Effects of Chronic Intermittent Hypoxia and Subsequent Normoxic Recovery on Renal Senescence and the PI3K/Akt/P21 Pathway in Rats

Introduction

Obstructive sleep apnea (OSA) is a sleep disorder characterized by chronic intermittent hypoxia (CIH). Affecting an estimated 1 billion individuals worldwide, it has emerged as a major public health concern¹ CIH induced by OSA accelerates the progression of cardiovascular disease,² metabolic disorders,³ neurological conditions,⁴ as well as cancer and aging-related processes,^5,6 while exacerbating cellular injury in these contexts. The kidney ranks among the organs most susceptible to premature aging and is particularly vulnerable to hypoxic stress. Recent evidence suggests that CIH can not only accelerate the progression of chronic kidney disease (CKD) but also induce renal senescence, a state of stress-induced premature cellular senescence, characterized by cell cycle arrest and aberrant secretory activity, mediated by mechanisms involving redox imbalance and dysregulated signaling pathways.^7,8 However, the pathogenesis of OSA-associated renal senescence remains multifaceted and incompletely understood.

CIH linked to OSA elicits oxidative stress and inflammatory responses,⁹ which likely constitute pivotal drivers of renal senescence.¹⁰ Studies have shown that IH activates the p53/p21 pathway via reactive oxygen species (ROS), thereby triggering cellular senescence and the senescence-associated secretory phenotype (SASP), with consequent disruption of the tissue microenvironment and fibrosis.¹¹ Other work has revealed that CIH exposure impairs mitochondrial function, yielding ROS that preferentially damage telomeric DNA; this targeted oxidative insult concurrently compromises telomere integrity and mitochondrial performance.¹² Moreover, ROS can suppress the PI3K/AKT pathway, thereby amplifying p21-mediated cell cycle arrest and reinforcing senescent phenotypes.^13,14 Notably, the anti-aging protein Klotho, whose deficiency is a hallmark of renal aging, has been shown to modulate insulin/IGF-1 signaling, a key upstream regulator of the PI3K/AKT pathway¹⁵ Reduced Klotho expression may therefore exacerbate CIH-induced PI3K/AKT suppression and p21 activation, creating a vicious cycle that promotes renal senescence.^16,17 To date, however, investigations have largely focused on adipose and vascular tissues, leaving the involvement of the PI3K/AKT/p21 axis and its interaction with Klotho in CIH-induced renal senescence largely unexplored. A preliminary version of this study has been presented as a preprint [https://doi.org/10.2139/ssrn.5244512].

Based on this premise, we conducted a longitudinal study employing a CIH rat model to systematically evaluate the dynamic progression and potential reversibility of CIH-induced renal senescence. Specifically, rats were subjected to 8 weeks of CIH followed by 4 weeks of normoxic recovery, modeling both injury and repair phases. We therefore aimed to: (1) determine whether CIH precipitates renal senescence; (2) assess the extent to which subsequent reoxygenation reverses these senescent phenotypes; and (3) elucidate the role of the PI3K/AKT/p21 signaling axis and its interaction with the anti-aging protein Klotho in this process. By dissecting these mechanisms, our study seeks to establish a causal link between OSA/CIH and accelerated renal aging, thereby identifying novel therapeutic targets and providing mechanistic insights for mitigating renal impairment in OSA patients.

Materials and Methods

Materials

Experimental Animals

Forty 5-week-old male Sprague-Dawley rats (150–180 g) were obtained from SpeFu (Beijing) Biotechnology Co., Ltd. (animal production license no. SCXK [Jing] 2019–001). Prior to experimentation, the rats underwent a 1-week acclimation period under controlled housing conditions: 22–23°C, 42–45% humidity, with feed provided at 25–30 g per 250 g body weight daily and water ad libitum. Cages contained clean, soft wood shavings as bedding, replaced every 3 days.

