccRCC); and Caki-1, derived from a ccRCC metastasis. As expected, upon reaching confluence, the pH of the conditioned media (pHe) of normal HK-2 cells was less acidic than that of the cancer cell lines (pHe: HK-2 = 7.37 ± 0.02, n = 26; 786-O = 7.04 ± 0.05, n = 24; Caki-1 = 7.15 ± 0.05, n = 17; one-way ANOVA followed by Tukey's multiple comparisons test: HK-2 vs. 786-O, p < 0.0001; HK-2 vs. Caki-1, p < 0.01). We investigated the effects of neutralizing the acidic microenvironment by adding different concentrations of NaOH to the media for 72 hours. We then analyzed apoptosis percentages using the acridine orange/ethidium bromide staining method. As shown in Fig. 1 A, NaOH concentrations (2.4–9.6 mM) caused minimal increases in cell death in HK-2 cells. In contrast, these concentrations led to a progressive increase in cell death in cancer cell lines. We selected 9.6 mM NaOH for 72-hour treatments in subsequent experiments. Although the mild alkaline treatment (9.6mM NaOH) did not significantly alter the HK-extracellular pH, it was sufficient to neutralize the acidic microenvironment of the cancer cell lines, as shown in Fig. 1 B. Next, we explored the combined effects of NHE1 inhibition and mild alkaline treatment. To specifically inhibit NHE1, we used the HOE 694 inhibitor (1 µM, [ 27 ]). Without NaOH, NHE1 inhibition only increased apoptosis in metastatic-derived Caki-1 cells. While NaOH alone slightly increased cell death in HK-2 cells, the combination of NHE1 inhibition and NaOH prevented NaOH-induced cell death in these cells. In contrast, NaOH-induced cell death was significantly higher in cancer cell lines compared to HK-2 cells, and NHE1 suppression did not alter this result (Fig. 1 C). Finally, annexin V/propidium iodide staining experiments confirmed that the cell death observed through acridine orange/ethidium bromide staining was primarily due to apoptosis (Figs. 1 D-E). Thus, the combination of mild alkali treatment and NHE1 inhibition selectively induced apoptosis in cancer cells with minimal damage to healthy cells. A- HK-2, 786-O, and Caki-1 cells were incubated for 72 hours with either vehicle or 1.2–9.6 mM NaOH. Apoptosis was quantified using the acridine orange/ethidium bromide staining method (AO/BE). B- Impact of NaOH on the pH of conditioned media after 72 hours with vehicle (-) or 9.6 mM NaOH (+). **p < 0.01, ****p < 0.0001; n = 103. One-way ANOVA followed by Tukey's multiple comparisons test vs. HK-2 without NaOH. C-E Cells were treated with vehicle or 1 µM HOE 694 (NHE1 inhibitor) in the presence or absence of 9.6 mM NaOH for 72 hours. C- Apoptosis was quantified by acridine orange/ethidium bromide staining. D- Apoptosis was assessed using the annexin V method. E- Necrosis measured using the annexin V method. One-way ANOVA followed by Tukey's multiple comparisons test for each cell line. +p < 0.05, ++p < 0.01, +++p < 0.001, vs vehicle. To further evaluate this effect under more physiologically relevant conditions, we examined the impact of alkaline treatment, with or without NHE1 inhibition, in a 3D culture model. For this purpose, we generated spheroids of Caki-1 cells, as they better recapitulate tumor architecture and microenvironment compared with conventional 2D cultures. Spheroids were produced using the hanging drop method, which facilitates cell self-aggregation in the absence of exogenous matrices, thereby providing a controlled, scaffold-free system to study intrinsic cell–cell interactions. Caki-1 cells were pre-incubated for 72 hours with vehicle, 9.6 mM NaOH, and/or 1 µM HOE 694 before spheroid formation, and treatments were maintained throughout the experimental period. Spheroids were monitored for 11 days. As shown in Fig. 2 A, at the beginning of the culture (day 0), all groups formed compact spheroids, indicating that none of the treatments interfered with the cells’ ability to self-aggregate. In conditions with vehicle, spheroids exhibited sustained and progressive growth up to day 11, maintaining a well-defined spherical shape and a constant increase in relative areas. In contrast, spheroids treated with NaOH or HOE grew until day 4 but subsequently remained smaller than both controls and the NaOH + HOE group. As shown in Fig. 2 B, by day 11, the spheroid area in these groups had decreased significantly. To comprehensively evaluate the effects of the treatments, we analyzed three key morphological parameters: relative area, as an indicator of growth; compactness, reflecting cell density and internal organization of the spheroid; and necrotic area, as a marker of cell viability [ 28 , 29 ]. The compactness analysis reinforced the observed differences. Whereas vehicle spheroids maintained high levels of density and internal organization throughout the experiment, those treated with NaOH exhibited a sustained decline starting on day 6. Both HOE conditions exhibited intermediate behavior, with compactness initially increasing up to day 4, followed by a marked decline toward the end of the experiment. As shown in Fig. 2 C, these differences were significant from day 6 onward. Necrotic area analysis also revealed a clear contrast. In vehicle spheroids, central necrosis was virtually absent throughout the experiment. Spheroids treated with HOE displayed increased necrosis, particularly in the final days. In contrast, those treated with NaOH (with or without HOE 694) showed an early and sustained increase in necrotic area from day 4. As illustrated in Fig. 2 D, these differences were highly significant. Taken together, these results demonstrate that while vehicle spheroids maintained sustained growth, structural organization, and low necrosis, extracellular alkalinization compromised these properties starting on day 4. In conclusion, our findings indicate that mild alkaline treatment, particularly when combined with NHE1 inhibition, compromises the survival of renal cancer cells in both 2D and 3D models, while exerting minimal cytotoxic effects on healthy renal epithelial cells in a 2D model. These results support the notion that modulation of pH homeostasis constitutes a promising therapeutic strategy to target the acidic tumor microenvironment selectively. A- Representative images of Caki-1 spheroids at day 0 and day 11, cultured in the presence or absence of 9.6 mM NaOH and/or 1 µM HOE 694. B- Time course of relative changes in total spheroid area under each condition. C- Time course of spheroid compactness. D- Time course of necrotic core area under each condition. One-way ANOVA followed by Tukey's multiple comparisons test for each cell line at day 11. **p < 0.01, ***p < 0.001, **** vs vehicle. Enhanced NHE1 activity in cancer cells compared to healthy cells is suppressed by mild alkaline treatment. We evaluated NHE1 activity in healthy and cancerous cell lines by monitoring intracellular pH (pHi) changes in BCECF-loaded cells. To quantify NHE1 activity, we employed the NH 4 Cl pre-pulse technique. One of the hallmarks of cancer is the reversal of the pH gradient, characterized by an elevated intracellular pH and a decreased extracellular pH [ 30 ]. As expected, 786-O cancer cells displayed higher pHi, increased buffering capacity, and enhanced NHE1 activity compared to healthy HK-2 cells (Figs. 3 A–D). In contrast, Caki-1 cells displayed elevated buffering capacity and NHE1 activity but did not exhibit an alkaline pHi. Previous studies have reported that NHE1 in cancer cells undergoes an alkaline shift in its pH-sensing mechanism [ 31 ]. To determine whether this phenomenon also occurs in renal cancer cells, we plotted NHE1-dependent H⁺ fluxes as a function of pHi after the NH₄⁺ pre-pulse (before adding Na + ). Figure 3 E reveals that H⁺ fluxes in 786-O cancer cells are shifted to the right, indicating an alkaline shift in the pH sensor of NHE1. In Caki-1 cells, which are metastatic, the shift to the right is less pronounced. A- Basal pHi. B- Buffering power. C- Representative NH₄⁺ pre-pulse recovery curves in the presence or absence of NHE1 inhibition with 1 µM HOE 694 for each cell line, expressed as the difference with steady-state pHi (ΔpHi). D- NHE1-dependent H + flux during the recovery of an ammonium pulse. One-way ANOVA followed by Tukey's multiple comparisons test for pHi, Buffering Power, or Flux H + . **** p < 0.0001, 786-O, or Caki-1 vs HK-2 cells. +++ p < 0.001, Caki-1 vs 786-O cells. E- The pH dependence of NHE1-mediated proton efflux was estimated for each cell line grown in standard culture medium. The lowest pHi after the NH₄⁺ pre-pulse before adding Na + was plotted against NHE1-dependent Flux H + (Flux H⁺). Data points were fitted with a linear regression curve to assess the relationship between basal pH i and NHE1 activity across cell lines. We subsequently investigated whether NaOH affected NHE1 activity. NHE1 possesses two pH sensors [ 32 ]. Alkali-induced modifications to NHE1 could result from allosteric changes, alterations in protein expression, or changes in the subcellular localization of NHE1. To explore these possibilities, we evaluated NHE1 activity in two conditions: cells grown in NaOH for three days to allow protein expression changes and cells exposed to NaOH for only five minutes before the NH 4 Cl pre-pulse to assess rapid effects. Figures 4 D-F show that all cell types experienced inhibition of NHE1 activity upon brief exposure to NaOH despite no changes in pHi (Figs. 4 A-C). However, after three days of growth in NaOH-containing media, NHE1 activity remained inhibited in cancer cells, while normal HK-2 cells exhibited a significant increase in NHE1 activity. We also assessed whether NaOH influenced the sensitivity of the NHE1 pH sensor (Figs. 4 G-I). In HK-2 cells, NaOH exposure did not alter the pH sensitivity of the NHE1 sensor. Conversely, even brief exposure to NaOH of cancer cells caused a leftward shift in NHE1 pH sensitivity in 786-O cells, resulting in values comparable to those of HK-2 cells (healthy model). In metastatic Caki-1 cells, the alkaline leftward shift is only evident after 72 hours of alkaline treatment and is much less pronounced. This shift likely explains the inhibition of NHE1 activity observed in cancer cells, which prompted us to further investigate its localization. Cells were grown with Vehicle or 9.6 mM NaOH (for 72h, or 5 min), and pHi was measured with BCECF. A-C pHi A- HK-2 cells. B- 786-O cells. C- Caki-1 cells. D-F-NHE1-dependent H + flux was estimated by the ammonium pre-pulse technique. D- HK-2 cells. E- 786-O cells. F- Caki-1 cells. G-I The pH dependence of NHE1-mediated proton efflux was calculated for each individual cell as described in Fig. 2 D. Cells were grown under control conditions (Vehicle) or treated with 9.6 mM NaOH for either 72 hours or 5 minutes. The lowest pHi after the NH₄⁺ pre-pulse before adding Na + was plotted against NHE1-dependent Flux H + (Flux H⁺), and data were fitted