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. 2019 Nov 6;173(2):362–372. doi: 10.1093/toxsci/kfz225

Proximal Tubular Vacuolization and Hypersensitivity to Drug-Induced Nephrotoxicity in Male Mice With Decreased Expression of the NADPH-Cytochrome P450 Reductase

Liang Ding 1,2,#, Lei Li 3,#, Senyan Liu 3,4,#, Xiaochen Bao 3, Kathleen G Dickman 5, Stewart S Sell 3,6, Changlin Mei 4, Qing-Yu Zhang 1,3,6, Jun Gu 3,6,✉, Xinxin Ding 1,2,✉
PMCID: PMC8000068  PMID: 31693140

Abstract

The effect of variations in the expression of cytochrome P450 reductase (CPR or POR) is determined in mice with decreased POR expression to identify potential vulnerabilities in people with low POR expression. There is an age-dependent appearance of increasing vacuolization in the proximal tubules of the renal cortex in 4- to 9-month-old male (but not female) Cpr-low (CL) mice. These mice have low POR expression in all cells of the body and upregulation of lysosome-associated membrane protein 1 expression in the renal cortex. Vacuolization is also seen in extrahepatic CL and extrarenal CL male mice, but not in mice with tissue-specific Por deletion in liver, intestinal epithelium, or kidney. The occurrence of vacuolization is accompanied by increases in serum blood-urea-nitrogen levels. Male CL mice are hypersensitive to cisplatin- and gentamicin-induced renal toxicity at 3 months of age, before proximal tubular (PT) vacuoles are detectable. At doses that do not cause renal toxicity in wild-type mice, both drugs cause substantial increases in serum blood-urea-nitrogen levels and PT vacuolization in male but not female CL mice. The hypersensitivity to drug-induced renal toxicity is accompanied by increases in circulating drug levels. These novel findings demonstrate deficiency of renal function in mice with globally reduced POR expression and suggest that low POR expression may be a risk factor for drug-induced nephrotoxicity in humans.

Keywords: cytochrome P450 reductase, kidney injury, drug toxicity, nephrotoxicity, vacuolization, proximal tubules

NADPH-cytochrome P450 reductase (CPR or POR, for P450 oxidoreductase) is an essential oxidoreductase located primarily in the endoplasmic reticulum and provides electrons needed for the catalytic activities of all microsomal cytochrome P450 (P450) monooxygenases (Black and Coon, 1987). The POR/P450 system is responsible for the biotransformation of many endogenous and exogenous compounds (Guengerich, 2008; Porter and Coon, 1991). In human patients, numerous mutant POR alleles have been identified, of which many are associated with inborn deficiencies in the synthesis of steroid hormones and various clinical manifestations, including skeletal malformation and reproductive defects (Fluck and Pandey, 2011; Fukami and Ogata, 2014; Miller, 2012). Other, more frequent, genetic variations in POR also occur and have been associated with changes in the metabolism of certain drugs (Gomes et al., 2009; Gong et al., 2017; Zhang et al., 2011).

Interindividual differences in human POR expression are well documented (Gomes et al., 2009; Hart et al., 2008; Kaminsky et al., 1984; Shephard et al., 1992; Yamano et al., 1989); however, few studies have examined the clinical consequences of altered POR expression in patients. Variations in POR expression can influence P450 function (Gu et al., 2003, 2007; Hart et al., 2008; Henderson et al., 2003; Kaminsky et al., 1984; Wei et al., 2010; Weng et al., 2005; Wu et al., 2005; Zhang et al., 2007, 2011). Decreased POR expression may affect the homeostasis of endogenous compounds and cause pathogenic changes, as illustrated in the Cpr-low (CL) mice, which show global suppression of POR expression (Wu et al., 2005), the liver-specific Cpr-null (liver-Cpr-null) mice (fatty liver) (Gu et al., 2003; Henderson et al., 2003), and the intestinal epithelium-specific Cpr-null (IE-Cpr-null) mice (hypersensitivity to ricin-induced intestinal damage) (Ahlawat et al., 2014).

