Testosterone contributes sex differences of urinary biomarkers for nephrotoxicity in rats

Abstract

Urinary biomarkers have been used widely in non-clinical toxicity studies to detect kidney dysfunction/injury caused by drugs under development. Although their usefulness to evaluate nephrotoxicity has been well studied, knowledge about sex differences in the urinary excretion levels of these biomarkers remains inadequate. We previously demonstrated the existence of sex differences in the excretion levels of urinary biomarkers and that these differences were associated with the endogenous testosterone levels. In this study, testosterone was repeatedly administered subcutaneously to female rats for 4 weeks along with male rats as a comparison control, to investigate how the blood levels of testosterone contribute to the sex differences in the urinary biomarker and renal cortical protein levels. The results showed that the urinary excretion of leucine aminopeptidase (LAP), gamma-glutamyltransferase (γ-GTP), cystatin C (Cys-C), liver-type fatty acid binding protein (L-FABP), and beta2-microglobulin (β2MG) were increased, and the urinary excretion of kidney injury molecule 1 (Kim-1) was decreased. The protein level of megalin, an endocytic receptor, in the renal cortex, was higher in female rats than in male rats, and testosterone treatment led to decrease in the level in the female rats. Our results suggest that the blood testosterone level might be responsible for the sex differences in the urinary excretion levels of low-molecular-weight proteins via regulating the expression level of megalin in the renal cortex.

INTRODUCTION

Urinary levels of proteins, electrolytes, glucose, etc., are measured, in addition to the serum creatinine (sCRE) and blood urea nitrogen (BUN) concentrations, to examine the condition of the kidneys in pharmacological and toxicological research. In non-clinical studies, these are known as useful biomarkers for detecting the efficacies and toxicities in a non-invasive way. A large number of proteins, including enzymes and small molecules, have been investigated as biomarkers of renal toxicity, and their specificities and sensitivities for kidney injury have been demonstrated in rodents. The Predictive Safety Testing Consortium (PSTC) proposed 7 urinary biomarkers (Kim-1, albumin, total protein, β2MG, Cys-C, clusterin, and trefoil factor 3) as being superior to sCRE and BUN to predict and detect kidney toxicity (Dieterle et al., 2010), and the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) in 2008, and the Japanese Pharmaceuticals and Medical Devices Agency (PMDA) in 2010 have qualified the proposal and recognized these 7 parameters as available urinary biomarkers correlated with glomerular and/or renal tubular injury. Other studies have reported L-FABP is potentially early biomarker of renal injury (Ferguson et al., 2010; Noiri et al., 2009). In addition, composite measurement of clusterin, Cys-C, Kim-1, N-acetyl-β-D-glucosaminidase (NAG), neutrophil gelatinase-associated lipocalin (NGAL), and osteopontin has been recognized as a qualified safety biomarker of renal tubular injury in clinical trials in normal healthy volunteers (Foundation for the National Institutes of Health, and Critical Path Institute, 2019).

The aforementioned urinary biomarkers have been reported to have the ability to detect kidney injury in experimental rat models. According to one report, urinary Cys-C and Kim-1 levels changed prior to proximal tubular damage in rats with cisplatin-induced acute kidney injury (Togashi et al., 2012). In another study, the levels of some of 15 biomarkers, including those mentioned above, changed depending on the damage to specific regions of the kidney following administration of nephrotoxins, puromycin, gentamicin, cisplatin, and 2-bromoethylamine (Sasaki et al., 2011). In a rat model of ischemia, the urinary level of Kim-1 increased early as compared with the urinary levels of Cys-C and monocyte chemoattractant protein-1 (Peng et al., 2015). Rouse et al. (2011) reported the time-course of variations in the urinary levels of biomarkers in rats after drug-induced kidney injury, including during the recovery period. Thus, while several studies have investigated urinary biomarkers in rats, most experiments were performed in male rats.