to evaluate shifts in the pH dependence of NHE1 activity. G- HK-2 cells. H- 786-O cells. I- Caki-1 cells. One-way ANOVA followed by Tukey's multiple comparisons test for each cell line. * p < 0.05, *** p < 0.001, **** p < 0.0001, vs vehicle. One-way ANOVA followed by Tukey's multiple comparisons test for each cell line. Differential NHE1 localization in cancer and healthy cells is altered by prolonged alkali exposure. We investigated whether changes in NHE1 activity following NaOH exposure correlated with alterations in protein localization. Immunofluorescence studies were performed using cells stained with the plasma membrane marker WGA, and we analyzed the colocalization of WGA and NHE1. Figure 5 presents representative images of stained cells and the Mander’s coefficient for WGA-NHE1 colocalization, reflecting plasma membrane-associated NHE1 under each condition. The Mander’s coefficient was higher in cancer cells, confirming increased plasma membrane localization of NHE1. This finding correlates with the higher NHE1 activity observed in cancer cells compared to the lower activity in healthy cells. After a 5-minute exposure to NaOH, plasma membrane NHE1 localization remained comparable to control conditions across all cell lines, indicating that rapid changes in NHE1 activity in cancer cells were not due to altered protein localization. However, prolonged exposure to NaOH resulted in significant changes in plasma membrane NHE1 localization. In healthy HK-2 cells, plasma membrane NHE1 levels increased after three days of NaOH treatment. In contrast, in 786-O cancer cells, plasma membrane NHE1 levels markedly decreased under the same conditions. Interestingly, metastatic Caki-1 cells did not exhibit changes in NHE1 localization following NaOH exposure. These findings suggest that alterations in NHE1 localization may partially account for the observed changes in NHE1 activity after prolonged alkaline treatment. Cells were cultured under three conditions: Vehicle, 9.6 mM NaOH for 72 hours, or 5 minutes before NHE1 immunofluorescence analysis. The plasma membrane was stained using the membrane marker WGA. Representative images are shown for each condition. Nuclei are stained blue, WGA (plasma membrane) is green, NHE1 is red, and areas of WGA-NHE1 colocalization appear white. A- HK-2 cells. B- 786-O cells. C- Caki-1 cells. D- Colocalization analysis was performed and Mander´s coefficient was calculated. One-way ANOVA followed by Tukey's multiple comparisons test for each cell line. * p < 0.05, vs vehicle. Discussion Growing experimental evidence indicates that cancer-related pH alterations play a fundamental role in the etiopathogenesis of the disease [ 30 ]. Targeting these altered extracellular pH dynamics represents a promising strategy to destabilize cancer cells. Neutralizing extracellular pH may offer a novel therapeutic approach for clear cell renal cell carcinoma (ccRCC), a cancer type where conventional treatments often fail. In this study, we demonstrate that neutralizing the extracellular microenvironment of ccRCC cells increases apoptosis. This finding aligns with previous reports showing that extracellular pH neutralization promotes apoptosis of cancer cells across various cancer types [ 7 – 10 ]. Specifically, in renal cell carcinoma, our results are consistent with earlier findings where a therapeutic strategy targeting cancer cell pH regulation achieved disease stabilization in a clinical case [ 16 ]. We confirmed this pro-apoptotic effect in both 2D cultures of primary (786-O) and metastatic (Caki-1), as well as in a 3D spheroid model of Caki-1 cells. Spheroids recreate the tumor microenvironment more accurately, including gradients of oxygen, nutrients, and pH. Under these conditions, mild alkaline treatment, either alone or in combination with NHE1 inhibition, reduced the spheroid area and compactness while enlarging the necrotic core. These effects reflect the increased apoptosis observed in monolayer cultures, indicating that pH modulation strategies may remain effective in more physiologically relevant contexts. Together, these observations support the potential of pH modulation as an effective adjunctive treatment for ccRCC and warrant further investigation. Previous studies on pH neutralization have not thoroughly examined how normal cells respond to alkaline treatment. In this study, we show that healthy proximal epithelial cells slightly increase apoptosis following mild alkaline treatment, a process dependent on the function of the NHE1 exchanger. This finding aligns with studies conducted on other cell types. For example, Cutaia et al. reported that human pulmonary arterial endothelial cells exhibit morphological changes and a marked increase in apoptosis under alkaline stress [ 33 ]. Similarly, we previously demonstrated that apoptosis in collecting duct cells correlates with reduced cell growth and G2/M phase arrest after alkaline exposure. [ 34 ]. Our results reveal that healthy cells initially exhibit low levels of plasma membrane NHE1 and minimal NHE1-dependent H + flux. However, after three days of mild alkaline treatment, we observed increased plasma membrane localization of NHE1 and elevated NHE1-dependent proton flux. Given that alkaline treatment-induced apoptosis in healthy proximal cells depends on NHE1 activity, we hypothesize that the re-localization of NHE1 to the plasma membrane is a prerequisite for the apoptotic process. Clear cell renal cell carcinoma (ccRCC) cells exhibit elevated levels of NHE1 at the plasma membrane, increased NHE1-dependent proton extrusion, and a characteristic inversion of the pH gradient, with extracellular acidosis and intracellular alkaline treatment. Previous studies have shown that an alkaline shift in the pH sensitivity of the NHE1 proton sensor is an early and critical event in the malignant transformation of fibroblasts and keratinocytes [ 31 ]. Our findings confirm that ccRCC cells display a similar shift in pH sensing. This alteration likely accounts for the sustained NHE1 activity observed even under alkaline intracellular conditions, thereby promoting persistent proton efflux, extracellular acidification, and the pH gradient reversal that typifies many cancer types. Following brief exposure to mild alkaline treatment we observed a marked decrease in NHE1-dependent proton flux. This reduction in activity may reflect allosteric inhibition, as the pH sensitivity of the NHE1 proton sensor in 786-O cells appears to shift toward values resembling those of healthy cells. Such a shift could impair NHE1 activation at elevated intracellular pH, thereby limiting proton extrusion despite the continued presence of NHE1 at the plasma membrane. Moreover, prolonged exposure to mild alkaline treatment reduced NHE1 localization at the plasma membrane, particularly in 786-O cells. In summary, the alkaline treatment-induced inhibition and altered subcellular localization of NHE1 in cancer cells may help explain why pharmacological inhibition of NHE1 alone has limited efficacy in inducing cell death when combined with alkalinizing treatments. We hypothesize that functional suppression of the Na⁺/H⁺ exchanger 1 (NHE1) by intracellular alkaline treatment is a key mechanism driving cancer cell death. Paradoxically, as described above, in healthy proximal tubule cells, alkalinization induces NHE1-dependent cell death. Therefore, the combination of alkaline treatment with direct NHE1 inhibition protects healthy cells while selectively inducing death in cancer cells. Figure 6 illustrates a proposed model. The scheme illustrates the effects of combined alkalinizing treatment and NHE1 inhibition on HK-2 cells (healthy proximal tubule cells), 786-O cells (primary ccRCC), and Caki-1 cells (metastatic ccRCC). The figure illustrates the proposed effects of alkalinizing treatment, alone or in combination with Na⁺/H⁺ exchanger isoform 1 (NHE1) inhibition, in healthy proximal tubule cells (HK-2), primary clear cell renal carcinoma cells (786-O), and metastatic clear cell renal carcinoma cells (Caki-1). In HK-2 cells, basal NHE1 activity is minimal, and the transporter shows little localization at the plasma membrane. Exposure to alkalinizing conditions induces a slight increase in cell death, which correlates with the re-localization of NHE1 to the plasma membrane and an increase in its activity. Interestingly, when NHE1 is inhibited under alkalinizing conditions, this minor apoptotic effect is no longer observed, suggesting that the small degree of apoptosis in healthy cells is mediated by NHE1 activity. In 786-O cells, NHE1 is upregulated under basal conditions, with increased localization at the plasma membrane. Alkalinization not only counteracts the acidic tumor microenvironment but also induces apoptosis while promoting NHE1 internalization and reducing activity. Consequently, the outcomes of alkalinization alone and alkalinization combined with NHE1 inhibition are similar, since in both scenarios NHE1 activity is suppressed and apoptotic responses are triggered. In Caki-1 cells, NHE1 is also upregulated and strongly localized at the plasma membrane in basal conditions. Upon alkalinization, the exchanger does not re-localize away from the membrane, but its activity is inhibited. Alkalinization induces marked apoptosis in these metastatic cells, an effect demonstrated in both two-dimensional (2D) and three-dimensional (3D) culture models. As in 786-O cells, the combined treatment does not further enhance the effect of alkalinization, as both conditions converge on NHE1 inhibition and apoptotic induction. Overall, this model proposes that alkalinization and NHE1 inhibition exert distinct effects depending on the cellular context. In healthy cells, NHE1 mediates a small apoptotic response to alkalinization, whereas in tumor cells, alkalinization itself induces apoptosis, either through NHE1 internalization (786-O) or inhibition of its activity at the membrane (Caki-1). This differential sensitivity underscores the potential of targeting pH regulation as a therapeutic strategy in clear cell renal carcinoma. What could explain the contrasting effects of NHE1 activity in healthy and cancer cells? NHE1 is involved in a wide range of cellular functions beyond ion transport, including volume regulation, actin remodeling, and cell migration. Its activity is also subject to multiple regulatory mechanisms, meaning that NHE1-dependent responses likely vary depending on the signals reaching the cell. In healthy cells, alkaline stress may activate NHE1, leading to cell death. In contrast, in cancer cells, the prolonged stress characteristic of this pathology, as described by Pedersen, may alter the response to alkaline stress [ 35 ]. NHE1 directly interacts with the cofactor proteins calcineurin B homologous protein 1 (CHP1) and CHP2 [ 36 ]. In vitro, studies on lung carcinoma