Primary Reagents and Instruments Instruments

Instruments

Ultrasonic cell disruptor (Thermo Scientific, USA); microplate reader (Bio-Rad); −4°C centrifuge (Thermo Scientific); 37°C incubator (Thermo Fisher Scientific); electrophoresis power supply (Bio-Rad); electrophoresis tank (DFRL); electroblotting tank (DFRL); orbital shaker (SCI-RS); gel imaging system (Thermo); NanoDrop ND-2000 spectrophotometer (NanoDrop); StepOnePlus real-time PCR system (Applied Biosystems). Inverted bright-field microscope with imaging capability (Nikon Eclipse Ci-L, Japan); image analysis software (Image-Pro Plus 6.0, Media Cybernetics, USA).

Reagents

4% paraformaldehyde (Solarbio); BCA protein assay kit (Solarbio); anti-PI3K antibody (41339, Solarbio, china, 1:1000); anti-AKT antibody (21054, Solarbio, china, 1:1000); anti-phospho-AKT (Ser473) antibody (11054, Solarbio, china, 1:500); anti-p21 antibody DF6423, Solarbio, china, 1:500); anti-Klotho antibody (43404, Solarbio, china, 1:1000); goat anti-rabbit secondary antibody (K1034, Solarbio, china, 1:10,000); anti-β-actin antibody (AF8294, Solarbio, china, 1:5000); ECL chemiluminescent substrate (Thermo Fisher Scientific); TRNzol Universal total RNA extraction reagent (TIANGEN); TaKaRa qPCR kit (TIANGEN). Serum creatinine (Cr, S03036), blood urea nitrogen (BUN, S03076), and cystatin C (CysC, H336-1-2) assay kits were purchased from Nanjing Jiancheng Bioengineering Institute, China.

Methods

Intermittent Hypoxia Protocol

We applied a validated CIH protocol that is well-established in the field to mimic the recurrent hypoxia/reoxygenation cycles characteristic of moderate-to-severe OSA, as described previously.¹⁸ The intermittent hypoxia chamber consisted of upper and lower platforms; the upper served as the operational platform, while the lower formed the oxygen chamber, constructed from glass panels, sheet metal, and rubber seals. The chamber door at the base was fully sealed, with casters fitted to the bottom for mobility. A plastic tubing for nitrogen infusion entered the chamber at the left base. Fixed gas detectors, automated electronic control panels, an AR-2000 pressure regulator, and a fan were mounted on the top, left, right, and rear walls, respectively. Solenoid valves for gas switching were positioned on the lower wall. Oxygen levels within the chamber were regulated by an electronic solenoid switch that governed input from a three-channel gas mixer delivering 100% N₂ and 100% O₂. Cycles lasted 120s: 25s of N₂ infusion reduced O₂ from 21% to a nadir of 7%, held for 10s, followed by 55s of ambient air infusion to restore O₂ to 21% and a 30s hold. In brief, experimental rats were housed in the IH environment for 8 h daily, with gas injectors and O₂/N₂ sensors ensuring cycle fidelity. CIH cycles coincided with the rats’ sleep phase (inactive period/lights on). Control rats were maintained at 21% O₂ in the same room for 8 weeks.