A number of studies have reported sex differences in the urinary levels of biomarkers in rats and mice (Sellers et al., 1950; Finlayson and Baumann, 1958; Koenig et al., 1978 and 1980; Grötsch et al., 1985) and investigated the mechanisms underlying the sex differences in the levels of traditional urinary biomarkers, e.g., proteins and enzymes such as γ-GTP and NAG, in castrated and testosterone-treated mice and rats. On the other hand, information on the sex differences in recently reported biomarkers, such as small molecules, is still limited. The urinary excretion of albumin have been reported to be higher in female rats than in male rats (Pinches et al., 2012) while those of β2MG, clusterin, and Cys-C have been reported to be higher in male rats than in female rats (Gautier et al., 2014). In addition, the mechanisms underlying the sex differences in the levels of these markers have not yet been clearly elucidated.

In the present study, we investigated the effect of testosterone on urinary excretion levels of biomarkers and on the protein levels in the renal cortex, and the mechanisms underlying the sex differences, by using male rats, female rats, and testosterone-treated female rats.

MATERIALS AND METHODS

Animals

Four-week-old male and female Sprague Dawley (SD) rats were obtained from The Jackson Laboratory Japan, Inc. (Kanagawa, Japan). They were quarantined and acclimatized for 10 days after receipt. Every 2 male rats or female rats were group-housed in cages with a bedding (White flakes, The Jackson Laboratory Japan, Inc., Kanagawa, Japan) under controlled temperature (23°C ± 3°C) and humidity (50% ± 20%) conditions and a 12-hour light/dark cycle. Basal diet (MF, Oriental Yeast Co., Ltd., Tokyo, Japan) and drinking water were provided ad libitum. During the urine sampling, the rats were housed individually in metabolic cages (Metabolica, Sugiyama-gen Co., Ltd., Tokyo, Japan). The experiments were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee at Taisho Pharmaceutical Co., Ltd (approved No.: AN13423-Z01).

Experimental design

After the acclimation, the male and female rats were allocated to three groups consisting of 6 rats each (n = 6), as follows: male-control (M/C), female-control (F/C), and female testosterone-treated (F/TES) groups. The control groups and testosterone treatment group received subcutaneous injection of corn oil (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and testosterone propionate (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), respectively, once daily for 4 weeks. The administration was started when the animals were 5 weeks old, that is, when the rats were still immature and there was little likelihood of the sex hormones exerting an influence on the systemic effects (Campion et al., 2013; Nazian, 1986), and continued up to 9 weeks of age, by which time the rats attained sexual maturity. The dose level of testosterone was set at 1 mg/kg/day from the initiation of administration to week 2 and at 2 mg/kg/day from week 3 to week 4, based on the following reasons. In some studies conducted in rats to investigate the sex differences in the urinary biomarker levels, a high dose level tended to be set for testosterone to allow the effects to be clearly determined. To determine the dose level of testosterone that would make the plasma testosterone level in female rats comparable to that in male rats, and not excessive, we referred to studies in which testosterone had been administered to female rats or castrated male rats. Yu (1989) demonstrated that administration of testosterone propionate by subcutaneous injection at the dose of 1.0 mg/kg twice weekly made plasma testosterone level in female rats (body weight range: 160 to 180 g) comparable to that in male rats. We demonstrated that following administration of testosterone propionate at the dose of 0.5 and 2.0 mg/kg by subcutaneous injection once daily, the plasma testosterone levels in castrated male rats were lower and 4 times higher, respectively, than those in sham-operated male rats (data not shown). Therefore, for this study, the initial and second dose levels of testosterone were set at 1 and 2 mg/kg, respectively. The report of Breedlove and Arnold (1981) that subcutaneous injection at 2 mg/kg daily of testosterone induced hormonal responses in female rats and castrated male rats would lend support to the dose level in our study being appropriate to observe hormonal responses.

