Establishment of a Rat Model for Renal Anemia Based on 5/6 Nephrectomized Rats

Abstract

Chronic kidney disease (CKD) progresses to renal fibrosis and anemia, ultimately leading to chronic renal failure (CRF). Renal anemia, primarily caused by impaired erythropoietin (EPO) production and dysregulated iron metabolism, reduces QOL and increases cardiovascular risk. To establish an experimental model of CRF-associated anemia, male Wistar rats underwent 5/6 nephrectomy (Nx). In a subset of Nx rats, tacrolimus (TAC, 1 mg/kg, subcutaneously every other day for two weeks starting at week 4) was administered to exacerbate renal injury. Renal function, hematological parameters, iron-related indices, and fibrotic changes were evaluated at defined postoperative time points. The Nx rats developed progressive renal dysfunction and interstitial fibrosis, accompanied by declining hematocrit and reduced plasma EPO levels and attenuation of renal hypoxia-inducible factor-2α expression. TAC administration further aggravated renal injury and anemia and resulted in a lower hematocrit despite detectable circulating EPO levels. This pattern may suggest a relative inadequacy of erythropoietic response under aggravated renal injury conditions rather than absolute EPO deficiency. These findings indicate that the Nx model reproducibly recapitulates key features of CKD-associated anemia. The addition of TAC accelerates pathological progression within this established model and facilitates the induction of advanced renal injury. This experimental system provides a practical preclinical platform for investigating the molecular mechanisms underlying CKD/CRF-related anemia and for evaluating therapeutic strategies targeting fibrosis, iron metabolism, or impaired erythropoietic response.

INTRODUCTION

Chronic kidney disease (CKD) is frequently accompanied by renal anemia, a complication primarily caused by insufficient erythropoietin (EPO) production from damaged renal tissue.¹⁾ Renal anemia is associated with reduced QOL, increased cardiovascular risk, and poor prognosis in patients with CKD.²⁾ Although recombinant EPO therapy has improved clinical management, the pathophysiological mechanisms underlying impaired erythropoiesis during progressive renal failure remain incompletely understood.

The 5/6 nephrectomy (Nx) rat model is widely used to reproduce progressive chronic renal failure (CRF) and has been reported to exhibit anemia during advanced stages of renal dysfunction.^3,4) However, the severity and onset of anemia vary depending on experimental conditions, and reductions in EPO production are not always consistently reproduced.⁵⁾ In addition, many existing models primarily focus on renal fibrosis and functional decline, whereas the temporal relationship between progressive renal injury and impaired erythropoiesis has not been sufficiently characterized.⁶⁾ Therefore, a well-defined and reproducible experimental model specifically suited for evaluating renal anemia associated with CRF progression remains necessary.

Tacrolimus (TAC), a calcineurin inhibitor widely used as an immunosuppressive agent in solid organ transplantation, is well known for its nephrotoxic properties.⁷⁾ Prolonged TAC exposure often exacerbates renal fibrosis and functional decline, contributing to chronic allograft dysfunction.⁸⁾ Because of its capacity to aggravate renal injury under conditions of reduced renal reserve, TAC has also been utilized experimentally to accelerate the progression of CKD in animal models.⁹⁾ From a pathophysiological perspective, TAC-induced aggravation of renal damage may further impair erythropoietic regulation in CKD. However, whether TAC accelerates the development of renal anemia during progressive renal failure has not been fully clarified.

In the present study, we aimed to establish and characterize a reproducible rat model of renal anemia based on Nx and to evaluate the impact of TAC on erythropoietic impairment during CRF progression. By longitudinally assessing renal function, hematological parameters, and renal fibrosis, we sought to clarify the relationship between progressive renal injury and anemia in this experimental setting.