Animal Grouping and Modeling

Following a 1-week acclimation, the 40 male 5-week-old Sprague-Dawley rats were processed as follows. At baseline (week 0), 8 rats were euthanized for blood and kidney collection (0-week NC and CIHs); the remainder were randomly assigned to either the normoxic control (NC) group or the chronic intermittent hypoxia (CIH) group using a random number table (n=16 per group). The NC group proceeded under normoxia, while the CIH group entered chronic intermittent hypoxia. To control for potential confounding effects of feeding and drinking cycles, both the CIH and NC groups were housed in identical chambers with no access to food or water during the daily 8-hour experimental period. During the chronic intermittent hypoxia phase (weeks 0–8), NC rats remained under normoxia, and CIH rats were placed in the automated hypoxia chamber. This protocol continued for 8 weeks. At week 8, 8 rats per group (n=8) were euthanized for sampling (8-week NC and CIHs). Arterial blood gas analysis at nadir (7% O₂) and recovery (20% O₂) points yielded saturations of 72% and 96%, respectively, confirming successful modeling. In the reoxygenation phase (weeks 8–12), remaining NC rats continued under normoxia, while CIH rats were reintroduced to the chamber with ambient air infusion, matching prior exposure duration and cycle frequency; housing aligned with NC conditions. At week 12, the remaining 8 rats per group (n=8) were euthanized for sampling (12-week NC and CIHs). All animal experimental procedures were approved by the Animal Research Ethics Committee of Baotou Medical College (Approval No. [2023]74) and were conducted in strict accordance with the Chinese “Guidelines for the Care and Use of Laboratory Animals” and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The experimental workflow is shown in (Supplementary Figure 1) To minimize bias, investigators responsible for outcome assessments (histological analysis, Western blot band quantification) were blinded to group allocation throughout the experiment.

Serum Biochemical Assays Serum concentrations of creatinine, blood urea nitrogen, and cystatin C were quantified using commercial biochemical kits (Nanjing Jiancheng Bioengineering Institute, China) per the manufacturers’ protocols. Results are expressed as μmol/L for Cr and mmol/L for BUN.

Hematoxylin-Eosin Staining of Renal Tissue and Morphometric Analysis Renal tissues were fixed in 4% paraformaldehyde for 48 h, paraffin-embedded, sectioned at 5 μm, and stained with hematoxylin-eosin. Coverslipped slides were imaged at 8× and 400× magnifications on an Eclipse Ci-L microscope, targeting representative regions with tissues occupying the full field of view under uniform background illumination. Images were analyzed with Image-Pro Plus 6.0 software, calibrated to millimeters as the unit of measure. For morphometric analysis, three non-consecutive sections from each kidney were evaluated. For cortex-to-medulla ratio, one entire cross-section at 8× magnification was analyzed per section. For tubular epithelial density, ten non-overlapping fields of the renal cortex were randomly captured at 400× magnification per section. Measurements from all fields/sections were averaged to yield a single value per animal, which was used as the unit for statistical analysis (n = 8). For 8× fields, cortical and medullary areas (mm²) were delineated and quantified per image, enabling calculation of the cortex-to-medulla ratio. In 400× fields, perimeters (mm) of five proximal tubules were measured, their epithelial cell counts recorded, and tubular epithelial density computed as cells per mm (cell count divided by perimeter).

Western Blot Analysis Renal tissues were lysed in RIPA buffer supplemented with protease inhibitors, and total protein concentrations were quantified using a BCA assay kit. Equal amounts of protein were resolved by electrophoresis on 5% SDS-PAGE gels, transferred to polyvinylidene difluoride membranes, blocked in 5% nonfat dry milk at room temperature, and washed in 1% TBST. Membranes were probed overnight at 4°C with primary antibodies directed against PI3K (1:1000), AKT (1:500), phospho-AKT (1:500), p21 (1:1000), Klotho (1:1000), and β-actin (1:5000). Following three washes in TBST, horseradish peroxidase-conjugated secondary antibody (1:10000) was applied, and immunoreactive bands were visualized by enhanced chemiluminescence (ECL). Band densities were quantified using ImageJ software (National Institutes of Health, USA). For each target protein, quantification was performed on three independent Western blot runs. The final value for each animal was the average of these technical replicates.

