Comparative study of renal filtration barrier structure and key gene expression between Lepus yarkandensis and Oryctolagus cuniculus

Abstract

The Lepusyarkandensis (L. yarkandensis) is an endemic species inhabiting the arid ecosystem of the Tarim Basin, characterized by extreme dryness and scarce water resources. In contrast, Oryctolagus cuniculus (O. cuniculus) is adapted to temperate environments rich in water. Over the course of long-term evolution, the L. yarkandensis has likely developed unique physiological and histological adaptations to cope with the survival challenges posed by such arid conditions. This study aims to investigate the differences between the L. yarkandensis and O. cuniculus in terms of hematological and biochemical blood parameters, renal histology, water-salt regulation capacity, and the expression of glomerular filtration barrier proteins, in order to investigate the adaptive strategies of the L. yarkandensis to drought environments. The results showed that the L. yarkandensis exhibited higher red blood cell counts, hematocrit levels, urine osmolality, and total urinary protein levels compared to O. cuniculus, while the urine pH was lower. Histological analysis revealed increased collagen content in the outer medulla and more complex tubular structures in the kidneys of the L. yarkandensis. Molecular analysis further demonstrated upregulated expression of glomerular filtration barrier proteins nephrin, podocin, and CD2AP, alongside downregulated expression of laminin and WT1. In summary, these findings provide insights into the potential physiological and molecular adaptations of L. yarkandensis, laying the groundwork for future research into the evolution of desert fauna.

Introduction

L. yarkandensis is a small mammal endemic to the Tarim Basin, capable of surviving in extremely arid, high-temperature, and saline-alkaline environments. It serves as an ideal model for investigating survival mechanisms in harsh habitat [1]. The Tarim Basin, located in southern Xinjiang, China, is characterized by an extreme climate, with annual precipitation below 200 mm and evaporation exceeding 2,000 mm, posing significant adaptive challenges for the ecosystem.

Over the course of evolution, the L. yarkandensis has developed efficient water retention, metabolic regulation, and stress-resistance mechanisms to cope with water scarcity and heat stress [2]. In contrast, O. cuniculus, a widely distributed and domesticated species, typically inhabits stable environments with greater water availability, and its physiological traits are adapted to milder climates [3]. Comparative studies between the L. yarkandensis and O. cuniculus in terms of physiological and histological features not only reveal the ecological adaptations of the L. yarkandensis but also offer valuable insights into animal physiology and conservation under extreme environmental conditions. In recent years, increasing attention has been given to the adaptive mechanisms of animals to extreme environments, particularly under conditions of drought, extreme cold, or high salinity [1, 4, 5].

Studies have shown that mammals living in arid environments often adapt through enhanced urinary concentration, optimized renal tubular structures, and regulation of metabolic enzyme activity. For example, desert rodents and camels exhibit elongated loops of Henle and efficient water reabsorption systems [1, 3]. However, as a region-specific species, the L. yarkandensis has been relatively understudied, especially in terms of systematic comparisons involving blood parameters, renal histology, and molecular regulation. While existing literature indicates that the L. yarkandensis exhibits strong dehydration tolerance and enhanced renal water reabsorption [3], the specific characteristics of its blood biochemistry, renal tissue structure, and glomerular filtration barrier (GFB) protein expression remain largely unexplored. O. cuniculus, as a well-studied laboratory model, provides a reliable reference for comparison. Its physiological traits are generally adapted to environments with more abundant water resources [2, 6], whereas the L. yarkandensis may exhibit unique physiological traits shaped by long-term arid selective pressures.

The kidney plays a vital role in maintaining water-salt balance, excreting metabolic waste, and regulating blood pressure, and it is central to adaptation in extreme environments [7,8,9]. The kidneys of the L. yarkandensis may have undergone structural and molecular optimization to enhance water reabsorption and electrolyte homeostasis. Moreover, blood parameters such as red blood cell count, hemoglobin concentration, and biochemical markers reflect metabolic status and environmental adaptation, while histological features provide microstructural evidence of organ function [10, 11].

While tubular adaptations for water conservation are well-documented in desert species [4], the role of the glomerular filtration barrier (GFB) in arid adaptation remains relatively underexplored. For an organism to survive on minimal water, its kidneys must produce highly concentrated urine without losing essential plasma proteins. This necessitates a filtration barrier of exceptional selectivity that can withstand the increased osmotic and hydraulic stresses associated with urine concentration. Therefore, we posit that the GFB is not merely a passive filter but a key actively adapted structure crucial for survival in arid environments (Fig. 1).

Fig. 1.

Research overview of this study and the research subjects, L. yarkandensis and O. cuniculus.

The GFB plays a key role in the formation of primary urine. It is composed of three layers: fenestrated endothelial cells, a thick glomerular basement membrane (GBM), and podocyte foot processes with slit diaphragms. These three layers collaboratively accomplish the filtration function. The fenestrated endothelium blocks blood cells, platelets, and macromolecules, while the glycocalyx on endothelial surfaces repels negatively charged plasma proteins. Thus, the endothelium and its glycocalyx act as a critical charge barrier [12,13,14,15]. The GBM, situated outside the vascular endothelium, consists of an inner lamina rara interna, a central lamina densa, and an outer lamina rara externa as seen under transmission electron microscopy. The dense lamina, the main barrier to macromolecules, is composed of interwoven 3-nm fibers forming a complex 3D mesh. The foot processes of podocytes extend secondary finger-like projections, between which lie 20–40 nm filtration slits, covered by slit diaphragms (SDs), forming the final layer of the GFB [16, 17].

Genes associated with the GFB include Nephrin, Podocin, CD2AP, Laminin, and WT1[18,19,20,21,22]. These genes are crucial for the L. yarkandensis’ adaptation to arid environments. Nephrin is a transmembrane protein with an immunoglobulin-like structure, localized mainly to the slit diaphragm and specific to podocytes. Podocin is another podocyte-associated membrane protein involved in signaling. CD2AP, a member of the immunoglobulin superfamily initially identified in T and NK cells, plays a role in immune adhesion and also maintains GFB integrity. Laminin, a non-collagenous glycoprotein in the extracellular matrix, maintains GBM structure and function alongside type IV collagen. WT1 is a key transcription factor regulating podocyte differentiation, playing roles in embryogenesis, cell differentiation, and tumorigenesis.

