1 Introduction
Acute kidney injury (AKI), also known as acute renal failure, refers to a rapid decline or interruption in kidney function [
1]. Renal ischemia-reperfusion injury (IRI) is a major cause of AKI, associated with high morbidity and mortality rates [
2,
3]. Ischemia is characterized by impaired or inhibited tissue perfusion, whereas reperfusion refers to the restoration of blood flow via pharmacological or mechanical interventions. During ischemia, cells are deprived of oxygen and rely on anaerobic glycolysis for energy production, which is particularly insufficient for energy-demanding renal tubules, such as proximal tubules [
4–
7]. Although reperfusion is expected to be beneficial, the sudden influx of oxygen leads to excessive reactive oxygen species (ROS) formation in ischemic tissues, triggering inflammatory cascades and exacerbating tissue damage due to inadequate antioxidant defenses [
8,
9]. The pathophysiology of IRI involves a complex interplay of oxidative stress, inflammation, and apoptosis, resulting in profound structural and functional renal damage.
The Wnt/β-catenin signaling pathway, an evolutionarily conserved regulator of cellular processes, plays a critical role in kidney development and injury [
10]. Dysregulation of this pathway has been reported to result in the progression of acute injury to chronic kidney injury, primarily through mechanisms involving oxidative stress, inflammation, apoptosis, and fibrosis [
11–
15]. Upregulation of the Wnt/β-catenin pathway has been shown to exacerbate renal injury, highlighting its importance in renal pathophysiology [
16–
18].
Spexin (SPX), a recently discovered neuropeptide, has attracted considerable attention for its diverse biological functions, including appetite regulation, maintenance of glucose homeostasis, and remarkable antioxidant and anti-inflammatory effects [
19–
21]. However, its dual role—whether protective or deleterious—in IRI has not been fully elucidated. While SPX’s anti-inflammatory and metabolic regulatory effects have been documented, its potential to exacerbate inflammation and tissue damage in certain contexts has largely been overlooked.
Several neuropeptides, as key modulators of physiologic and pathological processes, have been implicated in the exacerbation of ischemic kidney injury. For instance, neuropeptide Y (NPY) has been shown to worsen renal injury during ischemic events by enhancing vasoconstriction and inflammation [
22]. Patients with chronic kidney disease (CKD) exhibit increased circulating NPY levels, which correlate with proteinuria and reduced glomerular filtration rate (GFR). NPY, as a potent vasoconstrictor and sympathetic activator, contributes to the progression of hypertension and renal dysfunction [
23,
24]. Given the controversial effects of vasoconstrictive neuropeptides on renal function, the role of SPX in this context remains unclear.
This study aims to investigate the effects of exogenous SPX administration on IRI, with a particular focus on its interaction with the Wnt/β-catenin signaling pathway. Limited studies have explored SPX’s role in renal injury, and its effects on inflammatory and apoptotic mechanisms remain poorly understood. This study demonstrated that SPX not only aggravates renal injury induced by IRI but also induces marked toxic effects even in healthy kidneys by promoting inflammation, apoptosis, and fibrosis via the Wnt/β-catenin pathway. These results suggest that SPX may have intrinsic pro-inflammatory, pro-apoptotic and fibrotic properties, raising concerns about its therapeutic potential. This study provides the first detailed insight into the adverse effects of SPX on kidney injury, emphasizing the need for further research to clarify its role in renal pathophysiology.
2 Materials and methods
2.1 Animals and renal ischemia‒reperfusion injury model
Twenty-eight male BALB/c mice were obtained from the Multidisciplinary Animal Laboratory of Dokuz Eylül University. The animals were allowed a standard diet and housed at a controlled room temperature (22–25 °C) under a 12:12 h day/night cycle for the duration of the study. A bilateral renal ischemia/reperfusion model was established in male BALB/c mice [
25]. Anesthesia was administered via intraperitoneal injection of 0.02 mL ketamine (100 mg/mL) and 0.02 mL 2% xylazine. A ventral approach was used to expose the left and right kidneys. Atraumatic vascular clamps were applied to both renal arteries for 30 min. Following ischemia, the vascular clamps were removed, and the incisions were closed with surgical sutures. Reperfusion was allowed for 6 h. The mice were euthanized by cervical dislocation 6 h after the reperfusion period.