Quantitative Real-Time PCR Analysis Total RNA Extraction: Tissues were homogenized immediately after harvest, and total RNA was isolated using TRNzol Universal reagent (Tiangen) according to the manufacturer’s protocol. RNA integrity and concentration were evaluated with a NanoDrop spectrophotometer. Complementary DNA (cDNA) synthesis involved on-column genomic DNA digestion and reverse transcription using the TaKaRa kit. The resulting cDNA was diluted 1:5 and used as template for quantitative PCR under the following conditions: initial denaturation at 95°C for 2 min (1 cycle), followed by 40 cycles of denaturation at 95°C for 15s and annealing/extension at 60°C for 15–30 s. β-Actin served as the housekeeping gene for normalization. PCR product specificity was confirmed by melting curve analysis. Each sample was run in triplicate, and the mean Ct value was used for calculation. Relative expression levels were determined using the 2^−ΔΔCt method. Primers were custom-synthesized by Sangon Biotech (Shanghai) Co., Ltd. Primer sequences are provided in Table 1.

Table 1 Sequences of Primers for qPCR

Statistical Analysis Data were analyzed using SPSS 27.0 and GraphPad Prism 8.0. Continuous variables are reported as mean ± standard deviation. For comparisons across multiple time points (weeks 0, 8, 12), statistical significance was determined by two-way analysis of variance (ANOVA) with Tukey’s post hoc test, considering “Treatment” (Group: NC vs. CIH) and “Time” as the two independent factors. This analysis allowed for the assessment of main effects, interactions, and specific comparisons between groups at each time point as well as within the same group across different time points. P-value < 0.05 was considered statistically significant.

Results

Successful Establishment of the CIH Rat Model

At week 8, carotid arterial blood gas analysis showed arterial oxygen saturation levels of 72% at 7% O₂ and 96.4% at 21% O₂. The rats sustained 30 intermittent hypoxic episodes per hour, effectively recapitulating the pathophysiology of moderate-to-severe human OSA and verifying the efficacy of the CIH model. After 4 weeks of normoxic recovery, the saturation at 21% O₂ improved to 97%.The experimental workflow is shown in Figure 1.

Figure 1 Experimental workflow and animal grouping. Abbreviations: NC, normoxic control; CIH, chronic intermittent hypoxia-exposed group. Notes: Rats were exposed to chronic intermittent hypoxia (CIH, O2 nadir 7%, 30 cycles/h, 8 h/day) for 8 weeks, followed by 4 weeks of normoxic recovery. Blood and kidney tissues were collected at weeks 0, 8, and 12 for biochemical, histopathological, and molecular analyses (0/8/12weeks, n =4/8/8 point).

Effects of CIH and Reoxygenation on SCr, BUN, and CysC

At baseline (week 0), there were no significant differences in serum creatinine (SCr), blood urea nitrogen (BUN), or cystatin C (CysC) levels between the NC and CIH groups (P > 0.05). After 8 weeks of CIH exposure, the CIH group exhibited significantly higher levels of SCr (49.80 ± 4.42 μmol/L vs 41.07 ± 2.50 μmol/L in NC, P < 0.05), BUN (17.40 ± 1.26 mg/dL vs 14.03 ± 0.62 mg/dL, P < 0.05), and CysC (15.93 ± 0.64 mg/L vs 12.46 ± 1.60 mg/L, P < 0.05) compared to the NC group (Table 2). Following 4 weeks of normoxic recovery, these differences were no longer statistically significant (P > 0.05 vs NC for all markers; Table 2), indicating functional recovery.

Table 2 Serum Levels of Renal Function Markers

Effects of CIH and Reoxygenation on Renal Cortical and Medullary Areas

At week 0, no significant differences in cortical or medullary areas were observed between groups. At week 8, the CIH group showed significantly reduced cortical area, expanded medullary area, and decreased cortex-to-medulla ratio compared to the NC group (all P < 0.05; Figure 2A–C). These structural alterations persisted at week 12, with cortical area remaining significantly lower and medullary area significantly higher in the CIH group (both P < 0.01; Figure 2A–C).Representative whole-kidney sections are shown in Supplementary Figure 2.

Figure 2 Effects of CIH and Reoxygenation on Renal Cortical and Medullary Areas (HE staining, 8×). Notes: Representative HE-stained whole-kidney sections at 8× magnification showing cortico-medullary boundaries. a: cortex; b: medulla. Scale bar = 1 mm. (A) Quantitative analysis of cortical area (mm2). (B) Quantitative analysis of medullary area (mm2). (C) Cortex-to-medulla ratio. (n=8 per group). *P < 0.05, **P < 0.01 vs NC group at the same time point.

