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. 2025 Aug 21;8:100162. doi: 10.1016/j.crphys.2025.100162

Sex and dose differences in morphine administration on renal function, inflammatory and apoptotic markers in male and female Wistar rats

Olaoluwa Sesan Olukiran a,⁎, Oluwadare Joshua Ogundipe b, Stephen Taiye Adelodun c, Moses Agbomhere Hamed d, Olayemi Sola Babatunde e, Oluwole Ojo Alese f, Rufus Ojo Akomolafe a
PMCID: PMC12418877  PMID: 40933309

Abstract

This study examined the impact of morphine on renal function, antioxidant enzymes, and inflammatory and apoptotic markers in male and female Wistar rats, considering both sex- and dose-dependent effects. 40 Wistar rats (20 male, 20 female), each weighing 120–150 g were used in this study. The control group received distilled water (0.5 mL/100 g b.w), while experimental groups were given morphine orally at 20, 40 and 60 mg/kg daily for 30 days. Renal function, inflammatory, and apoptotic markers were assessed in the plasma and tissue homogenate. Kidneys were preserved in 10 % formo-saline for histological examination. Morphine significantly increased plasma creatinine in both male and female rats, with the increase being more pronounced in males. Caspase-3 and TNF-α were also elevated in both sexes, but with no significant difference between males and females. Male rats showed significantly higher catalase activity and elevated plasma sodium, potassium, phosphate, and chloride ion concentrations compared to females. Photomicrographs revealed that low and medium doses of morphine caused more severe kidney damage in both male and female rats, leading to atrophied glomeruli, widened Bowman's space, and loss of brush border in the tubules. Conversely, high-dose resulted in less pronounced damage, with only a few atrophied glomeruli and indistinct tubules. Morphine induced more pronounced lipid peroxidation and oxidative stress in female rats compared to males, as indicated by changes in their plasma electrolytes and antioxidant enzyme activities. Interestingly, lower dose caused more significant alterations in renal function, oxidative stress and apoptotic markers compared to medium and high doses.

Keywords: Morphine, Renal function, Sex-differences, Dose-dependent, Rats

Highlights

  • •

    Female rats showed greater changes in renal function than males.

  • •

    Females had better glomerular protection than males.

  • •

    Morphine's impact on renal function isn't dose dependent.

1. Introduction

Drugs, whether natural or synthetic, are used for medical purposes. However, using some of these drugs repeatedly can lead to transient or chronic dependency (Murthy et al., 2010). Globally, drug abuse has emerged as a major social problem. It is incredibly common, with an estimated 46 % of the general population reportedly exposed to drugs at some point in their lives. The National Drug Law Enforcement Agency (NDLEA) in Nigeria has reported that approximately 14.3 million Nigerians are involved in drug abuse. This figure, encompassing individuals between the ages of 15 and 64, also indicates a concerning trend of increasing involvement among women. Many abused substances, or their byproducts, are eliminated through the kidneys, making renal complications of drug abuse quite common. These complications can manifest as various glomerular, interstitial, and vascular diseases. Kidney damage from drug abuse can be acute and reversible, or it can progress to a chronic condition, potentially resulting in end-stage renal failure. This kidney involvement is typically due to either the elimination of the drugs through the kidneys or a direct nephrotoxic effect of the substances themselves (Kapusta, 1995). The repeated administration of opioid drugs may lead to the development of analgesic tolerance, adversely affecting renal functions. Opioids produce physiological changes in the kidney (Gupta and Weber, 2006; Weber et al., 2008), and endorphins along with other opioid peptides participate in the development of uremic syndrome (Trelewicz et al., 1993). Long-term opioid use is associated with undesirable consequences, including impaired renal function (Jalili et al., 2017). Studies have also suggested that chronic administration of clinically relevant doses of opioids causes structural abnormalities and renal dysfunction in a murine model of cancer (Arerangaiah, 2007).

Morphine, the primary psychoactive compound in opium, is a well-studied opioid analgesic drug (Salahshoor et al., 2016). It undergoes metabolism in the liver, forming morphine-3-glucuronide (M3G) (55 %), morphine-6-glucuronide (M6G) (10 %), and normorphine (4 %). In individuals with normal kidney function, these metabolites, along with about 10 % of the original morphine, are excreted by the kidneys. However, when the kidneys fail, morphine and its metabolites build up in the plasma, serum, brain, and cerebrospinal fluid, leading to myoclonic spasms and respiratory depression (Sjøgren et al., 1993; Dean, 2004). Morphine is addictive and can cause physiological dependence (Jalili et al., 2016a). Morphine addiction can cause progressive chronic renal failure (Perneger et al., 2001) and tubular epithelial cell degeneration (Sumathi et al., 2009). Addiction to opioid drugs, including morphine, constitutes a universal social and public health concern. Morphine, a powerful analgesic, is absorbed and metabolized within the hepatic and digestive systems, with subsequent excretion through the renal system (Lan et al., 2013). Morphine damages kidney cellular DNA by prompting macrophages and mesonephric cells to produce reactive oxygen species (ROS) and superoxide. It further increases free radical production through lipid peroxidation, which in turn blocks antioxidant enzymes and generates more free radicals or ROS. An overdose of morphine increases oxidative stress in renal epithelial cells, which leads to kidney injury (Senturk et al., 2009). The kidney plays a crucial role in maintaining a stable extracellular environment, which is essential for cells to function correctly. It achieves this by excreting metabolic waste products such as urea, creatinine, and uric acid. Additionally, the kidney precisely regulates the excretion of water and electrolytes to match the body's intake and endogenous production. A major disruption of kidney cellular activities can, in turn, impair the function of other organs. While a growing body of research explores the effects of opioids on kidney function, studies specifically investigating sex differences in morphine administration in rats remain scarce. This study investigates the impact of varying morphine doses on the kidneys of male and female Wistar rats, with a particular focus on sex-based differences.

