Interactions between kidney proteins and metabolism in Tibetan sheep at different altitudes

Introduction

Extreme environments are common natural conditions in high-altitude regions, characterized by low pressure, low oxygen levels, and low temperatures [1]. As altitude increases, atmospheric pressure decreases. This leads to a reduction in the partial pressure of oxygen at each node of the oxygen transport chain—from the environment to the mitochondria within tissues. Conversely, this decrease in oxygen partial pressure triggers corresponding changes in multiple organs and tissues. These adaptations help the body counteract the functional impacts of rising altitude and maintain normal physiological levels [2]. The duration of exposure at high altitudes has varying effects on kidney function. Short-term exposure can cause acid-base and sodium imbalances, while prolonged exposure allows the kidneys to gradually adapt and restore normal physiological function. Therefore, organisms living at high altitudes must develop corresponding adaptive mechanisms to ensure normal physiological functions in such environments. As plateau animals, Tibetan sheep have drawn widespread attention for their hypoxic adaptation to unique environments.

Hypoxia adaptation is a specialized physiological phenomenon, referring to the adaptive responses an organism adopts to sustain basic life functions when exposed to low-oxygen environments. In high-altitude regions, numerous species have evolved unique survival strategies and biological mechanisms for adapting to hypoxia due to the combined effects of long-term natural and artificial selection [3, 4]. The kidneys play a crucial role in the body’s adaptation to hypoxic conditions and in maintaining acid-base balance and electrolyte homeostasis. When oxygen supply is restricted, it can trigger a series of acid-base imbalances, such as pathological states like metabolic acidosis or alkalosis [5]. In response, the kidneys initiate compensatory mechanisms by precisely regulating the excretion of bicarbonate in urine, thereby helping to alleviate hypoxia-induced metabolic acidosis [6]. Specifically, under hypoxic conditions, renal proximal tubule cells can activate calcium and potassium ion channel mechanisms. This mechanism not only helps maintain appropriate cellular membrane potential stability, but this critical ion regulation also exerts a significant protective effect by inhibiting apoptosis and enhancing cellular tolerance to the hypoxic environment [7]. When confronted with hypoxic challenges, the kidneys also modulate their water and salt reabsorption mechanisms to maintain stable blood volume and blood pressure. The kidneys are highly sensitive to hypoxia, particularly the renal medulla, where the partial pressure of oxygen is low. Hypoxia activates renal afferent nerves, directly causing an increase in blood pressure and stimulating the production of erythropoietin, which increases the blood’s oxygen-carrying capacity. At the same time, the kidneys and the carotid bodies work together to detect hypoxia; mediated by the RAAS and HIF pathways, this creates a positive feedback loop that drives excessive sympathetic nervous system activation, ultimately leading to and maintaining hypertension [8]. Research has shown that the interaction between hypoxia and the renin-angiotensin system (RAS) is complex: under hypoxic conditions, the RAS is activated, and the activated RAS, in turn, exacerbates hypoxia [9]. This process is crucial for ensuring adequate oxygen and nutrient supply to the kidneys during hypoxia, enabling them to maintain normal physiological function under low-oxygen conditions. However, due to the extreme environment at high altitudes, when the kidneys’ adaptive mechanisms are disrupted, this can lead to kidney tissue damage. Prolonged hypoxia at high altitudes leads to increased cellular proliferation and impaired endothelial cell function [10]. Simultaneously, persistent hypoxia causes abnormal increases in serum uric acid levels [11]. This increase in uric acid may be linked to a decrease in ATP levels caused by adenine nucleotides and the subsequent activation of xanthine oxidase [12]. Research has also found that prolonged exposure to hypoxic environments leads to glomerular hypertrophy, a phenomenon observed in both humans and animals [13, 14]. This condition may be associated with a reduction in nephron number and glomerular hypertension [15, 16].

Therefore, this study selected Tibetan sheep as an ideal model for plateau research and conducted investigations on kidney tissue at three different altitudes. The adaptive mechanisms to hypoxic environments are complex, involving the coordinated action of multiple organ systems. Beyond core life-sustaining organs such as the heart, lungs, and central nervous system, the kidneys also play a crucial role in responding to hypoxic challenges. When confronted with hypoxic stress, the kidneys can effectively adapt to these adverse conditions through finely tuned adjustments to energy metabolism pathways-such as enhancing anaerobic glycolysis or regulating oxidative phosphorylation-while maintaining acid-base balance and other critical metabolic activities. This study aims to investigate the effects of long-term exposure to high-altitude environments on the kidneys, elucidate the role of kidney-related proteins in high-altitude hypoxia, and examine changes in metabolites. It also analyzes the protein-metabolite interactions involved in renal adaptation to high-altitude hypoxia, providing new insights into the adaptive mechanisms of kidneys in high-altitude animals and humans.