The main objective of this study is to investigate the physiological and molecular mechanisms underlying the L. yarkandensis’ adaptation to arid environments (Fig. 1). By comparing it with O. cuniculus, the study aims to identify differences in blood parameters, renal histology, and GFB functionality. Through comprehensive analysis of blood and urine biochemistry, protein expression, and gene regulation, we systematically compared the hematological and biochemical profiles, renal structural characteristics, osmoregulatory capacities, and GFB protein expression patterns of the two species. This study provides valuable evidence for the ecological adaptation of the L. yarkandensis and offers new perspectives for physiological research under extreme environmental conditions.

Materials and Methods

Experimental materials

Adult male L. yarkandensis were captured in the northwestern region of the Tarim Basin, specifically in Shaya County (Aksu Prefecture, 81°45′E, 39°31′N). All individuals were confirmed to be adults (skull length >75.50 mm), with an average age of approximately 8 months and body weights ranging from 1.5 to 1.8 kg [23]. Age-matched adult male O. cuniculus were obtained from the experimental station of the College of Animal Science and Technology, Tarim University. During the experimental period, animal survival and physiological conditions—including feeding behavior, activity levels, and signs of illness—were routinely monitored. The capture and all experimental procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Tarim University (Approval No. 2022015). All methods were performed in accordance with the relevant local animal welfare laws, guidelines, and policies.

Blood biochemical analysis

To minimize animal stress, rabbits were gently restrained using soft towels to ensure comfort and reduce struggling. Whole blood samples (n=8) were collected from the marginal ear vein and immediately transferred into lithium heparin-coated vacuum tubes using a syringe. Key biochemical parameters, including blood urea nitrogen (BUN), creatinine (Cr), aspartate aminotransferase (AST), total bilirubin (TBIL), globulin (GLOB), urea (U-REA), albumin/globulin ratio (A/G), and BUN/creatinine ratio (B/C), were measured using a fully automated multifunctional biochemical analyzer.

Plasma EPO concentrations were quantified using a commercial EPO ELISA kit. Archived plasma samples were thawed on ice, and assays were performed in duplicate according to the manufacturer’s protocol. Optical density was measured at 450 nm using a microplate reader, and concentrations (mIU/ml) were determined from a four-parameter logistic standard curve.

Hematological analysis

Whole blood was promptly transferred into EDTA-K2 anticoagulant vacuum tubes for hematological analysis. A fully automated five-part hematology analyzer was used to assess the following parameters: total white blood cell count (WBC), red blood cell count (RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), and mean corpuscular hemoglobin (MCH).

Urinalysis

Urine samples were collected using sterile trays placed under the rabbit cages. Urine test strips (URIT) were used to measure parameters such as total urinary protein (UTP), urine specific gravity (USG), and urine pH (UpH). Analyses were performed using a veterinary urine analyzer.

Urinary electrolytes (Na⁺, K⁺, Cl⁻, Ca²⁺) were measured from the same urine samples using an ion-selective electrode-based veterinary electrolyte analyzer (EX-300Vet, Guilin Urit Electronic Co., Ltd., Guilin, Guangxi, China). Samples were centrifuged at 1,500 rpm for 5 min prior to analysis to remove precipitates, and measurements were performed in duplicate for accuracy.

Osmolality measurement

Whole blood collected in EDTA-K2 tubes was centrifuged at 3,500 rpm for 12 min to separate into three layers: (1) red blood cells (bottom), (2) buffy coat (middle, containing leukocytes and platelets), and (3) plasma (top, a pale yellow transparent liquid). The supernatant plasma was collected. Urine was collected in additive-free plain tubes. The osmolality of plasma and urine samples was determined using a freezing point depression osmometer.

Paraffin embedding and immunohistochemistry

Kidney tissues were fixed in 4% paraformaldehyde for 24 h, dehydrated through graded ethanol, cleared in xylene, and embedded in paraffin. Sections of 6 µm thickness were prepared. For antigen retrieval, paraffin sections were heated in citrate buffer (pH 6.0) in a microwave oven, followed by treatment with 3% hydrogen peroxide to block endogenous peroxidase activity and pre-incubation with 5% goat serum to block non-specific binding. Sections were incubated overnight at 4°C with primary antibodies (dilution 1:200), followed by incubation at 37°C with HRP-conjugated secondary antibodies for 30 min. Diaminobenzidine (DAB) was used for color development, hematoxylin was used for counterstaining, and coverslips were mounted using neutral resin. Protein expression and localization were examined under a microscope.

Primary antibodies used were as follows: nephrin (Proteintech, Rosemont, IL, USA, 66970-1-Ig), podocin (UnivBio, Shanghai, China, 5863808), CD2AP (Affinity Biosciences, Cincinnati, OH, USA, DF2298), laminin 2α (Affinity Biosciences, DF13275), WT1 (Proteintech, 12609-1-AP). To validate antibody specificity for this non-model species, the immunizing peptide sequences for all primary antibodies were aligned with their orthologs from O. cuniculus using NCBI’s protein BLAST (blastp), which confirmed high sequence conservation at the target epitopes.

From each animal, three non-overlapping random fields containing well-preserved glomeruli or representative tubular structures were selected. Mean optical density (MOD) was measured within manually delineated regions of interest (ROIs) encompassing positively stained glomerular or tubular areas, using Image-Pro Plus software (version 6.0, Media Cybernetics, Rockville, MD, USA). Color deconvolution was applied to separate the DAB signal from the hematoxylin counterstain. The MOD values from the three independent fields were averaged to obtain a single representative value per animal. These animal-level means were then used for group comparisons and statistical analysis (unpaired Student’s t-test or one-way ANOVA as appropriate [24, 25].