2.2 Experimental design
A total of 28 male BALB/c mice (8–10 weeks and 30–40 g) were randomly divided into four groups as follows: the control group (
n = 7), the SPX group (SPX,
n = 7), the renal ischemia/reperfusion injury group (IRI,
n = 7), and the SPX + renal ischemia/reperfusion injury group (SPX + IRI,
n = 7). The mice in the control group were not subjected to any experimental interventions. The mice in the SPX and SPX + IRI groups received a subcutaneous injection of 50 µg/kg SPX (MedChem Express, HY-P1723) once a day for 4 weeks [
26–
28]. SPX administration in the SPX + IRI group was initiated 4 weeks prior to the induction of IRI to allow sufficient time for its potential systemic and renal effects to manifest. This preconditioning approach was designed to evaluate whether chronic SPX exposure could modulate baseline inflammatory, oxidative, and apoptotic pathways, thereby influencing the tissue’s response to subsequent ischemic stress. The study aimed to simulate a potential therapeutic scenario where SPX is used preventively rather than acutely, aligning with its hypothesized roles in regulating metabolic, anti-inflammatory, and antioxidative mechanisms. For the SPX group, SPX was administered to healthy animals to establish its baseline physiologic and molecular effects in the absence of any pathological conditions. The IRI group was maintained on a standard diet and water for 4 weeks before undergoing bilateral renal ischemia/reperfusion injury. The experimental design is summarized in Fig. 1.
2.3 Histological analysis
The left kidney tissues were fixed in a 10% neutral buffered formalin solution for 48 h. At the end of the fixation period, the tissues were dehydrated through a graded series of ethanol, clarified with xylene, and then embedded in paraffin. Sections of 5 µm thickness were stained with Mayer’s hematoxylin and eosin and Masson’s trichrome staining. Tissue damage was evaluated by the percentage of damaged tubules defined as tubular cell necrosis, tubular dilatation/atrophy, tubular cell flattening, loss of the proximal tubular brush border and intratubular cell debris. The development of fibrosis was evaluated via Masson’s trichrome staining. Tubular injury and fibrosis were evaluated using a semiquantitative scoring system by two blinded observers and scored as follows: 0 = absent (< 10%), 1 = minimal (10%–25%), 2 = mild (25%–50%), 3 = moderate (50%–75%), and 4 = severe (> 75%). Randomly selected 10 fields per mouse kidney at 20× magnification were scored and photographed under a light microscope (Carl Zeiss, Axiolab 5, Suzhou, China).
2.4 Measurement of renal biomarkers
Blood samples were centrifuged at 3000 rpm for 15 min. After centrifugation, the supernatants of all the blood samples were collected and stored at −80 °C until use. Blood urea nitrogen (BUN) and serum creatinine (SCr) levels were measured via an automated biochemical analyzer (Olympus Corporation, AU400, Japan).
2.5 Tissue collection and enzyme-linked immunosorbent assays (ELISAs)
The right kidney tissues were homogenized on ice in phosphate-buffered saline (PBS) via a glass homogenizer. The homogenates were subsequently centrifuged at 5000 g for 5 min to obtain the supernatant. The protein levels of kidney injury marker-1 (KIM-1, E0617Mo), neutrophil gelatinase-associated lipocalin (NGAL, EA0042Mo), Wnt family member-7b (Wnt-7b, E2405Mo), Wnt family member-4 (Wnt-4, E1495Mo), β-catenin (E0198Mo), p53 (E0409Mo), Bcl-2-associated X protein (Bax, E1612Mo), B cell lymphoma 2 (Bcl-2, E0476Mo), macrophage inflammatory protein 2 (MIP-2, E0647Mo), monocyte chemotactic protein 1 (MCP-1, E0566Mo), tumor necrosis factor-alpha (TNF-α, E0117Mo), interleukin-18 (IL-18, E0044Mo), interferon gamma (INF-γ, E0056Mo), inducible nitric oxide synthase (iNOS, E0386Mo), fibronectin (E0077Mo), and α-smooth muscle actin (α-SMA, E2769Mo) were quantified via ELISA kits (Bioassay Technology Laboratory, Zhejiang, China). Briefly, 50 μL of the supernatant was added to microplate wells precoated with specific antibodies for each analyte. Following an incubation period and subsequent washing steps, biotin-conjugated secondary antibodies specific to each analyte were added. Following further incubation and washing, streptavidin-horseradish peroxidase (HRP) was added to the wells to form an immune complex. After additional incubation and washing to remove unbound enzymes, a chromogenic HRP substrate solution was applied, resulting in a color change to blue. The reaction was stopped with a stop solution, and the absorbance was measured at 450 nm via a plate reader (Allsheng, AMR-100, China).