2. Materials and methods

2.1. Chemicals

Morphine hydrochloride was sourced from the Pharmacy Unit of the Obafemi Awolowo University Teaching Hospital Complex in Ile-Ife, Nigeria. We obtained biochemical assay kits from Randox Laboratories (Crumlin, Antrim, UK). ELISA kits were purchased from Elabscience Biotechnology Inc., USA. For plasma electrolyte assays, kits were sourced from Teco Diagnostic Laboratories (Anaheim, California, USA).

2.2. Animal management

This study utilized 40 Wistar rats (20 males and 20 females), each weighing between 120 and 150 g. All animals were acquired from the Animal Holdings of the College of Health Sciences, Obafemi Awolowo University (OAU), Ile-Ife, Osun State, Nigeria, where the research was carried out. The rats were maintained in accordance with the Animal Welfare Act and the Institutional Animal Ethical Committee's Guidelines for animal experimentation. Before the experiment began, the rats were acclimatized for two weeks under a natural light-dark cycle. They had free access to standard rodent pellets and water.

2.3. Experimental design

The rats were housed in four labeled plastic cages (A-D). Group A, serving as the control, received 0.5 mL/100 g of distilled water orally. Rats in Group B received 20 mg/kg/day of morphine, while Group C received 40 mg/kg/day, both administered orally. Lastly, rats in Group D administered 60 mg/kg/day of morphine orally. Each of these groups included both male and female rats. Morphine was administered for 30 days. After this period, blood samples were collected via cardiac puncture into lithium heparin bottles. These samples were then centrifuged at 3000 rpm for 10 min in a Cold Centrifuge (Centurium Scientific Model, 8881) to isolate the plasma. This plasma was subsequently used to assess renal function markers. Afterward, the rats' kidneys were carefully excised and weighed. The right kidney from each rat was homogenized with 10 mL of 0.25 M sucrose solution using an Electric Homogenizer (SI601001). The resulting homogenate was then centrifuged at 3000 rpm for 20 min, and the supernatant was collected for the analysis of antioxidant enzymes, inflammatory, and apoptotic markers. The left kidneys were preserved in 10 % formo-saline for subsequent photomicroscopic assessment.

2.4. Determination of plasma creatinine, Urea and Kidney Injury Molecule-1 (KIM-1)

The plasma creatinine and urea concentrations were determined by a standard colorimetric method using assay kits purchased from Randox Laboratories (Crumlin, Antrim, UK). Creatinine was assessed by following the method of Bartels and Bohmer (1972). To 10 μl of plasma, 1.0 ml of the working reagent (an equal-volume mix of picric acid and sodium hydroxide) was added. This mixture was then allowed to stand for 30 secs. Subsequently, the absorbance of both the sample and standard solution was measured spectrophotometrically at 492 nm against a reagent blank. Urea was assayed using the method of Fawcett and Scott (1960). To 10 μl of plasma, 100 μl of Reagent I (containing sodium nitroprusside and urease) was added. This mixture was then incubated at 37 ∘C for 10 min. Following incubation, 2.5 ml each of Reagent II (phenol) and Reagent III (sodium hypochlorite) were added to the mixture. This was thoroughly mixed and further incubated at 37∘C for another 15 min. Finally, the absorbance of the sample and standard was measured at 500 nm against a blank containing 10 μl of distilled water.

KIM-1 was determined using a KIM-1 sandwich enzyme-linked immunosorbent assay (ELISA) kit. First, 10 μL of the sample was combined with 40 μL of sample diluent in the sample well and gently mixed. In parallel, 50 μL of standard was added to the standard well. Next, 100 μL of Horseradish Peroxidase (HRP)-conjugate reagent was added to all wells, excluding the blank, and incubated for 60 min at 37 ∘C. Following incubation, each well was washed five times with washing buffer, allowing it to stand for 30-sec before draining. Subsequently, 50 μL of Chromogen Solution A and B was added to all wells and incubated for 15 min at 37∘C. The reaction was then stopped by adding 50 μL of stop solution. Finally, the developed yellow color was read spectrophotometrically at 450 nm against the blank and standard after 15 min. KIM-1 concentrations were expressed in pg/mL.

2.5. Measurement of Enzymatic and non-enzymatic antioxidants

2.5.1. Estimation of malondialdehyde

Malondialdehyde was determined according to the method of Ohkawa et al. (1979). Briefly, 500 μL of trichloroacetic acid (TCA) was added to 200 μL each of the sample supernatant and standard, and the mixture was centrifuged at 3000 rpm for 10 min. Thereafter, 1 mL of 0.75 % TBA was added to 0.1 mL of the supernatant, and the mixture was boiled in a water bath at 100 OC for 20 min and cooled on ice. The absorbance of the sample and standard was measured at 532 nm using a spectrophotometer, with a reagent blank as reference.

2.5.2. Determination of glutathione

Glutathione was estimated in the renal tissue by the method of Tietze (1969). Initially, glutathione was oxidized from 5,5′-diathio-bis-(2- nitrobenzoic acid) to a yellow 5′-thio-2-nitrobenzoic acid using an oxidizing agent i.e., sulfhydryl reagent. 500 μL renal supernatant was added to 4 % sulfosalicylic acid to obtain the precipitate. The mixture was allowed to incubate at 4 °C for at least an hour, followed by centrifugation for 20 min at 1200 g to obtain the supernatant. About 33 μL of the supernatant was mixed with potassium phosphate buffer (900 μL, 0.1 M, pH 7.4) and 66 μL of 100 mM 5,5′-diathio-bis-(2-nitrobenzoic acid). A yellow-colored complex was formed and its optical density was measured at 412 nm.

