Abstract
Objective
This study aimed to investigate erythropoietin receptor (EPOR) expression in human AKI and to examine its association with histopathological severity and clinical parameters.
Methods
Renal biopsy specimens from 21 patients with biopsy-confirmed acute kidney injury (AKI) and 12 control kidney tissues from nephrectomy samples were analyzed. EPOR expression was assessed by immunohistochemistry with AI-assisted digital quantification and by immunofluorescence, including co-staining with β-common receptor (βcR) and TUNEL.
Results
In human kidney biopsy specimens, EPOR expression was significantly higher in AKI kidneys compared to controls (p < 0.001). Immunofluorescence demonstrated co-localization of EPOR and βcR on tubular epithelial cell membranes. EPOR expression showed significant inverse correlations with stage of AKI (τ = −0.444, p = 0.012), tubular necrosis scores (τ = −0.485, p = 0.005), and acute pathology scores (τ = −0.475, p = 0.005). A significant positive correlation was found between EPOR expression and both red blood cell count (r = 0.490, p = 0.024) and Hemoglobin levels (r = 0.496, p = 0.022). Receiver operating characteristic analysis indicated moderate discriminatory performance of EPOR expression for differentiating AKI severity stages. Consistent with the human data, EPOR protein levels were significantly upregulated in mouse kidneys 24 h after ischemia–reperfusion injury. Single-cell transcriptomic analysis further demonstrated increased EPOR expression in tubular cell populations in AKI.
Conclusions
EPOR expression is upregulated in renal tubular epithelial cells in human AKI and is closely associated with histopathological injury severity and selected clinical parameters, highlighting that EPOR may serve as a marker of tubular stress and injury severity in AKI.
Keywords: Erythropoietin, erythropoietin receptor, acute kidney injury, tubular necrosis
Introduction
acute kidney injury (AKI) is a clinical syndrome characterized by a rapid decline in renal function, typically manifested by an increase in serum creatinine and/or a reduction in urine output [1]. According to the Kidney Disease: Improving Global Outcomes (KDIGO) criteria, AKI is defined by an increase in serum creatinine of ≥0.3 mg/dL within 48 h, a 1.5-fold rise from baseline within 7 d, or urine output <0.5 mL/kg/h for at least 6 h [1]. AKI is associated with substantial short- and long-term morbidity and mortality, and persistent or severe AKI markedly increases the risk of progression to chronic kidney disease (CKD) as well as cardiovascular complications, contributing to a significant global health burden [2–5].
In recent years, increasing attention has been directed toward the erythropoietin (EPO)/erythropoietin receptor (EPOR) signaling axis, not only for its established role in erythropoiesis but also for its involvement in cellular responses to hypoxia and tissue injury [6]. EPOR is a member of the type I cytokine receptor family and classically mediates erythropoiesis through homodimeric EPOR2 formation upon EPO binding. Beyond hematopoiesis, experimental studies have suggested that EPO may modulate apoptosis, inflammation, and oxidative stress in various organ injury models, including the kidney. These non-erythropoietic effects have been proposed to involve an alternative EPOR signaling configuration that includes the β-common receptor (βcR), distinct from the canonical EPOR homodimer pathway [7].
Despite extensive investigation in experimental systems, the expression pattern of EPOR in human AKI and its relationship to histopathological injury severity have not been systematically characterized. In particular, data derived from human renal biopsy specimens remain limited. Therefore, in this study, we examined EPOR expression in renal biopsies from patients with biopsy-confirmed AKI and analyzed its association with tubular injury severity and relevant clinicopathological parameters.
Methods
Patient enrollment and tissue sampling
This retrospective study included patients from Subei People’s Hospital between January 2016 and December 2024. Individuals who underwent renal biopsy and were diagnosed histopathologically with AKI formed the AKI group. Control samples were obtained from histologically normal renal cortex tissue adjacent to tumors in patients undergoing nephrectomy for renal neoplasms (> 5 cm from the neoplasm). A total of 21 patients were recruited from the inpatient services based on previously established eligibility criteria. Clinical trial number: not applicable. The paraffin-embedded renal tissues from enrolled patients were obtained from the Biobank (−80 °C) of Subei People’s Hospital, and subsequently sectioned into approximately 3 μm-thick slices using standard protocols in pathology department. We retrospectively analyzed renal tissue specimens obtained from patients who underwent renal biopsy for AKI, all of whom had provided written informed consent specifically for the biopsy procedure. All AKI biopsies were performed within one week after AKI diagnosis, while patients were still in the acute phase of the injury. Additionally, adjacent non-tumorous renal tissues from patients with renal cell carcinoma were selected as controls; these patients had provided informed consent for surgical intervention before nephrectomy. This study was approved by the Institutional Medical Ethics Committee (METC: No. 2022ky030). The study design and protocol are shown in a flow diagram (Figure 1), all in accordance with the Declaration of Helsinki.
