Objectives: Hypoxia is a key regulator of renal epithelial cell function and contributes to the pathogenesis of kidney diseases. Although two-dimensional cell cultures are commonly used to study hypoxic responses, three-dimensional spheroid cultures better represent the in vivo microenvironment, including cell-cell and cell-matrix interactions. In this study, we compared hypoxia-induced gene expression in two- and three-dimensional cultures of HK2 cells.
Materials and Methods: We induced hypoxia at 12 hours in two-dimensional cultures and at 24 hours in three-dimensional spheroids, with confirmation of hypoxia by hypoxia-inducible factor 1α protein expression and vascular endothelial growth factor mRNA upregulation. With quantitative real-time polymerase chain reaction analysis, we noted that long noncoding RNA H19 was significantly upregulated in both culture models, accompanied by modulation of NLRP³, Wnt¹, β-catenin, interleukin 1β, interleukin 6, tumor necrosis factor α, interleukin 10, microRNA-30a-5p, and microRNA-196a.
Results: Tumor necrosis factor α and microRNA-30a-5p expression were decreased, with strong interleukin 10 upregulation, in two-dimensional cultures. In three-dimensional cultures, expression levels of both microRNA-30a-5p and microRNA-196a were further upregulated, with less pronounced reduction of tumor necrosis factor α and limited increase of interleukin 10 expression.
Conclusions: Our findings suggested that microRNA-30a-5p and microRNA-196a may play a role in modulating hypoxia-induced cytokine responses, contributing to a more balanced cellular adaptation in three-dimensional cultures. Three-dimensional HK2 spheroids may provide a physiologically relevant model for studying hypoxic stress and may reveal the potential involvement of long noncoding RNA H19 and specific microRNAs in regulation of inflammatory and signaling pathways. Of note, this study represents one of the first investigations of hypoxia-induced gene expression in three-dimensional HK2 cultures, offering a novel contribution to understanding renal epithelial responses under low oxygen conditions.

Key words : Cell culture, Gene expression, Hypoxia, Proximal tubule cells

Introduction
Renal hypoxia is one of the most critical factors affecting kidney function. Renal hypoxia influences energy metabolism, inflammation, and cellular responses. The underlying mechanisms of renal hypoxia generally involve oxygen availability, blood flow, and metabolic processes. Under hypoxic conditions, the tricarboxylic acid cycle and oxidative phosphorylation in renal cells are disrupted, leading to decreased ATP production and subsequent cellular energy deprivation.¹ Prolonged renal hypoxia eventually leads to nephron cell death, increased fibrosis, and progression to chronic kidney disease.² Oxidative stress in hypoxic conditions initiates kidney injury and can lead to acute kidney injury.³ Hypoxia also activates inflammatory pathways, leading to elevated expression of proinflammatory cytokines including interleukin (IL)-1β and IL-12, which contribute to kidney damage.^1,4 One of the key regulators in hypoxic renal cells is hypoxia-inducible factor 1α (HIF-1α), which exerts a dual function by both promoting protective cellular responses against hypoxia and contributing to fibrosis and inflammation, particularly in chronic kidney injury.^4,5 A key downstream target of the transcription factor HIF-1α is vascular endothelial growth factor (VEGF), which plays a crucial role in angiogenesis under hypoxic conditions. By activating VEGF expression, HIF-1α promotes neovascularization and enhances oxygen delivery. Vascular endothelial growth factor also supports the viability and proliferation of renal cells under hypoxia.^6,7 Furthermore, HIF-1α enhances β-catenin expression, thereby increasing the expression of other target genes involved in cell proliferation, differentiation, and survival. This cascade triggers adaptive repair mechanisms following acute kidney injury and prevents the transition to chronic kidney disease. However, delayed HIF-1α activation in hypoxic conditions results in reduced Wnt/β-catenin signaling, which is associated with maladaptive repair processes.⁸ The long noncoding RNA H19 (lncRNA H19) is upregulated under hypoxic conditions and plays a regulatory role in various hypoxia-related processes, including cell viability, apoptosis, and inflammation.⁹ The inflammasome NLRP³ is involved in innate immune responses by recognizing microbial motifs, endogenous danger signals, and environmental stressors. Once activated, NLRP³ induces the release of proinflammatory cytokines such as IL-1β and IL-18 and can lead to pyroptosis-mediated cell death.¹⁰ Long noncoding RNA H19 stabilizes HIF-1α under hypoxic conditions, which in turn upregulates NLRP³ expression, contributing to tissue damage.^9,11 The microRNA miR-30a-5p plays a protective role against hypoxia-induced apoptosis and oxidative stress in kidney tubular cells. However, inhibition of miR-30a-5p expression by lncRNA H19 compromises this protective effect,¹² potentially exacerbating the severity of acute kidney injury. Another noncoding microRNA, miR-196a, is also involved in the regulation of cell viability, proliferation, and apoptosis. Although the direct regulatory relationship between lncRNA H19 and miR-196a in renal cells remains unclear, elucidating these mechanisms may offer new therapeutic targets.¹³ In this study, we aimed to elucidate the molecular mechanisms involved in renal processes under hypoxic conditions. Specifically, we investigated the expression levels of lncRNA H19 and its associated targets, including the proinflammatory cytokines NLRP³, Wnt¹, β-catenin, IL-1β, IL-6, and tumor necrosis factor α (TNF-α); the anti-inflammatory cytokine IL-10; and the hypoxia-induced microRNAs miR-30a-5p and miR-196a, in proximal tubular cells. Furthermore, we explored the relationship between the observed gene expression changes and acute kidney injury in both 2-dimensional (2D) and 3-dimensional (3D) cell culture models.