The CL mouse, produced via the insertion of a neomycin resistance gene in the last Por intron (Wu et al., 2005), is a valuable animal model for the identification of potential clinical consequences of a deceased POR expression in patients. The extent of decrease in POR expression in the CL mouse, approximately 70% to 90% in various tissues, is within the range of variations in POR levels found in human tissues (Gomes et al., 2009; Hart et al., 2008; Kaminsky et al., 1984; Shephard et al., 1992; Yamano et al., 1989). Like patients with rare POR mutations (Fluck and Pandey, 2011; Fukami and Ogata, 2014; Miller et al., 2011), CL mice show female infertility and disrupted steroid hormone homeostasis in both males and females (Wei et al., 2010; Wu et al., 2005). Cpr-low mice also display decreased plasma cholesterol levels, mild hepatic lipidosis, and reduced embryonic survival, phenotypes that are likely to also occur in humans with low POR expression.

The aim of this study was to further characterize the CL mouse, to identify additional phenotypes, which may predict potential clinical vulnerability in people with markedly reduced POR expression. The study focuses on the kidney, which had not been studied comprehensively in the CL mice, except for organ weights and POR/CYP expression. Cpr-low mice showed slightly decreased body weights in males and females, and small decreases in heart, lung, and kidney weights in males, at 3 months of age; kidney microsomes also showed significant increases in P450 levels in both males and females, in response to a large (approximately 80%) decrease in POR protein levels (Wu et al., 2005).

Histological examination of the kidneys of wild-type (WT) and CL mice revealed age- and sex-specific formation of abundant vacuoles in the renal proximal tubular (PT) epithelial cells of 4- to 9-month-old male CL mice, but not in female CL mice or WT mice. Subsequent studies were conducted to determine whether the PT vacuolization was caused by loss of Por in the kidney or elsewhere, using various tissue-specific Por knockout or knockdown mouse models, including IE-Cpr-null mice (Zhang et al., 2009), kidney-Cpr-null mice (Liu et al., 2013), liver-Cpr-null mice (Gu et al., 2003), extrahepatic-Cpr-low (xh-CL) mice (Wei et al., 2010), and extrarenal-Cpr-low (xr-CL) mice (Liu et al., 2013). The CL mice were further examined, to characterize the nature of the PT vacuoles, measure biomarkers of renal function, and determine sensitivities to drug-induced nephrotoxicity. The results of these studies suggest that low POR expression may be a risk factor for drug-induced nephrotoxicity in male patients.

MATERIALS AND METHODS

Mouse models

All studies with mice were approved by the Institutional Animal Care and Use Committee of the Wadsworth Center and the University of Arizona. Six mouse strains were used in this study, including WT, CL (Wu et al., 2005), kidney-Cpr-null (Liu et al., 2013), liver-Cpr-null (Gu et al., 2003), IE-Cpr-null (Zhang et al., 2009), xr-CL (Liu et al., 2013), and xh-CL (Wei et al., 2010), all on C57BL/6 background. Mice were allowed free access to water and food, and housed in an air flow-, temperature-, and light-controlled environment. For pharmacokinetics studies, blood samples (up to 20 µl each) from individual mice were collected from the tail vein at various time points.

Renal histopathology

The kidneys were dissected from mice immediately following euthanasia and fixed in 10% neutral buffered formalin. Paraffin-embedded tissues were sectioned at 4-μm thickness. Sections were stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS) reagents.

The severity of the renal vacuolization was assessed by grading as follows: −, no vacuoles detected; +/−, vacuoles affecting less than 5% of proximal tubules on a section, and are only luminal; +, affecting 10%–20% of the tubules, and vacuoles are larger but usually do not extend to basement membrane (BM); ++, affecting 20%–50% of the tubules, and some vacuoles now extend to BM; +++, affecting over 50% of the tubules, and most vacuoles extend to BM.