Figure 1.
Study design and number of patients in cohorts.
The inclusion criteria were defined separately for the AKI and control groups. For the AKI group, eligible patients were required to meet the KDIGO 2012 diagnostic criteria for acute kidney injury, have a biopsy-proven histopathological diagnosis of AKI, and be hospitalized for acute kidney dysfunction without clinical evidence of extrarenal organ failure. Control kidney tissues were obtained from patients with normal pre- and post-operative serum creatinine, no severe systemic disease or extrarenal organ involvement, and a histologically normal renal cortex located at a sufficient distance from tumor infiltration.
Immunofluorescence (human renal biopsies)
Immunofluorescence staining was performed on formalin‑fixed, paraffin‑embedded human renal biopsy sections. After standard deparaffinization and rehydration, heat-induced antigen retrieval was carried out for 30 min. Sections were then blocked with 3% bovine serum albumin (BSA) for 30 min and incubated overnight at 4 °C with the following primary antibodies: anti-EPOR (1:150 dilution, Origene, Rockville, MD, catalog # OTI4F12) and anti‑βcR (1:500 dilution, Servicebio, Wuhan, China, catalog # GB113265). Following washes, sections were incubated with appropriate fluorophore‑conjugated secondary antibodies, counterstained with DAPI, and imaged using a fluorescence microscope.
Immunohistochemistry (human renal biopsies)
Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded kidney sections. Sections were incubated with an anti-EPOR monoclonal antibody (1:350 dilution, Origene, catalog # OTI4F12), followed by incubation with a secondary antibody and visualization using a DAB (3,3′-diaminobenzidine) chromogenic substrate. Image acquisition and analysis were conducted using an artificial intelligence-based digital pathology platform (Servicebio®), which integrates deep learning algorithms trained on large histopathological datasets. EPOR expression levels were quantitatively assessed using average optical density (AOD), calculated as: AOD = Integrated optical density (IOD)/Area.
Histopathological evaluation
Renal biopsy specimens were semi-quantitatively assessed by experienced renal pathologists who were blinded to all clinical information. Four histopathological features – tubular atrophy, interstitial fibrosis, interstitial inflammation, and tubular epithelial necrosis – were evaluated on a standardized 0–4 scale reflecting the extent of involvement (0 = none, 1 = focal, 2 = multifocal, 3 = segmental, and 4 = diffuse). To capture the overall severity of acute injury, an Acute Pathology Score was generated by summing the scores for interstitial inflammation and tubular epithelial necrosis. All pathological assessments and digital quantification were performed by evaluators blinded to clinical data.
Single-cell sequencing
The Kidney Precision Medicine Project (KPMP) kidney tissue atlas (https://atlas.kpmp.org/explorer/dataviz) was used to access the single-cell RNA-seq dataset derived from renal biopsies from 28 healthy controls (HC) and 14 AKI patients.
Animal model of AKI
To establish an ischemia-reperfusion injury (IRI) model of AKI, male C57BL/6 mice aged 6–8 weeks from Shanghai Jihui Laboratory Animal Care Co., Ltd. were used and were raised in specific pathogen-free conditions of the Laboratory Animal Center of Naval Medical University for 12h day and night with food and water provided at will. All animals were anesthetized with 3% sodium pentobarbital. A unilateral nephrectomy was performed, followed by clamping of the renal artery of the remaining kidney to induce 25 min of ischemia. Reperfusion was initiated by removing the arterial clamp. Mice were euthanized 24 h after reperfusion to assess EPOR expression. Sham-operated animals underwent identical surgical exposure and handling without nephrectomy or vascular occlusion. All animal procedures were approved by the Institutional Animal Care and Use Committee and conformed to national guidelines on animal research ethics.
