Alendronate impairs key ion pumps and triggers structural damage in the upper airway mucosa and olfactory bulb

ABSTRACT

Introduction:  Alendronate (ALN) is bisphosphonate used in clinical practice to reduce the adverse consequences of bone pathological condition.

Objective:  This study aimed to investigate the effects of alendronate (ALN) administration on the immunoexpression of Na+K+ATPase and v-ATPase within the upper airway mucosa and olfactory bulb of rats.

Method:  Sixteen animals were equally distributed into control and experimental groups, with the latter receiving daily intraperitoneal doses of ALN (2.5 mg/kg). After 7 days, the rats were euthanized, and their heads were processed for histological evaluation and immunohistochemical quantification of both target proton pumps to assess tissue structural integrity.

Results:  On the seventh day, the animals were euthanized, and their heads were processed for histological and immunohistochemical examinations. Specimens treated with ALN exhibited a decreased presence of Na+K+ATPase and v-ATPase in both the respiratory epithelium and the olfactory bulb. These findings coincided with vacuolization and a loss of polarity within the respiratory epithelium, alongside profound cellular degeneration in the olfactory bulb.

Conclusion:  It can be concluded that the administration of alendronate may alter the histomorphology of the respiratory epithelial, additionally compromising olfactory bulb integrity due to the down-regulation of the expression of Na+K+ATPase and v-ATPase expression.

KEYWORDS:
Alendronate; Respiratory epithelium; Olfactory bulb; v-ATPase; Na+K+ATPase

VISUAL ABSTRACT

RESUMO

Introdução:  O alendronato (ALN) é um bisfosfonato usado na prática clínica para reduzir as consequências adversas de condições ósseas patológicas.

Objetivo:  Investigar os efeitos da administração de alendronato (ALN) na imunoexpressão de Na+K+ATPase e v-ATPase na mucosa das vias aéreas superiores e no bulbo olfativo de ratos.

Método:  Dezesseis animais foram distribuídos igualmente em grupos controle e experimental, com estes últimos recebendo doses diárias intraperitoneais de ALN (2,5 mg/kg). Após sete dias, os ratos foram eutanasiados e suas cabeças processadas para avaliação histológica e quantificação imunohistoquímica de ambas as bombas de prótons alvo para avaliar a integridade estrutural do tecido.

Resultados  -. Amostras tratadas com ALN apresentaram diminuição da presença de Na+K+ATPase e v-ATPase tanto no epitélio respiratório quanto no bulbo olfatório. Esses achados coincidiram com vacuolização e perda de polaridade dentro do epitélio respiratório, juntamente com profunda degeneração celular no bulbo olfatório.

Conclusão:  A administração de alendronato pode alterar a histomorfologia do epitelial respiratório, comprometendo adicionalmente a integridade do bulbo olfatório devido à regulação negativa da expressão de Na+K+ATPase e de v-ATPase.

PALAVRAS-CHAVE:
Alendronato; Epitélio respiratório; Bulbo olfativo; v-ATPase; Na+K+ATPase

RESUMO VISUAL

INTRODUCTION

Alendronate (ALN) is a common bisphosphonate used in clinical practice to reduce the adverse consequences of pathological condition that culminates in osteolysis such as osteoporosis, otospongiosis, Paget’s diseases, osteogenesis imperfect, multiple myeloma or bone metastasis due to invasiveness of neoplasms.^1-2

The effect of alendronate on bone modulation is vast and its fundamental action is associated with reducing the osteoclast number and viability.³ Besides that, alendronate seems to promote osteogenesis in craniofacial defects.^4-6 Despite its importance, the use of alendronate may cause numerous collateral effects, among them erosive mucositis and toxic effects on Schwann and neuronal cells.^7-9

Several anatomical areas are composed of bone and cartilage and may be affected by alendronate. Among them, includes the nasal septae and turbinates that support adjacent structures such as respiratory epithelium.¹⁰

The upper airway tract is covered by a thinner epithelium composed of ciliary pseudostratified columnar epithelium with goblet cells that possess an important action in respiratory organ physiology since they regulate inhaled air temperature and humidity and filter foreign particles.¹¹ However, a portion of the specialized olfactory epithelium known as the olfactory bulb consists of multiple cell types. One of these cells is a bipolar cell known as olfactory receptor neuron. At its basal surface, it gives rise to a small-diameter, unmyelinated axon that transmits smell signals between the olfactory epithelium and the primary olfactory brain.¹²