Total RNA extraction and cDNA synthesis

Total RNA was extracted from kidney tissues of L. yarkandensis and O. cuniculus using the Trizol method. Briefly, 80–100 mg of kidney tissue was ground in liquid nitrogen and lysed in 1 ml Trizol reagent, incubated at room temperature for 5 min, and subjected to phase separation by adding chloroform. After centrifugation, the aqueous phase was collected, RNA was precipitated with isopropanol, washed with 75% ethanol, dissolved in 30 µl of DEPC-treated water, and stored at −80°C. cDNA was synthesized using the EasyScript^® One-Step gDNA Removal and cDNA Synthesis SuperMix Kit.

qRT-PCR analysis

Primers were designed using Primer Premier 5, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as the reference gene. Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) was performed to analyze gene expression. Specific primers were designed based on the mRNA sequences of the target genes. Total RNA was reverse-transcribed into cDNA, which served as the template for amplification using SYBR Green I dye. Reactions were conducted on a real-time PCR instrument, and fluorescence signals were monitored in real time. Each reaction was performed in triplicate to ensure data reliability. Given the metabolic differences between the species, relative gene expression was quantified using the standard curve method to account for potential variations in GAPDH expression and PCR efficiency. Standard curves were generated for each gene from serial dilutions of pooled cDNA. PCR efficiencies (E) were calculated from the slope of each standard curve (E=10^(−1/slope)), and only assays with E between 90–110% and R²>0.99 were used. The relative expression ratio was calculated based on these efficiencies and the Ct values.

Western blot protein quantification

For protein extraction, kidney tissues were lysed at 4°C in RIPA buffer containing 1 mM PMSF and a protease inhibitor cocktail for 30 min with vortexing every 10 min. Lysates were centrifuged at 12,000 g for 15 min at 4°C, and the supernatant was collected as total protein. Protein concentration was determined using a BCA protein assay kit. Equal amounts of protein (30 µg per lane) were mixed with 5× SDS-PAGE loading buffer, denatured at 95°C for 5 min, and separated on 10% or 12% SDS-PAGE gels (80 V for 30 min, then 120 V until the dye front reached the bottom).

Proteins were transferred onto PVDF membranes using a wet transfer method (300 mA, 90 min). Membranes were blocked at room temperature with 5% skim milk in TBST for 1 h and incubated overnight at 4°C with primary antibodies (nephrin 1:1,000, podocin 1:800, CD2AP 1:200, laminin 1:200, WT1 1:200, diluted in TBST containing 5% BSA). After three washes with TBST (10 min each), membranes were incubated with HRP-conjugated secondary antibodies (1:5,000, diluted in TBST) for 1 h at room temperature. Following three additional TBST washes, signals were detected using ECL chemiluminescent reagents and visualized with the Tanon-5200 imaging system. Band intensities were quantified using ImageJ software, with GAPDH as the loading control. Primary antibodies were purchased from the following suppliers: nephrin antibody was from Proteintech (Cat. No. 66970-1-Ig). podocin antibody was from UnivBio (Cat. No. 5863808). CD2AP antibody was from Affinity Biosciences (Cat. No. DF2298). laminin 2α antibody was from Affinity Biosciences (Cat. No. DF13275). WT1 antibody was from Proteintech (Cat. No. 12609-1-AP).

Image quantification and data analysis

For all quantitative image analyses, including immunohistochemistry (MOD), H&E staining (interstitial area fraction), and morphometric measurements, three non-overlapping random fields of view containing representative structures were selected from each animal. Regions of interest (ROIs) were manually delineated to encompass the specific areas for quantification (e.g., glomerular or tubular cross-sections), carefully excluding artifacts or poorly preserved tissue. The quantitative parameters were measured in each field and averaged to yield a single representative value per animal for statistical analysis.

Statistical analyses were performed using GraphPad Prism software (version 8.0; GraphPad Software, San Diego, CA, USA). Data are presented as mean ± SD or mean ± SEM, as indicated in figure legends. The sample size (n) for all analyses represents the number of biological replicates (individual animals per species). Prior to analysis, data normality was assessed using the Shapiro-Wilk test, and homogeneity of variance was verified using the Brown-Forsythe test. As this study exclusively involved comparisons between two independent groups (L. yarkandensis vs. O. cuniculus), statistical significance was determined using an unpaired Student’s t-test for normally distributed data with equal variances. If the assumptions of normality or homogeneity of variance were not met, a non-parametric Mann-Whitney U test was employed. A P-value of <0.05 was considered statistically significant. Significance levels are indicated as: *P<0.05, **P<0.01, and ***P<0.001.

Results

Comparative analysis of blood parameters between L. yarkandensis and O. cuniculus

As shown in Table 1, there were no significant differences (P>0.05) between the two species in several indicators, including sodium (Na⁺), chloride (Cl⁻), total protein (TP), albumin (ALB), total cholesterol (TG), uric acid (UA), and the aspartate aminotransferase/alanine aminotransferase ratio (AST/ALT).

Table 1. Biochemical blood test results of O. cuniculus and L. yarkandensis.