Western blotting
Renal tissues from mice were homogenized and lysed for protein extraction. Equal quantities of protein were resolved by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skimmed milk for 1 h at room temperature, membranes were incubated overnight at 4 °C with anti-EPOR primary antibody (1:1,000, Abclonal, Woburn, MA, catalog # A2917). The following day, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Goat anti-Mouse IgG, 1:5,000, Abclonal, catalog # AS003) for 1 h at room temperature. Signal development was performed using an imaging system. Densitometric analysis was conducted using ImageJ software.
Statistical analysis
All statistical analyses were performed using SPSS version 29.0 (IBM Corp., Armonk, NY). Data distribution was evaluated using the Shapiro–Wilk test, Q–Q plots, and histograms. Continuous variables following a normal distribution were analyzed using independent sample t-tests, while non-normally distributed variables were compared using Kruskal–Wallis tests. Categorical variables were analyzed using chi-square or Fisher’s exact tests as appropriate. Correlation analyses were performed using Pearson’s correlation coefficient for normally distributed variables and Kendall’s Tau’s rank correlation coefficient for non-parametric date. A receiver operating characteristic (ROC) curve was constructed to evaluate the performance of EPOR. A p value < 0.05 was considered statistically significant.
Results
Baseline characteristics of study participants
A total of 33 human participants were included in the study, comprising 21 patients with biopsy-confirmed AKI and 12 control subjects who underwent nephrectomy for renal tumors. Compared with controls, patients with AKI exhibited significantly higher serum creatinine levels and significantly lower hemoglobin (Hb) and red blood cell (RBC) counts (Table 1). No significant differences were observed between the two groups with respect to age, sex distribution, body mass index, liver function parameters, or serum albumin levels.
Table 1.
Baseline characteristics of participants.
| Clinicopathological characteristics | Normal (N = 12) | AKI (N = 21) | p Value |
|---|---|---|---|
| Sex | |||
| Male | 6 | 10 | 1.000 |
| Female | 6 | 11 | |
| Age (years) | |||
| ≤45 | 4 | 3 | 0.440 |
| >45 | 8 | 18 | |
| BMI (kg/m2) | 23.36 ± 1.890 | 22.59 ± 2.530 | 0.366 |
| RBC (X1012) | 4.21 ± 0.500 | 3.11 ± 0.587 | 0.001 |
| Hemoglobin (g/L) | 128.25 ± 12.024 | 90.71 ± 18.940 | 0.001 |
| Serum creatinine (μmol/L) | 83.58 ± 10.317 | 556.52 ± 271.303 | 0.001 |
| Alanine aminotransferase (U/L) | 22.90 ± 10.147 | 22.74 ± 12.380 | 0.970 |
| Aspartate aminotransferase (U/L) | 21.22 ± 4.567 | 21.31 ± 8.642 | 0.973 |
| Albumin (g/L) | 32.01 ± 5.280 | 33.01 ± 4.429 | 0.564 |
| Serum calcium (mmol/L) | 2.05 ± 0.190 | 2.05 ± 0.157 | 0.121 |
| Serum phosphorus (mmol/L) | – | 1.82 ± 0.721 | |
| Parathyroid hormone (pg/mL) | – | 142.41 ± 92.361 | |
| Erythropoietin receptor (AOD) | 0.044 ± 0.009 | 0.093 ± 0.0164 | 0.001 |
| AKI | |||
| 1 | 0 | 7 | |
| 2 | 0 | 9 | |
| 3 | 0 | 5 | |
| Causes of AKI | |||
| ANCA-associated glomerulonephritis | 0 | 10 | |
| IgA Nephropathy | 0 | 3 | |
| Anti-GBM Disease | 0 | 1 | |
| Others | 0 | 7 | |
| Co-morbid conditions | |||
| Hypertension | 0 | 16 | |
| Diabetes | 0 | 3 |
‘–’ indicates parameters that were not measured rather than missing data.
Abbreviations: BMI: body mass index; RBC: red blood cell; AKI: acute kidney injury.