Several proteins act for appropriate histophysiology action of respiratory epithelium and olfactory bulb. In this context, Brezillon et al.¹³ highlight that proteins exhibiting ATPase activity seem to play a crucial role in the arrangement of the respiratory epithelium. This is because these ATP-dependent proteins are accountable for the formation of junctional complexes at the interface between basal and ciliated cells. Additionally, these proteins support the integrity of the interface basolateral epithelium on columnar cells, and ATP appears to aid in the anchoring of intermediate filaments like actin and cytokeratins.

Recognizably, the Na+K+ATPAse bump is an important protein whose ATPase activity has maintained an ionic composition of its cytoplasm, maintaining low sodium, and high potassium, and keeping a neutral Hidrogenionic potential (pH). Besides that, several studies have demonstrated the importance of Na+K+ATPAse on the modulation of tight junction development, permeability, and cell polarity14 and acts like an ion transporter either in neural axon and plasmatic membrane of the epithelium and neuroepithelial cells as occurs in olfactory bulb cells.¹⁵

Conversely, the v-ATPase is a transmembrane protein that possesses fundamental action either in epithelial or neuroepithelial cells. Intracellular v-ATPases play a crucial role in intracellular acid-base regulation, working on protein processing and degradation, vesicle trafficking, and the coupled transport of small molecules.¹⁶

Thus, the purpose of this study was to evaluate the effect of ALN on the respiratory epithelium of the nasal mucosa and olfactory bulb, while simultaneously verifying the immunoexpression of Na+K+ATPAse and v-ATPase in order to understand the cellular mechanisms underlying ALN-related mucosal and neuronal toxicity

METHOD

Animals

The Ethical Committee for Animal Research gave its approval to this work, which complied with the regulations of animal use and the Principles of Laboratory Animal Care (NIH Publication 85-23, revised in 1985). In this investigation, sixteen female rats (Rattus norvegicus albinus, Holtzman) weighing between 240 and 260 g at two months of age were used. The animals were housed in a room with a 12-hour light/dark cycle, regulated humidity (52%±5%), and ad libitum access to food and water.

Randomly, eight newborn rats received 1 ml of sterile 0.9% saline solution (control group); while eight rats received 2.5 mg/kg/day of alendronate trihydrate (Biolife, Curitiba, Brazil; lot number: 14042132C) for seven days (ALN group). Intraperitoneal injections of saline and alendronate solutions were given every day until the seventh day, when an isoflurane overdose led to euthanasia and immediately the entire heads of rats were removed.

Histological procedure

The surgical specimens were collected instantaneously after the euthanasia process and they were placed in a 4% formaldehyde fixative solution, which was made by mixing paraformaldehyde with 0.1 M sodium phosphate at a pH of 7.2, for 48 hours at a temperature between 18 and 20oC. Each sample was dehydrated in graded quantities of ethanol, clarified in xylene, and embedded in paraffin after being decalcified for 21 days in 7% Ethylenediaminetetraacetic Acid Tetrasodium (EDTA) containing 0.5% formaldehyde in 0.1 M sodium phosphate (pH 7.2).

From each specimen, sequential 3-μm-thick histological sections were cut in the anterior-posterior direction in accordance with the coronal anatomic plane. The slices were placed on silanized slides (Sigma-Aldrich Chemie, Steinheim, Germany) in order to detect Na+K+ATPase and v-ATPase using immunohistochemistry.