Animals were weighed once a week, including the initial day of dosing. Urine samples were collected pre-treatment (at 5 weeks old) and at week 2 and week 4 of dosing. Necropsy was performed on all the rats on the day after the last administration.

Urinalysis

Urine was continuously collected for approximately 24 hr using metabolic cages under cooling with circulating water set at 4°C by the cooling water circulation apparatus (EYELA CAP-3000, Tokyo Rikakikai Co., Ltd., Tokyo, Japan). All rats had access to food and water ad libitum throughout the urine collection period. After the 24-hr urine samples were collected, the urine samples were centrifuged (settings: 5°C, approximately 550 ×g, 5 min) and the supernatants were obtained for measurements of the urinary biomarkers. The urinary excretion levels of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), LAP, and γ-GTP were measured on the day of the urine collection, whereas those of the other biomarkers were measured after the samples had been stored in an ultra-low temperature freezer at -80°C. Urinalysis methods are shown in Table 1. The urinary excretion level of each of the urinary biomarkers was normalized to the urinary CRE concentration.

Table 1. Urinalysis methods.

Item	Method (Supplier)	Analyzer
LDH, ALP, LAP, γ-GTP, glucose	Enzymatic colorimetric method (A)	Hitachi 7180 biochemistry automatic analyzer (Hitachi High-Technologies Corporation, Tokyo, Japan)
NAG	Enzymatic colorimetric method (B)
CRE	Enzymatic colorimetric method (C)
Total protein	Colorimetric method (A)
L-FABP	ELISA, RFBP10 (D)	xMark™ Microplate Absorbance Spectrophotometer (Bio-Rad Laboratories, Inc., CA, USA)
Kim-1, clusterin	Milliplex MAP kit, Rat Kidney Toxicity Panel 1 (E)	Luminex 200 Instrument System (Thermo Fisher Scientific Inc., MA, USA)
Albumin, β2MG, Cys-C, NGAL	Milliplex MAP kit, Rat Kidney Toxicity Panel 2 (E)

ELISA: enzyme-linked immunosorbent assay

Supplier: A (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), B (KANTO CHEMICAL Co., Inc., Tokyo, Japan), C (Shino-Test Corporation, Tokyo, Japan), D (R&D systems Inc., MN, USA), E (Merck KGaA., Darmstadt, Germany)

Serum testosterone level

Blood samples to measure the testosterone level were collected twice from all the rats. The first sample was obtained from the jugular vein under the conscious condition just prior to the first administration of testosterone at the dose level of 2 mg/kg/day. The second sample was obtained from the abdominal aorta with the animals under isoflurane anesthesia on the day of the necropsy. The samples were centrifuged to obtain serum samples (settings: 4°C, approximately 2200 ×g, 15 min). The level of testosterone was determined using an enzyme immunoassay kit (ADI-900-065, Enzo Life Sciences, Inc., NY, USA).

Preparation of renal cortex homogenates

For necropsy, the rats were euthanized by exsanguination, under isoflurane anesthesia, via the abdominal aorta with a winged needle. To thoroughly wash the blood out from the kidneys, both the main artery and vein at the upper pole of the kidney were tightly pinched with forceps to prevent the wash solution from flowing into the tissues within the chest cavity, and the lower part of the posterior vena cava was cut for flow outlet. Twelve milliliters per animal of cooled phosphate buffered-saline (PBS) was infused from the tube line of the winged needle. The kidneys were extracted and placed in dishes on ice. The right kidney was used to obtain specimens of the renal cortex. Splitting the kidney in half lengthwise, the renal cortex was trimmed out. The renal cortex was minced, transferred to a microtube, and homogenized in cooled PBS (0.9 mL/0.1 g tissue) on ice. After centrifugation (settings: 4°C, approximately 5000 ×g, 5 min), the supernatants were stored in an ultra-low temperature freezer at -80°C until the measurements.