cells demonstrate that these cofactors regulate NHE1 activity. CHP1 exhibits ubiquitous localization, whereas CHP2 shows tissue-specific expression. Moreover, tumorigenic extracellular conditions, such as nutrient deprivation, modulate CHP2 expression [ 37 ]. Reflecting this regulatory complexity, Lacroix et al. proposed that NHE1 activation involves the oscillation of a dimeric NHE1 between two conformational states with distinct proton transport affinities, in agreement with the Monod–Wyman–Changeux allosteric model [ 38 ]. This regulatory behavior appears to depend on a cluster of histidine residues located in the C-terminal cytoplasmic domain, which serve as pH sensors. Remarkably, Dong et al. demonstrated that NHE1 functions as a symmetrical homodimer in complex with CHP1. CHP1 interacts differently with the distinct conformational states of each NHE1 monomer, suggesting that this differential binding contributes to the fine-tuning of NHE1’s pH sensitivity [ 36 ]. It is conceivable that, in cancerous tissues, the interaction of NHE1 with CHP2 rather than with CHP1 might alter its regulatory mechanism, potentially contributing to the aberrant pH sensitivity observed in tumors. However, this possibility remains to be experimentally validated. In conclusion, our study reveals that combining mild alkali treatment and NHE1 inhibition induces cancer cell death while minimizing damage to normal tissue. This therapeutic strategy offers a promising avenue for targeted therapy in clear cell renal cell carcinoma (ccRCC), potentially providing a novel approach for managing this challenging malignancy. Declarations Running title Alkali and NHE1 in ccRCC cell death Declarations Funding Declaration : This study was funded by grants from Fondo Nacional para la Ciencia y la Tecnología, Argentina [Grant Number: PICT 2020 − 01130]; Universidad de Buenos Aires, Argentina [Grant Number: UBACYT 20020220100022BA, UBACYT 20020130100697BA and UBACYT 20020220100029BA] and Consejo Nacional de Ciencia y Tecnología, Argentina [Grant Number: PIP 11220200102585CO] Competing Interests The authors of this manuscript certify that they have NO potential sources of conflict of interest (relationship, financial or otherwise) that might influence the author's objectivity in the subject matter or materials discussed in this manuscript. Data and/or Code availability The KNIME workflow used in this study to perform total cell count is openly available on Figshare at: Casal, Juan Jose; Rivarola, Valeria (2024). KNIME Workflow to count total cell number in photos of Acridine Orange/Ethidium Bromide experiments. Figshare. Software. https://doi.org/10.6084/m9.figshare.25913794.v2 The Python notebooks used in this study for automated quantification of necrotic areas in spheroid images are openly available on Figshare at: Funding Declaration This study was funded by grants from Fondo Nacional para la Ciencia y la Tecnología, Argentina [Grant Number: PICT 2020 − 01130]; Universidad de Buenos Aires, Argentina [Grant Number: UBACYT 20020220100022BA, UBACYT 20020130100697BA and UBACYT 20020220100029BA] and Consejo Nacional de Ciencia y Tecnología, Argentina [Grant Number: PIP 11220200102585CO] Author Contribution A.B.C conducted most of the validation, investigation, formal analysis and visualization.A.M., A.G., and G.Di G. contributed to some of the validation, investigation and formal analysis of pH experiments.N.B. was responsible for setting up the methodology of all cell models.J.J.C. developed the methodology, validation and software programming of KNIME workflow for automated cell counting from images and provided insights into all computer-based analyses and provided Data Curation.C.C. assisted in Funding acquisition and Writing - Review & Editing.P.F. and G.Di G. were involved in Funding acquisition, Conceptualization and Writing - Review & Editing.V.R. directed the project, so she is mainly involved in Conceptualization, Supervision, Formal analysis, Funding acquisition, Project administration and Writing - Original Draft and its Review & Editing. Acknowledgement The authors thank German La Iacona for his technical assistance and Matias Aduriz for his assistance in the development of the software to measure the necrotic area of spheroids. Data Availability The KNIME workflow used in this study to perform total cell count is openly available on Figshare at:Casal, Juan Jose; Rivarola, Valeria (2024). KNIME Workflow to count total cell number in photos of Acridine Orange/Ethidium Bromide experiments. Figshare. Software. https://doi.org/10.6084/m9.figshare.25913794.v2The Python notebooks used in this study for automated quantification of necrotic areas in spheroid images are openly available on Figshare at:-Individual images: Celi, Ana Beatriz; Aduriz, Matias; Casal, Juan Jose; Rivarola, Valeria (2025). Spheroid Necrotic Area Quantification for Individual Images. Figshare. Software. https://doi.org/10.6084/m9.figshare.30161296-Multiple images: Celi, Ana Beatriz; Aduriz, Matias; Casal, Juan Jose; Rivarola, Valeria (2025). 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Cite Share Download PDF Status: Published Journal Publication published 13 Mar, 2026 Read the published version in Molecular and Cellular Biochemistry → Version 2 posted Editorial decision: Revision requested 30 Nov, 2025 Reviews received at journal 29 Nov, 2025 Reviews received at journal 27 Nov, 2025 Reviewers agreed at journal 22 Nov, 2025 Reviewers agreed at journal 18 Nov, 2025 Reviews received at journal 15 Nov, 2025 Reviewers agreed at journal 20 Oct, 2025 Reviewers invited by journal 30 Sep, 2025 Editor assigned by journal 28 Sep, 2025 Submission checks completed at journal 27 Sep, 2025 First submitted to journal 25 Sep, 2025 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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13:39:03","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":121907,"visible":true,"origin":"","legend":"","description":"","filename":"913f5e524a674ddd97878b15782a324d1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7715921/v1/e33e982e051bbf08f876408d.xml"},{"id":92721534,"identity":"0363ee0d-f39e-48fa-a6a7-9647da9774fb","added_by":"auto","created_at":"2025-10-03 13:39:02","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":136739,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7715921/v1/b09799396503a0ae2f319c3a.html"},{"id":92721524,"identity":"798bcb3e-81b9-4de1-b2a8-1061cd477e4e","added_by":"auto","created_at":"2025-10-03 13:39:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":92697,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003epHi, buffering power and NHE1 activity in HK-2, 786-O, and Caki-1 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-\u003c/strong\u003eBasal pHi. \u003cstrong\u003eB-\u003c/strong\u003e Buffering power. \u003cstrong\u003eC-\u003c/strong\u003e Representative NH₄⁺ pre-pulse recovery curves in the presence or absence of NHE1 inhibition with 1 µM HOE 694 for each cell line, expressed as the difference with steady-state pHi (ΔpHi). \u003cstrong\u003eD-\u003c/strong\u003e NHE1-dependent H\u003csup\u003e+\u003c/sup\u003e flux during the recovery of an ammonium pulse. One-way ANOVA followed by Tukey's multiple comparisons test for pHi, Buffering Power, or Flux H\u003csup\u003e+\u003c/sup\u003e. **** p\u0026lt;0.0001, 786-O, or Caki-1 vs HK-2 cells. +++ p\u0026lt;0.001, Caki-1 vs 786-O cells. \u003cstrong\u003eE-\u003c/strong\u003e The pH dependence of NHE1-mediated proton efflux was estimated for each cell line grown in standard culture medium. The lowest pHi after the NH₄⁺ pre-pulse before adding Na\u003csup\u003e+\u003c/sup\u003e was plotted against NHE1-dependent Flux H\u003csup\u003e+\u003c/sup\u003e (Flux H⁺). Data points were fitted with a linear regression curve to assess the relationship between basal pHᵢ and NHE1 activity across cell lines.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7715921/v1/d5066862b91e35ed01ab4851.png"},{"id":92721526,"identity":"2520aec8-6b47-4736-a980-102bb99ba767","added_by":"auto","created_at":"2025-10-03 13:39:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":73941,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of mild alkali on pHi, NHE1-dependent H\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e flux and NHE1 pH sensitivity in HK-2, 786-O, and Caki-1 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were grown with Vehicle or 9.6 mM NaOH (for 72h, or 5 min), and pHi was measured with BCECF. \u003cstrong\u003eA-C pHi A-\u003c/strong\u003e HK-2 cells. \u003cstrong\u003eB-\u003c/strong\u003e 786-O cells. \u003cstrong\u003eC-\u003c/strong\u003e\u0026nbsp;Caki-1 cells. \u003cstrong\u003eD-F-NHE1-dependent H\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e flux\u003c/strong\u003e was estimated by the ammonium pre-pulse technique. \u003cstrong\u003eD- \u003c/strong\u003eHK-2 cells. \u003cstrong\u003eE-\u003c/strong\u003e 786-O cells. \u003cstrong\u003eF-\u003c/strong\u003e Caki-1 cells. \u003cstrong\u003eG-I \u003c/strong\u003eThe pH dependence of NHE1-mediated proton efflux was calculated for each individual cell as described in Figure 2D. Cells were grown under control conditions (Vehicle) or treated with 9.6 mM NaOH for either 72 hours or 5 minutes. The lowest pHi after the NH₄⁺ pre-pulse before adding Na\u003csup\u003e+\u003c/sup\u003e was plotted against NHE1-dependent Flux H\u003csup\u003e+\u003c/sup\u003e (Flux H⁺), and data were fitted to evaluate shifts in the pH dependence of NHE1 activity\u003cstrong\u003e. G-\u003c/strong\u003eHK-2 cells. \u003cstrong\u003eH-\u003c/strong\u003e 786-O cells. \u003cstrong\u003eI-\u003c/strong\u003e Caki-1 cells. One-way ANOVA followed by Tukey's multiple comparisons test for each cell line. * p\u0026lt;0.05, *** p\u0026lt;0.001, **** p\u0026lt;0.0001, vs vehicle. One-way ANOVA followed by Tukey's multiple comparisons test for each cell line.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7715921/v1/2606345f24d912e5afacc144.png"},{"id":92721528,"identity":"a5584fc2-deb9-4266-bf09-b8732d56390f","added_by":"auto","created_at":"2025-10-03 13:39:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":184015,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLocalization of NHE1 in HK-2, 786-O, and Caki-1 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were cultured under three conditions: Vehicle, 9.6 mM NaOH for 72 hours, or 5 minutes before NHE1 immunofluorescence analysis. The plasma membrane was stained using the membrane marker WGA. Representative images are shown for each condition. Nuclei are stained blue, WGA (plasma membrane) is green, NHE1 is red, and areas of WGA-NHE1 colocalization appear white. A- HK-2 cells. B- 786-O cells. C- Caki-1 cells. D- Colocalization analysis was performed and Mander´s coefficient was calculated. One-way ANOVA followed by Tukey's multiple comparisons test for each cell line. * p\u0026lt;0.05, vs vehicle.