Parameters	Reference Range	O.cuniculus (n=8)	L.yarkandensis (n=8)	t-value	P-value
Serum potassium / (mmol/l)	3.5~5.5	3.65 ± 1.02	7.42 ± 1.09**	3.618	0.002
Serum sodium / (mmol/l)	136~145	141.8 ± 3.25	147.59 ± 1.98	1.006	0.343
Serum chlorine / (mmol/l)	98~106	105 ± 2.28	113.76 ± 4.48	1.274	0.226
Serum calcium / (mmol/l)	2.2~2.7	3.3 ± 0.22	2.7 ± 0.48	1.318	0.209
Uric acid / (mg/dl)	3.6~8.0	11.07 ± 2.08	17.63 ± 2.39	0.775	0.448
UREA/ (mmol/l)	2.5~8.3	6.23 ± 0.32	16.85 ± 3.84*	2.809	0.012
Blood glucose / (mmol/l)	3.9~6.1	14.47 ± 3.71	4.61 ± 3.46**	3.235	0.007
Total cholesterol / (mmol/l)	<5.17	1.31 ± 0.47	1.6 ± 1.09	0.623	0.540
Triglyceride / (mmol/l)	<1.7	1.01 ± 0.56	3.28 ± 0.19*	2.113	0.045
Direct bilirubin / (μmol/l)	<3.4	0.45 ± 0.26	1.39 ± 1.04	1.766	0.096
Indirect bilirubin / (μmol/l)	3.4~20.5	0.44 ± 0.03	11.33 ± 1.27**	3.287	0.004
Total bilirubin / (μmol/l)	0~20.5	1.17 ± 0.93	2.59 ± 1.58*	2.185	0.043
Alkaline phosphatase / (U/l)	40~150	131.2 ± 12.69	58.66 ± 16.14	1.437	0.169
Glutamyltransferase / (U/l)	0~45	19.63 ± 2.41	17 ± 4.4	0.196	0.847
Total protein / (g/l)	60~80	51.99 ± 6.94	49.85 ± 11.88	0.407	0.689
Albumin / (g/l)	35~50	35.37 ± 4.7	34.66 ± 6.99	0.358	0.724
Globulin / (g/l)	20~35	15.66 ± 3.29	12.32 ± 4.91	1.136	0.271
White-sphere ratio (A/G)	1.5~2.5	2.4 ± 0.41	3.51 ± 2.28	1.463	0.160
Lactate dehydrogenase / (U/l)	120~250	149.2 ± 47.91	1,288.27 ± 345.42**	3.123	0.006
Creatine kinase / (U/l)	24~195	928.67 ± 468.54	3,954 ± 774.78***	5.137	0.000
Glutamic-pyruvic transaminase / (U/l)	9~50	38 ± 4.9	277.67 ± 78.48***	4.551	0.000
Glutamic oxalacetic transaminase / (U/l)	15~40	34.75 ± 16.45	354 ± 129.76***	5.239	0.000
Aspartate transaminase than alanine transaminase ( AST/ALT)	0.8~2.1	1.22 ± 0.86	1.67 ± 1.22	0.797	0.435
Urea nitrogen / (mmol/l)	1.8~7.1	7.14 ± 0.34	24.26 ± 12.64**	3.023	0.008
Creatinine / (umol/l)	44~115	71.7 ± 12.06	100.17 ± 18*	2.355	0.036
Urea nitrogen/creatinine (BUN/Cr)	10~20	161.61 ± 32.12	281.85 ± 93.92*	2.267	0.035

However, significant differences (P<0.05) were observed in several parameters, including urea (UREA), total bilirubin (TB), and creatinine (Cr). Further analysis revealed more pronounced differences in serum potassium (K⁺), glucose (Glu), lactate dehydrogenase (LDH), and blood urea nitrogen (BUN) levels (P<0.01). Notably, the expression levels of creatine kinase (CK), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) exhibited highly significant differences (P<0.001).

In addition, both AST and ALT levels in L. yarkandensis were significantly higher than those in O. cuniculus, although the ALT/AST ratio showed no significant difference. Furthermore, BUN, Cr, and the BUN/Cr ratio were all significantly elevated in L. yarkandensis (P<0.05).

The mean corpuscular volume (MCV) of L. yarkandensis was significantly lower than that of O. cuniculus (P<0.05). In contrast, red blood cell count (RBC), hematocrit (HCT), and mean corpuscular hemoglobin concentration (MCHC) were significantly higher in L. yarkandensis, exceeding the normal reference range for O. cuniculus, with highly significant differences observed (P<0.01, Table2).

Table 2. Results of routine blood tests.

Parameters	Reference Range	O.cuniculus	L.yarkandensis	t-value	P-value
White blood cell count / (10^9/l)	3~15	9.75 ± 3.8	1.96 ± 0.88**	4.370	0.004
Neutrophil numbe / (10^9/l)	0.50~6.60	6.3 ± 2.18	1.18 ± 0.23*	3.953	0.016
Lymphocyte count / (10^9/l)	1.00~6.80	4.68 ± 0.75	0.95 ± 0.49**	4.972	0.002
Monocyte count / (10^9/l)	0.08~1.51	0.69 ± 0.58	0.15 ± 0.11	2.315	0.082
Number of eosinophils / (10^9/l)	0.00~0.51	0.4 ± 0.34	0.07 ± 0.05	1.174	0.284
The number of basophils / (10^9/l)	0.00~0.76	0.03 ± 0.01	0.01 ± 0.01	2.041	0.076
Neutrophilic granulocyte percentage/ (%)	14.0~62.0	50.45 ± 8.25	51.18 ± 9.11	0.067	0.948
Lymphocyte percentage / (%)	25.0~82.0	41.4 ± 14.2	38.73 ± 13.64	0.316	0.761
Monocyte percentage / (%)	2.0~15.0	4.95 ± 3.85	6.33 ± 4.07	0.494	0.636
Percentage of eosinophils / (%)	0.0~6.0	2.9 ± 2.2	3.38 ± 1.98	0.250	0.809
Percentage of Basophil granulocyte / (%)	0.0~8.0	0.3 ± 0.1	0.4 ± 0.25	0.682	0.517
Erythrocyte count / (10^12/l)	3.4~6.5	5.12 ± 0.19	8.01 ± 1.23**	5.237	0.002
Hemoglobin / (g/l)	80~140	122.93 ± 14.26	165.4 ± 30.18**	3.773	0.009
Hematokrit / (%)	25~42	31.7 ± 2.35	42.98 ± 6.62**	3.892	0.007
Mean corpuscular volume / (fl)	60~80	62.07 ± 5.85	53.68 ± 1.42*	2.512	0.048
Mean erythrocyte hemoglobin content / (pg)	19.0-25.0	21.47 ± 1.53	20.94 ± 0.55	0.166	0.872
Mean corpuscular-hemoglobin concentration / (g/l)	300-360	340 ± 1.50	386.2 ± 8.08**	6.191	0.002
Red blood cell distribution width (RDW-SD)/ (fl)	28~65	29.95 ± 0.05	25.48 ± 0.83**	6.022	0.003

Conversely, white blood cell (WBC) and lymphocyte counts were notably lower in L. yarkandensis. While this could suggest a differences in basal immune surveillance or a response to chronic stress, the precise physiological implication requires further investigation into their immune ecology.