2.5.3. Determination of superoxide dismutase

Superoxide dismutase was determined using the method described by Misra and Fridovich (1972). An aliquot of 0.2 mL of the diluted sample was added to 2.5 mL of 0.05 M carbonate buffer (pH 10.2) to equilibrate in the spectrophotometer. The reaction was initiated by adding 0.3 mL of freshly prepared 0.3 mM adrenaline to the mixture, which was quickly mixed by inversion. The reference cuvette contained 2.5 mL buffer, 0.3 mL of substrate (adrenaline) and 0.2 mL of water. The increase in absorbance at 480 nm was monitored every 30 secs for 150 secs.

2.5.4. Estimation of catalase

Catalase activity was estimated using the method described by Aebi (1984). In a cuvette, 1000 μL of supernatant was mixed with 450 μL of phosphate buffer (50 mM, pH 7.4) and 2000 μL of 10 mM H2O2. The decrease in absorbance was recorded at 240 nm for 1 min using a spectrophotometer. The molar extinction coefficient of H2O2 (0.0436 mM−1 cm−1) was used to calculate catalase activity. One unit of activity is equal to 1 mmol of H2O2 degraded per minute, which is expressed as units/mg.

2.5.5. Determination of nitric oxide

Nitric oxide (NO) was measured using the Griess Reaction, following a previously documented method by Ridnour et al. (2000). Briefly, 300 μL of the nitrate-containing sample was mixed with 100 μL of Griess reagent. After 30 min of incubation at room temperature, the mixture was measured spectrophotometrically against a blank, which consisted of 100 μL of Griess reagent and 2.9 mL of distilled water.

2.6. Assay of plasma biochemical and electrolytes

Plasma calcium concentrations were measured using biochemical kits from Randox Laboratories (Crumlin, Co. Antrim, UK). Plasma sodium, potassium, chloride, and phosphate concentrations were determined using kits from Teco Diagnostic Laboratories (Anaheim, California, USA). All assays were performed according to the manufacturers’ instructions.

2.7. Assessment of markers of inflammation and apoptosis

Tumor necrosis factor-alpha (TNF-α) and Caspase-3 levels were measured using ELISA kits, following the same methodology as the KIM-1 assay. TNF-α concentrations are reported in pg/mL, while Caspase-3 concentrations are expressed in ng/mL.

2.8. Histopathological assessment

The kidneys were prepared for microscopic examination by dehydrating them in a graded alcohol series, clearing them with xylene, and embedding them in paraffin wax. Sections 5–6 μm thick were then cut using a microtome, mounted on slides, and stained with hematoxylin and eosin. Finally, the kidney tissues were examined under an Olympus CH light microscope (Olympus, Tokyo, Japan), and photomicrographs were captured at ×100 and ×400 magnifications using a Leica DM 750 Camera.

2.9. Statistical analysis

Results are presented as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA), followed by Newman–Keuls post hoc test, was used to assess significant differences among the control and morphine-treated groups for both male and female rats. An unpaired Student's t-test was used to compare male and female groups across all morphine doses. Although the sample sizes were not statistically predetermined, they were comparable to those commonly used in this research area. It is worth noting that data collection did not involve randomization. All statistical analyses were performed using GraphPad Prism version 5.03 (GraphPad Software, La Jolla, CA, USA). A p-value of less than 0.05 was considered statistically significant.

3. Results

3.1. Sex and dose -related Variations in Kidney Biomarkers (creatinine, Urea and Kidney Injury Molecule-1) Following Morphine Treatment in Rats

Male and female rats treated with 20 mg/kg of morphine showed significantly elevated plasma creatinine levels compared to the control group (male: F = 2.084; p = 0.027; female: F = 4.218; p = 0.030). However, at higher doses of 40 and 60 mg/kg, plasma creatinine in both sexes remained unchanged relative to the control group. The male rats consistently exhibited significantly higher plasma creatinine levels than females across all tested morphine doses: 20 mg/kg (t = 2.092; p = 0.041), 40 mg/kg (t = 1.918; p = 0.048), and 60 mg/kg (t = 2.947; p = 0.013) (Fig. 1A).

Fig. 1.

Fig. 1

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A, B and C: Show Sex and Dose -related Variations in Kidney Biomarkers (Creatinine, Urea and Kidney Injury Molecule-1) Following Morphine Treatment in Rats. (A) Creatinine, (B) Urea, (C) Kidney Injury Molecule −1). Bars represent mean ± SEM (n = 5). ∗ = significantly different from the Control group (p < 0.05); α = significantly different from the Males (p < 0.05) (One-way ANOVA followed by Newman-keuls’ post-hoc).

Across all tested doses, the drug had no significant effect on plasma urea in either male or female rats when compared to their respective control groups. Additionally, there was no significant difference in plasma urea between male and female rats at any given morphine dose (20, 40, or 60 mg/kg) (Fig. 1B).

Morphine treatment at all three doses had no significant effect on plasma KIM-1 levels in either male or female rats compared to their respective control groups (male: F = 1.190; p = 0.345; female: F = 1.188; p = 0.346). Similarly, no significant differences were observed in plasma KIM-1 levels between male and female rats across any of the morphine doses (20 mg/kg: t = 2.084; p = 0.071; 40 mg/kg: t = 1.154; p = 0.282; 60 mg/kg: t = 1.748; p = 0.059). However, overall, males exhibited considerably lower plasma KIM-1 levels than females (Fig. 1C).

3.2. Sex and dose-related variations in TNF-α and Caspase-3 Following Morphine Treatment in Rats

In male rats treated with 20 mg/kg of morphine, a significant increase in tumor necrosis factor-alpha (TNF-α) levels was observed in kidney tissue (F = 23.17, p < 0.001), which was notably higher than both the control and other treatment groups. Similarly, female rats at the same 20 mg/kg dose exhibited a significant elevation in TNF-α levels compared to the control group (F = 4.713, p = 0.015). However, no significant differences in TNF-α levels were observed between male and female rats at any of the tested doses (20 mg/kg: t = 0.719, p = 0.246; 40 mg/kg: t = 0.723, p = 0.234; 60 mg/kg: t = 0.299, p = 0.386) (Fig. 2A).