Transmission electron microscopy

Kidney samples were fixed in phosphate buffered 4% glutaraldehyde and post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.2, for 1 h, and processed using standard techniques. Sections (60–90 nm thick) were stained using uranyl acetate and lead citrate and examined on a Zeiss 910 TEM operating at 80 kV.

Immunohistochemical analysis

Formalin-fixed, paraffin-embedded, 4-µm sections were treated with an epitope retrieval solution (Biogenex, San Ramon, California) for 30 min at 96°C and a peroxidase-blocking solution (Dako, Carpinteria, California), and then incubated at room temperature with a rabbit anti-mouse lysosome-associated membrane protein 1 (LAMP-1) antibody (Santa Cruz, Burlingame, California) at 1:1000 for 60 min. The bound antibody was detected by peroxidase-conjugated secondary antibody and visualized with DAB substrate (Invitrogen, Carlsbad, California), followed by hematoxylin counterstaining. Negative-control sections were incubated with normal rabbit serum (Biogenex) in place of the primary antibody.

Biochemical analysis of the serum

Blood was taken from the heart immediately after euthanasia. The levels of BUN (Blood Urea Nitrogen) and Cre (Creatinine) were determined in serum samples, using commercial kits (for BUN, Diagnostic Chemicals, Oxford, Connecticut; for Cre, WAKO, Richmond, Virginia).

Drug-induced nephrotoxicity

Cpr-low and WT mice (3 months old, male and female) were studied. For cisplatin, mice were treated once with either saline (0.9% USP, sterile, RMBIO, Missoula, Montana) or cisplatin (≥ 98% USP, Spectrum Chemical, Gardena, California) in saline at 5 mg/kg via IP injection, and blood and kidney were collected 72 h later for BUN and Cre assays and pathological examination of the kidney (with H&E staining), as described above. For gentamicin, mice were treated with either saline or gentamicin (Sigma-Aldrich) in saline, at 60 mg/kg, once daily for 6 consecutive days, via IP injection, and blood and kidney were collected on 7th day (24 h after the last injection) for analysis.

Pharmacokinetic study of cisplatin and gentamicin

Cpr-low and WT mice (3 months old, male and female) were treated with a single dose of cisplatin (5 mg/kg) or gentamicin (60 mg/kg) via IP injection. Blood samples were collected from the tail vein for detection of drug levels. Pharmacokinetic parameters were calculated using PK solver (Microsoft, Redmond, Washington), by assuming a noncompartmental model.

Detection of gentamicin and cisplatin

Gentamicin and cisplatin were determined in serum samples from mice used for toxicity studies and in tail vein blood from the pharmacokinetics studies. Gentamicin was measured by using an ELISA Kit, according to the manufacturer’s protocol (BioVision, Inc, K4206). The absorbance values were read on a Versa Max tunable microplate reader (Molecular Devices, Sunnyvale, California). Cisplatin was represented by Pt, which was determined using a Perkin Elmer model ELAN DRC-II inductively coupled plasma mass spectrometer (ICP-MS) (Shelton, Connecticut), using the ICP-MS-68A-C certified standards (High-Purity Standards, Charleston, California). The diluent used for analyzing blood samples by ICP-MS contained 0.01% ammonium 1-pyrrolidinedithiocarbamate, 0.4% tetramethylammonium hydroxide, 1% ethanol, and 0.05% Triton X-100. The Pt data was collected in NH3-gas mode.

Statistical analysis

All data are expressed as means ± SD. A 2-way analysis of variance (ANOVA), followed by Tukey's test for pairwise comparisons, was used for comparison among various groups, whereas a paired t test was used for comparisons between two groups, with use of GraphPad Prism (GraphPad Software, La Jolla, California). p < .05 was considered statistically significant.