Materials and Methods

Ethical statement
This study involved in vitro experiments using established cell lines. In accordance with institutional and national regulations, ethical committee approval was not required for cell culture-based experimental studies. All experimental procedures were conducted in compliance with relevant laboratory safety and ethical guidelines.

Two-dimensional cell culture and maintenance
In this study, we used the proximal tubular epithelial cell line HK2 (ATCC, CRL-2190), with cells cultured by using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS; GIBCO), 1% penicillin/streptomycin (GIBCO), and 1% L-glutamine (GIBCO). For the 2D cultures, we maintained cells in medium containing 10% FBS. Cells were subcultured every 2 days, with cultures maintained at 37 °C in an incubator with a controlled 5% CO2 environment. We conducted experiments using cells up to passage 8 to ensure consistency and viability.

Three-dimensional cell culture and maintenance
The 3D culture model was established using the hanging drop method. We suspended cells at a concentration of 100 cells/μL in DMEM/F12 medium supplemented with 10% FBS. We carefully pipetted 27 μL drops onto the inner surface of sterile 100-mm Petri dish lids. To maintain humidity, we added 3 mL of phosphate-buffered saline to the bottom of each dish. Lids were then inverted, and cultures were incubated at 37 °C in a 5% CO2 atmosphere. After 3 days of incubation, we microscopically confirmed spheroid formation.

Determination of cell viability and number in two-dimensional cell cultures
Cells were subjected to hypoxic conditions once they reached approximately 80% confluency and demonstrated a viability rate of >90%. For 2D cultures, we seeded cells into 6-well plates at a density of 150 000 cells per well in 2 mL of medium to ensure an adequate cell number for subsequent RNA isolation.

Determination of cell viability and number in three-dimensional cell cultures
We prepared approximately 80 hanging drops by dispensing 27 μL drops containing 2700 cells each onto lids of 100-mm Petri dishes. We used the trypan blue exclusion method for cell counting and to assess cell viability at a final concentration of 0.4% (Figure 1). Cells that appeared blue and morphologically distorted were considered nonviable and were excluded from the total cell count.¹⁴

Induction of hypoxic conditions
To establish hypoxia, we placed culture plates inside the hypoxia chamber, which we then sealed by using a clip. We introduced a gas mixture of 5% CO2 and 95% N2 into the chamber for 4 minutes. We monitored oxygen levels inside the chamber in real time using an oximeter. Once the oxygen concentration dropped to 0% to 1%, the chamber was transferred to an incubator set at 37 °C with 5% CO2 and controlled humidity. For 2D cultures, we incubated cells under hypoxic conditions for 4, 8, 12, and 24 hours. For 3D cultures, we tested exposure durations of 12, 24, and 48 hours. The optimal hypoxia duration was determined based on VEGF expression levels. Table 1 lists the primer sequences.