2.6 Reverse transcription‒quantitative polymerase chain reaction (RT‒qPCR)
Total RNA was isolated from right kidney tissue sample via Hybrid-RTM (305-101, GeneAll, Korea) according to the manufacturer’s instructions. The concentration of the extracted RNA was determined via spectrophotometry. The RNA (500 ng) from each sample was reverse transcribed to cDNA via a cDNA synthesis kit (C03-01-05, A.B.T.™, Turkey). The cDNA was amplified via an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA) following the manufacturer’s protocol. For real-time PCR, cDNA was amplified via SYBR Green (Q03-01-05, A.B.T. SYBR Green Mastermix, Turkey) with the following parameters: denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 60 s, and elongation at 95 °C for 45 s. The 20 μL reaction mixture contained 10 μL of 2× MasterMix with SYBR Green, 4 μL of cDNA, 4 μL of H2O, and 1 μL of each forward or reverse primer (10 μmol/L concentration). Each sample was run in triplicate to ensure statistical accuracy. GAPDH was used as a housekeeping gene for normalization. The fold change values were calculated via the 2−ΔΔCt relative expression formula. The selected primers are shown in Table 1.
2.7 Statistical analysis
Normality and homogeneity of variances were assessed using the Shapiro–Wilk test and Levene’s test, respectively. Data with a normal distribution (Shapiro–Wilk test, P ≥ 0.05) were analyzed using parametric one-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons. Non-normally distributed data (Shapiro–Wilk test, P < 0.05) were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. P < 0.05 was considered statistically significant. All results are presented as the mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism (version 10.4.2; GraphPad Software, San Diego, CA, USA).
3 Results
3.1 SPX impairs renal function in both IRI-induced injured and healthy kidneys
To evaluate the effects of SPX on renal function impaired by IRI, we measured renal function biomarkers 6 h after IRI in mice. Both BUN and SCr levels were significantly elevated in the IRI group compared to the control group (Fig. 2A and 2B). Although BUN levels did not show significant changes in the SPX group compared to control mice, SCr levels were significantly higher in the SPX and SPX + IRI groups compared to the control and IRI groups. NGAL levels were significantly elevated in the IRI and SPX + IRI groups compared to the controls; however, there was no significant difference between the IRI and SPX + IRI groups (Fig. 2C). KIM-1 levels were significantly higher in the SPX, IRI, and SPX + IRI groups than those in the control group, with no significant difference was found between the IRI and SPX + IRI groups (Fig. 2D). These findings suggest that spexin impairs renal function by increasing SCr and KIM-1 levels in healthy kidneys and further aggravates IRI-induced kidney injury by enhancing SCr levels.
3.2 SPX induces pathological alterations in healthy kidneys
Kidney sections from the control group exhibited normal histological architecture. In contrast, moderate to severe histopathological changes, including sclerotic glomeruli, tubular cell necrosis, tubular dilation/atrophy, tubular cell flattening, intratubular cell debris, and tubular casts, were more prominent in the SPX, IRI, and SPX + IRI groups (Fig. 3A). Intratubular and interstitial inflammatory infiltration, along with vascular congestion, were widely observed in both the cortical and medullary regions of the kidneys in the IRI and SPX + IRI groups. The mean tubular injury score (TIS) was significantly higher in the SPX, IRI, and SPX + IRI groups than that in the control group (Fig. 3B). Additionally, the TIS in the SPX + IRI groups was higher than that in the IRI group, but it was not statistically significant. The results indicate that SPX exerts detrimental effects on renal tissue even under normal physiologic conditions.