RESULTS

Age-Dependent and Sex-Specific PT Vacuolization in CL Mice

Marked vacuolization in proximal renal tubules is seen in the kidneys of male CL mice at 4, 6, and 9 months of age, but not in female CL mice or WT mice of both sexes at these ages, or in 2-month-old mice (Figure 1). The vacuoles were discrete, oval, and of various sizes, and found only in the S1/S2 segments of the proximal tubules, which are located primarily in the renal cortex (Figure 2).

Figure 1.

Figure 1.

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Age- and sex-dependent occurrence of PT vacuolization in Cpr-low (CL) mice. Light micrographs of representative hematoxylin and eosin-stained paraffin sections (4 μm) of kidneys from 6-month-old wild-type (WT) mice and 2- to 9-month-old CL mice are shown. Vacuoles (arrow) were detected in renal proximal tubules of male CL mice, but not in female CL mice or male or female WT mice. ×400 magnification. Scale bar: 50 μm.

Figure 2.

Figure 2.

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Regional distribution of vacuoles in the kidney. Representative images of periodic acid Schiff stained sections of kidney cortex and medulla from wild-type (WT) and Cpr-low (CL) mice (male, 6 months old) are shown. Arrowheads point to examples of brush borders on proximal tubules. ×600 magnification. Vacuoles (arrows) are abundant in the PT brush boarder cells of the renal cortex of the CL mouse, but they are not observed in the medulla of either WT or CL mice, or in the cortex of the WT mice. Scale bar: 25 μm.

The developmental changes in the extent of renal vacuolization in male CL mice were assessed by grading its severity. In male mice, mild vacuolization (+/− to +) was found in 75% of the slides from 4-month-old mice, and moderate to severe vacuolization (+ to +++) were found in all slides from 6- and 9-month-old mice (Table 1).

Table 1.

Extent of Renal Vacuolization in Male CL Mice

Age (months) Fraction of slides in grade (%)
− +/− to + + to +++
4 25 75 0
6 0 0 100
9 0 0 100
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For each group, 5 mice were studied, with more than 10 slides randomly selected from each mouse and analyzed for presence and severity of the renal vacuolization, as described in Materials and Methods. The values presented are percentages of slides that fall under a particular grade for each mouse group.

Association of the Renal PT Vacuoles With Lysosomes

Further analysis using a transmission electron microscope (TEM) (Figure 3A) showed that the PT vacuoles were clear, “empty,” and localized in intracellular cytoplasm of the proximal tubules. Although some vacuoles appeared as singlets of various sizes, others appeared in groups or were associated with lysosome-like osmophilic structures.

Figure 3.

Figure 3.

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Apparent association of vacuoles with lysosome. A, Transmission electron microscopy analysis of vacuoles in the renal proximal tubules. Representative sections from 6-month-old male wild-type (WT) and Cpr-low (CL) mice are shown. Vacuoles (examples indicated by arrows) were detected in the cytosol of tubular cells and ranged from small, clear structures to large clusters associated with osmophilic bodies consistent with lysosomes. Scale bar: 2 µm. B, Immunohistochemical localization of lysosome-associated membrane protein 1 (LAMP-1) in renal cortex. Paraffin sections (4 μm) of kidney cortex from WT and CL mice, male, 6 months old, were analyzed. In CL mice, the vacuolated proximal tubules showed elevated LAMP-1 expression (examples indicated by arrows). The negative controls (with normal rabbit serum) were also shown. ×200 magnification. Scale bar: 50 μm.

Immunohistochemical analysis for LAMP-1 (Figure 3B) showed that, in 6-month-old CL male mice, the boarders of the vacuoles were clearly positive for LAMP-1, and the vacuole-containing PTs showed much more prominent overall staining for LAMP-1 than other areas, which suggested occurrence of lysosomal expansion. Additional histochemical analysis confirmed that the vacuoles were negative for lipids (by Oil-red O stain or Luxol Fast Blue stain; Supplementary Figs. 1 and 2), or glycogen (by PAS stain; Figure 2).