However, the mean corpuscular volume (MCV) of L. yarkandensis was significantly lower than that of O. cuniculus (P<0.05). A lower MCV indicates the presence of smaller red blood cells (microcytosis), which can be associated with conditions such as iron deficiency or chronic inflammation, but may also represent a species-specific trait optimized for blood flow dynamics in a dehydrated state.

The plasma concentration of arginine vasopressin (ADH) was significantly elevated in O. cuniculus compared to that in L. yarkandensis (Fig. 2A). This difference was statistically significant (P<0.001), suggesting a distinct physiological regulation of water conservation or osmotic balance between the two species. Plasma EPO levels were slightly but significantly higher in L. yarkandensis (14.2 ± 0.4 mIU/ml) compared to O. cuniculus (11.8 ± 0.7 mIU/ml; P<0.05, Fig. 2B).

Fig. 2.

(A) Comparison of plasma ADH concentrations (B) EPO concentrations between O. cuniculus and L. yarkandensis.

As shown in Table 3, plasma osmolality did not differ between species, but urine osmolality was three-fold higher in L. yarkandensis compared to O. cuniculus (2,159.0 ± 235.8 vs. 715.9 ± 198.5 mOsm/kg, P<0.001). Comprehensive urinalysis (Table 4) confirmed this enhanced concentration ability, revealing significantly higher specific gravity (P<0.001) and total protein (P<0.001) in L. yarkandensis, alongside a lower pH (P<0.001). L. yarkandensis urine also tested positive for glucose, urobilinogen, and bilirubin, with elevated WBC counts. Electrolyte analysis showed markedly lower urinary Na⁺ and Cl⁻ (both P<0.001), but higher K⁺ (P<0.001) and Ca²⁺ (P<0.01) excretion in L. yarkandensis.

Table 3. Results of osmolality measurements.

Parameters	Reference Range	O.cuniculus	L.yarkandensis	t-value	P-value
Posm/(mmol/l) Plasma osmolality	340–360	339.7 ± 23.4	336.4 ± 24.5	0.369	0.718
/(mmol/l) Urine osmolality	50–1,400	715.9 ± 198.5	2,159.0 ± 235.8***	10.293	0.000

Table 4. Results of comprehensive routine urinalysis and urinary electrolyte measurements.

Parameters	Reference Range	O. cuniculus (n=8)	L. yarkandensis (n=8)	t-value	P-value
White blood cells (WBC)/(cell/μl)	0-5	3.7 ± 0.04	7.3 ± 0.2***	5.752	0.000
Ketones (KET)	“-” — “4+”	“-”	“-”	-	-
Nitrite (NIT)	“-/+”	“-”	“-”	-	-
Urobilinogen (URO)	“-” — “4+”	“-”	“2+”	-	-
Bilirubin (BIL)	“-” — “3+”	“-”	“2+”	-	-
Total protein (UTP)/(g/l)	0–0.15	0.5 ± 0.2	2.9 ± 0.5***	7.958	0.000
Glucose (GLU)	“-” — “4+”	“-”	“+”	-	-
Specific gravity (USG)/(g/cm³)	1.003–1.035	1.012 ± 0.004	1.028 ± 0.008***	6.151	0.000
Occult blood (BLD)	“-” — “4+”	“-”	“-”	-	-
pH (UpH)	0–14	8.2 ± 0.5	6.0 ± 0.89***	5.654	0.000
Vitamin C (VC)	“-” — “3+”	“+”	“+”	-	-
Sodium (Na⁺)/(mmol/l)	50–150*	128.4 ± 15.2	62.7 ± 12.8***	7.412	<0.001
Potassium (K⁺)/(mmol/l)	20–80*	58.6 ± 9.3	112.4 ± 14.7***	8.156	<0.001
Chloride (Cl⁻)/(mmol/l)	80–140*	132.1 ± 11.5	78.3 ± 10.9***	6.893	<0.001
Calcium (Ca²⁺)/(mmol/l)	5–20*	12.8 ± 2.4	18.5 ± 3.1**	4.215	0.003

Comparative H&E staining of renal cortex and medulla in O. cuniculus and L. yarkandensis

In O. cuniculus, relatively intact histological structures can be observed, including the distal tubule (DT), parietal layer (WL), glomerulus (G), outer medullary collecting duct (OMCD), descending thin limb (DTL), ascending thin limb (ATL), inner medullary collecting duct (IMCD), proximal convoluted tubule (PCT), macula densa (MD), and proximal straight tubule (PST). The renal cortex of O. cuniculus exhibits deeper eosin staining compared to L. yarkandensis, potentially attributable to the stronger acidophilia of the cytoplasm in O. cuniculus. In contrast, L. yarkandensis demonstrate a higher density of glomeruli per unit area than O. cuniculus.

Morphometric analysis revealed distinct patterns of renal interstitial expansion between O. cniculus and L. yarkandensis across different kidney zones (Fig. 3). In the outer medulla (OM), the interstitial area fraction was significantly higher in L. yarkandensis compared to O. cuniculus. A similar trend was observed in the inner medulla (IM), although the difference did not reach statistical significance. In contrast, the interstitial area in the renal cortex (C) was comparable between the two species. These results suggest a species-specific adaptation in the medullary region of L. yarkandensis, which may be associated with its enhanced capacity for water conservation.

Fig. 3.

Comparative histological and morphometric analysis of renal structure in O. cuniculus and L. yarkandensis. (A) Representative H&E-stained micrographs of renal tissues from O. cuniculus and L. yarkandensis. (B) Quantitative comparison of renal interstitial area fraction between O. cuniculus and L. yarkandensis across the cortex, outer medulla, and inner medulla. Scale bars: 50 µm.

In L. yarkandensis, relatively intact histological structures are observable, including the distal tubule (DT), parietal layer (WL), glomerulus (G), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), descending thin limb (DTL), ascending thin limb (ATL), and inner medullary collecting duct (IMCD). The glomerular tufts, composed of capillary networks, exhibit a distinct pink hue under staining. Renal tubules are also visible, displaying densely packed arrangements with regularly shaped cells, where nuclei are stained blue.