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7715921/v1/c6085bfc818453d7c2502b25.png"},{"id":92721529,"identity":"955d2357-abce-4f70-9a4e-412b9c553cf7","added_by":"auto","created_at":"2025-10-03 13:39:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":159119,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed model of the effects of combined alkaline treatment and NHE1 inhibition treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe scheme illustrates the effects of combined alkalinizing treatment and NHE1 inhibition on HK-2 cells (healthy proximal tubule cells), 786-O cells (primary ccRCC), and Caki-1 cells (metastatic ccRCC).\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7715921/v1/c905a8640db283bb3a23a1f1.png"},{"id":104739487,"identity":"eb4c9775-9da0-4183-bcda-e9b57faa80b0","added_by":"auto","created_at":"2026-03-16 16:07:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1748210,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5875263/v2/8688c320-8881-4291-b839-facedfe2ea97.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeting pH Regulation in Cancer: Combined Mild Alkaline treatment and NHE1 Inhibition as a Potential Therapy for Clear Cell Renal Cell Carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOne of the hallmarks of cancer cells is their acidic microenvironment. Cancer cells develop adaptive mechanisms that allow them to evade acid-mediated toxicity, promoting their growth while compromising the viability of surrounding normal cells. Localized acidosis further enhances tumor progression by facilitating extracellular matrix degradation, which promotes invasion and suppresses immune responses to tumor antigens [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among the key regulators of intracellular pH (pHi) in cancer cells is the isoform 1 of the Na⁺/H⁺ exchanger (NHE1) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. NHE1 plays a vital role in physiological processes such as epithelial salt transport, cell volume regulation, proliferation, and apoptosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, its influence on apoptosis is context-dependent, as it can either promote or inhibit cell death depending on the specific cell type and conditions studied [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn recent years, researchers have proposed neutralizing the acidic microenvironment of tumors to slow the progression of certain cancers. In silico simulations, which model changes in microenvironmental pH, suggest that increasing pH can reduce cancer progression rate [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Experimental studies in breast, prostate, and melanoma cancers further support this approach [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, maintaining stable intracellular pH is critical for proper cell proliferation and death mechanisms under physiological conditions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, to establish optimal conditions for safely applying this strategy, the potential adverse effects of pH neutralization on healthy tissues must also be evaluated.\u003c/p\u003e\u003cp\u003eRenal cell carcinoma (RCC) includes a diverse group of cancers originating from renal epithelial cells and ranks among the ten most common cancers worldwide. Clear cell renal cell carcinoma (ccRCC) is the most prevalent subtype, accounting for 70\u0026ndash;80% of RCC cases [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. When detected at advanced stages or following metastatic spread, ccRCC exhibits high mortality in both men and women, primarily owing to its resistance to chemotherapy and radiotherapy [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Understanding the role of the cancer microenvironment and its interaction with transporters like NHE1 could offer new therapeutic strategies for ccRCC. For instance, Koltai et al. describe a case where a patient\u0026rsquo;s ccRCC stabilized following treatment with repurposed drugs that neutralized the inverted pH gradient [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study investigates the effects of mild alkaline treatment combined with NHE1 inhibition on cell death in normal and cancerous renal cells. We use three human renal cell models: HK-2, representing healthy proximal tubular epithelial cells; 786-O, derived from a primary clear cell renal cell carcinoma; and Caki-1, derived from a ccRCC metastasis. Our findings demonstrate that the combination of mild alkaline treatment and NHE1 inhibition selectively induces cell death in ccRCC cells while minimally affecting normal renal cells. Moreover, prolonged exposure to alkaline treatment downregulates NHE1 expression in ccRCC cells, suggesting a potential mechanism for this selective effect. These results provide insights into the interplay between extracellular pH modulation and NHE1 activity, offering a promising avenue for therapeutic strategies targeting ccRCC.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell culture\u003c/h2\u003e\u003cp\u003eWe cultured HK-2, 786-O, and Caki-1 cells using standard periodic subcultures on plastic supports. The culture media used were DMEM-F12 for HK-2 cells, RPMI for 786-O cells, and McCoy's 5A for Caki-1 cells, all supplemented with fetal bovine serum and antibiotics.\u003c/p\u003e\u003cp\u003eThe non-tumorigenic human proximal tubular epithelial cell line HK-2 (ATCC Cat# CRL-2190, RRID: CVCL_0302) was cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. HK-2 cells were kindly provided by Dr. Mar\u0026iacute;a Marta Amaral (Laboratorio de Fisiopatogenia, IFIBIO-Houssay, CONICET\u0026ndash;UBA, Argentina), who acquired them directly from the American Type Culture Collection (ATCC). The human renal epithelial cell lines 786-O (ATCC Cat# CRL-1932, RRID: CVCL_1051) and Caki-1 (ATCC Cat# HTB-46, RRID: CVCL_0234) were maintained on plastic supports using standard subculturing protocols. Caki-1 cells were cultured in McCoy\u0026rsquo;s 5A medium, and 786-O cells in RPMI 1640 medium both supplemented with 10% FBS and 1% penicillin/streptomycin [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These cell lines were kindly provided by Dr. Mariana Callero (Instituto de Oncolog\u0026iacute;a \u0026ldquo;Dr. \u0026Aacute;ngel Roffo\u0026rdquo;, UBA, Argentina), who obtained them from the National Cancer Institute cell repository. All cell lines are not listed as misidentified or problematic in the ICLAC or Cellosaurus databases. All cell lines were handled under consistent culture conditions and were routinely tested and confirmed negative for mycoplasma contamination throughout the course of the experiments.\u003c/p\u003e\u003cp\u003eTo create alkaline conditions, we adjusted the culture media by adding NaOH. For NHE1 blockade experiments, we added 1 \u0026micro;M HOE 694 (Aventis Pharma Deutschland GmbH, Germany) or vehicle (water, diluted 1:1000) to the medium. The osmolarity of the culture media and solutions, measured with a Vapro 5520 vapor pressure osmometer (Wescor), was not affected by the addition of NaOH, the vehicles, or the inhibitors.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell Death analysis using Acridine Orange and Ethidium Bromide staining\u003c/h3\u003e\n\u003cp\u003eTo quantify cell death, we washed the cells with 1X PBS and stained them with the fluorescent DNA-binding dyes acridine orange (AO, 15 \u0026micro;g/ml) and ethidium bromide (EB, 500 \u0026micro;g/ml) for 1 minute at room temperature. AO, which emits green fluorescence, penetrates both viable and dead cells, while EB, emitting red fluorescence, selectively marks dead cells. After staining, we washed the cells twice with PBS and immediately captured micrographs using an Olympus OPTIPHOT IMT-2 microscope equipped with an SPlan 10 PL 10X/0.30 NA objective. A Sony Alpha 3000 camera digitized the captures. We analyzed at least six fields from three independent coverslips for each condition.\u003c/p\u003e\u003cp\u003eTo evaluate the percentage of cells displaying morphological features of cell death, we performed each experiment in triplicate and scored between 1,000 and 5,000 cells for each condition. We identified dead cells using established criteria such as nuclear morphological changes, chromatin condensation, and staining [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], ImageJ software facilitated image analysis and counting. We designed a KNIME workflow to perform total cell counts (see Data and/or Code availability).\u003c/p\u003e\n\u003ch3\u003eCell Death Analysis using Annexin V and Propidium iodide staining\u003c/h3\u003e\n\u003cp\u003eTo assess cell death using an alternative method, we performed Annexin V/Propidium Iodide (PI) staining as described by S\u0026aacute;nchez et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. We stained the cells with FITC-conjugated Annexin V (AV, BD Pharmingen) and Propidium Iodide, following the manufacturer's protocol. We captured micrographs with an Olympus OPTIPHOT IMT-2 microscope equipped with an SPlan 10 PL 10X/0.30 NA objective and digitized them using a Sony Alpha 3000 camera attached to the microscope. For each condition, we analyzed at least six fields from a minimum of three independent coverslips.\u003c/p\u003e\u003cp\u003eWe classified cells based on staining patterns: AV-/PI- cells were considered viable, AV+/PI- cells were in early apoptosis, AV+/PI\u0026thinsp;+\u0026thinsp;cells were in late apoptosis, and AV-/PI\u0026thinsp;+\u0026thinsp;cells were necrotic.\u003c/p\u003e\n\u003ch3\u003eSpheroid Formation and Image Analysis\u003c/h3\u003e\n\u003cp\u003eCaki-1 cells were pre-exposed to vehicle or experimental treatments for 72 hours before spheroid generation. We assembled spheroids using the hanging drop method (2,000 cells in 30 \u0026micro;L of McCoy\u0026rsquo;s medium supplemented with 10% FBS and antibiotics) maintaining the same treatment conditions throughout the process.\u003c/p\u003e\u003cp\u003eAfter 4 days, we transferred the spheroids to 96-well plates containing fresh medium with either vehicle or treatments, which were maintained for the entire 11-day follow-up period. Images were acquired using a Sony Alpha 3000 camera and analyzed in ImageJ for area and compactness, with values normalized to day 0. For the quantification of areas of cell death, a custom Python script was developed in Google Colab, utilizing adaptive thresholding based on image intensity histograms (see Data and/or Code availability). Analyses were performed in three independent experiments, evaluating 20\u0026ndash;26 spheroids per treatment.