Tissue Origin of the PT Vacuolization Phenotype

As a first step toward identifying the mechanisms of PT vacuolization, we determined whether the vacuolization is due to decreased POR expression in the kidney, or in the portal-of-entry organs that control dietary intake of nutrients—the liver and the intestine—which may impact the workload of the renal tubules responsible for waste excretion. As shown in Figure 4, PT vacuolization was not detected in 6-month-old male mice with tissue-specific Por deletion in hepatocytes (liver-Cpr-null), enterocytes (IE-Cpr-null), or kidney PT epithelial cells (kidney-Cpr-null); but it is observed in 6-month-old male CL mice, as well as in xh-CL and xr-CL mice, where POR expression was rescued in the liver and proximal tubules, respectively. These results suggest that the PT vacuolization found in the male CL mice was not dependent on the suppression of POR/P450 activities in any of the 3 tissues (liver, intestine, or renal proximal tubules), but was due to systemic changes resulting from the suppression of POR expression in 1 or more other organs.

Figure 4.

Figure 4.

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Association of proximal tubular (PT) vacuolization with Cpr-low (CL) status. PT vacuolization (examples are indicated by arrows) was observed in CL (A), extrahepatic-CL, (B) and extrarenal-CL (C) mice (6 months old, male), but not in IE- (D), liver- (E), or kidney-Cpr-null (F) mice (6 months old, male). Light micrographs of representative hematoxylin and eosin-stained paraffin sections (4 μm) are shown. ×400 magnification. Scale bar: 50 µm.

Association of the PT Vacuolization With Changes in Renal Function

Serum BUN levels and BUN/Cre ratios were significantly higher in male CL mice than in male WT mice at 6–7 months of age (by 25%–27%; Figure 5A). Serum BUN levels were also compared between WT and xh-CL mice (Figure 5B), and found to be similar in 2-month-old WT and xh-CL mice, male or female, but significantly higher in male xh-CL mice than in male WT mice or female xh-CL mice at 6 months (53%–68%) or 9 months (38%–39%) of age. Thus, the PT vacuolization in CL and xh-CL male mice was accompanied by mild changes in renal function.

Figure 5.

Figure 5.

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Effects of Por status and drug treatment on renal function. A, Serum levels of BUN and Cre in 6- to 7-month-old male wild-type (WT) and Cpr-low (CL) mice. Serum samples of male mice (n = 5 per group) were analyzed for BUN and Cre levels. **p < .01; ****p < .0001; versus WT male; paired t test. B, Serum BUN levels of 2-, 6-, and 9-month-old WT and extrahepatic-Cpr-low (xh-CL) mice. *p < .05; ***p < .001; versus WT male; &p < .05; &&p < .01; versus xh-CL female; n = 5. C, Cisplatin effects on renal function. The levels of BUN and Cre were determined in serum samples of WT and CL mice (3 months old) obtained at 72 h after a single intraperitoneal injection of cisplatin (5 mg/kg), as described in Materials and Methods. ****p < .0001; versus cisplatin-treated WT male or CL female, or saline-treated CL male; n = 3. D, Gentamicin effects on renal function. The levels of BUN and Cre were determined in serum samples of WT and CL mice (3 months old) at 24 h after 6 intraperitoneal injections of gentamicin (once daily at 60 mg/kg), as described in Materials and Methods. ****p < .0001; versus gentamicin-treated WT male or CL female, or saline-treated CL male; n = 3. Two-way ANOVA followed by Tukey’s test for multiple comparisons (B–D).