MATERIALS AND METHODS

Test Substances and Reagents

The TAC (FK-506) was purchased from LC Laboratories, Inc. (Boston, MA, U.S.A.). Midazolam for preparing the mixed anesthetic solution was obtained from Sandoz, Inc. (Tokyo, Japan), while medetomidine and butorphanol were purchased from Meiji Seika Pharma Co., Ltd. (Tokyo, Japan) and combined according to standard protocols. Cremophor EL (20% [w/v]; Nacalai Tesque, Inc., Kyoto, Japan) dissolved in physiological saline was used as the vehicle for drug administration. All other reagents used in this study were of analytical grade.

Experimental Animals

Male Wistar rats (Japan SLC, Hamamatsu, Japan), 6–7 weeks old and weighing 150–180 g, were purchased and acclimatized before the experiment. The animals were maintained in controlled environmental conditions consisting of a 12-h light/dark cycle, a temperature of 23 ± 2°C, and 55 ± 10% humidity, with free access to standard chow and filtered tap water. All animal procedures were conducted in accordance with the Animal Experimentation Guidelines of Himeji Dokkyo University and were approved by the Institutional Animal Care and Use Committee (Approval No. R5-06).

In Vivo Rat Experiments

Establishment of Anemia Models with or without Blood Sampling

An Nx model was employed to reproduce renal anemia associated with CRF. Mixed anesthetic agents were administered subcutaneously at 1 mL/kg body weight, followed by right Nx and the partial ligation of the left renal artery using a previously described method.^4,10–12) To evaluate (i) the development of renal anemia after Nx and (ii) the effect of TAC under reduced renal reserve, animals were allocated into seven predefined groups (n = 5 per group), based on surgery (Sham vs. Nx), pharmacological treatment (none/vehicle/TAC), and whether serial blood sampling (BC) was performed.

The seven experimental groups were defined as follows: (1) Sham (BC+): sham-operated rats with serial blood sampling at weeks 3, 6, and 9; (2) Nx (BC+): Nx rats with serial blood sampling at weeks 3, 6, and 9; (3) Sham + vehicle (BC−): sham-operated rats receiving vehicle, no interim tail-vein sampling, and terminal sampling at week 6; (4) Nx + vehicle (BC−): Nx rats receiving vehicle, no interim tail-vein sampling, and terminal sampling at week 6; (5) Nx + TAC (BC−): Nx rats receiving TAC, no interim tail-vein sampling, terminal sampling at week 6; (6) Sham (BC−): sham-operated rats evaluated only at week 9 without interim blood sampling; and (7) Nx (BC−): Nx rats evaluated only at week 9 without interim blood sampling.

For BC (+) groups, blood sampling was performed from the tail veins of two groups at weeks 3, 6, and 9 after surgery (groups 1 and 2). The cumulative sampling volume was kept below 0.3% of total body weight. For BC (−) groups, samples were obtained only at the terminal time point without interim blood sampling (week 6 for groups 3–5; week 9 for groups 6–7).

Based on our previous study showing that TAC (5 mg/kg/d for two weeks) did not significantly affect renal function in normal rats,⁹⁾ TAC was administered after a four-week recovery period following Nx. TAC (1 mg/kg, every other day for two weeks) was administered subcutaneously to accelerate progressive renal dysfunction in Nx rats (group 5). Vehicle was administered on the same schedule in the corresponding vehicle groups (groups 3 and 4). At week 6 after surgery, hematological parameters were compared among groups 3–5.

Evaluation of Anemia-Related Parameters and Renal Function

After sacrifice by aortic bleeding under deep anesthesia, information on kidney weight, body weight, urine, and blood was collected at each sampling point. The hematocrit (Hct) was determined by microhematocrit centrifugation (12000 rpm, 5 min) and read using an Hct reader. The plasma EPO levels were measured using a Rat EPO enzyme-linked immunosorbent assay Kit (Abcam plc, Cambridge, U.K.). To evaluate temporal changes in EPO levels within the same individuals, the change from baseline (ΔEPO) was calculated as Δi,t = Xi,t − Xi,0, where Xi,t represents the EPO concentration (pg/mL) of individual i at postoperative week t, and Xi,0 represents the baseline value (week 0) of the same individual. The plasma iron and unsaturated iron-binding capacity (UIBC) were measured spectrophotometrically using the ferrozine and bathophenanthroline methods to assess the iron metabolism. Total iron-binding capacity (TIBC) was calculated, and transferrin saturation (TSAT) was derived using the following equation:

  

$$TSAT\left(\text{\%}\right)=\frac{Ironconcentration\left(\raisebox{1ex}{$\mu g$}\!\left/ \!\raisebox{-1ex}{$dL$}\right.\right)}{TIBC\left(\raisebox{1ex}{$\mu g$}\!\left/ \!\raisebox{-1ex}{$dL$}\right.\right)}\times 100$$

All measurements were performed using Metallogenics Iron (Fe) Assay Kit and Microassay UIBC Assay Kit (Metallogenics Co., Ltd., Chiba, Japan). Results were compared with those of the Sham group to validate the anemia model.

Assessment of Renal Function and Renal Injury

The blood samples were centrifuged immediately after collection. Then, the plasma was used to measure the plasma creatinine levels using the LabAssay^TM Creatinine Kit (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Creatinine concentration (Ccr) (mL/min) was calculated according to the following equation, and corrected for the body weight of each animal (mL/min/kg):

  

$$Ccr=\frac{UCr\times UV}{PCr\times Ct}$$

where UCr is the urinary creatinine concentration (mg/dL), UV is the urine volume (mL), PCr is the plasma creatinine concentration (mg/dL), and Ct is the collection period (min).

The blood urea nitrogen (BUN) levels were measured with the Invitrogen^TM Urea Nitrogen (BUN) Colorimetric Detection Kit (Thermo Fisher Scientific, Waltham, MA, U.S.A.), respectively. The plasma and urinary albumin concentrations were determined using the QuantiChrom^TM BCG Albumin Assay Kit (BioAssay Systems, Hayward, CA, U.S.A.). The urinary albumin-to-creatinine ratio (UACR) was calculated as an indicator of renal damage.

Morphological Evaluation of the Renal Lesions

The remnant left kidney was fixed in Carnoy’s solution (ethanol : chloroform : acetic acid = 6 : 3 : 1), embedded in paraffin, and sectioned into 3–4-μm-thick pieces. Masson’s trichrome (MT) and Picro-Sirius Red (PSR) staining were performed to evaluate renal interstitial fibrosis.^4,10–12) PSR staining was used for quantitative assessment of total collagen deposition, whereas MT staining was used to visualize interstitial fibrotic remodeling within the heterogeneous renal tissue architecture. Periodic acid–Schiff (PAS) staining was additionally performed in the TAC-treated rats to assess glomerular structural changes and the tubular brush-border injury.

The stained specimens were observed under a BZ9000 microscope (KEYENCE Ltd., Osaka, Japan), while the ImageJ software (National Institutes of Health, Bethesda, MD, U.S.A.) was employed for the quantitative image analysis of the fibrotic and PAS-positive areas.

Western Blot Analysis

The left remnant kidney was rapidly excised, frozen in liquid nitrogen, and stored at −80°C until use. Approximately 100 mg of frozen tissue was homogenized using Sepasol^®-RNA I Super G (Nacalai Tesque), and the protein fraction was prepared following acid guanidine thiocyanate–phenol extraction according to the manufacturer’s protocol. Protein concentrations were determined by the Bradford assay.¹³⁾ Equal amounts (20 μg) of denatured protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes using a semi-dry transfer system.

The membranes were blocked to prevent nonspecific binding and incubated with primary antibodies: anti-α-smooth muscle actin (α-SMA, 1 : 1000; Sigma-Aldrich, Tokyo, Japan) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1 : 20000–1 : 40000; Proteintech, Chicago, IL, U.S.A.). Goat anti-mouse immunoglobulin G (Abcam, Cambridge, U.K.) was used as the secondary antibody. The chemiluminescent signals were visualized using ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) and detected with LAS3000 (FUJIFILM Healthcare, Tokyo, Japan). The band intensities were normalized to GAPDH and quantified using ImageJ.