Immunohistochemistry

Each slide composed of specimens with a thickness of three micrometers was deparaffinized in xylene and after ten minutes in each of ninety-five and seventy percent alcohol, they were hydrated in 70% alcohol. A 1% pepsin solution (pH 1.8) (Sigma-Aldrich, Germany) was used for the antigen retrieval process, which lasted for one hour at 37°C. After 20 minutes of room temperature cooling, they were given a 10-minute soak in distilled water and subsequently, they were immersed in 0.1% hydrogen peroxide (H2O2) (Thermo Fisher Scientific, US) for fifteen minutes, to endogenous peroxidase activity was inhibited. After five minutes of running tap water washing, the specimens were immersed in phosphate-buffered saline (Sigma-Aldrich, Germany) for a further five minutes. The primary antibodies anti-α1-Na+K+ATPase (Santa Cruz Biotechnology Inc., sc-166848, CA, USA, dilution factor: 1:180) and v-ATPase (Santa Cruz Biotechnology Inc., sc-166848, CA, USA, dilution factor: 1:150) were incubated overnight for the samples. In order to identify the primary antibodies, a labeled streptavidin biotin antibody-binding detection system (Universal HRP immunostaining kit - Diagnostic Byosystem Universal HRP Immunostaining Kit, Diagnostic Byosystem, Foster City, CA, USA) was employed for 30 minutes. Following this, the sample was immersed in dimethane chromogen (Universal HRP immunostaining kit - Diagnostic Byosystem Universal HRP Immunostaining Kit, Diagnostic Byosystem, Foster City, CA, USA) for a duration of 15 minutes, resulting in a brownish precipitate at the antigen site. Harris’s hematoxylin was used for two minutes as a counterstain on the specimens. For every sample that omitted, a negative control was carried out.

Image analyses

Using a digital camera (Samsung, South Korea) and a light microscope, images of the histological and immunohistochemical sections were taken at magnifications of ×100 and ×200. At a resolution of 300 dpi, 110×65 cm digital photographs were taken and stored.

For the histological and immunohistochemical evaluation, the microscopy images were processed using Adobe Photoshop version 11.0 for Mac. For each image obtained, all area of the nasal cavity was considered for evaluation and it was composed of entire nasal septae recovered by respiratory epithelium. In serial posterior slices, it was considered areas that presented integral internal capsular region of the olfactory bulb for analysis. However, since the histological is a limited technique used to obtain the olfactory bulb in surgical pieces, it was possible to analyze only areas of the olfactory bulb posteriorly to the ethmoid bone, and all findings were presented in accordance with the layers that the original Cajal depiction of the olfactory bulbs frontal region described.¹⁷

Each micrograph was submitted to the Image J software (https://imagej.nih.gov/ij), and positivity for Na+K+ATPase and v-ATPase cells (detected by brownish color in membrane and cytoplasm of ells of interest) were manually counted and tagged. It was considered 5,000 cells for specimens and the area accounted for was randomized chosen. The results were transformed in the percentage of positivity.

Statistical analysis

The Shapiro-Wilk test was used to establish normalcy, and the nonparametric Kruskal-Wallis test was used to identify statistically significant group differences. A significance criterion of p<0.05 was established.

RESULTS

The Na+K+ATPase and v-ATPase expression immunohistochemical data are represented respectively present on Figure 1, while the immunohistochemistry illustration is given in Figure 2.

FIGURE 1
Box plot diagram revealing the median, maximum, and minimum values of Na+K+ATPase as well as v-ATPase on respiratory epithelium and olfactory bulb in the course of the study. The same superscript character (asterisk) is not statistically different, p>0.5.

FIGURE 2
Micrographs A and B demonstrate the pattern of immunoexpression of Na+K+ATPase (in brownish color) in respiratory on nasal turbinates for control and specimens that received alendronate respectively. Note the presence of the immunoexpressions (arrows) for either for Na+K+ATPase on either control group or alendronate, however, the test group demonstrated areas of loss of immunopositivity for protein (chevron). Micrographs C and D reveal similarly pattern for v-APTase on control and ALN group. E and G demonstrate immunoexpression of Na+K+ATPase and v-ATPase positivity (arrows-brownish color) for control, while F and H reveal loss of positivity for Na+K+ATPase and v-ATPase on respiratory epithelium (arrows) of ALN group. Note loss of adhesiveness and intraepithelial vacuolization in the alendronate group (chevron). Micrographs I and J show the expression for Na+K+ATPase (in brownish color) for the control and alendronate groups respectively. Note the intense positivity in glomerular cells (arrow) in the control group, while in the test group cellular degeneration (chevron). Yet, there is an expression for protein (Notched arrow) in the axonal area. Note the organized expression occurred in the control group while in alendronate specimens occur a disorganized and infrequent expression. Micrographs K and L demonstrate that v-ATPase reveals a similar pattern of Na+K+ATPase. (A to D and I to L- Original magnification ´100; E to H- Original magnification ´200; positivity area occurs in brownish color).