3.3 SPX activates Wnt/β-catenin signaling independently of renal IRI
We investigated the effects of SPX on the Wnt/β-catenin signaling pathway. Wnt-4 mRNA and protein expression levels were markedly upregulated in the SPX, IRI, and SPX + IRI groups compared to the control group. However, no statistically significant difference was observed between the IRI and SPX + IRI groups (Fig. 4A and 4B). In contrast, Wnt-7b mRNA and protein expression levels were significantly elevated in the SPX and SPX + IRI groups compared to the control and IRI groups (Fig. 4C and 4D). Similarly, β-catenin mRNA and protein expression levels were markedly higher in the SPX and SPX + IRI groups than those of the control and IRI groups (Fig. 4E and 4F). According to these results, Wnt-4 expression is primarily upregulated in response to IRI, SPX independently induces the upregulation of Wnt-4, Wnt-7b, and β-catenin, irrespective of IRI. The present findings indicated that SPX induces the activation of Wnt-4, Wnt-7b, and β-catenin under physiologic conditions, as well as in response to renal IRI.
3.4 SPX promotes inflammation in both IRI-induced injured and healthy kidneys
The levels of INF-γ, iNOS, MIP-2, and TNF-α were significantly increased in the IRI group compared to the control group, whereas IL-18 and MCP-1 levels did not differ significantly (Fig. 5). Administration of SPX alone resulted in a significant elevation of INF-γ, iNOS, MCP-1, MIP-2, TNF-α, and IL-18 levels when compared to the control group. Moreover, the levels of all inflammatory markers, except TNF-α, were significantly higher in both the SPX and SPX + IRI groups compared to the IRI group. These results suggest that SPX elicits a pronounced pro-inflammatory response by upregulating multiple inflammatory cytokines and chemokines under both ischemic and physiologic conditions.
3.5 SPX induces pro-apoptotic and anti-apoptotic responses in both IRI-induced injured and healthy kidneys
The mRNA and protein expression levels of p53 were significantly increased in the SPX, IRI, and SPX + IRI groups compared to the control group. However, no significant differences were observed between the IRI and SPX + IRI groups (Fig. 6A and 6B). Similarly, Bax mRNA and protein expression levels were significantly elevated in the SPX, IRI, and SPX + IRI groups compared to the controls, with no significant differences between the IRI and SPX + IRI groups (Fig. 6C and 6D). As for the anti-apoptotic Bcl-2, its mRNA expression levels were significantly higher in the SPX, IRI, and SPX + IRI groups compared to the control group. Notably, Bcl-2 mRNA levels in the IRI group were found to be significantly lower than in the SPX and SPX + IRI groups (Fig. 6E). Despite the upregulation of Bcl-2 mRNA, its corresponding protein levels remained statistically unchanged among the groups (Fig. 6F). These findings indicate that SPX promotes apoptotic signaling while simultaneously activating compensatory anti-apoptotic mechanisms in renal tissue, both in IRI-induced injured and healthy kidneys.
3.6 SPX induces renal fibrosis independently of renal IRI
The kidneys in the control group exhibited a normal histological appearance, as confirmed by Masson’s trichrome staining (Fig. 7A). In contrast, mild to severe fibrosis was detected in the SPX and SPX + IRI groups compared to the control group. Although the mean fibrosis score was higher in the SPX and SPX + IRI groups, it was not different from the IRI group (Fig. 7B). Fibronectin levels were markedly elevated in the SPX and SPX + IRI groups compared to the control group (Fig. 7C). Fibronectin levels remained comparable among the SPX, IRI, and SPX + IRI groups. α-SMA levels were significantly higher in the SPX, IRI, and SPX + IRI groups than that in the control group. Notably, α-SMA levels in SPX and SPX + IRI groups were significantly higher than that in the IRI group (Fig. 7D). These results show that SPX promotes renal fibrosis, thereby contributing to renal injury, and underscore its pro-fibrotic effects in both IRI-induced injured and healthy kidneys.
4 Discussion
IRI is a major contributor to AKI in various clinical contexts, including trauma, multiple organ failure, renal transplantation, partial nephrectomy, sepsis, and urological procedures [
29]. IRI poses a significant public health burden, reducing patients’ quality of life and straining healthcare systems. Early intervention to prevent IRI is essential for minimizing long-term renal damage, prompting ongoing research into renoprotective strategies targeting the acute phase of injury [
4,
5,
7].