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence studies\u003c/h3\u003e\n\u003cp\u003eWe conducted immunofluorescence studies on all cell lines to detect NHE1 expression. First, we seeded the cells onto coverslips and allowed them to grow in the presence or absence of 9.6 mM NaOH. In some experiments, we incubated the cells with 5 \u0026micro;g/ml wheat germ agglutinin Alexa 488 conjugate (WGA, Thermo Fisher Scientific, Cat# W11261). We then fixed the cells with 3% paraformaldehyde for 15 minutes and permeabilized them with 0.2% saponin at room temperature. Next, we incubated the samples overnight at 4\u0026ordm;C with monoclonal anti-NHE1 (dilution 1:200, Santa Cruz Biotechnology, Cat# sc-518041, RRID: AB_3481247) and followed with a 2-hour incubation at room temperature with secondary antibodies (anti-mouse Alexa 594 conjugate, dilution 1:200, Thermo Fisher Scientific, Cat# A-11032, RRID: AB_2534091). To stain the nuclei, we used Hoechst (5 \u0026micro;g/ml) for 3 minutes and then mounted the coverslips with Vectashield mounting medium (Vector Laboratories Inc., CA, USA) at room temperature.\u003c/p\u003e\u003cp\u003eWe captured and digitized the images using Fluoview FV1000 software on an Olympus FV1000 confocal microscope (objective UPLSAPO 40X/1.30 NA). We analyzed at least five fields from three independent coverslips per condition. Finally, we performed 3D reconstruction and colocalization analysis using Imaris software, calculating the amount of colocalization with the Mander\u0026rsquo;s coefficient.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eIntracellular pH Measurement and NHE1 Activity Assessment Using BCECF-AM and NH\u003csub\u003e4\u003c/sub\u003eCl Pre-Pulse Technique\u003c/h2\u003e\u003cp\u003eFor intracellular pH (pHi) measurements, we grew cells on glass coverslips and loaded them with 13 \u0026micro;M 2\u0026rsquo;,7\u0026rsquo;bis(2-carboxyethyl)-5-(and\u0026ndash;6) carboxyfluorescein acetoxymethyl ester (BCECF-AM, Thermo Fisher Scientific, Cat# B1170) for 15 minutes at 37\u0026deg;C. Afterward, we washed away the excess dye. We then placed the chamber on a Nikon TE-200 epifluorescence inverted microscope (Nikon Plan Fluor 40X oil immersion objective lens) with a micro-incubator system and temperature controller (Harvard Apparatus Inc, MA, USA). Fluorescence data were collected every 10 seconds from a small circular region in the cells using a charge-coupled-device camera (Hamamatsu C4742-95) connected to a computer running the Metafluor acquisition program (Universal Imaging Corporation, PA, USA).\u003c/p\u003e\u003cp\u003eWe determined intracellular pH by calculating the ratio of BCECF emission intensity at 535 nm. The dye was excited at ~\u0026thinsp;490 nm, and its isosbestic point was set at ~\u0026thinsp;440 nm. We calibrated the 490/440 BCECF fluorescence ratio by incubating the cells in a high K\u003csup\u003e+\u003c/sup\u003e solution (140mM KCl, 4.6mM, NaCl, 1mM MgCl2, 2mM CaCl\u003csub\u003e2\u003c/sub\u003e, 10mM Hepes, 5mM glucose), followed by permeabilization with 5mM nigericin to equilibrate extracellular pH (pHo) with pHi. We then stepped the pH-bathing solution between 6.6 and 8.5. The 490/440 ratio remained linear within this pH range (r\u0026thinsp;=\u0026thinsp;0.96, n\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComposition of solutions used for determination of pH\u003csub\u003ei\u003c/sub\u003e recovery.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSolution\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0 Na\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003eCl\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNaCl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e----\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e127\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNaHCO\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e----\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e----\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e----\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHepes\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eKCl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCaCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMgCl\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e----\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e----\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e----\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMgSO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eKH\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003ePO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eK\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eSO\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eNH\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eCl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e----\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e----\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTMA\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e----\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e147\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e----\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eglucose\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAll concentrations are given in mM; pH was adjusted to 7.4 at 37\u0026deg;C.\u003c/p\u003e\u003cp\u003eIn some experiments, we monitored NHE1 activity by evaluating the recovery of pHi after an acid load, using the NH\u003csub\u003e4\u003c/sub\u003eCl pre-pulse technique as previously described [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. We first exposed the cells to a control solution, followed by a 20 mM NH\u003csub\u003e4\u003c/sub\u003eCl pulse (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and then Na\u003csup\u003e+\u003c/sup\u003e removal (0 Na\u003csup\u003e+\u003c/sup\u003e in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Afterward, we reintroduced Na\u003csup\u003e+\u003c/sup\u003e to the bath and evaluated Na\u003csup\u003e+\u003c/sup\u003e-dependent pHi recovery mechanisms, see Online Resource 1.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eOnline Resource 1 pH experiments A-\u003c/b\u003e Representative of pinhole placement. \u003cb\u003eB-\u003c/b\u003e Representative intracellular pH (pHi) vs time in control 786-O cells\u003c/p\u003e\u003cp\u003eWe calculated acid fluxes using the following equation Eq. (A.1):\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:JH=\\beta\\:i\\times\\:\\frac{dpH}{dt}\\)\u003c/span\u003e\u003c/span\u003e Eq. (A.1)\u003c/p\u003e\u003cp\u003eH\u003csup\u003e+\u003c/sup\u003e represents the H\u003csup\u003e+\u003c/sup\u003e flux, dpH/dt is the recovery rate from the acid load, and βi is the intracellular buffering power. We estimated intracellular H\u003csup\u003e+\u003c/sup\u003e buffering power (βi) using the application of external permeant weak bases, as described by Roos, Boron, and Saleh [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this study, we used NH\u003csub\u003e4\u003c/sub\u003eCl, which permeates the cell membrane in its uncharged form but exists mainly in the ionized form once inside the cell. The ionization of this weak base absorbs H\u003csup\u003e+\u003c/sup\u003e ions. Thus, NH\u003csub\u003e4\u003c/sub\u003eCl induces changes in pHi, which we used to estimate βi using the following equation Eq. (A.2):\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\beta\\:i\\left(mM\\right)=\\left(\\right[NH\\_4\\:]\\_o\\cdot\\:10^((pHo-pHip)\\left)\\right)/\\varDelta\\:pHi\\)\u003c/span\u003e\u003c/span\u003e Eq. (A.2)\u003c/p\u003e\u003cp\u003eWhere ΔpHi is the change in pHi following the ammonium load, [NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e]\u003csub\u003e0\u003c/sub\u003e is the concentration of external NH4\u003csup\u003e+\u003c/sup\u003e, pHo is the pH of the external solution, and pHip is the intracellular pH after the ammonium load.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eStatistics\u003c/h3\u003e\n\u003cp\u003eWe presented the values as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM), with \"n\" indicating the number of cells compared. To compare data between the two groups, we used an unpaired Student's t-test, considering differences significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. For comparisons among multiple groups, we performed a one-way analysis of variance (ANOVA) followed by a Tukey post-hoc test, defining significance as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eCancer cell death induced by alkali and NHE1 inhibition with minimal impact on healthy cells.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe used three human renal cell models: HK-2, representing healthy proximal tubular epithelial cells; 786-O, derived from primary clear cell renal cell carcinoma (ccRCC); and Caki-1, derived from a ccRCC metastasis. As expected, upon reaching confluence, the pH of the conditioned media (pHe) of normal HK-2 cells was less acidic than that of the cancer cell lines (pHe: HK-2\u0026thinsp;=\u0026thinsp;7.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, n\u0026thinsp;=\u0026thinsp;26; 786-O\u0026thinsp;=\u0026thinsp;7.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;24; Caki-1\u0026thinsp;=\u0026thinsp;7.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;17; one-way ANOVA followed by Tukey's multiple comparisons test: HK-2 vs. 786-O, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; HK-2 vs. Caki-1, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003cp\u003eWe investigated the effects of neutralizing the acidic microenvironment by adding different concentrations of NaOH to the media for 72 hours. We then analyzed apoptosis percentages using the acridine orange/ethidium bromide staining method. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, NaOH concentrations (2.4\u0026ndash;9.6 mM) caused minimal increases in cell death in HK-2 cells. In contrast, these concentrations led to a progressive increase in cell death in cancer cell lines. We selected 9.6 mM NaOH for 72-hour treatments in subsequent experiments.