Protein levels of renal biomarkers in renal cortex

The renal cortical levels of total protein, LDH, ALP, LAP, γ-GTP, NAG, and megalin were measured in the renal cortical extracts. Protein assay (Bio-Rad Laboratories, Inc., CA, USA) was performed for measuring the total protein level. The level of megalin was measured using an ELISA method (LS-F33155, LifeSpan BioSciences, Inc., CA, USA). The levels of LDH, ALP, LAP, γ-GTP, and NAG were measured using the Hitachi 7180 biochemistry automatic analyzer, in the same manner as the urinalysis. The level of each protein in the renal cortex was normalized to the total protein concentration.

Statistical analysis

Statistical analyses were performed for testosterone level, normalized urinary biomarkers, and normalized proteins in the renal cortex, to compare the levels among the 3 groups at each point of examination. First, the homogeneity of variance was analyzed by the Bartlett test. If the variance was homogeneous (p≥0.05), Tukey’s test was performed; if the variance was heterogeneous (p<0.05), the Tukey test (Joint-ranking) was performed. In all the analyses, the levels of significance were set at p<0.05, p<0.01 and p<0.001.

RESULTS

Serum Testosterone levels

Serum testosterone levels in the M/C, F/C, and F/TES groups are shown in Fig.1. The serum testosterone level in the M/C group increased by maturation from week 2 to week 4. In the F/C group, the testosterone level was lower than that in the M/C group and showed no change from week 2 to week 4. In the F/TES group as compared to the M/C group, the testosterone level at week 2 (after dosing of 1 mg/kg/day of testosterone for 2 weeks) was slightly high and at week 4 (after following dosing of 2 mg/kg/day of testosterone for 2 weeks) was almost comparable.

Fig. 1

Serum testosterone levels at week 2 and week 4 (n=6). Male-control (M/C), female-control (F/C), and female testosterone-treated (F/TES) groups are indicated by black, red, and green, respectively. Marks and bars represent the average and S.D., respectively. Significant differences were found, as follows. (* p<0.05 [between M/C and F/C]; ## p<0.01, ### p<0.001 [between F/C and F/TES]; $$$ p<0.001 [between M/C and F/TES])

Urinary excretion levels of biomarkers in the testosterone-administered female rats (F/TES group) were quite similar to those in the male rats

Significant differences in the urinary excretion levels of several biomarkers were observed between the M/C and F/C groups. The urinary levels of LAP, γ-GTP, β2MG, and L-FABP were higher in the M/C group than in the F/C group at all time-points (Fig. 2). The urinary levels of total protein at week 2 and week 4, Cys-C at week 2, and ALP at week 4 were higher, or tended to be higher (total protein at week 4, p = 0.0683), in the M/C group than in the F/C group (Fig. 3). The urinary levels of Kim-1 and albumin were higher in the F/C group than in the M/C at all time-points and at week 4, respectively. The levels of the other parameters were comparable between the M/C and F/C groups, with no significant sex differences in these parameters (Figs. 3 and 4).

Fig. 2

Urinary biomarker levels of LAP, γ-GTP, L-FABP, and β2MG at pre-treatment, week 2, and week 4 (n=6). Mark and bar represent the average and S.D., respectively. Significant differences were found, as follows. (* p<0.05, ** p<0.01, *** p<0.001 [between M/C and F/C]; # p<0.05, ### p<0.001 [between F/C and F/TES]; $ p<0.05, $$ p<0.01, $$$ p<0.001 [between M/C and F/TES])

Fig. 3

Urinary biomarker levels of total protein, albumin, Cys-C, ALP, Kim-1, and NGAL at pre-treatment, week 2, and week 4 (n=6). Mark and bar represent the average and S.D., respectively. Significant differences were found, as follows. (* p<0.05, ** p<0.01, *** p<0.001 [between M/C and F/C]; # p<0.05, ### p<0.001 [between F/C and F/TES]; $ p<0.05, $$ p<0.01, $$$ p<0.001 [between M/C and F/TES])

Fig. 4

Urinary biomarker levels of LDH, NAG, glucose, and clusterin at pre-treatment, week 2, and week 4 (n=6). Mark and bar represent the average and S.D., respectively.