Immunohistochemical Examination

The remnant kidney fixed in Carnoy’s solution, embedded in paraffin, and sliced to a thickness of 3–4 μm was used for subsequent immunohistochemical staining. After deparaffinization, heat-induced antigen retrieval was performed using microwave treatment (pH 9, 15 min). Endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide in methanol at room temperature for 30 min, followed by washing with phosphate-buffered saline. Sections were then incubated with anti-hypoxia–inducible factor-2α (HIF-2α) antibody (1 : 500; #NB100-122, Novus Biologicals, LLC, Centennial, CO, U.S.A.) for 60 min at room temperature. After primary antibody incubation, the sections were treated with a secondary antibody (EnVision+ System-HRP Labelled Polymer Anti-Rabbit, Agilent Technologies, Inc., Santa Clara, CA, U.S.A.) and visualized using DAB+ Liquid (Agilent Technologies, Inc.). Nuclear counterstaining was performed with hematoxylin. Finally, the sections were dehydrated, mounted with a mounting medium (PathoMount^®, FUJIFILM Wako Pure Chemical Corporation), and examined using a BZ9000 microscope (KEYENCE Ltd.). Immunohistochemical staining was quantified using the H-score (histochemical-score) method according to the following equation, with a maximum score of 200. Because staining intensity was quantified using grayscale values in ImageJ (higher values indicate stronger 3,3′-diaminobenzidine [DAB] signal), nuclei were categorized as strongly positive (60–119), weakly positive (30–59), or negative (0–29).¹⁴⁾

  

$$\text{H-score\hspace{0.17em}}=(1\times \text{\hspace{0.17em}weak }[\%])+(2\times \text{\hspace{0.17em}strong\hspace{0.17em}\hspace{0.17em}}[\%])$$

Nuclei were segmented for scoring, and glomeruli were excluded from the analysis; only nuclei within tubular and interstitial regions were evaluated. Data are summarized as mean ± standard deviation (S.D.) consistently, in line with international reporting guidelines (SAMPL and ICMJE). For each animal, one representative paraffin section was randomly selected for analysis. Approximately five non-overlapping fields within the tubular and interstitial regions were evaluated per section, and the mean value per animal was calculated. Because HIF-2α immunostaining showed considerable heterogeneity in staining intensity and distribution, the analysis was regarded as semi-quantitative and exploratory in nature. Therefore, the results are presented descriptively as mean ± S.D. (n = 5 per group) without formal statistical comparison.

Statistical Analysis

Data are expressed as mean ± S.D. unless otherwise specified in the figure or table legends. Statistical analyses were performed using one- or two-way ANOVA, followed by appropriate post hoc multiple comparison tests, or other appropriate tests as specified in each figure and table legend. A p-value <0.05 was considered statistically significant. Data analysis was conducted using JMP^® Pro version 15.2.0 (SAS Institute Japan Ltd., Tokyo, Japan).

RESULTS

Changes in Body Weight, Kidney Weight Ratio, and Renal Function Parameters

To evaluate the time-dependent progression of renal anemia, serial blood sampling was intentionally performed in this experimental series. First, body weight, left kidney weight, the kidney weight-to-body weight ratio, and renal function-related biochemical parameters were evaluated for the validity of the CRF model (Table 1). At week 9 after surgery, the Nx group exhibited significantly lower body weight and significantly higher left kidney weight and kidney weight-to-body weight ratio compared with the Sham group. The preoperative body weights were comparable among animals, but a significant increase was observed from week 3 to week 9. PCr and BUN levels were significantly elevated in the Nx group at week 9 compared with the Sham group (Table 1). The PCr and BUN levels in the Nx group increased in a time-dependent manner. The median UACR was elevated approximately 11-fold compared to the Sham group at 9 weeks after surgery (Table 1).