\u003c/p\u003e\u003cp\u003eAlthough the mild alkaline treatment (9.6mM NaOH) did not significantly alter the HK-extracellular pH, it was sufficient to neutralize the acidic microenvironment of the cancer cell lines, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. Next, we explored the combined effects of NHE1 inhibition and mild alkaline treatment. To specifically inhibit NHE1, we used the HOE 694 inhibitor (1 \u0026micro;M, [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]). Without NaOH, NHE1 inhibition only increased apoptosis in metastatic-derived Caki-1 cells. While NaOH alone slightly increased cell death in HK-2 cells, the combination of NHE1 inhibition and NaOH prevented NaOH-induced cell death in these cells. In contrast, NaOH-induced cell death was significantly higher in cancer cell lines compared to HK-2 cells, and NHE1 suppression did not alter this result (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Finally, annexin V/propidium iodide staining experiments confirmed that the cell death observed through acridine orange/ethidium bromide staining was primarily due to apoptosis (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E). Thus, the combination of mild alkali treatment and NHE1 inhibition selectively induced apoptosis in cancer cells with minimal damage to healthy cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eA-\u003c/b\u003e HK-2, 786-O, and Caki-1 cells were incubated for 72 hours with either vehicle or 1.2\u0026ndash;9.6 mM NaOH. Apoptosis was quantified using the acridine orange/ethidium bromide staining method (AO/BE). \u003cb\u003eB-\u003c/b\u003e Impact of NaOH on the pH of conditioned media after 72 hours with vehicle (-) or 9.6 mM NaOH (+). **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; n\u0026thinsp;=\u0026thinsp;103. One-way ANOVA followed by Tukey's multiple comparisons test vs. HK-2 without NaOH. \u003cb\u003eC-E\u003c/b\u003e Cells were treated with vehicle or 1 \u0026micro;M HOE 694 (NHE1 inhibitor) in the presence or absence of 9.6 mM NaOH for 72 hours. \u003cb\u003eC-\u003c/b\u003e Apoptosis was quantified by acridine orange/ethidium bromide staining. \u003cb\u003eD-\u003c/b\u003e Apoptosis was assessed using the annexin V method. \u003cb\u003eE-\u003c/b\u003e Necrosis measured using the annexin V method. One-way ANOVA followed by Tukey's multiple comparisons test for each cell line. +p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ++p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, +++p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, vs vehicle.\u003c/p\u003e\u003cp\u003eTo further evaluate this effect under more physiologically relevant conditions, we examined the impact of alkaline treatment, with or without NHE1 inhibition, in a 3D culture model. For this purpose, we generated spheroids of Caki-1 cells, as they better recapitulate tumor architecture and microenvironment compared with conventional 2D cultures. Spheroids were produced using the hanging drop method, which facilitates cell self-aggregation in the absence of exogenous matrices, thereby providing a controlled, scaffold-free system to study intrinsic cell\u0026ndash;cell interactions.\u003c/p\u003e\u003cp\u003eCaki-1 cells were pre-incubated for 72 hours with vehicle, 9.6 mM NaOH, and/or 1 \u0026micro;M HOE 694 before spheroid formation, and treatments were maintained throughout the experimental period. Spheroids were monitored for 11 days. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, at the beginning of the culture (day 0), all groups formed compact spheroids, indicating that none of the treatments interfered with the cells\u0026rsquo; ability to self-aggregate.\u003c/p\u003e\u003cp\u003eIn conditions with vehicle, spheroids exhibited sustained and progressive growth up to day 11, maintaining a well-defined spherical shape and a constant increase in relative areas. In contrast, spheroids treated with NaOH or HOE grew until day 4 but subsequently remained smaller than both controls and the NaOH\u0026thinsp;+\u0026thinsp;HOE group. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, by day 11, the spheroid area in these groups had decreased significantly.\u003c/p\u003e\u003cp\u003eTo comprehensively evaluate the effects of the treatments, we analyzed three key morphological parameters: relative area, as an indicator of growth; compactness, reflecting cell density and internal organization of the spheroid; and necrotic area, as a marker of cell viability [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The compactness analysis reinforced the observed differences. Whereas vehicle spheroids maintained high levels of density and internal organization throughout the experiment, those treated with NaOH exhibited a sustained decline starting on day 6. Both HOE conditions exhibited intermediate behavior, with compactness initially increasing up to day 4, followed by a marked decline toward the end of the experiment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, these differences were significant from day 6 onward.\u003c/p\u003e\u003cp\u003eNecrotic area analysis also revealed a clear contrast. In vehicle spheroids, central necrosis was virtually absent throughout the experiment. Spheroids treated with HOE displayed increased necrosis, particularly in the final days. In contrast, those treated with NaOH (with or without HOE 694) showed an early and sustained increase in necrotic area from day 4. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, these differences were highly significant.\u003c/p\u003e\u003cp\u003eTaken together, these results demonstrate that while vehicle spheroids maintained sustained growth, structural organization, and low necrosis, extracellular alkalinization compromised these properties starting on day 4.\u003c/p\u003e\u003cp\u003eIn conclusion, our findings indicate that mild alkaline treatment, particularly when combined with NHE1 inhibition, compromises the survival of renal cancer cells in both 2D and 3D models, while exerting minimal cytotoxic effects on healthy renal epithelial cells in a 2D model. These results support the notion that modulation of pH homeostasis constitutes a promising therapeutic strategy to target the acidic tumor microenvironment selectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eA-\u003c/b\u003e Representative images of Caki-1 spheroids at day 0 and day 11, cultured in the presence or absence of 9.6 mM NaOH and/or 1 \u0026micro;M HOE 694. \u003cb\u003eB-\u003c/b\u003e Time course of relative changes in total spheroid area under each condition. \u003cb\u003eC-\u003c/b\u003e Time course of spheroid compactness. \u003cb\u003eD-\u003c/b\u003e Time course of necrotic core area under each condition. One-way ANOVA followed by Tukey's multiple comparisons test for each cell line at day 11. **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, **** vs vehicle.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEnhanced NHE1 activity in cancer cells compared to healthy cells is suppressed by mild alkaline treatment.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe evaluated NHE1 activity in healthy and cancerous cell lines by monitoring intracellular pH (pHi) changes in BCECF-loaded cells. To quantify NHE1 activity, we employed the NH\u003csub\u003e4\u003c/sub\u003eCl pre-pulse technique. One of the hallmarks of cancer is the reversal of the pH gradient, characterized by an elevated intracellular pH and a decreased extracellular pH [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. As expected, 786-O cancer cells displayed higher pHi, increased buffering capacity, and enhanced NHE1 activity compared to healthy HK-2 cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;D). In contrast, Caki-1 cells displayed elevated buffering capacity and NHE1 activity but did not exhibit an alkaline pHi.\u003c/p\u003e\u003cp\u003ePrevious studies have reported that NHE1 in cancer cells undergoes an alkaline shift in its pH-sensing mechanism [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. To determine whether this phenomenon also occurs in renal cancer cells, we plotted NHE1-dependent H⁺ fluxes as a function of pHi after the NH₄⁺ pre-pulse (before adding Na\u003csup\u003e+\u003c/sup\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE reveals that H⁺ fluxes in 786-O cancer cells are shifted to the right, indicating an alkaline shift in the pH sensor of NHE1. In Caki-1 cells, which are metastatic, the shift to the right is less pronounced.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eA-\u003c/b\u003eBasal pHi. \u003cb\u003eB-\u003c/b\u003e Buffering power. \u003cb\u003eC-\u003c/b\u003e Representative NH₄⁺ pre-pulse recovery curves in the presence or absence of NHE1 inhibition with 1 \u0026micro;M HOE 694 for each cell line, expressed as the difference with steady-state pHi (ΔpHi). \u003cb\u003eD-\u003c/b\u003e NHE1-dependent H\u003csup\u003e+\u003c/sup\u003e flux during the recovery of an ammonium pulse. One-way ANOVA followed by Tukey's multiple comparisons test for pHi, Buffering Power, or Flux H\u003csup\u003e+\u003c/sup\u003e. **** p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, 786-O, or Caki-1 vs HK-2 cells. +++ p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Caki-1 vs 786-O cells. \u003cb\u003eE-\u003c/b\u003e The pH dependence of NHE1-mediated proton efflux was estimated for each cell line grown in standard culture medium. The lowest pHi after the NH₄⁺ pre-pulse before adding Na\u003csup\u003e+\u003c/sup\u003e was plotted against NHE1-dependent Flux H\u003csup\u003e+\u003c/sup\u003e (Flux H⁺). Data points were fitted with a linear regression curve to assess the relationship between basal pH\u003csub\u003ei\u003c/sub\u003e and NHE1 activity across cell lines.\u003c/p\u003e\u003cp\u003eWe subsequently investigated whether NaOH affected NHE1 activity. NHE1 possesses two pH sensors [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Alkali-induced modifications to NHE1 could result from allosteric changes, alterations in protein expression, or changes in the subcellular localization of NHE1. To explore these possibilities, we evaluated NHE1 activity in two conditions: cells grown in NaOH for three days to allow protein expression changes and cells exposed to NaOH for only five minutes before the NH\u003csub\u003e4\u003c/sub\u003eCl pre-pulse to assess rapid effects.\u003c/p\u003e\u003cp\u003eFigures \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F show that all cell types experienced inhibition of NHE1 activity upon brief exposure to NaOH despite no changes in pHi (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). However, after three days of growth in NaOH-containing media, NHE1 activity remained inhibited in cancer cells, while normal HK-2 cells exhibited a significant increase in NHE1 activity.\u003c/p\u003e\u003cp\u003eWe also assessed whether NaOH influenced the sensitivity of the NHE1 pH sensor (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-I). In HK-2 cells, NaOH exposure did not alter the pH sensitivity of the NHE1 sensor. Conversely, even brief exposure to NaOH of cancer cells caused a leftward shift in NHE1 pH sensitivity in 786-O cells, resulting in values comparable to those of HK-2 cells (healthy model). In metastatic Caki-1 cells, the alkaline leftward shift is only evident after 72 hours of alkaline treatment and is much less pronounced. This shift likely explains the inhibition of NHE1 activity observed in cancer cells, which prompted us to further investigate its localization.