Respiratory epithelium

On the 7th day of evaluation, the control group exhibited a respiratory epithelium with normal histological characteristics. In contrast, numerous regions of intraepithelial vacuolation and alterations in epithelial polarity were observed in the specimens that received ALN. Furthermore, the ALN-treated specimens demonstrated a transition from columnar to cuboidal epithelium, as well as areas showing a loss of basolateral adhesion within the epithelium. Moreover, the regions where these microscopic modifications were detected coincided with the loss of expression of Na+K+ATPase and v-ATPase proteins (Fig. 1). In the control group, these proteins were expressed throughout the membrane and cytoplasm of the ciliated pseudostratified columnar epithelium, as well as on the membrane of goblet cells lining the respiratory tract. Both Na+K+ATPase (Figs. 2A and E) and v-ATPase (Figs. 2B and F) shared the same expression pattern in the alendronate-treated specimens (Figs. 2B and F for Na+K+ATPase; Figs. 2C and G for v-ATPase); however, the number of positive cells was significantly lower (Fig. 1). Interestingly, the epithelial lining at the apex of the nasal turbinates was negative for both evaluated proteins, whereas other topographical regions of the respiratory tract demonstrated substantial areas with a loss of protein positivity. A similar expression pattern was observed in the maxillary sinus; however, the negative epithelial cells of the maxillary sinus displayed a distinct morphology, characterized by squamous to cuboidal epithelial cells.

Olfactory bulb

Significant differences also were seen in the olfactory bulb for Na+K+ATPase and v-ATPase proteins and histomorphology. On control, all cells that compounded the glomerular layer of the olfactory bulb were positive for Na+K+ATPase (Fig. 2I) and v-ATPase (Fig. 2K). The glomeruli exhibited immunopositivity for Na+K+ATPase and v-ATPase either in cytoplasm or membrane while the peripheral tufted cells, that surrounded each glomeruli body, demonstrated positivity for v-ATPase in membrane axon and dendrite, while Na+K+ATPase was restricted to neuronal prolongation areas. In the group that received alendronate, the presence of Na+K+ATPase (Fig. 2J) and v-ATPase (Fig. 2L) was found in few anucleated glomerulus cells, while other cellular bodies of glomeruli were negative. In addition, only sparse axonal and dendritic areas exhibited focal positivity on regions occupied by peripheral tufted cells.

Yet, in the control group, both v-ATPase and Na+K+ATPase also were well expressed in the inner plexiform layer, an area compatible with grains and white matter layer on the cellular body, axon, and dendrite; however, the mitral cells layer exhibited only expression for v-ATPase. In contrast, the specimens that received ALN demonstrated a loss of positivity for both v-ATPase and Na+K+ATPase when compared to the control group.

DISCUSSION

In the present study, we demonstrated that the expression of v-ATPase and Na+K+ATPase were significantly decreased in specimens that received ALN. These results were consistent with areas that revealed cell degeneration on the olfactory bulb, particularly in its glomerulus bodies of the olfactory bulb, and histological changes of the respiratory lining epithelial tissue.

The previous discovery that ALN inhibits farnesyl pyrophosphate synthase in the mevalonate cascade metabolic pathway may help to explain the loss of membrane ATPase-protein.¹⁸ This inhibitory event may lead to an accumulation of non-prenylated GTP-binding proteins spread in the cytoplasm of the cell due to suppression of cholesterol. Thus, the binding protein’s ability to facilitate protein-protein interaction in the cellular membrane is eliminated in its non-prenylated state, condition that may provide protein denaturation.^19-20 It have to be highlighted that when a protein that works as a transmembranic receptor protein is spread throughout the cytoplasm, it may exhibit changes in its tertiary or quaternary protein configuration, and the modification of the level protein organization simulating the denaturation of protein and inhibit either its usual functional action as well as its immunohistochemical detection.^21-22