Histopathological features in AKI biopsies
Renal histology from AKI patients exhibited characteristic tubulointerstitial injury with varying degrees of severity. Acute tubular necrosis (ATN) was absent in 28.6% of cases, focal in 42.8%, multifocal in 19.0%, and segmental or diffuse in the remaining 9.6%. Interstitial inflammation was diffuse in 76.2% of patients, with the rest showing segmental (14.3%) or multifocal (9.5%) patterns. Regarding chronic changes, interstitial fibrosis ranged from none to diffuse, with 47.6% showing multifocal fibrosis and 14.3% exhibiting diffuse fibrosis. Tubular atrophy was most commonly multifocal (52.4%), while 14.3% of cases showed diffuse atrophy. (Table 2).
Table 2.
Histopathological scores of AKI patients.
| Pathological descriptions | Pathological scores (n = 21) |
|---|---|
| Acute Tubular Necrosis | |
| 0 | 6 |
| 1 | 9 |
| 2 | 4 |
| 3 | 1 |
| 4 | 1 |
| Inflammatory infiltration | |
| 0 | 0 |
| 1 | 0 |
| 2 | 2 |
| 3 | 3 |
| 4 | 16 |
| Interstitial fibrosis | |
| 0 | 1 |
| 1 | 3 |
| 2 | 10 |
| 3 | 4 |
| 4 | 3 |
| Tubular atrophy | |
| 0 | 0 |
| 1 | 3 |
| 2 | 11 |
| 3 | 4 |
| 4 | 3 |
Co-localization of EPOR and βcR in human AKI kidneys
Immunofluorescence analysis revealed co-localized expression of EPOR and the βcR on the plasma membrane of renal tubular epithelial cells in AKI biopsy specimens. In comparison with control kidney tissue, AKI samples exhibited more prominent membrane-associated co-localization of EPOR and βcR, indicating a spatial association of these receptors in tubular epithelial cells during AKI (Figure 2).
Figure 2.
Co‑localization of EPOR and βcR on tubular epithelial cell membranes.
Upregulation of EPOR in human AKI
Immunohistochemical analysis demonstrated that EPOR expression was significantly increased in AKI biopsy specimens compared with control kidney tissue (p < 0.001). While chromogenic immunohistochemistry suggested predominantly cytoplasmic EPOR immunoreactivity, high-magnification immunofluorescence analysis revealed membrane-associated localization of EPOR in tubular epithelial cells. Quantitative digital image analysis showed a significant increase in the AOD of EPOR staining in the AKI group relative to controls across all AKI stages (Stages 1–3) (p < 0.05; Figure 3(A,B)). Although no statistically significant differences in EPOR expression were observed among different AKI stages, a gradual downward numerical trend was noted with increasing AKI severity.
Figure 3.
EPOR expression in human AKI. (A) Representative photomicrographs of kidney sections from renal patients (Control and AKI Stage 1–3) stained by immunohistochemistry for EPOR. (B) Staining scoring of EPOR of the four groups (Control, n = 12; AKI Stage 1, n = 7; AKI Stage 2, n = 9; AKI Stage 3, n = 5). Data were presented as means ± SD. *p < 0.05, Student’s t-test. (C) Representative photomicrographs of kidney sections from renal patients (Control and AKI Stage 1–3) stained by Immunofluorescence for EPOR and TUNEL. (D) Differential expression of EPOR in kidney cell clusters from HC and AKI in the Kidney Precision Medicine Project kidney tissue atlas: a higher expression of EPOR was observed compared to HC in AKI patients; EPOR expression of AKI was higher in the PTCs, PCs, and CNTs, compared to HC. HC, n = 28. AKI, n = 14. Data link: https://atlas.kpmp.org/explorer/dataviz.
To further examine the relationship between EPOR expression and tubular epithelial cell injury, immunofluorescence co-staining for EPOR and TUNEL was performed. In advanced AKI stages, EPOR signals were reduced in areas exhibiting extensive tubular epithelial cell apoptosis, whereas residual intact tubular epithelial cells retained detectable EPOR expression (Figure 3(C)).
Consistent with these findings, analysis of KPMP demonstrated higher EPOR expression in AKI samples compared with healthy controls. However, KPMP analysis is presented for qualitative visualization and contextual support only, and no inferential statistical comparisons were intended. Increased EPOR expression was observed predominantly in proximal tubular cells, principal cells, and connecting tubule cells (Figure 3(D)).