Total RNA extraction and complementary DNA synthesis
After we determined the optimal exposure times to hypoxia for both the 2D- and 3D-cultured cells, we isolated total RNA, including microRNA and lncRNA, from these cells. For this purpose, we used the GeneAll Hybrid-R microRNA kit (GeneAll), performed according to the manufacturer’s instructions. After isolation, we measured the absorbance ratios at 260/280 nm spectrophotometrically by using a NanoDrop instrument (Thermo Fisher Scientific). Samples with a 260/280 ratio >2.0 and concentration >15 ng/μL were used for complementary DNA (cDNA) synthesis. For cDNA synthesis from isolated RNA samples, we used a commercial kit (ELK Biotechnology) in accordance with the manufacturer’s instructions. Specifically, a reaction mix was prepared by combining 2 µL of 5× genomic DNA eraser buffer, 1 µL of genomic DNA eraser, 4 µL of template RNA, and 4 µL of nuclease-free water. The mixture was incubated at 42 °C for 2 minutes. Next, we added 1 µL of oligo-dNTP, 2 µL of primer, and 2 µL of nuclease-free water, incubated samples at 70 °C for 5 minutes, and then immediately transferred samples to ice. We next added 4 µL of 5× RT buffer and 1 µL of EntiLink reverse transcriptase to each reaction. The final mixture was incubated at 37 °C for 60 minutes, followed by incubation at 95 °C for 5 minutes. We stored synthesized cDNA samples at -20 °C until further use for quantitative real-time polymerase chain reaction analysis (qRT-PCR).

Quantitative real-time polymerase chain reaction analysis for determination of molecular expression levels
To assess hypoxia, we analyzed expression levels of VEGF. In addition, expression levels of lncRNA H19 and its target genes (NLRP³, Wnt¹, β-catenin, IL-1β, IL-6, IL-10, TNF-α, miR-30a-5p, and miR-196a) were quantified in control and hypoxia-exposed cell groups by using qRT-PCR with SYBR green dye (Amplicon) on a PicoReal-Time PCR instrument (Thermo Fisher Scientific). We performed the procedure according to the manufacturer’s instructions for SYBR green.

Results

Temporal differences in hypoxia induction between two-dimensional and three-dimensional cultures
HK2 cells grown under 2D conditions were subcultured every 2 days, whereas spheroid formation was observed in 3D cultures by day 3. Induction of hypoxia occurred earlier in 2D cultures (12 h) compared with 3D cultures (24 h). As shown with use of qRT-PCR, VEGF expression also peaked earlier in 2D cultures (12 h) than in 3D cultures (24 h), further confirming the delayed onset of hypoxia in 3D conditions (Figure 2).

Comparative gene expression profiles of two-dimensional and three-dimensional cultures under hypoxic conditions
As shown by qRT-PCR results, under hypoxic conditions, lncRNA H19 expression increased approximately 6-fold compared with the control group. This upregulation was accompanied by significant alterations in the expression of NLRP³, Wnt¹, β-catenin, IL-1β, IL-6, TNF-α, miR-30a-5p, IL-10, and miR-196a. In 2D cultures, 12 hours of hypoxia resulted in a decrease in TNF-α and miR-30a-5p expression levels relative to the control group, whereas all other genes showed an upregulation. Among these, the highest increase was detected in IL-6 expression (8.2-fold), whereas the lowest increase was observed in NLRP³ expression (0.89-fold). Conversely, the greatest reduction was found in TNF-α expression (2.62-fold), and the smallest reduction was observed in miR-30a-5p expression (0.42-fold). In 3D cultures, after 24 hours of hypoxia, a similar expression pattern was observed, with TNF-α and miR-30a-5p levels decreased compared with the control group, whereas all other genes were upregulated. The most prominent increase was detected in Wnt¹ expression (8.1-fold), whereas the lowest increase was observed in IL-1β expression (0.46-fold). Among the downregulated genes, TNF-α showed the largest decrease (2.38-fold), and miR-30a-5p exhibited the smallest reduction (0.30-fold) (Figure 3). Overall, these results indicated that hypoxia induces comparable patterns of gene expression in both 2D and 3D cultures, with differences in the timing and magnitude of the response.