Electron micrographs of the renal cortex and medulla between O. cuniculus and L. yarkandensis

Transmission electron microscopy (TEM) was used to examine the deeper ultrastructure of the cortex and medulla (Figs. 4A–L and M–R). In the renal cortex, tubular epithelial cells of both species contained numerous mitochondria (Mt) (Figs. 4A–B and D–E). However, mitochondria in L. yarkandensis appeared more numerous, densely packed, and exhibited highly compacted cristae compared to those in O. cuniculus.

Fig. 4.

Comparative ultrastructural morphology of renal cortex and medulla in O. cuniculus and L. yarkandensis by transmission electron microscopy and scanning electron microscopy. Cortex: Transmission electron micrographs illustrating the ultrastructure of the renal cortex in O. cuniculus (A–C, G–I) and L. yarkandensis (D–F, J–L). Scanning electron microscopy illustrating the ultrastructure of the renal cortex in O. cuniculus (S–V) and L. yarkandensis (W–Z). Medulla: Transmission electron micrographs of the renal medulla in O. cuniculus (M–O) and L. yarkandensis (P–R). Images depict the structure of medullary tubules and interstitium, with annotations for mitochondria (Mt) and nuclei (N). Scalebars: 2 µm, except where otherwise indicated on individual panels.

Analysis of the glomerular filtration barrier (GFB) confirmed intact structures in both species, composed of fenestrated endothelium, GBM, and podocyte foot processes (Figs. 4G–L). High-magnification TEM revealed distinct differences in the slit diaphragms. While the GBM thickness appeared comparable, the filtration slits between interdigitating foot processes were notably narrower and appeared tighter in L. yarkandensis (Fig. 4L) compared to O. cuniculus (Fig. 4I).

In the renal medulla, similar trends in mitochondrial morphology were observed. Medullary tubular cells in L. yarkandensis (Figs. 4P and Q) displayed a higher density of mitochondria with intricately folded, dense cristae relative to O. cuniculus (Figs. 4M and N), suggesting an enhanced metabolic capacity for active transport in the medulla of the desert-adapted species.

Scanning electron microscopy (SEM) provided a three-dimensional view of the glomerular surface architecture (Figs. 4S–Z). In both species, glomeruli exhibited well-defined, generally spherical to oval morphologies (Figs. 4S and W). Podocyte cell bodies (PC) extended primary and secondary processes that wrapped around glomerular capillaries (Figs. 4T and X). High-magnification SEM revealed that the interdigitation of podocyte foot processes appeared more regular, tighter, and more densely arranged in L. yarkandensis (Figs. 4Y and Z) compared to the slightly looser arrangement observed in O. cuniculus (Figs. 4U and V).

Distribution ofPodocyte- Associated and Glomerular Basement Membrane Markers in the Kidneys of O. cuniculus and L. yarkandensis

Immunohistochemical (IHC) analysis revealed the expression of podocyte-associated and glomerular basement membrane (GBM) markers in the renal cortex of both species (Fig. 5). In the cortex, these proteins were primarily localized to the glomeruli (Figs. 5A, B, D, E, G, H, J, K, M and N). Qualitatively, L. yarkandensis exhibited markedly stronger staining signals for nephrin, podocin, and CD2AP compared to O. cuniculus, whereas the staining for laminin and WT1 was more intense in O. cuniculus. Quantitative analysis of mean optical density confirmed that the protein expression of nephrin, podocin, and CD2AP was significantly upregulated in the cortex of L. yarkandensis relative to O. cuniculus (P<0.001), while the expression of laminin and WT1 was significantly downregulated (P<0.001) (Figs. 5C, F, I, L and O).

Fig. 5.

Histological and molecular characterization of renal cortex tissues in O. cuniculus and L. yarkandensis. (A–O) Immunohistochemical staining of renal cortex sections and quantitative bar graphs comparing expression levels of key podocyte-associated proteins (nephrin, podocin, CD2AP, laminin, and WT1). Scale bars: 50 µm.

Expression of mRNA and Protein in the Kidneys of O. cuniculus and L. yarkandensis

To delineate the molecular basis of the observed histological differences, we quantified the expression of key glomerular filtration barrier genes and proteins in renal tissues using qRT-PCR and Western blot, respectively, utilizing GAPDH as the internal control.

The findings revealed coordinated upregulation of several podocyte-associated markers in L. yarkandensis relative to O. cuniculus. Specifically, both mRNA and protein levels of Nephrin (Fig. 6A) and Podocin (Fig. 6B) were significantly increased (all P<0.01). CD2AP expression also followed this pattern, showing significant upregulation at both the transcriptional (P<0.01) and translational (P<0.05) levels (Figs. 6C, H and M).

Fig. 6.

Comparative expression of podocyte-associated and glomerular basement membrane (GBM) markers in the renal cortex of O. cuniculus and L. yarkandensis. (A–E) Relative mRNA expression levels of Nephrin (A), Podocin (B), CD2AP (C), Laminin (D), and WT1 (E) measured by qRT-PCR. Data are normalized to GAPDH. (F–J) Representative Western blot images showing protein bands for nephrin (F), podocin (G), CD2AP (H), laminin (I), and WT1 (J), with GAPDH as the loading control. (K–O) Densitometric quantification of protein levels relative to GAPDH for nephrin (K), podocin (L), CD2AP (M), laminin (N), and WT1 (O).

In contrast, both Laminin and WT1 exhibited concordant downregulation in L. yarkandensis compared to O. cuniculus. Their mRNA (Laminin: P<0.05, Fig. 6D; WT1: P<0.05, Fig. 6E) and protein (laminin: P<0.01, Figs. 6I and N; WT1: P<0.01, Figs. 6J and O) levels were significantly decreased.

Discussion

The L. yarkandensis possesses unique biological characteristics, including exceptional tolerance to hunger and thirst, a strong ability to produce highly concentrated urine, and effective water retention. These traits are closely linked to its distinctive physiological mechanisms and the harsh environment in which it survives. The special living conditions have endowed its body fluids with physiological and nutritional functions that differ markedly from those of other animals. Studying the physiology and pathology of the L. yarkandensis requires comprehensive reference to its complete hematological parameters. Biochemical indicators reflect the animal’s internal metabolic processes and the functional status of certain tissues and organs, while various physiological blood parameters serve as important criteria for evaluating normal physiological functions and are crucial diagnostic markers in clinical examinations.