Materials and methods

Results

Discussion

High-altitude animals, living long-term in the low-oxygen environment of plateaus, undergo adaptive changes in the morphological structure of their tissues and organs, as well as in their physiological and biochemical indicators. This study aims to investigate kidney tissues from Tibetan sheep at different altitudes using proteomic and metabolomic approaches. The goal is to elucidate the hypoxic adaptation-related protein-metabolite interaction mechanisms in high-altitude animal kidneys, thereby providing novel insights into renal health in such environments. Using proteomics techniques to study the kidney, we identified a total of 78 differentially expressed proteins, which revealed proteins closely associated with kidney function. Research has found that ALDH2 is primarily expressed in kidney tissue and localized to renal tubular epithelial cells. Additionally, studies in rats indicate that ALDH2 expression contributes to reduced damage in kidney tissue [23]. Research has also revealed a relationship between ALDH2 and renal tissue fibrosis, specifically that enhancing ALDH2 expression can inhibit the progression of fibrosis [24]. Another study indicates that suppressing ALDH2 expression exacerbates damage to kidney tissue [25]. This also demonstrates that ALDH2 exerts a protective effect on the kidneys. Research has demonstrated that COX5A plays a crucial regulatory role in mitochondrial energy metabolism. Furthermore, a study in mice revealed that COX5A exerts a protective effect during mitochondrial oxidative stress [26]. Reactive oxygen species (ROS) serve as the primary source of oxidative stress in high-altitude hypoxia, and ROS imbalance can trigger various physiological and pathological conditions. Research has revealed that COX5A modulates the expression of antioxidant genes by regulating YAP1, thereby exerting resistance to oxidative stress mechanisms [27]. This also indicates that COX5A plays a crucial role in defending against oxidative stress within the hypoxic adaptation mechanism of Tibetan sheep kidney tissue. Additionally, another study found that miR-30a-5p can target and inhibit GLUD1, thereby further suppressing oxidative stress and apoptosis in renal tubular epithelial cells under hypoxic conditions [28]. A study on humans found that disrupting NDUFS2 leads to decreased glycolytic capacity and ATP production in renal cells, while conversely increasing ROS levels [29]. This finding bears similarities to NUDFS8: silencing NUDFS8 reduces ATP production in endothelial cells, leading to increased ROS production and oxidative stress [30]. SLC17A1 is highly expressed in renal epithelial cells and has been demonstrated to interact with Lac-Phe. Knockout of SLC17A1 in mice resulted in reduced urinary Lac-Phe levels [31]. This also confirms that SLC17A1 participates in renal hypoxic adaptation mechanisms through metabolic processes, thereby protecting kidney health. The above studies confirm that the hypoxic adaptation mechanism identified in Tibetan sheep kidney tissue involves not a single protein but a synergistic action of multiple proteins. Specifically, ALDH2 and GLUD1 protect the kidneys by reducing inflammation; COX5A counters oxidative stress; NDUFS2 and NDUFS8 supply ATP energy to the kidneys; and SLC17A1 participates in metabolic processes. Collectively, these proteins ensure kidney health in animals living in high-altitude environments. Due to the kidneys’ high energy demands, they are highly dependent on mitochondrial function, with the TCA cycle serving as the core of energy metabolism, primarily responsible for energy production [32]. Meanwhile, mitochondria, acting as powerhouses, produce adenosine triphosphate (ATP) through oxidative phosphorylation via the electron transport chain to supply cells [29]. This finding is consistent with the results of this study, which indicate that Tibetan sheep kidneys significantly enrich key components of the TCA cycle and oxidative phosphorylation pathways in their hypoxic adaptation mechanisms, playing a crucial role in these processes. Additionally, the study revealed that NDUFS8 and NDUFS2 are core components of the ubiquinone oxidoreductase (NADH) complex, playing a pivotal role in energy production within the oxidative phosphorylation pathway. Their primary function is to facilitate the transfer of electrons from NADH to ubiquinone [33–35]. These studies also demonstrate that NDUFS2 and NDUFS8 provide energy through the oxidative phosphorylation pathway, while GLUD1 and ALDH2 participate as key enzymes in the TCA cycle (supplying acetyl-CoA and NADH). Meanwhile, SLC17A1 indirectly influences energy metabolism and oxidative stress status by regulating urate levels within the body. However, research on sheep kidneys has certain limitations. Much of the evidence cited above comes from models other than sheep and the kidney—primarily from studies on humans, rats, and mice—and this evidence cannot serve as direct confirmation of hypoxic adaptation in Tibetan sheep kidneys. Therefore, in future studies, we will conduct direct experimental research on sheep kidneys to validate our findings.