Neuropeptides have emerged as candidate modulators of AKI due to their diverse roles in modulating inflammation, oxidative stress, and cellular repair. These small signaling molecules interact with specific receptors to regulate kidney damage and recovery. Some neuropeptides exhibit anti-inflammatory and anti-apoptotic effects, mitigate tubular damage, and preserve renal function in experimental AKI models [
30,
31]. Conversely, neuropeptide Y exacerbates renal injury during ischemia by increasing vasoconstriction and inflammation [
22]. Patients with CKD exhibit increased circulating neuropeptide Y levels, correlating with worsening renal function, proteinuria, and reduced glomerular filtration rate [
19,
20]. SPX, a 14-amino acid endogenous peptide, is widely distributed in peripheral and central tissues [
32,
33]. It influences food intake, weight regulation, and metabolism [
15–
17], but its role in renal pathology physiologic or pathological roles remain to be fully elucidated [
32,
33]. To our knowledge, this is the first study to comprehensively investigate the renal effects of chronic SPX administration in a murine model of IRI.
AKI biomarkers, consisting of small molecules released into the circulation and urine as a result of tubular damage or reduced glomerular filtration rate, serve as critical indicators to clinically identifying renal injury [
34]. We comprehensively investigated the effect of SPX on renal dysfunction induced by IRI. Our results reveal that SPX markedly elevates BUN and SCr levels in healthy mice, implicating a possible intrinsic nephrotoxic effect of SPX on renal function independent of IRI. Moreover, SPX further elevated SCr levels in the IRI group, supporting its role in exacerbating renal injury. Interestingly, the effects of SPX in CKD models have been inconsistent. While SPX normalized BUN and SCr levels in obese rats [
27], it failed to produce a restorative effect in adenine-induced CKD [
32]. These inconsistencies suggest that there may be an interaction between SPX activity and the metabolic or inflammatory environment in which it is administered.
KIM-1 and NGAL are widely recognized early biomarkers of AKI [
34–
36]. NGAL, also known as lipocalin-2, is a glycoprotein and a highly sensitive biomarker of ischemia-induced injury to both proximal and distal tubules [
35]. KIM-1 is a type I transmembrane glycoprotein, its expression is typically low in healthy renal tissue but significantly upregulated in differentiated proximal tubular cells following ischemic injury [
36]. Previous data suggested that SPX reduced KIM-1 levels without affecting NGAL in chronic renal failure models [
32]. However, in our study, moderate to severe tubular damage was observed in the SPX, IRI, and SPX + IRI groups, accompanied by increased KIM-1 expression. Although NGAL levels were elevated in both the IRI and SPX + IRI groups, there was no significant difference between the groups. This suggests that SPX does not further exacerbate NGAL-associated tubular injury in the presence of IRI. The divergence in KIM-1 and NGAL responses implies that SPX may selectively activate specific injury pathways or cellular stress responses predominantly reflected by KIM-1 expression. These findings suggest that SPX may compromise renal epithelial integrity through mechanisms involving early cellular stress, inflammatory signaling or epithelial dysfunction, independent of renal IRI. Furthermore, the contrasting results in AKI and CKD models highlight the importance of the underlying pathophysiology in modulating the renal effects of SPX. Given that AKI involves rapid cellular injury and immediate dysfunction, whereas CKD progresses gradually with compensatory adaptations, the detrimental influence of SPX appears to be more pronounced in the acute setting, possibly mediated by combined cellular stress, hemodynamic alterations, and different injury mechanisms.
The inflammatory response is defined as a key early response to renal injury. It has been determined that renal tubular epithelial cells and infiltrating macrophages release pro-inflammatory cytokines and recruit additional inflammatory cells to the site of injury [
37]. While inflammation is necessary to promote tissue repair within physiologic limits, excessive or prolonged inflammation can lead to fibrosis and the development of CKD [
38]. In the present study, we observed extensive interstitial and peritubular infiltration, particularly in the SPX, IRI, and SPX + IRI groups. Notably, levels of INF-γ, IL-18, iNOS, MCP-1, and MIP-2 were significantly elevated in SPX-treated mice compared to both IRI and control groups. SPX not only exacerbated inflammation induced by renal IRI, but also initiated significant inflammatory activation in healthy kidneys. A previous study showed that administration of 300 µg of SPX to rats on a high-fat diet from week 17 to 20 significantly reduced interstitial inflammation and exerted anti-inflammatory effects by downregulating NF-κB activity and associated inflammatory markers [
27]. In contrast, another study using 35 µg/kg SPX for 4 weeks in adenine-induced chronic kidney injury rats failed to normalize inflammatory markers, suggesting no significant anti-inflammatory effect [
32]. However, in contrast to these findings, our study suggests that daily administration of SPX at a dose of 50 µg/kg for 4 weeks induces renal tubular inflammation and elevates multiple inflammatory markers in the kidneys of mice, thus exerting a nephrotoxic effect. The observed discrepancies between our results and previous studies could be attributed to differences in disease models, metabolic background, target tissues, or baseline inflammatory states. Moreover, despite comparable doses and treatment durations, the differential interactions between SPX administration and acute versus chronic pathophysiological processes may further explain the renal damage observed in our experimental model. The divergent effects of SPX observed between obese and healthy animals may be explained by differences in their underlying immunometabolic environments.