Table 1. Body Weight and Renal Injury Markers in Sham and Nx Rats

Treatment	Sham	Nx
Time after the surgery (weeks)	9	3	6	9
	n = 5	n = 5	n = 5	n = 5
Body weight (g)	307 ± 12	179 ± 21	225 ± 23††	229 ± 16***, ††
Left kidney (g)	1.1 ± 0.1	0.6 ± 0.11	0.8 ± 0.05†††	0.7 ± 0.04***, †
Left kidney/Body weight (%)	0.4 ± 0.02	0.3 ± 0.04	0.4 ± 0.03†	0.3 ± 0.01‡
Plasma creatinine (mg/dL)	0.6 ± 0.0	1.0 ± 0.1	1.2 ± 0.2	1.5 ± 0.1***
BUN (mg/dL)	26 ± 0	50 ± 3	66 ± 3 ††	86 ± 3***, †††, ‡‡
UACR (mg/g)	2 (2–3)	7 (5–10)	14 (10–39)	23 (18–29) §§

Animals were euthanized at weeks 3, 6, and 9 after surgery, and all measurements were obtained from different cohorts at each time point (i.e., not longitudinal measurements in the same animals). Time-matched Sham controls were prepared only for anemia-related indices, whereas additional terminal Sham cohorts at weeks 3 and 6 were not prepared for other endpoints to minimize animal use. Body weight and kidney weight (including kidney-to-body weight ratio) are expressed as mean ± S.D. (n = 5 per group), and UACR is expressed as median IQR. Statistical analysis was performed using two-way ANOVA (factors: surgical group and time), followed by Tukey’s multiple comparison test. ***p < 0.001, vs. Sham rats at 9 weeks; ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001 vs. Nx rats at 3 weeks; ^‡p < 0.05, ^‡‡p < 0.01 vs. Nx rats at 6 weeks. For UACR at 9 weeks after surgery, Sham and Nx rats were compared using the Mann–Whitney U test. ^§§p < 0.01, significantly different from Sham rats at 9 weeks after surgery (nonparametric test). Additional terminal Sham cohorts at weeks 3 and 6 were not included to minimize animal use. BUN: blood urea nitrogen; S.D.: standard deviation; UACR: urinary albumin-to-creatinine ratio; IQR: interquartile range; Nx: nephrectomy.

Similarly, body weight, left kidney weight, the kidney weight-to-body weight ratio, and renal function-related biochemical parameters in the Nx rats receiving subcutaneous administration of TAC (1 mg/kg, every other day for 2 weeks) were compared with those in the Nx rats receiving the same volume of vehicle (20% Cremophor EL) (Table 2). Although the administered dose was relatively low, renal function was further deteriorated by additional TAC administration. In addition, the renal functional data in the rats with TAC administration (Table 2) were similar to those of Nx rats at 9 weeks after surgery (Table 1).

Table 2. Body Weight and Renal Injury Markers in Nx Rats with or without TAC Administration

Treatment	Vehicle (n = 5)	TAC (n = 5)
Body weight (g)	228 ± 17	183± 24
Left kidney (g)	0.8 ± 0.05	0.6 ± 0.11
Left kidney/body weight (%)	0.4 ± 0.06	0.3 ± 0.03
Plasma creatinine (mg/dL)	0.8 ± 0.1	1.3 ± 0.1**
BUN (mg/dL)	51 ± 4	91 ± 4***
UACR (mg/g)	5 (2–13)	13 (7–22)†

Tacrolimus (1 mg/kg) was subcutaneously administered every other day for two weeks, starting at week 4 after Nx surgery. Vehicle (20% Cremophor EL) was administered at 0.1 mL/kg on the same schedule. Data are expressed as mean ± S.D. (n = 5 per group), except for UACR, which is presented as median (IQR). Comparisons between Vehicle and TAC groups were performed using an unpaired two-tailed Student’s t-test for parametric variables. UACR was analyzed using the Mann–Whitney U test. **p < 0.01, ***p < 0.001, significantly different from vehicle-treated rats. ^†p < 0.05, significantly different from vehicle-treated rats (nonparametric test). TAC: Tacrolimus.