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCells were grown with Vehicle or 9.6 mM NaOH (for 72h, or 5 min), and pHi was measured with BCECF. \u003cb\u003eA-C pHi A-\u003c/b\u003e HK-2 cells. \u003cb\u003eB-\u003c/b\u003e 786-O cells. \u003cb\u003eC-\u003c/b\u003e Caki-1 cells. \u003cb\u003eD-F-NHE1-dependent H\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eflux\u003c/b\u003e was estimated by the ammonium pre-pulse technique. \u003cb\u003eD-\u003c/b\u003e HK-2 cells. \u003cb\u003eE-\u003c/b\u003e 786-O cells. \u003cb\u003eF-\u003c/b\u003e Caki-1 cells. \u003cb\u003eG-I\u003c/b\u003e The pH dependence of NHE1-mediated proton efflux was calculated for each individual cell as described in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD. Cells were grown under control conditions (Vehicle) or treated with 9.6 mM NaOH for either 72 hours or 5 minutes. The lowest pHi after the NH₄⁺ pre-pulse before adding Na\u003csup\u003e+\u003c/sup\u003e was plotted against NHE1-dependent Flux H\u003csup\u003e+\u003c/sup\u003e (Flux H⁺), and data were fitted to evaluate shifts in the pH dependence of NHE1 activity. \u003cb\u003eG-\u003c/b\u003eHK-2 cells. \u003cb\u003eH-\u003c/b\u003e 786-O cells. \u003cb\u003eI-\u003c/b\u003e Caki-1 cells. One-way ANOVA followed by Tukey's multiple comparisons test for each cell line. * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, **** p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, vs vehicle. One-way ANOVA followed by Tukey's multiple comparisons test for each cell line.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDifferential NHE1 localization in cancer and healthy cells is altered by prolonged alkali exposure.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe investigated whether changes in NHE1 activity following NaOH exposure correlated with alterations in protein localization. Immunofluorescence studies were performed using cells stained with the plasma membrane marker WGA, and we analyzed the colocalization of WGA and NHE1. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e presents representative images of stained cells and the Mander\u0026rsquo;s coefficient for WGA-NHE1 colocalization, reflecting plasma membrane-associated NHE1 under each condition.\u003c/p\u003e\u003cp\u003eThe Mander\u0026rsquo;s coefficient was higher in cancer cells, confirming increased plasma membrane localization of NHE1. This finding correlates with the higher NHE1 activity observed in cancer cells compared to the lower activity in healthy cells. After a 5-minute exposure to NaOH, plasma membrane NHE1 localization remained comparable to control conditions across all cell lines, indicating that rapid changes in NHE1 activity in cancer cells were not due to altered protein localization.\u003c/p\u003e\u003cp\u003eHowever, prolonged exposure to NaOH resulted in significant changes in plasma membrane NHE1 localization. In healthy HK-2 cells, plasma membrane NHE1 levels increased after three days of NaOH treatment. In contrast, in 786-O cancer cells, plasma membrane NHE1 levels markedly decreased under the same conditions. Interestingly, metastatic Caki-1 cells did not exhibit changes in NHE1 localization following NaOH exposure.\u003c/p\u003e\u003cp\u003eThese findings suggest that alterations in NHE1 localization may partially account for the observed changes in NHE1 activity after prolonged alkaline treatment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCells were cultured under three conditions: Vehicle, 9.6 mM NaOH for 72 hours, or 5 minutes before NHE1 immunofluorescence analysis. The plasma membrane was stained using the membrane marker WGA. Representative images are shown for each condition. Nuclei are stained blue, WGA (plasma membrane) is green, NHE1 is red, and areas of WGA-NHE1 colocalization appear white. A- HK-2 cells. B- 786-O cells. C- Caki-1 cells. D- Colocalization analysis was performed and Mander\u0026acute;s coefficient was calculated. One-way ANOVA followed by Tukey's multiple comparisons test for each cell line. * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, vs vehicle.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eGrowing experimental evidence indicates that cancer-related pH alterations play a fundamental role in the etiopathogenesis of the disease [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Targeting these altered extracellular pH dynamics represents a promising strategy to destabilize cancer cells. Neutralizing extracellular pH may offer a novel therapeutic approach for clear cell renal cell carcinoma (ccRCC), a cancer type where conventional treatments often fail.\u003c/p\u003e\u003cp\u003eIn this study, we demonstrate that neutralizing the extracellular microenvironment of ccRCC cells increases apoptosis. This finding aligns with previous reports showing that extracellular pH neutralization promotes apoptosis of cancer cells across various cancer types [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Specifically, in renal cell carcinoma, our results are consistent with earlier findings where a therapeutic strategy targeting cancer cell pH regulation achieved disease stabilization in a clinical case [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. We confirmed this pro-apoptotic effect in both 2D cultures of primary (786-O) and metastatic (Caki-1), as well as in a 3D spheroid model of Caki-1 cells. Spheroids recreate the tumor microenvironment more accurately, including gradients of oxygen, nutrients, and pH. Under these conditions, mild alkaline treatment, either alone or in combination with NHE1 inhibition, reduced the spheroid area and compactness while enlarging the necrotic core. These effects reflect the increased apoptosis observed in monolayer cultures, indicating that pH modulation strategies may remain effective in more physiologically relevant contexts. Together, these observations support the potential of pH modulation as an effective adjunctive treatment for ccRCC and warrant further investigation.\u003c/p\u003e\u003cp\u003ePrevious studies on pH neutralization have not thoroughly examined how normal cells respond to alkaline treatment. In this study, we show that healthy proximal epithelial cells slightly increase apoptosis following mild alkaline treatment, a process dependent on the function of the NHE1 exchanger. This finding aligns with studies conducted on other cell types. For example, Cutaia et al. reported that human pulmonary arterial endothelial cells exhibit morphological changes and a marked increase in apoptosis under alkaline stress [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Similarly, we previously demonstrated that apoptosis in collecting duct cells correlates with reduced cell growth and G2/M phase arrest after alkaline exposure. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Our results reveal that healthy cells initially exhibit low levels of plasma membrane NHE1 and minimal NHE1-dependent H\u003csup\u003e+\u003c/sup\u003e flux. However, after three days of mild alkaline treatment, we observed increased plasma membrane localization of NHE1 and elevated NHE1-dependent proton flux. Given that alkaline treatment-induced apoptosis in healthy proximal cells depends on NHE1 activity, we hypothesize that the re-localization of NHE1 to the plasma membrane is a prerequisite for the apoptotic process.\u003c/p\u003e\u003cp\u003eClear cell renal cell carcinoma (ccRCC) cells exhibit elevated levels of NHE1 at the plasma membrane, increased NHE1-dependent proton extrusion, and a characteristic inversion of the pH gradient, with extracellular acidosis and intracellular alkaline treatment. Previous studies have shown that an alkaline shift in the pH sensitivity of the NHE1 proton sensor is an early and critical event in the malignant transformation of fibroblasts and keratinocytes [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Our findings confirm that ccRCC cells display a similar shift in pH sensing. This alteration likely accounts for the sustained NHE1 activity observed even under alkaline intracellular conditions, thereby promoting persistent proton efflux, extracellular acidification, and the pH gradient reversal that typifies many cancer types. Following brief exposure to mild alkaline treatment we observed a marked decrease in NHE1-dependent proton flux. This reduction in activity may reflect allosteric inhibition, as the pH sensitivity of the NHE1 proton sensor in 786-O cells appears to shift toward values resembling those of healthy cells. Such a shift could impair NHE1 activation at elevated intracellular pH, thereby limiting proton extrusion despite the continued presence of NHE1 at the plasma membrane. Moreover, prolonged exposure to mild alkaline treatment reduced NHE1 localization at the plasma membrane, particularly in 786-O cells. In summary, the alkaline treatment-induced inhibition and altered subcellular localization of NHE1 in cancer cells may help explain why pharmacological inhibition of NHE1 alone has limited efficacy in inducing cell death when combined with alkalinizing treatments. We hypothesize that functional suppression of the Na⁺/H⁺ exchanger 1 (NHE1) by intracellular alkaline treatment is a key mechanism driving cancer cell death. Paradoxically, as described above, in healthy proximal tubule cells, alkalinization induces NHE1-dependent cell death. Therefore, the combination of alkaline treatment with direct NHE1 inhibition protects healthy cells while selectively inducing death in cancer cells. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e illustrates a proposed model.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe scheme illustrates the effects of combined alkalinizing treatment and NHE1 inhibition on HK-2 cells (healthy proximal tubule cells), 786-O cells (primary ccRCC), and Caki-1 cells (metastatic ccRCC).\u003c/p\u003e\u003cp\u003eThe figure illustrates the proposed effects of alkalinizing treatment, alone or in combination with Na⁺/H⁺ exchanger isoform 1 (NHE1) inhibition, in healthy proximal tubule cells (HK-2), primary clear cell renal carcinoma cells (786-O), and metastatic clear cell renal carcinoma cells (Caki-1). In HK-2 cells, basal NHE1 activity is minimal, and the transporter shows little localization at the plasma membrane. Exposure to alkalinizing conditions induces a slight increase in cell death, which correlates with the re-localization of NHE1 to the plasma membrane and an increase in its activity. Interestingly, when NHE1 is inhibited under alkalinizing conditions, this minor apoptotic effect is no longer observed, suggesting that the small degree of apoptosis in healthy cells is mediated by NHE1 activity. In 786-O cells, NHE1 is upregulated under basal conditions, with increased localization at the plasma membrane. Alkalinization not only counteracts the acidic tumor microenvironment but also induces apoptosis while promoting NHE1 internalization and reducing activity. Consequently, the outcomes of alkalinization alone and alkalinization combined with NHE1 inhibition are similar, since in both scenarios NHE1 activity is suppressed and apoptotic responses are triggered. In Caki-1 cells, NHE1 is also upregulated and strongly localized at the plasma membrane in basal conditions. Upon alkalinization, the exchanger does not re-localize away from the membrane, but its activity is inhibited. Alkalinization induces marked apoptosis in these metastatic cells, an effect demonstrated in both two-dimensional (2D) and three-dimensional (3D) culture models. As in 786-O cells, the combined treatment does not further enhance the effect of alkalinization, as both conditions converge on NHE1 inhibition and apoptotic induction. Overall, this model proposes that alkalinization and NHE1 inhibition exert distinct effects depending on the cellular context. In healthy cells, NHE1 mediates a small apoptotic response to alkalinization, whereas in tumor cells, alkalinization itself induces apoptosis, either through NHE1 internalization (786-O) or inhibition of its activity at the membrane (Caki-1). This differential sensitivity underscores the potential of targeting pH regulation as a therapeutic strategy in clear cell renal carcinoma.