Fig. 2.

Fig. 2

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A and B: Sex and Dose-related Variations in TNF-α and Caspase-3 Levels Following Morphine Treatment in Rats. (A) Tumor Necrosis Factor-alpha, (B) Caspase-3. Bars represent mean ± SEM (n = 5). ∗ = significantly different from the Control group (p < 0.05); # = significantly different from rats treated with 20 mg/kg of Morphine (p < 0.05) (One-way ANOVA followed by Newman-keuls’ post-hoc).

Male rats treated with 20 mg/kg of morphine showed a significant elevation in caspase-3 levels (F = 7.830; p = 0.002) compared to both the control group and other treated groups. Similarly, female rats treated with the same morphine dose also had significantly higher caspase-3 levels (F = 6.333; p = 0.005) than the control group and female rats treated with 60 mg/kg of the drug. However, no significant sex-related differences in caspase-3 levels were observed across the three morphine doses (20 mg/kg: t = 0.825, p = 0.217; 40 mg/kg: t = 0.319, p = 0.379; 60 mg/kg: t = 0.771, p = 0.231) (Fig. 2B).

3.3. Sex and dose-related Variations in Malondialdehyde, reduced glutathione, superoxide dismutase following morphine treatment in rats

Across all morphine doses, no significant differences in malondialdehyde (MDA) levels were observed between treated male rats and their control group (F = 0.697, p = 0.567). The same held true for female rats, where no significant differences were observed between treated groups and their respective controls (F = 2.266; p = 0.120). However, a significant sex-based difference was observed: male rats exhibited higher MDA levels across all three morphine doses than their female counterparts (t = 2.187; p = 0.036; t = 2.280; p = 0.031; and t = 2.183; p = 0.047, respectively) (Fig. 3A).

Fig. 3.

Fig. 3

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A, B and C: Show the Sex and Dose-related Variations in Malondialdehyde, Reduced glutathione and Superoxide dismutase Following Morphine Treatment in Rats. (A) Malondialdehyde, (B) Reduced glutathione, (C) Superoxide dismutase. Bars represent mean ± SEM (n = 5). ∗ = significantly different from the Control (p < 0.05); α = significantly different from the Males (p < 0.05) (One-way ANOVA followed by Newman-keuls’ post-hoc).

In both male and female rats, morphine administration at all doses did not significantly alter reduced glutathione levels in the renal homogenate compared to their respective control groups. For the male rats, the difference was not significant (F = 0.985; p = 0.427), and the same was observed in females (F = 1.248; p = 0.325). However, a significant sex-based difference in reduced glutathione levels emerged at the higher morphine doses: male rats treated with 40 mg/kg and 60 mg/kg of morphine exhibited significantly higher levels than their female counterparts (40 mg/kg: t = 3.536, p = 0.004; 60 mg/kg: t = 3.284, p = 0.006). At the 20 mg/kg dose, no significant difference was observed between the sexes (t = 1.495, p = 0.093) (Fig. 3B).

Superoxide dismutase (SOD) activity in male rats treated with morphine did not differ significantly from that of the control group (F = 2.176; p = 0.234). However, in female rats, SOD activity was significantly higher in those treated with 40 and 60 mg/kg of morphine compared to the control group (F = 5.620; p = 0.012). No significant differences in SOD activity were observed between male and female rats at any of the three morphine doses (20 mg/kg: t = 0.025, p = 0.490; 40 mg/kg: t = 0.636, p = 0.274; 60 mg/kg: t = 1.860, p = 0.056) (Fig. 3C).

3.4. Sex and dose-related variations in catalase and nitric oxide following morphine treatment in rats

In male rats, catalase activity significantly increased with higher morphine doses. Specifically, those treated with 40 and 60 mg/kg of morphine exhibited significantly higher catalase activity compared to both the control group and those treated with 20 mg/kg (F = 9.879, p = 0.002). Conversely, female rats showed no significant changes in catalase activity across any of the morphine doses when compared to their control group (F = 1.528, p = 0.258). When comparing sexes, male rats treated with 20 mg/kg of morphine displayed significantly higher catalase activity than their female counterparts (t = 3.029, p = 0.012). However, at the 40 and 60 mg/kg doses, no significant differences in catalase activity were observed between male and female rats (40 mg/kg: t = 1.277, p = 0.124; 60 mg/kg: t = 1.684, p = 0.072) (Fig. 4A).

Fig. 4.

Fig. 4

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A and B: Show the Sex and Dose-related Variations in Catalase and Nitric Oxide Following Morphine Treatment in Rats. (A) Catalase, (B) Nitric Oxide. Bars represent mean ± SEM (n = 5). ∗ = significantly different from the control. α = significantly different from the Males. # = significantly different from rats treated with 20 mg/kg of Morphine (p < 0.05) (One-way ANOVA followed by Newman-keuls’ post-hoc).

In male rats, nitric oxide (NO) levels remained unchanged across all three morphine doses compared to the control group (F = 0.171; p = 0.914). However, female rats treated with 20 mg/kg of morphine exhibited significantly elevated NO levels compared to their control counterparts (F = 4.861; p = 0.014). When comparing sexes, no significant differences in nitric oxide levels were observed between male and female rats at any of the morphine doses (20 mg/kg: t = 0.235, p = 0.410; 40 mg/kg: t = 1.174, p = 0.137; 60 mg/kg: t = 1.264, p = 0.121) (Fig. 4B).