Hct, EPO, and Iron Metabolism

Hct values were measured preoperatively (0W) and at 3, 6, and 9 weeks after surgery (corresponding to 11–17 weeks of age) (Fig. 1A). Compared with the Sham group, Nx rats exhibited a significant and progressive decrease in Hct beginning at week 3. In the Sham group, the change from baseline at 9 weeks was +1.6 ± 1.7% (mean increase (%) ± S.D.), indicating no marked alteration during the experimental period. In contrast, the Nx group showed a change from baseline of −11 ± 0.9% at week 9, with a significant difference compared to the Sham group (Fig. 1A). Two-way ANOVA was employed to evaluate the interaction between surgical condition (Sham vs. Nx) and time after surgery (Supplementary Fig. 1A). The plasma EPO levels were measured preoperatively (week 0) and at 3, 6, and 9 weeks after surgery (Fig. 1B), and the time-dependent difference in the plasma EPO level (ΔEPO) was examined (Supplementary Fig. 1B). At week 3, no significant difference in ΔEPO (pg/mL) was observed between groups (Sham: +0.6 ± 1.7; Nx: −6.2 ± 2.1). At week 6, a significant difference in ΔEPO (pg/mL) was observed between the Sham (2.4 ± 3.2) and the Nx (−9.7 ± 1.7) groups. At week 9, the change in plasma EPO levels from baseline in the Sham group was −6.2 ± 2.5 (pg/mL), whereas the Nx group showed −9.5 ± 0.7 (pg/mL).

Fig. 1. Time-Course Changes in Hct and Plasma EPO Levels in Nx Rats with Serial Blood Sampling

Measurements were performed immediately before surgery (week 0), and at 3, 6, and 9 weeks postoperatively. (A) Hct levels in Sham-operated (black circle) and Nx (red circle) rats. (B) Plasma EPO concentrations in Sham-operated (black circle) and Nx (red circle) rats. Data are expressed as mean ± S.D. (n = 5 per group). Statistical analysis was performed using two-way ANOVA (factors: surgical group and time), followed by Tukey’s multiple comparison test. (*p < 0.05, **p < 0.01, ***p < 0.001 compared with Sham rats at the same time point; ^†p < 0.05, ^†††p < 0.001 compared with Nx rats at baseline (week 0) within the same group).

In the Nx group, a trend toward correlation between EPO levels and Hct was observed. Furthermore, despite the progressive decline in Hct, no compensatory increase in EPO was observed (Fig. 2). At week 6 after surgery, Hct in the TAC-treated group (expressed as mean ± S.D., n = 5) was significantly reduced to 31 ± 1.1%, compared with 44 ± 0.6% in the Sham group and 36 ± 1.8% in the Vehicle group. Plasma EPO levels were significantly decreased in the Vehicle group (12.4 ± 1.33 pg/mL) compared with the Sham group (32.0 ± 4.50 pg/mL), whereas the TAC-treated group showed significantly higher levels (22.7 ± 2.74 pg/mL). Statistical analysis was performed using one-way ANOVA, followed by Tukey’s multiple comparison test. Furthermore, it was confirmed that even when blood sampling from the tail vein on weeks 3 and 6 after surgery was not performed, there was no difference in renal function from the data in Table 1 (Supplementary Table 1), and there was no difference in Hct values or plasma EPO concentrations (Supplementary Fig. 2).