Hypersensitivity of Male CL Mice to Cisplatin- and Gentamicin-Induced Nephrotoxicity

Treatment with cisplatin at 5 mg/kg (a single IP injection), or gentamicin at 60 mg/kg (6 IP injections, once daily), caused significant increases in serum BUN level and BUN/Cre ratio in male CL mice (approximately 2-fold for cisplatin and approximately 3-fold for gentamicin, over saline control), but not in female CL mice or in male or female WT mice, all at 3 months of age (Figs. 5C and 5D). The drug-induced BUN increases were accompanied by marked histopathological changes in the kidney of male CL mice, which included moderate to marked, acute, diffuse, vacuolar degeneration of proximal tubules with infrequent tubular epithelial necrosis and intratubular proteinaceous casts (Figs. 6A and 6B). No vacuoles or other pathological changes were observed in the other groups.

Figure 6.

Figure 6.

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Histopathological evidence for hypersensitivity of male Cpr-low (CL) mice to cisplatin- and gentamicin-induced renal toxicity. Light micrographs of representative hematoxylin and eosin-stained paraffin sections (4 μm) of kidneys from cisplatin (A) or gentamicin (B) treated wild-type (WT) and CL mice (male and female, 3 months old). Tissues were obtained at 72 h after a single intraperitoneal injection of cisplatin (5 mg/kg) or 24 h after 6 intraperitoneal injections of gentamicin (once daily at 60 mg/kg), as described in Materials and Methods. Extensive vacuolization was observed in the drug-treated CL male mice (examples indicated by arrows), but not in WT male or female or CL female mice. Vacuoles were not observed in any of the saline-treated groups (examples are shown for saline-treated male and female CL mice). Other drug-induced pathological changes included tubular epithelial necrosis and intratubular proteinaceous casts (examples indicated by arrowheads and stars, respectively). ×400 magnification. Scale bar: 50 µm.

Pharmacokinetic Profiles of Gentamicin and Cisplatin in Male and Female CL Mice

The serum drug concentrations determined at the time of tissue harvesting for pathological analysis (72 h after a single IP injection of cisplatin or 24 h after the last of the 6 IP injections of gentamicin) of 3-month-old WT and CL mice are shown (Figs. 7A and 7B). Both drugs were at higher levels in CL mice than in WT mice, either male or female. Circulating drug levels were further determined for WT and CL mice at various times after a single IP injection at 5 mg/kg (cisplatin) or 60 mg/kg (gentamicin) (Figs. 7C and 7D). No difference was observed among the 4 groups (male, female, WT, CL) in the maximal drug levels achieved, but significant differences were found in the elimination phase, where higher drug levels (slower elimination) were found in CL mice (both male and female) than in WT mice. Pharmacokinetic analysis indicated significant decreases in clearance rate (CL/F; 50%–67% for cisplatin and 25%–67% for gentamicin) and increases in area under the concentration-time curve (40%–53% for cisplatin and 29%–33% for gentamicin) in both male and female CL mice than in corresponding WT mice (Supplementary Table 1).

Figure 7.

Figure 7.

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Pharmacokinetic profiles of cisplatin and gentamicin. A, Cisplatin levels in serum at 72 h after treatment. Wild-type (WT) and Cpr-low (CL) mice (3 months old) were treated with a single intraperitoneal injection (5 mg/kg) of cisplatin. ***p < .001; CL male versus WT male; &p < .05; CL male versus CL female; #p < .05; CL female versus WT female; n = 3. B, Gentamicin levels in serum at 24 h after a 1-week treatment. Wild-type and CL mice (3 months old) were treated with gentamicin at 60 mg/kg for 6 consecutive days by intraperitoneal injection. Serum samples were collected on 7th day (24 h after the last injection). *p < .05; CL male versus WT male; #p < .05; CL female versus WT female; n = 3. C, Cisplatin whole blood concentration-time curve after a single injection. Wild-type and CL mice (3 months old) were treated with a single intraperitoneal injection (5 mg/kg) of cisplatin. Blood samples were collected from the tail vein at various times after the injection. ***p < .001; ****p < .0001; CL male versus WT male; #, ##, ####p < .05, .01, .0001, respectively; CL female versus WT female; n = 3. D, Gentamicin plasma concentration-time curve after a single injection. Wild-type and CL mice (3 months old) were treated with a single intraperitoneal injection (60 mg/kg) of gentamicin. Blood samples were collected from the tail vein at various times after the injection. *, **, ****p < .05, .01, .0001, respectively; CL male versus WT male; #, ##, ###, ####p < .05, .01, .001, .0001, respectively; CL female versus WT female; n = 3. Two-way ANOVA followed by Tukey’s test for pairwise comparisons (A–D).