Association of EPOR expression with histopathological severity
Kendall’s tau-b correlation analysis demonstrated that EPOR expression was significantly and inversely correlated with the severity of acute tubular epithelial necrosis (tau = −0.485, p = 0.005) and the composite Acute Pathology Score, defined as the sum of interstitial inflammation and necrosis scores (tau = −0.475, p = 0.005) (Figure 4(A,B)). No statistically significant correlations were found between EPOR expression and either interstitial fibrosis or tubular atrophy scores.
Figure 4.
Association of EPOR expression with clinical and pathological Indicators. (A,B) Kendall’s tau-b correlations between EPOR and ATN or Acute Pathology Score in the AKI groups. (C,D) Pearson correlations between EPOR and RBC or Hb in the AKI groups. (E) Kendall’s tau-b correlations between EPOR and Stage of AKI. (F) ROC curves to evaluate the discriminative capacity of EPOR expression between different AKI severity groups.
Correlation between EPOR expression and clinical indicators
Pearson correlation analysis revealed a statistically significant positive association between renal EPOR expression and RBC count (r = 0.490, p = 0.024) as well as Hb levels (r = 0.496, p = 0.022) (Figure 4(C,D)). The positive association between EPOR expression and Hb/RBC may reflect systemic activation of the hypoxia–EPO axis during acute stress, but this requires further mechanistic investigation. Notably, EPOR levels were inversely correlated with stage of AKI (tau = −0.444, p = 0.012), indicating that higher EPOR expression is linked to milder histological injury. (Figure 4(E)). Furthermore,
ROC curve analysis was performed to evaluate the discriminative capacity of EPOR expression between different AKI severity groups. Area under the curve (AUC) for distinguishing AKI stage 1 from stages 2–3 was 0.786 (95% CI: 0.586–0.985, whereas the AUC for differentiating combined stage 1–2 from stage 3 was 0.850 (95% CI: 0.681–1.00), indicating moderate-to-good diagnostic performance (Figure 4(F)).
EPOR expression elevated in murine AKI model
To support the observations from human biopsies, EPOR upregulation was confirmed in a mouse IRI model. Renal tissue harvested 24 h post-reperfusion exhibited significantly increased EPOR protein levels compared to sham-operated controls (Figure 5). These findings confirm that EPOR is upregulated in response to ischemic stress in vivo.
Figure 5.
EPOR expression was elevated in the murine AKI Model. (A) Representative microphotographs of kidney sections from the experimental mice indicated above were stained by HE. (B) Renal tissue serum creatinine (SCr) and blood urea nitrogen (BUN) were examined from the experimental mice indicated above. Data were presented as Box-and-scatter plots. *p < 0.05, n = 3, Student’s t-test. (C) Western blotting. The renal tissues from sham and IRI were assayed for EPOR and β-actin. (d) Quantification of WB Data was presented as mean ± SD, n = 3. *p < 0.05, Student’s t-test.
Discussion
This study systematically characterized EPOR expression in human AKI renal tissues and examined its association with clinicopathological factors. We demonstrate that EPOR expression is significantly increased in renal tubular epithelial cells in AKI compared with control kidney tissue. Moreover, EPOR expression was inversely associated with AKI stage, acute tubular epithelial necrosis, and a composite acute pathology score, and positively associated with Hb and RBC levels. Together, these findings indicate that EPOR upregulation is a consistent feature of human AKI and is closely related to the severity of tubular injury.
Immunofluorescence staining confirmed the co-localized expression of EPOR and βcR in renal tubular epithelial cells in AKI tissues. Furthermore, Immunohistochemical staining revealed significantly higher EPOR expression in AKI tissues. However, a progressive decline in EPOR levels was noted with advancing histological injury, a trend further supported by EPOR/TUNEL co-staining. The study suggests a significant inverse correlation between EPOR expression and the Stage of AKI, acute tubular epithelial necrosis, the composite Acute Pathology Score, alongside a positive correlation with RBC and Hb. EPOR showed favorable discriminative ability in distinguishing between different stages of AKI.
EPOR, a member of the type I cytokine receptor family, mediates erythropoiesis through homodimeric (EPOR)2 assembly upon EPO binding [8]. In normal adult rats, renal tubular epithelial cells exhibit minimal EPOR expression [9]. βcR serves as the shared signaling subunit for IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors [10–12]. Co-expression of EPOR and βcR has been documented.