There were no significant differences in the levels of any of the urinary biomarkers between the F/TES group and F/C group prior to the treatment period, confirming the absence of any biases between these 2 groups of female rats (Figs. 2, 3, and 4). On the other hand, there were significant differences in the urinary levels of LAP, γ-GTP, β2MG, and L-FABP between the M/C and F/TES group, just like between the M/C and F/C groups, prior to the start of the treatment period (Fig. 2). The urinary levels of ALP and Cys-C were significantly lower and those of Kim-1 tended to be higher (p = 0.088) in the F/TES group as compared with those in the M/C group prior to the treatment period (Fig. 3).

In the F/TES group, the urinary excretion levels of LAP, γ-GTP, β2MG, and L-FABP increased at week 4, but not at week 2 (Fig. 2). The levels were statistically significantly different or tended to be different (p = 0.0683 [γ-GTP], p = 0.0524 [β2MG], and p = 0.0597 [L-FABP]), as compared with those in the F/C group (Fig. 2). At week 2, the urinary excretion level of Cys-C increased, while that of Kim-1 decreased (Fig. 3). The levels of both parameters were statistically significantly different as compared with those in the F/C group and comparable to those in the M/C group. At week 4, the Cys-C level in the F/TES group remained higher than that in the F/C group. The urinary level of Kim-1 in the F/TES group remained comparable to that in the M/C group; however, it was not significantly different from that in the F/C group, due to a slight decline of the urinary Kim-1 excretion level in the F/C group. The urinary level of ALP was statistically significantly higher in the F/TES group at week 4 as compared with that in the F/C group and comparable with that in the M/C group (Fig. 3). The urinary levels of total protein and albumin in the F/TES group were comparable to those in the F/C group throughout the experimental period. There was a significant increase in the urinary NGAL excretion level in the F/TES group as compared with the M/C group at week 4. There were no remarkable differences in the urinary excretion levels of LDH, NAG, glucose, or clusterin among the groups (Fig. 4).

Effect of testosterone on the protein levels in the renal cortex

The LDH, ALP, and γ-GTP levels in the renal cortex were lower in the M/C group than those in the F/C group (Fig. 5). The ALP level was also lower in the M/C group than that in the F/TES group. There were no significant differences in the LAP or NAG levels among the 3 groups.

Fig. 5

Enzyme activities in the renal cortex at week 4. Bars represent the average and S.D. (n=6). Significant differences were found, as follows. (* p<0.05, ** p<0.01, *** p<0.001 [between M/C and F/C]; $$ p<0.01 [between M/C and F/TES])

The megalin levels in renal cortices of the M/C and F/TES were lower than the level in the F/C group, and the level in the M/C group was comparable with that in the F/TES group (Fig. 6).

Fig. 6

Megalin protein expression in the renal cortex at week 4. Bars mean the averages and S.D. (n=6). Significant differences were found, as follows. (** p<0.01 [between M/C and F/C]; ### p<0.001 [between F/C and F/TES])

DISCUSSION

Sex differences in physiological parameters have been investigated for a long time, in varied fields, such as the cardiovascular systems, immunological, lipid metabolism, and metabolic enzyme fields. In the field of drug development, the toxicities of candidate drugs have been investigated in toxicity studies conducted in rodents and non-rodents, both male and female. The toxicities of drug candidates should be judged taking into consideration the sex differences in physiological parameters. Therefore, investigation of sex differences in animals used in non-clinical studies would be useful to arrive at an appropriate toxicological judgement. We have previously reported that the existence of sex differences in the urinary excretion levels in rats, specifically male rats was higher than female rats for LAP, γ-GTP, total protein, β2MG, L-FABP, Cys-C, and female rats was higher than male rats for Kim-1, suggesting that testosterone might be one of the factors responsible for the sex differences (Tsuji et al., 2017).