Our metabolomics study of Tibetan sheep at different altitudes identified 24 key DEMs. Trend analysis of these metabolites revealed dynamic expression patterns. The detected differentially expressed metabolites included Wedelolactone, Diphenylamine, and Bioresmethrin; these compounds may originate from environmental exposure, pesticide residues, or feed, and their biological significance requires further validation. Research has found that, as a natural product, Wedelolactone possesses excellent antioxidant properties [36]. Its presence in sheep kidneys may be attributed to feed (such as sheep grazing on Asteraceae plants containing this compound) or other exogenous sources. However, further experimental verification is needed to determine whether Wedelolactone exerts anti-inflammatory or antioxidant effects on sheep kidneys. Cytidine is a component of RNA and a precursor to uridine. Research has shown that metabolites produced by the breakdown of uridine enable cells to grow normally in glucose-deficient environments. Additionally, studies indicate that uridine can provide energy in cases of mitochondrial oxidative phosphorylation defects [37]. These studies also corroborate our findings that metabolites participate in the establishment of renal hypoxic adaptation mechanisms in Tibetan sheep at high altitudes, providing antioxidant, anti-inflammatory, and energy supply functions. Further KEGG enrichment analysis of DEMs revealed that the HMG-CoA reductase inhibitors signaling pathway and the Biosynthesis of unsaturated fatty acids pathway primarily contribute to renal hypoxic adaptation mechanisms. Research has found that the HMG-CoA reductase inhibitors signaling pathway primarily functions to reduce the occurrence of inflammation, particularly by blocking inflammatory mediators in microglia following hypoxia [38]. Our research confirms that changes in metabolites influence alterations in metabolic pathways, leading to the upregulation of anti-inflammatory pathways. This represents one of the mechanisms by which Tibetan sheep kidneys maintain renal health under hypoxic conditions. At the same time, after conducting an integrated analysis of the positive and negative ion patterns in the metabolome, we found that the results were consistent with those of previous studies, which confirms the reliability of our analysis. We further investigated the relationship between proteins and metabolites, revealing correlations between the renal proteome and metabolome. Subsequent analysis of DEPs and DEMs demonstrated significant positive/negative correlations between proteins and metabolites. At the same time, research has shown that most key proteins influence metabolites by participating in metabolic pathways. This indicates that renal hypoxic adaptation mechanisms are not driven by isolated proteins or metabolites, but rather by synergistic interactions between proteins and metabolites. Together, these interactions influence adaptive mechanisms, ensuring normal renal function in high-altitude environments. However, the lack of independent experimental validation is a limitation of this study. Furthermore, we would like to emphasize that future research will focus on conducting further functional validation of the identified key candidate molecules to elucidate their causal mechanisms in high-altitude hypoxic adaptation. In addition, an ideal study design should include multiple independent sampling points at each elevation gradient and, to the extent possible, control for or record key covariates (such as rearing methods and genetic background) to reduce confounding bias and enhance the reliability of causal inferences. Future studies will further optimize their designs in this direction.

Conclusion

This study identified altitude-associated proteomic and metabolomic patterns and screened for key DMPs (ALDH2, COX5A, GLUD1, NDUFS2, NDUFS8, and SLC17A1) as well as key metabolites (Wedelolactone, Cytidine, and 3-hydroxybenzoic acid). These findings pertain to energy metabolism, oxidative phosphorylation, and candidate antioxidant/anti-inflammatory pathways. These findings require validation in controlled or replicable study designs.

Supplementary Information

Authors' contributions

KL performed the data analysis and wrote the manuscript. JZ performed investigation and collected the samples. XY performed the procedures and formal analysis using software. PZ did the project administration and revised the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This study was financially supported by the 2024 Gansu Province Universities Industrial Support Plan Projects (2024CYZC-59), 2024 Gansu Province Youth Science and Technology Fund (24JRRP006), 2025 Gansu Province Universities Young Doctor Support Projects (2025QB-098).

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD075530.

Declarations