The Wnt/β-catenin signaling pathway, a key regulator of kidney development and repair, is reactivated in response to renal injury [
10]. This epithelial pathway modulates essential cellular processes, including proliferation, adhesion, differentiation, and survival particularly through modulation of apoptosis and regeneration [
10,
14,
18,
39]. While the Wnt/β-catenin signaling pathway plays a protective role in the early stages of acute kidney injury, its sustained activation contributes to chronic fibrotic progression [
11,
12,
40]. Wnt-4, which is upregulated during embryonic tubular development, shows low expression in normal healthy tubules but is rapidly re-expressed in damaged proximal tubules, and induces apoptosis during the early stages of renal IRI and cisplatin induced AKI [
40,
41]. Wnt-7b is secreted by macrophages accumulating damaged renal parenchyma during IRI and is involved in kidney repair by regenerating epithelial cells [
42].
In vitro, β-catenin signaling reduces Bax-mediated apoptosis and induces cell survival in proximal tubular cells [
40,
43]. Silencing or overexpressing the Wnt/β-catenin pathway has been demonstrated to prevent the activation of repair mechanisms in the kidney, induce excessive apoptosis by modulating the expression levels of pro-apoptotic and anti-apoptotic proteins in human and animal kidneys, and exacerbate renal injury. The process of abnormal apoptosis has been shown to result in tubular atrophy, fibrosis, and, consequently, loss of renal function [
43,
44]. It is also known that apoptosis-related genes are expressed in the early stages of ischemia and exacerbate renal damage induced by IRI [
45–
47]. These findings collectively suggest that precise modulation of Wnt/β-catenin signaling, rather than its full inhibition or activation, is essential for balanced renal repair.
The present study demonstrates that SPX has significant effects on renal tissue through modulation of the Wnt/β-catenin signaling pathway, with consequent effects on both apoptotic and fibrotic processes under physiologic and IRI conditions. The marked upregulation of Wnt-4 in response to IRI, together with the independent elevation of Wnt-4, Wnt-7b, and β-catenin following SPX administration, suggests potent IRI-independent activation of this pathway. This molecular activation was accompanied by increased expression of the pro-apoptotic markers p53 and Bax in SPX, IRI, and SPX + IRI groups, suggesting that both IRI and SPX induce intrinsic apoptotic signaling in renal tissue. Despite the increase in anti-apoptotic Bcl-2 mRNA expression, its protein levels remained unchanged. The elevation of Bcl-2 mRNA, particularly in the SPX, IRI, and SPX + IRI groups, suggests a compensatory cellular response aimed at counteracting pro-apoptotic signaling. Notably, mRNA levels of Bcl-2 were significantly higher in the SPX and SPX + IRI groups compared to the IRI groups. However, the lack of a corresponding increase in Bcl-2 protein levels may indicate post-transcriptional regulatory mechanisms or rapid degradation under stress conditions. It may reflect the complex dynamics of apoptotic regulation in acute kidney stress. Taken together, these findings suggest that while SPX appears to activate apoptotic pathways similar to IRI, it also modulates anti-apoptotic gene expression at the transcriptional level. The overall biological effect likely reflects a balance between these opposing signals. These findings suggest that SPX may exert context-dependent effects in renal pathology, acting as both a stressor and an adaptive modulator depending on the underlying physiologic state.
Prolonged or aberrant activation of the Wnt/β-catenin pathway contributes to tubulointerstitial fibrosis by promoting epithelial-to-mesenchymal transition (EMT), activating interstitial fibroblasts, and enhancing extracellular matrix (ECM) deposition [
11,
40]. Chronic Wnt/β-catenin signaling also downregulates epithelial markers while upregulating profibrotic genes such as fibronectin, vimentin, and α-SMA [
12]. These molecular alterations impair tubular architecture, leading to irreversible scarring, nephron loss, and progressive renal dysfunction. Although transient Wnt/β-catenin activation may aid in acute repair, its sustained stimulation drives fibrosis and contributes to CKD pathogenesis [
11,
12].