In the present study, the serum testosterone levels in the F/TES group at week 2 and week 4 showed comparable or slightly high levels as compared to the M/C group. In addition, the testosterone level in the F/TES group was clearly high as compared to the F/C group. Therefore, we considered that the animals in the F/TES group were systemically exposed to the target testosterone level we expected throughout the study period.

Low-molecular-weight proteins filtered by the glomeruli and excreted in the urine have been recognized as biomarkers to detect damages in the renal tubules, especially in the proximal tubules. The present result of increased urinary excretion of β2MG, L-FABP, and Cys-C in the F/TES group compared to the F/C group was consistent with the previous result observed in castrated male rats, where blood levels of these proteins were comparable to those in sham-operated rats (Tsuji et al., 2017). From the thing suggested that testosterone did not have any major influence on the regulation of the levels of these proteins in the blood, we speculate that the urinary excretion levels of β2MG, L-FABP, and Cys-C can vary according to absorption in proximal tubules. In regard to NGAL, the F/TES group showed increased excretion level with a wide variability at week 4, which was different from the direction observed in the M/C group. The difference in direction of change was probably caused by variation of urinary CRE from week 2 to week 4. In the M/C group, the urinary CRE concentration increased by body weight gain, and urinary biomarker levels corrected by CRE were lower at week 4 than those at week 2 (Fig. 7). In contrast, variation of urinary CRE concentration in the F/TES was lower than that in the M/C group. Gautier et al. (2014) reported equivocal evidence of a sex difference in the urinary excretion levels. Difficulty of research of sex difference in urinary NGAL could be due to secretion mechanism from renal tubular cells as well as CRE variation.

Fig. 7

Curve of body weight change (A) and urinary CRE concentration (B) (n=6). Mark and bar represent the average and S.D., respectively.

Numerous receptors and transporters localized in the kidney regulate the urinary excretions of substances filtered through the glomeruli. Among these receptors, megalin and cubilin are known to mediate uptake of proteins by the proximal tubules (Nielsen et al., 2016). Leheste et al. (1999) reported that megalin-knockout mice developed low-molecular-weight proteinuria. Jensen et al. (2017) and Kaseda et al. (2007) reported that megalin played roles in the regulation of urinary Cys-C excretion and endocytosis of Cys-C in in-vivo and in-vitro test systems. Orlando et al. (1998) had demonstrated that megalin bound to β2MG. Oyama et al. (2005) revealed evidence of megalin-mediated L-FABP uptake in the proximal tubules. Hvidberg et al. (2005) reported that megalin bound to NGAL and contributed to its cellular uptake. On the other hand, cubilin shows specificities for different ligands, such as apolipoprotein A-I, high-density lipoprotein, and fibroblast growth factor (Nielsen et al., 2016). Based on the specificities of the receptors, we focused on megalin as a target receptor potentially associated with the sex differences in the urinary excretion levels of low-molecular-weight proteins. In the present study, the protein level of megalin in the renal cortex measured on the day after the last testosterone dosing was significantly lower in the M/C group than that in the F/C group. In addition, the megalin level in the F/TES group was significantly lower than that in the F/C group and comparable to that in the M/C group. There has been scant research on the sex difference in renal megalin expression. Veiras et al. (2017) reported that the expression of megalin protein in the renal cortex was higher in male rats than that in female rats. The experimental condition in that study was different from that in the present study in terms of the age of the rats. We examined the megalin protein expression level in rats aged 9 weeks old, by which time male rats seem to become sexually mature, while Veiras et al. examined the level in male rats with body weights of 200–225 g. Therefore, the reason for the conflicting results was speculated as being related to the difference in the degree of sexual maturation of the male rats between the two studies. It has been reported that the blood testosterone level gradually increases from 7 weeks of age and peaks by 9 to 10 weeks of age (Nazian, 1986). In our data, the testosterone level was markedly elevated at 9 weeks of age. From these reports and our data, we contend that the renal level of megalin could be regulated by testosterone, and that the decline in the expression level of megalin by testosterone treatment could explain the increased urinary excretion levels of urinary β2MG, L-FABP, and Cys-C.