DISCUSSION

Proximal tubular vacuolization has been observed in many renal diseases and xenobiotic-induced renal injuries (Andoh et al., 1994; Brady et al., 1990; Christensen and Maunsbach, 1979; dos Santos et al., 2012; Isik et al., 2006; Kambham et al., 2007; Maunsbach et al., 1962; Visweswaran et al., 1997). Though PT vacuolization is not a specific diagnostic finding in renal pathology, many ischemic (Smith et al., 2006) or toxic injuries to the kidney can lead to PT vacuolization. The molecular mechanism for PT vacuolization in the male CL mice is unclear, but it likely reflects changes in the abundances of certain circulating compounds that are caused directly or indirectly by the chronic suppression of POR expression. The age dependency of PT vacuolization suggests that it may be a result of gradual accumulation of certain endogenous or dietary compounds in the PT cells; whereas the male-specific occurrence of the vacuolization implies that it is directly or indirectly related to androgen homeostasis. Cpr-low mice are known to have elevated levels of serum testosterone compared with WT mice, whereas males of either genotype have much higher testosterone levels than females of corresponding genotype (Weng et al., 2010).

Cytoplasmic vacuolization may result from accumulation of a variety of substances or cellular components, such as lipid droplets, water, glycogen, plasma, and lysosome (Decleves et al., 2014; Dickenmann et al., 2008; Morishita et al., 2005; Obert et al., 2007). The PT vacuoles in the CL mice appeared to be clear upon TEM examination (Figure 3). They did not contain fat, based on results of oil-red O stain, which detects neutral triglycerides and lipids (Obert et al., 2007), or glycogen, based on results of PAS stain, which detects structures containing a high proportion of carbohydrates (Ulusoy and Eren, 2006). However, the TEM result also suggests that the vacuoles may have a lysosomal origin, an idea further confirmed by immunohistochemical detection of enrichment of a lysosomal marker, LAMP-1, at sites of vacuolization. In addition, studies using a unique set of mouse models with tissue-specific Por knockout or rescue showed that the PT vacuolization is not caused by loss of POR function in the liver, intestine, or renal PT cells. It remains to be determined whether decreases in POR expression and androgen metabolism in the testis led to the renal PT vacuolization.

The antibiotic drug gentamicin and the anticancer drug cisplatin, both commonly used in clinical therapy, are frequently associated with drug-induced renal toxicity in patients; these 2 drugs are also widely used in animal models of acute kidney injury in mice and rats (Singh et al., 2012). Our finding that CL male mice are hypersensitive to gentamicin and cisplatin-induced renal toxicity suggests that low POR expression is a risk factor for renal toxicity induced by these drugs. Notably, the prevalence of gentamicin nephrotoxicity in men was found to be greater than in women (Bertino et al., 1993; Moore et al., 1984); whereas, a male selectivity in cisplatin renal toxicity has not been demonstrated in clinical studies (Bhat et al., 2015; Chen et al., 2017; de Jongh et al., 2003; Latcha et al., 2016). In mice, the male specificity in drug-induced renal toxicity is consistent with the male specificity of the age-dependent appearance of PT vacuoles. It is, however, unclear whether the PT vacuolization is the cause of the increased drug sensitivity, as the mice that we tested for drug toxicity were 3 months old, an age when PT vacuolization did not occur without the drug treatments (Figure 6). Further studies are needed to determine whether ultrastructural changes in the PT cells occurred in the CL male mice, which may correlate with the male-specific drug hypersensitivity.