Effects of CIH and Normoxic Recovery on Renal Tubule Morphology

HE staining revealed normal renal architecture in both groups at week 0. At week 8, the CIH group exhibited significant tubular dilation (increased tubular perimeter), reduced epithelial cell count per tubule, and decreased tubular epithelial density compared to the NC group (all P < 0.05; Figure 3A–C). By week 12, no significant intergroup differences were observed in tubular perimeter or epithelial density (both P > 0.05). Epithelial cell count per tubule remained slightly lower in the CIH group, but the difference was not statistically significant (P > 0.05; Figure 3A–C).Additional HE-stained images are available in Supplementary Figure 3.

Figure 3 Effects of CIH and normoxic recovery on renal tubule morphology. Notes: Representative high-magnification (400×) hematoxylin-eosin stained images of renal tubules from NC and CIH groups at weeks 0, 8, and 12. (A) Quantitative analysis of tubular perimeter (mm). (B) Quantitative analysis of tubular epithelial cell count per tubule. (C) Quantitative analysis of tubular epithelial density (cells/mm). Representative HE-stained images are shown in Supplementary Figure 3 (n=8 per group). *P < 0.05 vs NC group at the same time point; blue arrows indicate renal tubules; green arrows indicate tubular epithelial cells. Scale bar = 20 μm.

Effects of CIH and Normoxic Recovery on the PI3K/Akt/p21 Pathway

At week 8, CIH exposure was associated with significant downregulation of the PI3K/Akt pathway, as evidenced by decreased PI3K protein and mRNA levels (both P < 0.05), reduced p-Akt/Akt ratio (P < 0.01), and concurrent upregulation of p21 protein and mRNA expression (both P < 0.05) (Figure 4A–F). Following 4 weeks of normoxic recovery, PI3K/Akt signaling was restored to levels comparable to the NC group (P > 0.05), accompanied by significant suppression of p21 expression compared to the CIH group at week 8 (P < 0.05; Figure 4A–F).Original Western blot images for PI3K/Akt/p21 are provided in Supplementary Figure 4.

Figure 4 Effects of CIH and normoxic recovery on the PI3K/Akt/p21 pathway. Notes: (A) PI3K protein expression with representative Western blots. (B) Phosphorylated Akt (p-Akt) protein expression normalized to total Akt. (C) p21 protein expression with representative Western blots. (D) PI3K mRNA expression. (E) Akt mRNA expression. (F) p21 mRNA expression. (n=8 per group). *P < 0.05, **P < 0.01 vs NC group at the same time point.

Effects of CIH and Reoxygenation on p21 and Klotho Expression

At week 8, CIH exposure significantly suppressed Klotho expression at both protein and mRNA levels (P < 0.05) and upregulated p21 expression (P < 0.01) compared to the NC group (Figure 5A and B). Following 4 weeks of normoxic recovery, p21 expression returned to baseline levels (P > 0.05), whereas Klotho expression remained significantly suppressed (P < 0.05; Figure 5B and D).Western blot images for Klotho and p21 are shown in Supplementary Figure 5, and raw gray value data are available in Supplementary Table 1.

Figure 5 Effects of CIH and normoxic recovery on p21 and Klotho expression. Notes: (A) Representative Western blots for P21 and β-actin. (B) Quantitative analysis of Klotho protein expression. (C) p21 mRNA expression. (D) Klotho mRNA expression. (n=8 per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs NC group at the same time point.