Among the ligands of megalin, the excretion levels of albumin and clusterin showed no clear changes following testosterone treatment. Absorption of albumin in the proximal tubules is mediated by proteins such as megalin, cubilin, and clathrin (Molitoris et al., 2022). Therefore, it is speculated that uptake mediated by the other proteins could compensate for the reduced albumin absorption mediated by megalin. In regard to clusterin, we showed no sex difference in the urinary excretion of clusterin among the 3 groups. It has been reported that urinary clusterin level was higher in male rats than in female rats, and the urinary clusterin level in male rats showed greater variability compared to female rats (Gautier et al., 2014). That might suggest urinary clusterin level is affected or regulated by lots of factors or conditions.

There were differences in the degree of changes in the urinary excretion levels among low-molecular-weight proteins observed following testosterone treatment. Further studies are necessary to clarify the degree of contribution of megalin to the absorption of each protein.

The enzymes LDH, ALP, γ-GTP, and LAP excreted in the urine are useful biomarkers to detect damage of the renal tubules, where these enzymes are localized (Endou, 1978; Koseki et al., 1980). It has been reported that the urinary excretion of γ-GTP and LAP was higher in male rats than in female rats and castrated male rats (Grötsch et al., 1985; Girolami et al., 1989; Tsuji et al., 2017). In the present study, the F/TES group showed increased urinary excretion levels of LAP and γ-GTP as compared to the F/C group. The renal γ-GTP concentration was higher in the F/C group than that in the M/C group, and comparable between the M/C and F/TES groups. In regard to the renal LAP concentration, there were no significant differences among the 3 groups. It has been suggested that the increase in urinary LAP and γ-GTP excretion levels might result from promotion of cell death by testosterone (Verzola et al., 2004; Muraoka, 2001); however, we observed no changes in the excretion levels of the cell injury markers LDH and NAG in the present study. Considering the difference of the renal γ-GTP concentration mentioned above, we speculate that testosterone might promote the release of γ-GTP from the brush border membrane. There was no significant change in the renal concentration of LAP, which is known to be localized in the same part of the cell as γ-GTP. We presume that the mechanisms by which testosterone regulates the urinary excretion and renal expression of proteins might differ between γ-GTP and LAP.

Kim-1 is a well-known renal-injury-specific biomarker (Bonventre, 2008). Sex differences of urinary Kim-1 excretion level have been considered to be due to the difference of urinary CRE between male and female rats (Pinches et al., 2012). On the other hand, it has been reported that the castrated male rats showed higher urinary Kim-1 level than the sham-operated male rats, without no significant difference in urinary CRE levels (Tsuji et al., 2017). Contribution of urinary CRE level on sex differences of urinary Kim-1 has not been concluded.

In the present study, the F/TES group showed lower urinary Kim-1 level than the F/C group although there was no difference in the urinary CRE excretion level between them. The result suggested additional information on the mechanism of sex differences in urinary Kim-1 unrelated to urinary CRE excretion level and that testosterone contributes to urinary Kim-1 excretion. Further investigation is necessary to clarify the mechanism underlying the sex difference in urinary Kim-1 excretion.

In conclusion, exogenous testosterone changed the urinary excretion levels of biomarkers and renal megalin expression level in female rats to levels comparable to those in male rats. Our results suggest that the mechanism of sex differences in the urinary excretion levels of low-molecular-weight proteins such as β2MG, L-FABP, and Cys-C is possibly related to reabsorption via megalin which expression is regulated by testosterone.

Conflict of interest

The authors declare that there is no conflict of interest.

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