Interpretation of biochemical profiles and metabolic adaptations

In this study, the elevated serum potassium (K⁺) observed in the L. yarkandensis may be attributed to multiple causes of hyperkalemia, including but not limited to renal insufficiency, metabolic acidosis, tissue damage, and endocrine disorders. Renal insufficiency is one of the most common causes because the kidneys are the primary organs responsible for potassium excretion. When renal function is impaired, potassium excretion decreases, leading to elevated blood potassium levels. However, it cannot be excluded that potassium release may also result from extensive cellular damage during capture, causing intracellular potassium to shift extracellularly and increase serum potassium concentration. Elevated total bilirubin may indicate hemolytic anemia, a common cause of hyperbilirubinemia, where red blood cell rupture releases large amounts of hemoglobin, whose metabolic products contain abundant bilirubin.

Increased serum urea, blood urea nitrogen (BUN), and creatinine (Cr) levels can result from pre-renal, renal, or post-renal factors. Pre-renal causes are due to reduced renal blood flow or systemic conditions such as dehydration, hypotension, and heart failure, all of which reduce kidney perfusion and impair urea excretion. Renal causes include intrinsic kidney damage such as acute renal failure, chronic kidney disease, and tubular necrosis, which directly compromise the filtration function. Post-renal factors mainly involve urinary tract obstruction—such as urethral stones, tumors, or prostatic hypertrophy—that block urine outflow, causing urea accumulation in the blood. The data suggest that the elevated urea in L. yarkandensis is primarily due to pre-renal factors, although they do not exhibit dehydration symptoms, possibly related to their long-term adaptation to desert environments.

The integrity of the GFB and the reabsorptive capacity of the renal tubules are critically reflected in urinary solute composition. While specific measurements of urinary albumin, glucose, and amino acids were not conducted in this study, our data provide functional insights. The markedly elevated UTP in L. yarkandensis (Table 4) strongly indicates altered glomerular permselectivity, likely encompassing albuminuria, which is a hallmark of barrier dysfunction [26]. This finding aligns with our molecular data showing differential expression of GFB proteins, suggesting a adaptive trade-off that permits extreme urine concentration while potentially allowing a low level of protein leakage. Conversely, the profound urine concentration ability (Table 3) implies highly efficient tubular reabsorption of solutes, including glucose and amino acids, as their osmotic loss would otherwise impede the achievement of such high osmolality.

The plasma glucose concentration in L. yarkandensis is lower than in O. cuniculus, possibly as an adaptive energy conservation strategy in food-scarce environments, where lower blood glucose levels reduce energy wastage. Additionally, LDH and creatine kinase (CK) activities in L. yarkandensis are significantly higher. Living in the high-altitude, hypoxic Tarim region requires efficient energy metabolism. Elevated CK activity facilitates rapid ATP regeneration, supporting muscle and cardiac function under low oxygen conditions. LDH plays a crucial role in anaerobic metabolism by converting lactate to pyruvate to produce energy. The high LDH activity suggests enhanced anaerobic metabolic capacity,which may contribute to the ability of L. yarkandensis to concentrate urine more efficiently. The elevated CK and LDH levels in L. yarkandensis reflect long-term evolutionary adaptation, indicating genetic selection across generations to cope with extreme environmental conditions distinct from those of O. cuniculus.

Stress states, such as evading predators in the wild, may cause transient elevations in AST and ALT. These enzymes catalyze transamination reactions essential in the pyruvate-glucose cycle. The surge in their levels may explain the lower blood glucose observed in L. yarkandensis, indicating higher biochemical metabolic rates than O. cuniculus. Their elevation may also indicate hepatic cellular injury, causing leakage of these enzymes into the bloodstream. The liver, as the primary metabolic organ, is involved in detoxification, protein synthesis, and lipid and glucose metabolism. Liver dysfunction can disrupt these processes, leading to metabolic disorders, decreased vitality, anorexia, and impaired behavior and activity.

Hematological characteristics and immune status indicative of arid adaptation

The elevated red blood cell count, hemoglobin, and hematocrit in L. yarkandensis (Table 2) were accompanied by mildly increased plasma EPO levels compared to O. cuniculus (Fig. 2B). Although statistically significant, the modest elevation (~20%) remains within normal physiological limits and is unlikely to fully account for the pronounced erythrocytosis observed. This suggests that the hematopoietic adaptations in L. yarkandensis are not primarily driven by a classic hypoxia-inducible EPO response (as seen in high-altitude polycythemia). Instead, the slight EPO increase may reflect species-specific renal regulation, potentially linked to the arid, saline-alkaline environment of the Tarim Basin, where enhanced kidney function (e.g., for water conservation) could influence basal EPO production. Alternative mechanisms, such as improved iron metabolism, altered hemoglobin-oxygen affinity, or direct environmental influences on erythropoiesis, likely contribute more substantially to the observed erythrocyte elevations.

Conversely, white blood cell (WBC) and lymphocyte counts are notably lower in L. yarkandensis. This discrepancy may result from different environmental pressures influencing their immune systems, sampling time and methodology, or inherent differences in immunoregulation mechanisms. Accurate interpretation requires further research, including comprehensive data on immune status, environmental stress, genetics, and health condition, as well as additional laboratory and field studies.

Renal functional adaptations: osmoregulation and electrolyte handling

The profound urine concentrating ability observed in L. yarkandensis, evidenced by a three-fold higher osmolality and significantly elevated specific gravity compared to O. cuniculus, constitutes a critical adaptation for survival in the hyper-arid Tarim Basin. This superior water conservation is closely linked to specific electrolyte handling strategies. The marked reduction in urinary Na⁺ and Cl⁻ excretion indicates highly efficient renal conservation of these major extracellular ions, likely mediated by enhanced reabsorption in the distal nephron and collecting ducts to minimize osmotic water loss.