It should be taken into consideration that damage of Na+K+ATPase in the epithelium is also associated with degenerative processes, that inducing hydropic or vacuolar degeneration due to accumulation of Na+ in the intracellular compartment, as well as leads to a loss of epithelial cell surface membrane polarity.²³ In addition, there is evidence that associates the loss of Na+K+ATPase to detachment of the cytoskeleton and surface membrane structures whose results lead to numerous cellular physiopathological adaptations including damage of cell-cell contact as well as cell-extracellular matrix adhesion.²⁴ Consequently, loss of Na+K+ATPase seems to be also relevant to the functional action of respiratory epithelium, since this protein also collaborates for mucociliary clearance and airway lining fluid. It should be highlighted that ciliated epithelial cells that compound the respiratory epithelium are important in propelling mucus up the airway, thereby removing particulate material.¹¹

In addition, either suppression of Na+K+ATPase or v-ATPase not only may induce damage in respiratory histophysiology, but also seems to facilitate secondary infections since this epithelium also is associated with sinus development and also It controls the flow of water and ions into the mucus in the airways, secretes lactoferrin, lysozyme, and antimicrobial peptides (cathelicidins and β-defensins), and releases reactive oxygen and nitrogen species to annihilate pathogens that invade the respiratory tract.^25-26

Conversely, loss of Na+K+ATPase as well as v-ATPase on bulb olfactory coincided with structural changes in the morphology of this organ. It is noteworthy that Na+K+ATPase is important for depolarization and repolarization of the membrane, a condition that is also important for neurotransmission.²⁷

Specifically, the v-ATPase is a biomarker of specialized proton-secreting cells. This protein is present in lateral membrane in a subpopulation of olfactory sensory cells and the microvilli of the apical plasma membrane of sustentacular cells. It was also discovered that, when produced in olfactory neurons, v-ATPase typically assumed a greater subapical location in olfactory sustentacular cells. Typically, v-ATPase may be associated with CO2 detection when expressed in olfactory neurons, aiding in signal transduction that supports respiratory tract homeostasis and cellular integrity.²⁸ Supporting this hypothesis, the lack of expression of v-ATPse seems to have contributed to the intracellular acidity of the sensory epithelium of the olfactory bulb, and this situation may be suggested herein since the integrity of the glomerular cells showed an evident degenerating cellular situation.²⁹

CONCLUSION

Herein, we demonstrated that Alendronate induces loss on Na+K+ATPase and v-ATPase expression, situations that may modify the histophysiology of the respiratory epithelium of the nasal cavity, as well as degeneration of the olfactory bulb.

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• Central Mensage
  Alendronate compromises local cellular homeostasis due to energy-ionic suppression (loss of ATPases), negatively affecting the histomorphology of the nasal turbinates, the nasal septum, and the integrity of the olfactory bulb.
• Perspective
  It is opportune to evaluate possible alterations in protein expression and histomorphological characteristics of the upper airways and central nervous system in the long term after interruption or prolongation of alendronate treatment, with emphasis on the distribution of lesion severity, patterns of cellular degeneration, and tissue recovery capacity. Thus, it will be possible to assess the extent to which chronic alendronate administration may have influenced the prevalence and characteristics of cellular and structural alterations in the respiratory epithelium and olfactory bulb, with emphasis on dosage distribution, histological patterns, and ATP-dependent ion transport mechanisms.
• How to cite this article
  Gomes SP, Gonzaga CC, Polanski JF, Isolan GR, Neukamp MB, Lall A, Giovanini AF. O alendronato prejudica as bombas de íons chave e desencadeia danos estruturais na mucosa das vias aéreas superiores e no bulbo olfativo. BioSCIENCE. 2026;84:e00007. https://doi.org/10.55684/2026.84.pt.e00007
• Funding:
  None
• Data availability:
  Data are available from the corresponding author upon reasonable request.

Edited by

• Associate Editor:
  Ramiro Colleoni Neto https://orcid.org/0000-0002-5429-3692

Data availability

Data are available from the corresponding author upon reasonable request.

Publication Dates

• Publication in this collection
  21 Aug 2026
• Date of issue
  2026

History

• Received
  11 Dec 2025
• Accepted
  25 Feb 2026
• Published
  20 Mar 2026