3.5. Sex and dose-related Variations in Sodium, potassium, Calcium ions following morphine treatment in rats

Plasma sodium ion concentrations in both male and female rats remained unchanged across all three morphine doses when compared to their respective control groups (males: F = 0.430, p = 0.743; females: F = 0.775, p = 0.525). Although male rats exhibited consistently higher plasma sodium ion concentrations than females at the 20, 40, and 60 mg/kg doses, these differences were not statistically significant (20 mg/kg: t = 0.934, p = 0.377; 40 mg/kg: t = 1.597, p = 0.078; 60 mg/kg: t = 1.794, p = 0.055) (Fig. 5A).

Fig. 5.

Fig. 5

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A, B and C: Sex and Dose-related Variations in Sodium, Potassium and Calcium Following Morphine Treatment in Rats. (A) Sodium, (B) Potassium, (C) Calcium. Bars represent mean ± SEM (n = 5). ∗ = significantly different from the Control (p < 0.05) α = significantly different from the Males. (One-way ANOVA followed by Newman-keuls’ post-hoc).

Plasma potassium concentrations in male rats treated with all three morphine doses (20, 40, and 60 mg/kg) were significantly elevated compared to the control group (F = 15.35; p = 0.012). Female rats also showed higher plasma potassium levels with morphine treatment, although this increase was not statistically significant compared to their control group (F = 2.122; p = 0.138). No significant sex-based differences in plasma potassium ion concentrations were observed between male and female rats at any of the morphine doses (20 mg/kg: t = 0.455, p = 0.332; 40 mg/kg: t = 0.158, p = 0.439; 60 mg/kg: t = 0.559, p = 0.296) (Fig. 5B).

Plasma calcium concentrations in both male and female rats did not differ significantly from their respective control groups across all morphine doses (males: F = 0.399; p = 0.755; females: F = 0.304; p = 0.821). Similarly, there was no significant difference in plasma calcium concentration between male and female rats at any of the three morphine doses administered (t = 0.629; p = 0.273; t = 0.832; p = 0.215; and t = 0.843; p = 0.212, respectively) (Fig. 5C).

3.6. Sex and dose-related variations in phosphate and chloride ions following morphine treatment in rats

Plasma phosphate concentrations in both male and female rats treated with all three morphine doses did not significantly differ from their respective control groups (males: F = 1.654; p = 0.217; females: F = 1.103; p = 0.377). While plasma phosphate concentrations in male rats treated with 20 and 60 mg/kg of morphine did not significantly differ from those in female rats (t = 1.681; p = 0.066 and t = 0.967; p = 0.181, respectively), male rats given 40 mg/kg of the drug showed significantly higher plasma phosphate levels compared to their female counterparts (t = 3.552; p = 0.006) (Fig. 6A).

Fig. 6.

Fig. 6

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A and B: Show the Sex and Dose-related Variations in Plasma Phosphate and Chloride Following Morphine Treatment in Rats. (A) Phosphate, (B) Chloride. Bars represent mean ± SEM (n = 5). α = significantly different from the Males (p < 0.05) (One-way ANOVA followed by Newman-keuls’ post-hoc).

In both male and female rats, plasma chloride concentrations remained unchanged across all morphine doses when compared to their respective control groups (males: F = 1.353; p = 0.293; females: F = 0.227; p = 0.876). At the highest dose, male rats treated with 60 mg/kg of morphine had significantly higher plasma chloride concentrations than their female counterparts (t = 2.057; p = 0.043). However, at the 20 and 40 mg/kg doses, no significant differences in plasma chloride ion concentration were observed between male and female rats (t = 0.922; p = 0.192 and t = 1.149; p = 0.142, respectively) (Fig. 6B).

3.7. Sex and dose-related variations in relative kidney Weight following morphine treatment in rats

Morphine treatment did not significantly alter relative kidney weight in either male or female rats. Male rats treated with varying doses of morphine showed no significant difference compared to the control group (F = 1.836; p = 0.181). Similarly, female rats administered the three morphine doses also had relative kidney weights that were not significantly different from their control group (F = 3.083; p = 0.150). At lower doses, male rats showed a significantly higher relative kidney weight compared to females (t = 3.634; p = 0.003). A similar significant difference was observed at 40 mg/kg, where male rats had higher relative kidney weights compared to female rats (t = 3.583; p = 0.004). However, at the highest dose of 60 mg/kg of morphine, there was no significant difference in relative kidney weight between male and female rats (t = 0.101; p = 0.461) (Fig. 7).

Fig. 7.

Fig. 7

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Shows the Sex and Dose-related Variations in the Relative Kidney Weight Following Morphine Treatment in Rats. Bars represent mean ± SEM (n = 5). α = significantly different from the Males (p < 0.05) (One-way ANOVA followed by Newman-keuls’ post-hoc).

3.8. Kidney Photomicrographs of male rats treated with Graded Doses of Morphine

A light micrograph of a representative kidney from the control group revealed a normal renal histoarchitecture. The glomeruli appeared healthy, and both the proximal and distal convoluted tubules were clearly defined. In rats given 20 mg/kg of morphine, the glomeruli showed atrophy, resulting in widened urinary spaces and a loss of the brush border in the proximal convoluted tubules. Rats treated with 40 mg/kg of morphine also exhibited kidney damage, including a loss of glomeruli and an indistinct appearance of the proximal convoluted tubules. Conversely, in rats treated with 60 mg/kg of morphine, the histology of the proximal and distal tubules was poorly visualized, with only the glomeruli being clearly discernible (Fig. 8).

Fig. 8.

Fig. 8

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Shows the Kidney Photomicrographs of Male Rats Treated with Graded Doses of Morphine. Light micrograph of the representative kidney of the control group showed normal histoarchitecture of the kidney with the glomeruli (G) appearing normal and the proximal tubule (blue arrow) and distal tubule (black arrow) are well delineated. The glomerulus of the rats treated with 20 mg/kg of morphine showed atrophy leading to widened urinary space (yellow arrow), with loss of brush border in the proximal convoluted tubules. The rats treated with 40 mg/kg of the drug also presents loss of glomeruli (star symbol), and indistinctive appearance of the proximal convoluted tubules. On the other hand, rats treated with 60 mg/kg of morphine showed poor demonstration of the histology of the proximal and distal tubules as only the glomeruli can be appreciated. AM – Control Male rats; BM – 20 mg/kg of morphine; CM – 40 mg/kg of morphine; DM – 60 mg/kg of morphine.