Conversely, the elevated urinary K⁺ and Ca²⁺ excretion in L. yarkandensis reflects necessary physiological trade-offs. Higher K⁺ output may result from consuming potassium-rich halophytic vegetation typical of saline desert environments or represent an obligatory loss driven by mechanisms prioritizing Na⁺ retention. Similarly, increased Ca²⁺ excretion might be required to manage high mineral intake from saline-alkaline soil and water sources, preventing systemic hypercalcemia. Most notably, the significant proteinuria observed alongside extreme urine concentration suggests an adaptive compromise in glomerular permselectivity. We posit that this represents a functional trade-off, where limited leakage of macromolecules is tolerated to achieve the immense solute concentration gradients necessary for maximal water recovery under severe dehydration stress.

It is important to note that while the elevated urinary protein and extreme osmolality in L. yarkandensis are consistent with a trade-off model for water conservation, our current data do not definitively distinguish between adaptive physiologic protein handling and potential pathological leakage. Functional assessments, such as measuring GFR and albumin-creatinine ratios, are necessary in future studies to fully characterize renal function in this species.

Molecular remodeling of the glomerular filtration barrier

By comparing the expression of genes related to the renal filtration barrier, this study explores the adaptive mechanisms of L. yarkandensis to arid environments. In studies of Podocin and Nephrin expression, it has been shown that mature podocytes exhibit a stellate shape with processes, and that immunolocalization of Nephrin and Podocin suggests a linear distribution on the surface of podocyte membranes in the kidney [27]. Previous research on Nephrin and Podocin expression in rat kidneys demonstrated that both proteins are localized in the glomeruli of the renal cortex [28,29,30], which is consistent with the findings of the present study.

Regarding CD2AP, its expression in the kidney is not limited to podocytes; it is also found in the epithelial cells of the collecting ducts, displaying a diffuse cytoplasmic distribution, with lower levels of expression in epithelial cells of distal and proximal tubules [31]. Studies on the molecular mechanisms underlying the dysfunction of the slit diaphragm in glomerular podocytes have shown that CD2AP is expressed in the glomerular basement membrane [32,33,34,35], which aligns with the present study’s observations in the renal cortex. Furthermore, research has demonstrated that CD2AP, Nephrin, and Podocin are co-localized in the slit diaphragm region, with CD2AP serving as a critical component that links Nephrin and Podocin to the podocyte cytoskeleton. These three proteins interact to form the Nephrin–CD2AP–Podocin complex, which anchors the slit diaphragm to the actin cytoskeleton of podocyte foot processes, thereby maintaining the normal filtration function of the glomerular membrane [36].

In previous studies, Laminin has been shown to be located in the basement membrane of the filtration barrier [37,38,39], while WT1 is expressed in glomerular podocytes and localized within the nucleus [40, 41]. The results of this study on the comparative expression of filtration barrier-related genes in the kidneys of L. yarkandensis and O. cuniculus revealed that the expression of Nephrin, Podocin, and CD2AP was significantly upregulated in L. yarkandensis, whereas Laminin and WT1 expression was markedly downregulated.

Nephrin, Podocin, and CD2AP play key roles in the structure of podocyte foot processes and in the maintenance of their interconnections. The upregulation of these genes may enhance the stability of the inter-podocyte junctions, keeping the gaps between foot processes narrow and regular. This contributes to the effective filtration of macromolecules, preventing their leakage into the urine. The downregulation of Laminin and WT1 may affect the regulation of filtration barrier permeability. WT1 plays a critical role in podocyte function and basement membrane formation, and its downregulation could increase the permeability of the barrier. However, the upregulation of Nephrin and related proteins may partially counteract this effect, maintaining a relatively stable filtration barrier.

As L. yarkandensis inhabits arid environments, the upregulation of Nephrin, Podocin, and CD2AP may represent an adaptive change. This pattern may be consistent with differences in filtration barrier–related components, which could contribute to urine-concentrating capacity and reduced water loss under water-scarce conditions. However, functional barrier performance and macromolecule handling were not directly assessed in this study.

Limitations and future directions

Finally, although the primary antibodies used in this study exhibited high sequence conservation with Oryctolagus cuniculus orthologs (as validated by NCBI protein BLAST), minor deviations from 100% homology at the epitope level cannot be excluded. Such differences may slightly reduce antibody-antigen affinity, potentially influencing signal intensity in Western blot and IHC assays. Consequently, while the observed expression trends (upregulation of nephrin, podocin, and CD2AP; downregulation of laminin and WT1 in L. yarkandensis) are robust and consistent across methods, absolute quantitative comparisons should be interpreted with caution. Future studies using species-specific antibodies or antibody-independent approaches (e.g., mass spectrometry) would further strengthen these findings.

A primary limitation of this study is the use of antibodies not specifically generated against L. yarkandensis proteins. Although NCBI BLAST analysis confirmed high epitope sequence conservation with O. cuniculus orthologs, minor differences in binding affinity cannot be ruled out. Therefore, the observed quantitative differences in protein expression should be interpreted as relative interspecies trends rather than measures of absolute abundance. Future validation using species-specific antibodies or targeted proteomics would strengthen these findings.

Conclusion

The expression patterns of nephrin, podocin, CD2AP, laminin, and WT1 suggest distinct molecular differences in glomerular filtration barrier components in L. yarkandensis. These patterns may be consistent with adaptation to arid and water-scarce environments; however, renal filtration efficiency and tubular reabsorption function were not directly examined in this study. Further functional experiments are needed to verify these adaptive interpretations. These key slit diaphragm proteins may contribute to glomerular barrier stability, precisely regulating metabolic waste and electrolytes, which could represent an adaptive response to the need for urine concentration in arid habitat. The downregulation of Laminin and WT1 could potentially reflect remodeling or reduced developmental regulation of the glomerular basement membrane, which might represent a trade-off to optimize filtration efficiency. Immunohistochemical analyses suggest increased expression of these proteins in glomerular capillary endothelial and collecting duct cells, which appears consistent with heightened renal filtration and reabsorption activity. Consistent qPCR and Western blot results imply that transcriptional regulation might partly underlie these expression differences.

Funding

This study was funded by the National Natural Science Foundation of China (32160112), Xinjiang Production & Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin (BRFW2402).