Renal fibrosis is typically a late-stage consequence of ischemic injury [
29]. However, previous studies have reported that early fibrotic markers such as α-SMA and fibronectin can be upregulated within a short timeframe following renal injury [
48]. In our study, mild histological fibrosis and a significant early induction of fibrogenic markers were observed as early as 6 h post-reperfusion in SPX-treated groups. This is particularly noteworthy given that SPX was administered chronically for four weeks prior to IRI induction, likely priming the renal tissue toward a profibrotic state.
Consistent with these findings, SPX-induced activation of the Wnt/β-catenin pathway was associated with increased α-SMA and fibronectin expression, and histologically evident fibrosis, even in the absence of IRI. Notably, α-SMA levels were significantly higher in SPX-treated kidneys than in the IRI group, underscoring the fibrogenic potential of SPX independent of ischemic injury. These results suggest that SPX may exacerbate renal injury by promoting fibrotic remodeling through Wnt/β-catenin pathway activation.
These findings raise important questions regarding the specificity and sufficiency of Wnt/β-catenin signaling in mediating SPX-induced renal injury. Although our findings demonstrate a strong association, direct causality remains to be established. Given the observed upregulation of pro-apoptotic markers (e.g., p53, Bax) alongside compensatory Bcl-2 expression, it is plausible that SPX-induced renal injury arises from a multifactorial process involving Wnt/β-catenin signaling, intrinsic apoptotic pathways, inflammatory mediators such as NF-κB, and potentially oxidative stress. Future studies employing pharmacological inhibition or genetic silence of Wnt/β-catenin components will be necessary to delineate its specific contribution relative to other interacting mechanisms.
Several limitations should be acknowledged. The complex nature of IRI and its interactions with inflammatory and apoptotic pathways posed methodological challenges that became more apparent as the study progressed. Although we carefully designed our experiments to assess the effects of chronic SPX exposure on renal function, inflammation and apoptosis, certain limitations were not fully anticipated at the outset—an inherent challenge in experimental research where unforeseen biological and technical variables often emerge during data analysis. A major limitation of our study is the single time point at which the experiments were conducted (6 h post-IRI), which limits our ability to assess the long-term effects of SPX on renal injury and recovery induced by renal IRI. Neuropeptides, including SPX, are known to exhibit time-dependent effects, with chronic exposure potentially leading to receptor desensitization, downregulation, and the activation of alternative intracellular signaling pathways. Given that SPX is a candidate therapeutic agent for chronic metabolic diseases, understanding its prolonged effects is clinically relevant. While it is acknowledged that acute SPX exposure might elicit distinct physiologic responses, the present study specifically aimed to simulate the sustained exposure scenario, which is highly relevant for potential chronic therapeutic applications.
Another limitation is the exclusive use of a mouse model, which raises questions about the translatability of our findings to human renal physiology. Although rodent models provide valuable mechanistic insights, differences in renal structure and function between species require further validation in human kidney cells or clinical samples. Additional studies investigating the role of SPX in human renal disease will be crucial to determine its potential clinical relevance.
Finally, although we observed marked activation of inflammatory and Wnt/β-catenin signaling, we did not directly assess the molecular intermediates or upstream receptors involved. In addition, central inflammatory regulators such as IL-6 and NF-κB were not quantified. These omissions are acknowledged as limitations and will inform the design of future experiments. Follow-up studies incorporating dose–response, gene silencing, pharmacological inhibition, or receptor blockade strategies will be instrumental in establishing mechanistic causality. Given the novelty of SPX in renal pathophysiology and the limited available literature, direct comparisons remain challenging. Nonetheless, our findings provide a valuable foundation for future research exploring the mechanistic link between SPX-mediated Wnt/β-catenin activation and inflammation.
Despite these limitations, this study is the first to demonstrate that chronic administration of SPX promotes inflammation, apoptosis, and fibrotic remodeling in the kidney, even in the absence of ischemic injury. These findings highlight the context-dependent and potentially nephrotoxic effects of SPX, underscoring the need for caution when considering its use in therapeutic settings involving renal vulnerability. Future studies are essential to determine the long-term renal safety profile of SPX.
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