3.9. Kidney Photomicrographs of female rats treated with Graded Doses of Morphine

A light micrograph of a representative kidney from the control group revealed an intact glomerulus with no signs of atrophy. The proximal convoluted tubules appeared normal with intact brush borders, and the distal convoluted tubules were also well-delineated. Rats administered 20 mg/kg of morphine showed signs of glomerular atrophy, with few intact glomeruli and preserved proximal and distal convoluted tubules. The glomeruli in rats treated with 40 mg/kg of the drug showed signs of glomeruli atrophy, while the tubules appearing well-defined. On the other hand, rats treated with 60 mg/kg of the drug show intact glomeruli but indistinctive proximal and distal convoluted tubules (Fig. 9).

Fig. 9.

Fig. 9

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Shows Kidney Photomicrographs of Female Rats Treated with Graded Doses of Morphine. Light micrograph of the representative kidney of the control group showed glomerulus (G) appearing intact with no sign of atrophy, the proximal convolute tubules (blue arrow) appeared normal with intact brush borders and the distal convoluted tubules (black arrow) also well delineated. Rats administered with 20 mg/kg of morphine showed sign of atrophied glomerulus resulting in widened urinary space (yellow arrow) with intact proximal and distal convoluted tubules. The glomeruli in rats treated with 40 mg/kg of the drug showed sign of glomeruli atrophy with the tubules appearing well-defined. On the other hand, rats treated with 60 mg/kg of the drug show intact glomeruli but indistinctive proximal and distal convoluted tubules. AF – Control Female rats; BF – 20 mg/kg of morphine; CF– 40 mg/kg of morphine; DF – 60 mg/kg of morphine.

4. Discussion

This study investigated the sex- and dose-dependent effects of morphine administration on renal function, antioxidant enzymes, and inflammatory and apoptotic markers in male and female Wistar rats. Morphine has been shown to impair renal glomeruli and podocytes, affecting their filtration capabilities, as well as their number, diameter, and structure. This damage is thought to occur through superoxide production by macrophages and DNA damage (Singhal et al., 1994). Our findings indicate that a low dose of morphine significantly increased plasma creatinine in both male and female rats compared to control rats. This aligns with Sumathi and Devaraj's report, which also demonstrated that chronic morphine administration elevates serum levels of urea, uric acid, and creatinine. The photomicrograph illustrating this group's rat kidney revealed glomerular atrophy and a widened Bowman's space. This suggests that morphine administration leads to impairment and dysfunction of the renal glomeruli.

Hormones significantly influence the structure and function of the kidneys. Studies conducted in animal models have showed that estradiol, a key hormone, can slow the progression of glomerulosclerosis (scarring of the kidney's filtering units) and help maintain overall kidney size in females (Wilson et al., 1996; Jelinsky et al., 2003). Conversely, testosterone appears to contribute to the rapid progression of glomerulosclerosis (Gava et al., 2002). Animal studies also suggest that testosterone stimulates apoptosis (programmed cell death) in proximal tubular cells, which are important for kidney function (Verzola et al., 2009). Across various morphine doses, male rats consistently exhibited significantly higher plasma creatinine levels compared to female rats. However, no significant difference in plasma urea was found between the sexes. Our findings indicate that differences in sex hormones, particularly testosterone, may likely contribute to the observed variations in plasma creatinine levels between male and female rats. This is further supported by reports that estrogen plays a protective role in females. Another potential reason for the higher plasma creatinine levels observed in the male rats may result from a significant reduction in their urine output. This reduction could stem from a secondary decrease in renal hemodynamics and the inhibition of baroreflex pathways (Mercadante and Arcuri, 2004), leading to elevated concentrations of substances in the plasma.

Kidney Injury Molecule-1 (KIM-1) has been identified as an early and sensitive biomarker for kidney injury, showing significant urinary elevation within 24 h, prior to any substantial increase in serum creatinine (Shao et al., 2014). In this study, Morphine treatment did not significantly alter plasma KIM-1 levels in either male or female rats when compared to their respective control groups. Similarly, no significant difference was observed in plasma KIM-1 levels between male and female rats at any specific morphine dose, although, males exhibited considerably lower overall plasma KIM-1 levels than females. This study's finding of significantly increased plasma creatinine levels in morphine-treated rats, compared to control, further confirms KIM-1 as a predictive marker of renal tubular injury rather than glomerular dysfunction. Evidently, elevation of KIM-1 is particularly associated with kidney tubular injury (Sabbisetti et al., 2014). A limitation of this study is that urinary KIM-1 level was not measured in the rats.

Tumor necrosis factor-alpha (TNF-α) is a primary mediator in pro-inflammatory processes, influencing necrosis, apoptosis, and proliferation (DeLeo et al., 2000; Kawasaki et al., 2008). Largely, TNF-α is produced by macrophages and T-lymphocytes in response to damaged tissue, and it may therefore serve as a systemic inflammatory marker for tissue injury. Tumor necrosis factor-alpha (TNF-α) plays a vital role in acute kidney injury (AKI) through its action on renal endothelial TNF receptor 1 (TNFR1) (Al-Lamki et al., 2001; Cunningham et al., 2002), which is predominantly expressed in the glomerular endothelium (Al-Lamki et al., 2001). Also, the injurious effect of TNF-α on renal endothelial cells (ECs) has been previously reported (Wu et al., 2009). Our results showed that TNF-α of the male and female rats at low dose of morphine was significantly higher when compared with the corresponding controls and other treated groups. This could be attributed to increased expression of TNFR receptor 1 and/or injury to the renal epithelial cells resulting from up-regulation of inflammatory process. Also, intravenous administration of tumor necrosis factor has been reported to reduce glomerular filtration rate (GFR), leading to loss of glomerular endothelial cell fenestrae, increased fenestrae diameter, and damage to the glomerular endothelial surface layer (Xu et al., 2013). This further corroborates the increased plasma creatinine observed in rats treated with a low dose of morphine, as plasma creatinine concentrations primarily depend on glomerular filtration rate or function. However, no significant differences were observed in TNF-α levels between males and females at any of the tested doses. This indicates that the expression of Toll-Like Receptors, which trigger inflammatory responses in morphine-treated rats, is not elevated to a greater extent in males compared to females.