In multiple organs, including the kidney, heart, and nervous system [13,14]. Immunocytochemical analyses in spinal cord neurons and EPO-protected cardiomyocytes demonstrate cellular co-localization of βcR and EPOR, which is associated with cytoprotection [7]. In a murine unilateral ureteral obstruction (UUO) model, carbamylated erythropoietin (CEPO), a non-erythropoietic EPO derivative that selectively activated the EPOR/βcR heteroreceptor and suppressed apoptosis [15]. In this study, immunofluorescence analysis of human AKI biopsy specimens revealed co-localized expression of EPOR and βcR. Compared with normal renal tissue, AKI kidneys displayed prominent co-expression of EPOR and βcR on the plasma membrane of tubular epithelial cells.
AKI is classified as prerenal, intrinsic, and postrenal. Prerenal AKI and intrinsic AKI represent the most common causes for AKI in hospitalized patients. Our findings support the hypothesis that EPOR expression is upregulated in Prerenal AKI and intrinsic AKI. Several findings align with our study’s observation of elevated EPOR expression, providing further support for EPOR’s role in AKI pathophysiology. For instance, Yang et al. used Western blot analysis to measure EPOR expression in renal tissues 48 h after IRI in mice, showing significantly elevated EPOR levels [16]. Similarly, Shi et al. reported increased EPOR expression in AKI mice within the first 4 d post-injury, which peaked on day 2 and returned to baseline by day 7 [17]. In contrast, Heitrich et al. reported no significant difference in EPOR expression between septic AKI and normal renal tissues 18 h post-injury [18]. Additionally, studies using lipopolysaccharide-induced AKI models found no change in EPOR expression at 24 h post-injury [19]. The discrepancies in these results may be attributed to differences in timing and severity of modeling. In this study, all biopsy samples were obtained within one week after hospital admission during the acute phase characterized by rising serum creatinine levels. Integrating evidence from animal experiments and human tissue analyses, the early elevation of EPOR represents a marker of tubular stress response in AKI. Taken together, these findings indicate that EPOR regulation in AKI is highly context-dependent, and differences between ischemic/hypoxic and inflammatory injury models should be carefully considered when interpreting EPOR expression patterns across studies.
We observed that in human AKI, the decline in EPOR expression was synchronized with an increase in tubular epithelial cell apoptosis (TUNEL-positive). The concurrent reduction of EPOR expression and increase in tubular epithelial apoptosis suggest an association between EPOR expression and preserved epithelial integrity during AKI progression. Our study also explored correlations between EPOR expression and clinical and histopathological indicators. Current research suggests that EPO or EPO derivatives may reduce serum creatinine and ameliorate tissue injury in AKI [20], consistent with our findings of an inverse correlation between EPOR expression and the Stage of AKI, acute tubular epithelial necrosis, and the composite Acute Pathology Score. This observation can be interpreted in two possible ways. First, EPOR upregulation may reflect an endogenous stress-adaptive response that is associated with preserved epithelial integrity, potentially through activation of pro-survival signaling pathways (e.g., JAK2/STAT5, PI3K/Akt) [21–23]. Second, EPOR expression may be lost in severely injured tubules due to metabolic collapse and structural disruption; thus, reduced EPOR may also represent a consequence of advanced injury. These explanations are not mutually exclusive and should be tested in future mechanistic studies.
In AKI, renal tissue hypoxia is a core driver of pathological changes. The hypoxia-inducible factor (HIF), serving as the central regulator for cellular sensing and adaptation to hypoxia, has been demonstrated to be rapidly upregulated in expression and activity in various AKI animal models [24]. In human kidneys, limited but important evidence indicates that in AKI-related conditions, such as ischemic renal injury and acute rejection of renal allografts, HIF-1ɑ protein accumulates in the nuclei of renal tubular epithelial cells [25]. This suggests that the upregulation of EPOR observed in this study is likely driven by local HIF activation at the injury site, given that EPOR is a classic transcriptional target of HIF. However, systematic investigations of HIF expression in human AKI tissues of specific etiologies (e.g., ANCA-associated glomerulonephritis) during the acute phase remain scarce. Therefore, the precise expression patterns of HIF, its spatial co-localization with EPOR, and its interplay with inflammatory signaling warrant further elucidation in future studies.