The CL status is associated with higher serum levels of cisplatin and gentamicin in both males and females, compared with WT mice. Although an increase in drug bioavailability could potentially contribute to an increase in toxicity, the lack of a robust sex specificity of this increase in circulating drug levels suggests that this result alone does not explain the male-specific hypersensitivity. The CL mice have compensatory upregulation of P450 expression in the kidney, which occurred in both males and females (Wu et al., 2005). In that connection, the pathogenic mechanisms of renal toxicity by cisplatin and gentamicin are believed to involve overproduction of reactive oxygen metabolites (Liu and Baliga, 2003; Quintanilha et al., 2017). Though cisplatin and gentamicin do not require bioactivation by P450 enzymes to cause toxicity, the enzymes may be a source of the catalytic iron for oxidant production in gentamicin- and cisplatin-induced nephrotoxicity (Baliga et al., 1998, 1999; Huang and Schacht, 1990). However, although an induction in P450 expression may contribute to the renal toxicity induced by these drugs, it would not explain the male-specific hypersensitivity in the CL mice. It is possible that the elevation in drug bioavailability or the upregulation in P450 expression, combined with certain male-specific cellular or molecular changes in CL kidney, was responsible for the renal toxicity.

The mechanism for the decreased excretion of the 2 drugs in the CL mice has not been determined. Given that these drugs are not P450 substrates, the decrease in POR expression would not directly lead to a decrease in their disposition. A previous genomic analysis of the CL mouse liver did not reveal significant changes in the expression of major drug transporters (Weng et al., 2005); but it remains to be determined whether the expression of relevant drug transporters was changed in the kidney of these mice, leading to the drug hypersensitivity.

The functional impact of a low POR expression in mice and humans will likely be similar (Riddick et al., 2013). Known phenotypes of human POR deficiency (due to loss-of-function mutations of the POR protein), including disordered steroidogenesis and skeletal malformation, are also found in the CL mice (Wu et al., 2005), and a bone-specific Cpr-null mouse (Panda et al., 2013), respectively. A clinical association between POR deficiency and renal diseases has not been reported; though, corresponding to the male-specificity of the PT vacuolization in the CL mouse, the incidence of chronic kidney diseases in humans is also greater in males than in females (Halbesma et al., 2008; Iseki, 2008).

In summary, we report widespread vacuolization in renal PT epithelial cells of 4- to 9-month-old male mice with chronic suppression of POR expression. These vacuoles appeared to be of a lysosomal origin. Their formation was not triggered by loss of POR expression in liver, intestine, or kidney, but is associated with mild increases in serum BUN levels. Furthermore, male CL mice were remarkably hypersensitive to cisplatin- and gentamicin-induced renal toxicity at 3-months of age, before PT vacuoles were detectable. These novel findings provide a unique opportunity to further identify molecular mechanisms underlying the in vivo functions of POR-dependent enzymes and implicate potential renal disease risks in people with low POR expression, especially in males.

DECLARATION OF CONFLICTING INTERESTS

The author/authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Supplementary Material

kfz225_Supplementary_Data
Click here for additional data file. (586.9KB, zip)

ACKNOWLEDGMENTS

We gratefully acknowledge the use of the Biochemistry, Advanced Light Microscopy, Electron Microscopy, and Histopathology Core facilities of the Wadsworth Center, the University of Arizona Cancer Center Tissue Acquisition and Cellular/Molecular Analysis Shared Resource, and the Arizona Laboratory for Emerging Contaminants. We also thank Ms Weizhu Yang for assistance with mouse breeding.

FUNDING

This research was supported in part by National Institutes of Health (ES018884, GM082978, ES006694, ES020867, CA023074, and CA092596).

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