This study showed that rats treated with the low dose of morphine had significantly elevated caspase-3 level compared to the control and other experimental groups. Consistent with this result is the finding of Luo et al. (2013) who reported caspase-3 activation in the kidney and liver of mice following morphine administration for 7 days. A study published by Jalili et al. (2019) reported that the oxidative stress generated by morphine is due to the stimulated cytochrome P450 that in turn produces excessive free radicals. As a result, these free radicals damage the proteins and DNA of renal cells and induce cellular apoptosis. The elevated caspase-3 level observed in rats treated with a low dose of morphine suggests that morphine activates the apoptotic pathway. This needs to be further explored. No significant sex-related difference was found in caspase-3 levels between male and female rats across all three morphine doses. This implies caspase-3 expression wasn't upregulated in rats of either sex.

The formation of free radicals leads to oxidative stress and lipid peroxidation, both of which contribute to renal tissue damage (Atici et al., 2005). Formation of excess free radicals or antioxidant depletion may result in oxidative stress, a potential mechanism of the toxicity of opioid drugs. Reduced glutathione (GSH) production is considered the first line of defense against oxidative damage and free radical generation, where GSH functions as a scavenger and co-factor in metabolic detoxification (Nozal et al., 1997). Superoxide dismutase (SOD) is an enzymatic antioxidant that is present almost in all oxygen-metabolizing cells. This enzyme functions to protect cells from excessive superoxide. Malondialdehyde (MDA) is a by-product of oxidative damage by free radicals, that act as a marker for oxidative stress, notably lipid peroxidation (Li Pomi et al., 2025). Morphine promotes the production of free radicals by activating lipid peroxidation, thereby obstructing the antioxidant enzymes and forming free radicals or reactive oxygen species (Ahmadizadeh et al., 2012). In this study, morphine substantially raised the activities of SOD and CAT in the morphine treated rats compared to the control. This is indicative of an adaptive response by the body to get rid of excess superoxide and/or upregulation of the body antioxidant defense mechanism. This agrees with the findings of Perez-Casanova et al. (2008), who reported an increase in the activities of SOD and GSH-Px after 20 weeks of morphine treatment in morphine-dependent rhesus macaques. Also, morphine has been reported to inhibit the production of ROS in human neuroblastoma cell line induced by doxorubicin, an antitumor drug (Lin et al., 2007). However, several studies suggested that activities of these enzymes are decreased after morphine exposure (Payabvash et al., 2006; Abdel-Zaher et al., 2010; Sumathi et al., 2011; Zhou et al., 2011; Rozisky et al., 2013). Furthermore, the impact of chronic morphine treatment on the activities of CAT seems to be undetermined (Payabvash et al., 2006; Perez-Casanova et al., 2008; Zhou et al., 2011). The effect of morphine on antioxidant enzymes may be influenced by many different factors, including the duration of exposure, dosage and species involved.

A number of studies have demonstrated the sex difference in the activity of antioxidant defense enzymes in different tissues of rats. Gene transcription and RNA production are under the selective influence of sex hormones, which explain their regulation in the biosynthesis of specific proteins (Azevedo et al., 2001; Kasapovic et al., 2001; Tam et al., 2003). Accordingly, these hormones can endogenously regulate the pattern of expression of antioxidant defense enzyme. In this study, malondialdehyde and reduced glutathione levels were not significantly different in the males and females when compared with their respective controls. However, their levels were significantly higher in the male rats compared with the females. Also, catalase activity in the renal homogenate of the male rats was significantly higher than that of the female rats. This corroborated by Finley and Kincaid (1991), who reported that plasma and kidney cytosol selenium content, an endogenous antioxidant, and GSH-Px activity were increased in the males compared to the females. It has also been established that males exhibited significantly higher levels of endogenous antioxidant defense parameters in kidney, including Mn, Cu, Zn, SOD, glutathione peroxidase, and catalase than females. Moreover, estrogen's interaction with its receptor (ER) has been linked to oxidative stress-mediated pathways (Mobley and Brueggemier, 2002). Some studies suggest that estrogen exposure can induce DNA damage under acute conditions in cell culture, particularly in ER-positive cells (Mobley and Brueggemier, 2002). Estrogen is known to induce oxidative stress, which may explain the reduced antioxidant enzyme activity observed in female rats compared to their male counterparts.

Nitric oxide (NO), a free radical produced in mammalian cells, plays a crucial role in regulating various physiological processes. However, elevated NO levels are associated with numerous pathological conditions (Jalili et al., 2016b). Morphine can stimulate the release of noradrenaline in the paraventricular and amygdala nuclei and directly influence the solitary nuclei to enhance NO production. It increases NO levels by regulating intracellular calcium and activating calcium/calmodulin-dependent nitric oxide synthase (NOS). This surge in NO facilitates a massive influx of calcium into the cytosol, leading to cellular toxicity. The presence of NOS isoforms, such as inducible nitric oxide synthase (iNOS), has been associated with thickening of the proximal and distal tubules as well as the collecting ducts, thereby accelerating the progression of nephrotoxicity, nephritic, and nephrotic diseases (Jalili et al., 2019). Our findings revealed a significant increase in NO levels in female rats treated with morphine compared to controls. This supports previous evidence suggesting that estrogen can induce oxidative stress in females. However, no significant difference in NO levels was observed between male and female rats at any of the three morphine doses.