Interestingly, EPOR expression in our cohort also exhibited a positive correlation with Hb and RBC counts. Although the EPOR detected in this study was localized to renal tubular cells and is not directly involved in hematopoiesis, this relationship may reflect coordinated systemic responses. The systemic activation of the EPO/EPOR axis could enhance erythroid progenitor sensitivity to circulating EPO, thereby promoting erythropoiesis. Thus, higher renal EPOR expression may serve as a surrogate marker for generalized EPO responsiveness and elevated erythropoietic activity, which in turn manifests as increased RBC and Hb levels.
Although the potential of EPOR in distinguishing AKI severity was observed through ROC curve analysis, another important limitation of this study is the lack of long-term clinical follow-up data. Consequently, we are unable to assess the association between the observed EPOR expression patterns and patients’ key clinical outcomes, such as complete renal recovery, progression to CKD, or the need for long-term dialysis. This prevents a full exploration of EPOR’s potential as a prognostic biomarker. Precisely this point highlights a critical direction for future research: systematically collecting baseline renal tissue specimens in established prospective AKI cohorts and correlating them with long-term follow-up data to determine whether EPOR expression can predict renal repair outcomes.
Several limitations of this study should be acknowledged. First, control tissues were derived from tumor-adjacent kidney samples, which may not fully represent normal renal tissue. Second, the relatively small sample size and observational design limit the ability to draw causal conclusions. Third, functional outcomes, such as long-term renal recovery or progression to CKD, were not assessed. Finally, mechanistic experiments directly addressing the functional role of EPOR in tubular epithelial cells were beyond the scope of this study. Even within the first week after AKI onset, inter-individual differences in the timing of injury evolution, severity, and underlying pathophysiological processes may contribute to variability in EPOR expression levels. Future investigations with larger cohorts, longitudinal follow-up, and experimental modulation of EPOR signaling are warranted to clarify further the biological and clinical significance of EPOR in AKI.
In conclusion, this study demonstrates that EPOR expression is upregulated in renal tubular epithelial cells in human AKI and is closely associated with histopathological injury severity and selected clinical parameters. These findings suggest that EPOR may serve as a marker of tubular stress and injury severity in AKI, providing a foundation for future mechanistic and translational studies.
Supplementary Material
Acknowledgments
The author thanks the patients and doctors who participated in the research. K.X. Conception and design of the study; the acquisition of data and data analysis, interpretation of data; drafting of and substantive revision of the manuscript. C.X. The acquisition of data and data analysis; substantive revision of the manuscript. C.L.M. The acquisition of data, substantive revision of the manuscript. D.L.X. Design of the study, statistical data analysis, and interpretation. G.Z. The acquisition of data and data analysis, in particular regarding histopathological evaluation. M.J.L. The acquisition of data and data analysis, in particular regarding I/R. L.L.F. Conception and design of the study; interpretation of data; substantive revision of the manuscript. L.M.Z. Conception and design of the study; data analysis, interpretation of data; Drafting of and substantive revision of the manuscript. R.W. Conception and design of the study; data analysis, interpretation of data; All authors have approved the submitted version and agreed both to be personally accountable for their and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.
Funding Statement
This work is supported by funds from the Jiangsu Provincial Health Commission (Z2022068) and Northern Jiangsu People’s Hospital (SBQN22006). Shanghai Science and Technology Innovation Action Plan of Scientific Instruments and Chemical Reagents Project (24142201800), and China Scholarship Council (202408310237).
Ethics approval and consent to participate
This study adhered throughout to the principles outlined in the revised Declaration of Helsinki. This internationally recognized statement provides ethical guidance for physicians and researchers conducting human subject research. Written informed consent was obtained from all participants. This ensured their comprehension of the study’s nature and their right to withdraw at any point. Confidentiality of participants’ personal information was guaranteed. Furthermore, participants were explicitly informed about the measures taken to ensure anonymity and confidentiality of their data. For added ethical oversight, approval for the study was granted by the Research Ethics Committees of Jiangsu Subei People’s Hospital (Approval No.2022ky030).
Consent for publication
Not applicable.
Disclosure statement
The authors declare no competing interests.
Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.