Discussion

Effects of CIH and Reoxygenation on Renal Function Moderate-to-severe OSA impairs glomerular filtration rate (GFR) in patients relative to non-OSA individuals and elevates chronic kidney disease (CKD) risk.^18,19 CIH rodent models corroborate this: Arnaud et al reported that prolonged moderate-to-severe exposures (FiO₂ 6.5–10%, 20–30 cycles/h, 8 h/day, 35–60 days) raise Scr, BUNand albuminuria while boosting remodeling and fibrotic markers.²⁰ Our protocol (FiO₂ 7%, 30 cycles/h, 8 h/day, 8 weeks) mirrors these intensities; accordingly, 8-week CIH here spiked rat serum Cr, BUN, and CysC, denoting glomerular and tubular deficits. CIH-linked renal injury traces to increased generation of reactive oxygen species (ROS),²¹ which—coupled with stabilized hypoxia-inducible factor (HIF) and chronic inflammation—contributes to pathogenesis.²² This process can erode antioxidant defenses, fostering ROS accrual, inflammasome activation, and fibrogenesis, which manifest as GFR decline and azotemia. ROS and cytokines can directly damage tubular epithelia, provoking senescence and apoptosis; senescent cells then exacerbate inflammation and fibrosis via senescence-associated secretory phenotype (SASP),^23,24 potentially creating a vicious cycle of remodeling and dysfunction. Our data are consistent with the hypothesis that tubular epithelial senescence plays a pivotal role in CIH-driven injury and fibrotic progression.

Prior studies have emphasized CIH’s functional impact, yet the restorative potential of reoxygenation remains less explored. Here, 4 weeks of normoxia normalized CIH-induced metric aberrations (P > 0.05 versus controls), underscoring its functional salvage potential. Functional gains notwithstanding, structural reversal—specifically, the clearance of senescent cells or reversal of tissue-level senescence signatures—lacks documentation. Histopathological scrutiny thus emerges as essential for prognostic insight beyond these functional gains.

Effects of CIH and Reoxygenation on Renal Histopathology Histopathological interrogation here delineates CIH’s impact on renal architecture and its reversibility. 8-week CIH provoked overt remodeling: hematoxylin-eosin sections evidenced cortical contraction with medullary hypertrophy (P < 0.05 versus controls). Ultrastructurally, CIH kidneys bore glomerular sclerosis, tubular dilation, and epithelial sloughing. Morphometry affirmed tubular perimeter expansion and epithelial sparsification (cells/mm; P < 0.05), underpinning CIH’s functional sabotage. Such epithelial attrition and cortico-medullary overhaul are consistent with senescence hallmarks. Oxidative stress hastens telomere erosion; critical shortening then engages p53 to enforce durable arrest. Viewed in this context.^25,26 Our findings implicate CIH in promoting a senescent phenotype in tubular epithelia via oxidative stress. SASP from these cells unleashes proinflammatory and profibrotic payloads,²⁷ deranging niche equilibrium and promoting nephron loss via unrelenting inflammation-fibrosisa fulcrum for CIH-steered CKD.²⁸

A key novel aspect of our study is the demonstration of an asymmetry in recovery: 4 weeks of normoxia rectified functional metrics but scarcely budged cortical atrophy or structural entrenchment. The epithelial density rebound post-reoxygenation may signal edema resolution with viable cell compaction or modest repopulation, yet avails little against CIH’s nephron toll. Gains in function, then, may hinge on heightened performance from residual units amid quelled oxidative-inflammatory siege, underscoring renal compensatory prowess. Irrevocably, though, senescent/apoptotic nephrons, colonized by collagen, defy reoxygenation’s regenerative pull. Reoxygenation may rescue stressed cells but appears insufficient to reverse entrenched scars.

In sum, these data attest to CIH’s structural renal damage, linked to accelerated senescence and fibrotic recasting.²⁹ For OSA-linked nephropathy, they spotlight the importance of early intervention to forestall structural lock-in.