Proximal tubule cells are involved in reabsorption of 60 % of water and salt filtered by the glomerulus (Wagner, 2025). Additionally, they reabsorb nearly all filtered glucose and amino acids, as well as a substantial part of filtered phosphate, bicarbonate and low molecular weight proteins. These are enriched with mitochondria which makes them highly vulnerable to oxidative stress. It has been shown that mitochondrial activity, reactive oxygen species (ROS) generation, and antioxidant activity differ between mitochondria from the heart and brain of male and female mice. Morphine enhances tubular sodium reabsorption via an opiate receptor-dependent mechanism primarily occurring in the proximal tubule (el-Awady and Walker, 1990). Female iron-deficient kidneys exhibited complex II down-regulation and increased mitochondrial oxidative stress in a study by Woodman et al. (2018). Our results demonstrated that morphine caused a significant decrease in plasma sodium and potassium levels in females compared to males. Similarly, females had significantly lower plasma phosphate and chloride ion concentrations than males. This suggests either heightened oxidative stress within the mitochondria of the proximal tubule which is crucial for reabsorbing nearly all filtered solutes, or an increased expression of opiate receptors in the male proximal tubule compared to females.

As sodium ions move out of the renal tubule, the tubular fluid becomes negatively charged relative to the renal interstitial fluid. This electrical gradient drives the passive reabsorption of chloride and phosphate ions out of the tubular lumen. Therefore, the active reabsorption of sodium and potassium is linked to the passive reabsorption of chloride and phosphate ions. Male rats treated with all three morphine doses showed significantly elevated plasma potassium concentrations compared to the control group, while female rats also exhibited higher plasma potassium levels with morphine treatment when compared to their control group. This suggests morphine administration reduces potassium excretion in both males and females, partly by enhancing proximal tubular reabsorption of potassium. This effect is likely mediated by morphine's activation of opiate receptors.

In the present study, the relative kidney weight of male rats treated with different doses of morphine was significantly higher than that of female rats. This finding aligns with the report by Denic et al. (2017), which stated that male kidneys are not only larger in size but also contain a greater number of nephrons. Similarly, Weber and colleagues (2012) reported an increase in kidney weight and glomerular volume in C57BL/6 WT mice following chronic morphine administration. Their study showed that morphine treatment induced glomerular expansion, tubular dilatation, intraglomerular and peritubular congestion, and increased kidney mass after 3 and 6 weeks of treatment. The observed increase in relative kidney weight among male rats in this study may therefore suggest morphine-induced renal hypertrophy or expansion, consistent with previous findings.

In conclusion, our study demonstrated that female rat kidneys are more susceptible to morphine-induced oxidative stress and lipid peroxidation than male kidneys. This susceptibility was supported by the more pronounced alterations in plasma electrolytes and antioxidant activities, both enzymatic and non-enzymatic, observed in females. Conversely, females showed better glomerular protection than males, as evidenced by the higher plasma creatinine levels in males compared to females, a difference likely influenced by sex steroid hormones. Additionally, rats administered a low dose of morphine exhibited greater changes in renal function, plasma electrolytes, oxidative stress, and apoptotic markers than those treated with medium and high doses. These findings suggest that the adverse renal effects of morphine are not strictly dose-dependent.

It's important to acknowledge the limitations of this study. Given the significant influence of hormones on kidney structure and function, future research should incorporate measurements of sex steroid hormones to better understand their potential sex-specific impact on the kidneys of morphine-treated rats. Although this study demonstrated a significant morphine-induced reduction in plasma sodium and potassium levels in females compared to males, further investigation is warranted to explore sex-based differences in opiate receptor expression on proximal tubules. Techniques such as polymerase chain reaction (PCR) and Western blot analysis would be valuable for this purpose. Another limitation is the absence of data on Tumor Necrosis Factor Receptor 1 (TNFR1) expression on renal epithelial cells. Our findings showed significantly elevated TNF-α levels in both male and female rats administered a low dose of morphine compared to controls and other treatment groups. Future studies should expand on this by assessing TNFR1 expression in renal epithelial cells to better understand the mechanism underlying TNF-α–mediated renal damage.

Informed consent statement

Not Applicable.

Author contributions

OSO conceptualized the work, collection, analysis and interpretation of data. He also participated in writing of the manuscript and reviewing it critically for intellectual content. DJO was involved collection and interpretation of data. He also participated in revising the article critically for important intellectual content. STA was involved in the preparation and interpretation of the histological slides. He also participated in revising the article critically for important intellectual content. MAH was involved in data collection and analysis. He also participated in revising the article critically for important intellectual content. OSB was involved collection and interpretation of data. He also participated in revising the article critically for important intellectual content. OOA was involved in drafting the article and reviewing it critically for important intellectual content. ROA supervised various stages of the work and proof read the article for intellectual content.

Institutional review board statement

The animal study protocols described in this study were reviewed and approved by the Health Ethics Research Committee, College of Health Sciences, Obafemi Awolowo University in accordance with the EU Directive 2010/63/EU.

CRediT author statement

Olaoluwa Olukiran: Conceptualization, Methodology, Writing- Original draft preparation, Funding acquisition. Oluwadare Ogundipe: Investigation, Writing- Reviewing and Editing. Stephen Adelodun: Investigation. Moses Hamed: Investigation. Olayemi Babatunde: Investigation. Oluwole Alese: Investigation, Writing- Reviewing and Editing. Rufus Akomolafe: Supervision, Writing- Reviewing and Editing, Funding acquisition.

Funding statement

This research was supported by Institution-based Research Intervention Allocation (TETFUND) (IBR/2023/Vol.1/035).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available upon request from the corresponding author.

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