Discussion
In this study, we demonstrated that hypoxia was induced earlier in 2D HK2 cell cultures (12 h) compared with 3D spheroid cultures (24 h), as confirmed by elevated VEGF mRNA levels. These findings indicated that the 3D microenvironment, characterized by enhanced cell-cell and cell-matrix interactions, delays the onset of hypoxic responses relative to conventional 2D cultures. Such temporal differences highlight the physiological relevance of 3D spheroid models in mimicking tissue-like oxygen gradients.¹⁵ Importantly, this study represents one of the first investigations of hypoxia-induced gene expression in 3D HK2 cell cultures, providing a novel contribution to the literature on renal epithelial responses to low oxygen conditions. The concurrent upregulation of H19 and changes in inflammatory and signaling genes suggest that H19 may act as a regulatory molecule under hypoxic stress.¹⁶ Previous studies have implicated H19 in the modulation of inflammation by NLRP³/IL-1b pathway,^17,18 IL-6 expression,⁹ IL-10 expression,¹⁹ TNF-a expression,⁹ Wnt/β-catenin signaling,²⁰ and hypoxia-responsive pathways, supporting the notion that H19 could orchestrate multiple downstream targets in response to low oxygen tension. Notably, the differential upregulation of IL-6 in 2D versus Wnt¹ in 3D cultures may reflect context-dependent regulation influenced by microenvironmental cues. The consistent downregulation of TNF-α and upregulation of IL-10 across both models might represent an adaptive mechanism to limit proinflammatory signaling and protect cells from hypoxia-induced stress. The divergent cytokine responses observed between 2D and 3D cultures may be mediated, at least in part, by miR-30a-5p and miR-196a. In 2D cultures, decreased miR-30a-5p and moderately increased miR-196a may trigger a compensatory anti-inflammatory response, resulting in pronounced IL-10 upregulation and strong TNF-α downregulation. In contrast, in 3D cultures, both miR-30a-5p and miR-196a are upregulated more strongly, which could contribute to a more balanced modulation of TNF-α and IL-10, supporting controlled cellular adaptation under hypoxic stress.^21-23 These findings also suggest that, although hypoxia induces a broadly similar transcriptional response in both culture models, the magnitude and timing of gene expression changes are influenced by the 3D architecture.

Conclusions
Our results suggest that lncRNA H19 may serve as a central regulator of hypoxia-associated signaling networks, setting the stage for future studies aimed at elucidating its mechanistic contributions to cellular adaptation under low oxygen conditions. The findings also emphasize the value of 3D culture systems for modeling physiologically relevant hypoxic microenvironments. Although 2D cultures allow rapid induction of hypoxia-responsive genes, 3D spheroids offer a delayed yet sustained response that better reflects in vivo conditions. Despite these insights, the study remains limited to mRNA-level assessment of VEGF, since HIF1α protein levels could not be reliably detected. The direct regulatory role of H19 remains to be established. Future mechanistic studies that use gene silencing (eg, siRNA or CRISPR-mediated knockdown) or overexpression strategies are necessary to determine whether H19 directly controls the expression of NLRP³, Wnt¹, β-catenin, IL-1β, IL-6, IL-10, TNF-α, and the relevant microRNAs under hypoxic conditions.