\u003c/p\u003e\u003cp\u003eWhat could explain the contrasting effects of NHE1 activity in healthy and cancer cells? NHE1 is involved in a wide range of cellular functions beyond ion transport, including volume regulation, actin remodeling, and cell migration. Its activity is also subject to multiple regulatory mechanisms, meaning that NHE1-dependent responses likely vary depending on the signals reaching the cell. In healthy cells, alkaline stress may activate NHE1, leading to cell death. In contrast, in cancer cells, the prolonged stress characteristic of this pathology, as described by Pedersen, may alter the response to alkaline stress [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNHE1 directly interacts with the cofactor proteins calcineurin B homologous protein 1 (CHP1) and CHP2 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In vitro, studies on lung carcinoma cells demonstrate that these cofactors regulate NHE1 activity. CHP1 exhibits ubiquitous localization, whereas CHP2 shows tissue-specific expression. Moreover, tumorigenic extracellular conditions, such as nutrient deprivation, modulate CHP2 expression [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Reflecting this regulatory complexity, Lacroix et al. proposed that NHE1 activation involves the oscillation of a dimeric NHE1 between two conformational states with distinct proton transport affinities, in agreement with the Monod\u0026ndash;Wyman\u0026ndash;Changeux allosteric model [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This regulatory behavior appears to depend on a cluster of histidine residues located in the C-terminal cytoplasmic domain, which serve as pH sensors. Remarkably, Dong et al. demonstrated that NHE1 functions as a symmetrical homodimer in complex with CHP1. CHP1 interacts differently with the distinct conformational states of each NHE1 monomer, suggesting that this differential binding contributes to the fine-tuning of NHE1\u0026rsquo;s pH sensitivity [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. It is conceivable that, in cancerous tissues, the interaction of NHE1 with CHP2 rather than with CHP1 might alter its regulatory mechanism, potentially contributing to the aberrant pH sensitivity observed in tumors. However, this possibility remains to be experimentally validated.\u003c/p\u003e\u003cp\u003eIn conclusion, our study reveals that combining mild alkali treatment and NHE1 inhibition induces cancer cell death while minimizing damage to normal tissue. This therapeutic strategy offers a promising avenue for targeted therapy in clear cell renal cell carcinoma (ccRCC), potentially providing a novel approach for managing this challenging malignancy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eRunning title\u003c/h2\u003e\u003cp\u003eAlkali and NHE1 in ccRCC cell death\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eDeclarations\u003c/h2\u003e\u003cp\u003e\u003cb\u003eFunding Declaration\u003c/b\u003e: This study was funded by grants from Fondo Nacional para la Ciencia y la Tecnolog\u0026iacute;a, Argentina [Grant Number: PICT 2020\u0026thinsp;\u0026minus;\u0026thinsp;01130]; Universidad de Buenos Aires, Argentina [Grant Number: UBACYT 20020220100022BA, UBACYT 20020130100697BA and UBACYT 20020220100029BA] and Consejo Nacional de Ciencia y Tecnolog\u0026iacute;a, Argentina [Grant Number: PIP 11220200102585CO]\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eThe authors of this manuscript certify that they have NO potential sources of conflict of interest (relationship, financial or otherwise) that might influence the author's objectivity in the subject matter or materials discussed in this manuscript.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eData and/or Code availability\u003c/h2\u003e\u003cp\u003eThe KNIME workflow used in this study to perform total cell count is openly available on Figshare at:\u003c/p\u003e\u003cp\u003eCasal, Juan Jose; Rivarola, Valeria (2024). KNIME Workflow to count total cell number in photos of Acridine Orange/Ethidium Bromide experiments. Figshare. Software. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.6084/m9.figshare.25913794.v2\u003c/span\u003e\u003cspan address=\"10.6084/m9.figshare.25913794.v2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eThe Python notebooks used in this study for automated quantification of necrotic areas in spheroid images are openly available on Figshare at:\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding Declaration\u003c/h2\u003eThis study was funded by grants from Fondo Nacional para la Ciencia y la Tecnolog\u0026iacute;a, Argentina [Grant Number: PICT 2020\u0026thinsp;\u0026minus;\u0026thinsp;01130]; Universidad de Buenos Aires, Argentina [Grant Number: UBACYT 20020220100022BA, UBACYT 20020130100697BA and UBACYT 20020220100029BA] and Consejo Nacional de Ciencia y Tecnolog\u0026iacute;a, Argentina [Grant Number: PIP 11220200102585CO]\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.B.C conducted most of the validation, investigation, formal analysis and visualization.A.M., A.G., and G.Di G. contributed to some of the validation, investigation and formal analysis of pH experiments.N.B. was responsible for setting up the methodology of all cell models.J.J.C. developed the methodology, validation and software programming of KNIME workflow for automated cell counting from images and provided insights into all computer-based analyses and provided Data Curation.C.C. assisted in Funding acquisition and Writing - Review \u0026amp; Editing.P.F. and G.Di G. were involved in Funding acquisition, Conceptualization and Writing - Review \u0026amp; Editing.V.R. directed the project, so she is mainly involved in Conceptualization, Supervision, Formal analysis, Funding acquisition, Project administration and Writing - Original Draft and its Review \u0026amp; Editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank German La Iacona for his technical assistance and Matias Aduriz for his assistance in the development of the software to measure the necrotic area of spheroids.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe KNIME workflow used in this study to perform total cell count is openly available on Figshare at:Casal, Juan Jose; Rivarola, Valeria (2024). KNIME Workflow to count total cell number in photos of Acridine Orange/Ethidium Bromide experiments. Figshare. Software. https://doi.org/10.6084/m9.figshare.25913794.v2The Python notebooks used in this study for automated quantification of necrotic areas in spheroid images are openly available on Figshare at:-Individual images: Celi, Ana Beatriz; Aduriz, Matias; Casal, Juan Jose; Rivarola, Valeria (2025). Spheroid Necrotic Area Quantification for Individual Images. Figshare. Software. https://doi.org/10.6084/m9.figshare.30161296-Multiple images: Celi, Ana Beatriz; Aduriz, Matias; Casal, Juan Jose; Rivarola, Valeria (2025). Spheroid Necrotic Area Quantification for Multiple Images. Figshare. Software. https://doi.org/10.6084/m9.figshare.30159781The data that support the findings of this study are available from the authors upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFais S, Venturi G, Gatenby B (2014) Microenvironmental acidosis in carcinogenesis and metastases: new strategies in prevention and therapy. Cancer Metastasis Rev 33:1095\u0026ndash;1108. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10555-014-9531-3\u003c/span\u003e\u003cspan address=\"10.1007/s10555-014-9531-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKoltai T (2016) Cancer: Fundamentals behind pH targeting and the double-edged approach. 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EMBO Rep 5:91\u0026ndash;96. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/sj.embor.7400035\u003c/span\u003e\u003cspan address=\"10.1038/sj.embor.7400035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5875263/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5875263/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAcid-base homeostasis is critical for maintaining physiological functions. An acidic tumor microenvironment, driven by altered cellular metabolism, plays a pivotal role in tumor progression by fostering aggressive phenotypes, immune evasion, and resistance to therapy, often at the detriment of surrounding normal tissues. The Na⁺/H⁺ exchanger isoform 1 (NHE1) is a key regulator of intracellular pH and a critical factor in cancer cell survival and proliferation. This study aimed to evaluate the effect of mild alkaline treatment, combined with NHE1 inhibition, on cell viability in normal renal cells and clear cell renal cell carcinoma (ccRCC) cells. Our findings reveal that this therapeutic combination selectively induces cell death in ccRCC cells while sparing normal renal cells. Mechanistically, we demonstrate that NHE1 activity is higher in ccRCC cells than in normal cells. In our experimental model, mild alkaline treatment differentially affected NHE1 activity, stimulating it in normal cells but suppressing it in cancer cells. Furthermore, prolonged alkaline exposure alters the subcellular localization of NHE1 in the plasma membrane, with distinct patterns observed between normal and cancer cells. These results suggest that targeting NHE1 activity in conjunction with alkaline treatment represents a promising strategy for ccRCC treatment, providing a potential therapeutic avenue to exploit the differential pH regulation between cancerous and normal cells.\u003c/p\u003e","manuscriptTitle":"Targeting pH Regulation in Cancer: Combined Mild Alkaline treatment and NHE1 Inhibition as a Potential Therapy for Clear Cell Renal Cell Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":"","date":"2026-01-28 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