Fig. 2. Hct Level as a Function of Plasma EPO Levels in Rats at 6 Weeks after Surgery

The Hct levels at 6 weeks after surgery are shown for the Sham group (black), the Nx with Vehicle group (red), and the Nx with TAC-treated group (blue), without tail vein blood collection throughout the period.

The plasma iron concentration and UIBC decreased significantly in Nx rats at 9 weeks after surgery (Figs. 3A and 3B). In addition, both parameters exhibited a downward trend compared with those in Nx rats at weeks 3 and 6. The TSAT calculated from plasma iron and UIBC values of rats at 9 weeks after surgery did not differ significantly between Nx and Sham groups (Fig. 3C). Similar to the Hct and plasma EPO

Fig. 3. The Changes in the Iron Metabolism Markers in the Sham (Week 9) and Nx Rats

(A) The plasma iron concentration, (B) the UIBC, and (C) the TSAT are shown. Blood samples were collected at regular three-week intervals. Bar graphs represent group means, and error bars indicate S.D. The individual data points are shown as dots (n = 5 per group). Data were analyzed by one-way ANOVA, followed by Dunnett’s multiple comparison test, comparing each group with Nx rats at week 9. *p < 0.05, **p < 0.01, significantly different from Nx rats at 9 weeks after surgery, respectively. UIBC: unsaturated iron-binding capacity; TSAT: transferrin saturation.

levels (Fig. 2), the difference between Sham and Nx at 9 weeks after surgery without serial blood sampling at weeks 3 and 6 after surgery showed a comparable trend with the rats with serial blood sampling (Supplementary Fig. 3).

For iron-related parameters, plasma iron tended to increase in the TAC-treated group compared with Sham, while UIBC was significantly decreased compared with both Sham and Vehicle groups. In contrast, TSAT was significantly higher in the TAC-treated group than in both the Sham group and the vehicle-treated Nx group (Fig. 4).

Fig. 4. A Comparison between the Iron Metabolism Parameters at Week 6 after Surgery

(A) The plasma iron concentration, (B) the UIBC, and (C) the TSAT are shown. The bar graphs represent group means, and error bars indicate SD. Individual data points are shown as dots (n = 5 per group). All measurements were performed at 6 weeks after surgery, comparing the Sham group and the experimental group without serial blood collection at 3 weeks after surgery. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s multiple comparison test. *p < 0.05, significantly different from the Sham group; ^†p < 0.05, significantly different from the Nx + vehicle group.

Evaluation of Renal Fibrosis

The progression of renal interstitial fibrosis was assessed by PSR and MT staining. In comparison with the Sham group at postoperative week 9, the Nx group at the same time point exhibited a significant 32.4-fold increase in fibrotic area (Fig. 5). Moreover, the fibrotic area at postoperative week 9 was 8.7- and 2.0-fold greater than that at weeks 3 and 6, respectively. Although the quantitative analysis was not performed for the BC (−) Sham group, the histological examination revealed a similarly minimal degree of fibrosis as observed in the BC (+) Sham group, indicating that blood sampling did not exert a discernible impact on fibrotic changes. It was revealed that the fibrotic area was markedly increased in the TAC-treated Nx group compared with the Sham group and the vehicle-administered Nx groups of rats at 6 weeks after surgery (Fig. 6).

Fig. 5. The PSR Staining Images and Renal Fibrosis Quantification in Sham and Nx Rats

(A) Representative images of PSR staining in kidney sections from Sham (week 9) and Nx rats at 3, 6, and 9 weeks after surgery. Scale bar: 100 μm. (B) Each bar shows the mean ± S.D. (n = 5 per group). Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s multiple comparison test, with Nx rats at week 9 used as the reference group. *p < 0.05, significantly different from Nx week 9 as a reference. PSR: The Picro-Sirius Red.