Effects of CIH and Reoxygenation on Renal Senescence-Associated Pathways and Molecules

The PI3K/AKT cascade steers cell cycle dynamics, metabolism, and senescence.³⁰ Fang et al showed CIH is associated with reduced AKT phosphorylation, thereby affecting downstream p21.³¹ Our Western blots mirror this pattern: 8-week IH was associated with reduced renal p-AKT and increased expression of the cell cycle inhibitor p21 (P < 0.05). p21’s upregulation, as a central mediator of senescence, can promote tubular epithelial cell cycle arrest below.³² Thus, the CIH-associated suppression of the PI3K/Akt pathway may contribute to p21-driven senescence, providing a molecular rationale for the observed renal dysfunction and structural changes. Four-week reoxygenation, in turn, restored pathway activity and tempered p21 elevation—a change that may facilitate functional and cellular recovery.

We further assayed the renoprotective Klotho, kidney-abundant and a senescence sentinel whose loss heralds aging;³¹ depletion ramps p21, p16, and γ-H2AX.³³ CIH here significantly reduced renal Klotho, consonant with reports; its downregulation may exacerbate a self-reinforcing cycle with oxidative stress and inflammation to spur senescence.³⁴ The most intriguing finding regarding Klotho is its persistent suppression after reoxygenation, despite the recovery of the PI3K/Akt/p21 axis. This dissociation suggests that Klotho regulation may occur at a deeper, more stable level of control, possibly involving epigenetic mechanisms such as promoter hypermethylation or enduring transcriptional repression established during the chronic hypoxic insult.^16,35 The failure to restore Klotho, a key anti-aging and renoprotective factor, could be a critical determinant of the irreversible structural components observed and may explain the sustained risk of CKD progression in OSA patients even after treatment.

In conclusion, our study provides evidence linking CIH to renal senescence phenotypes via the modulation of the PI3K/Akt/p21 pathway and Klotho. The partial reversibility of functional and signaling abnormalities upon reoxygenation contrasts with the persistence of structural damage and Klotho downregulation. This highlights that CIH-induced renal injury involves both dynamic, stress-responsive pathways and more stable, potentially epigenetically regulated components like Klotho. This notion is supported by recent evidence demonstrating that epigenetic regulators such as the IRF8-RUNX1 complex can drive renal cell senescence through upregulation of LINC01806, a long non-coding RNA.³⁶ Future studies employing direct pathway manipulation and a broader panel of senescence markers (eg., SA-β-gal, additional SASP factors, and progenitor cell senescence as highlighted by Ma et al) are warranted to establish causality and explore Klotho as a therapeutic target for mitigating irreversible renal damage in OSA.

Conclusion

This study demonstrates that CIH is associated with renal senescence phenotypes in rats, characterized by suppression of the PI3K/Akt pathway, upregulation of p21, downregulation of Klotho, and concomitant functional and structural renal impairment. Normoxic recovery effectively restored renal function and normalized the PI3K/Akt/p21 axis, but failed to reverse established structural damage or sustained Klotho suppression.

The central finding is the dissociation between reversible functional/molecular changes and persistent structural/anti-aging factor deficits after reoxygenation, suggesting that CIH-induced renal injury involves both dynamic stress-responsive pathways and more stable, potentially epigenetically anchored alterations. The persistent loss of Klotho may be a critical determinant of irreversible tissue damage, even after removal of the hypoxic insult.

These findings provide mechanistic insights linking OSA/CIH to accelerated renal senescence and highlight Klotho restoration and senescent cell clearance as potential therapeutic strategies to prevent long-term renal sequelae in OSA patients.

This study has several limitations. First, the correlative nature of our findings necessitates future interventional studies (eg., using PI3K/Akt activators or Klotho supplementation) to establish direct causality. Second, our assessment of senescence relied on a limited set of markers; a more comprehensive panel (eg., SA-β-gal, additional SASP factors) would strengthen future conclusions. Third, the observation period may have been insufficient to capture the full progression of fibrosis. Future research should employ longer timelines and direct fibrosis quantification to elucidate the complete trajectory from senescence to irreversible scarring. Addressing these points will be crucial for translating these mechanistic insights into effective therapies for OSA-associated kidney disease.