Fig. 6. The Representative Images of Renal Tissue Stained with MT and Evaluation of the Renal Fibrosis Area in the Sham and Nx Rats

(A) Representative MT-stained images of renal tissue in Sham and Nx rats at 6 weeks after surgery with or without TAC treatment. Scale bar: 100 μm. (B) Each bar shows the mean ± S.D. (n = 5 per group). Statistical analysis was performed using one-way ANOVA, followed by Tukey’s multiple comparison tests. *p < 0.05 compared with Sham rats. ^† p < 0.05 compared with the Nx rats receiving vehicle treatment. MT: Masson’s Trichrome.

The histological analysis with PAS staining demonstrated significant enlargement of both glomerular and Bowman’s capsule diameters in the TAC-treated group relative to the Sham and vehicle-treated Nx groups (A). In addition, mesangial cell proliferation and thickening of glomerular capillary tufts were observed (Figs. 7A and 7B).

Fig. 7. The PAS Staining Images and the Mesangial Area in the Sham and Nx Rats at 6 Weeks after Surgery

(A) Representative PAS-stained images of glomeruli at 6 weeks after surgery. Arrows indicate mesangial cell proliferation. Scale bar: 100 μm. (B) Each bar represents the mean ± S.D. (n = 5 per group). Statistical analysis was performed using one-way ANOVA, followed by Tukey’s multiple comparison tests. *p < 0.05 compared with Sham rats. The arrow indicates a highly sclerotic mesangial area. PAS: Periodic acid–Schiff.

Western blot analysis demonstrated that the expression of α-SMA, a myofibroblast marker associated with renal sclerosis and fibrosis, was significantly increased in the Nx group 9 weeks after surgery compared with that in the Sham group at the same postoperative week. Furthermore, within the Nx group, α-SMA expression was significantly higher at 6 and 9 weeks compared with 3 weeks, indicating a progressive increase over time and reaching the highest level at week 9 after surgery (Fig. 8).

Fig. 8. Western Blot Analysis of α-SMA in Sham and Nx Rats at 3, 6, and 9 Weeks after Surgery

(A) Representative Western blot images for α-SMA (upper) and GAPDH (lower) as a housekeeping protein are shown. Molecular weights are indicated in kilodaltons. (B) The quantitative analysis of α-SMA protein expression in renal tissue from Sham (week 9) and Nx rats at 3, 6, and 9 weeks after surgery. Each bar shows the mean ± S.D. (n = 5 per group). Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s multiple comparison test, with Nx rats at week 9 used as the reference group. *p < 0.05, significantly different from the Nx rats at week 9 as a reference. α-SMA: α-smooth muscle actin. GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

The expression of HIF-2α, a major transcription factor mediating the induction of renal EPO,¹⁵⁾ was examined using a specific antibody against HIF-2α in the kidney tissue from Sham and Nx groups (Fig. 9A). To evaluate HIF-2α expression, semi-quantitative H-scores were calculated. At 9 weeks after surgery, the mean H-score in the Nx group was 15.3 ± 6.1, substantially lower than that in the Sham group (62.7 ± 6.2). Tissue staining using hematoxylin and DAB showed a tendency toward scattered weak nuclear staining from the cortex to the outer medulla, with heterogeneous and low-intensity signals.

Fig. 9. Distribution of HIF-2α-Positive Nuclei and Mean H-Score in Sham and Nx Rat Kidneys

(A) The representative immunohistochemistry image for HIF-2α staining with a specific antibody in the kidney tissue. Each “G” indicates a glomerulus, and (B) each bar shows the mean H-score for HIF-2α-positive nuclei in the blood sampling groups (Sham and Nx rats). (C) Summarizes the mean H-score ± S.D. and percentages of strongly positive, weakly positive, and blank nuclei for each group. Data are presented descriptively; no statistical testing was performed. HIF-2α: hypoxia–inducible factor-2α.

DISCUSSION

CKD is a progressive disorder accompanied by multiple complications that markedly impair patient prognosis. Among these, renal anemia—characterized by decreased hemoglobin levels and insufficient EPO production—requires individualized management and timely therapeutic intervention. The establishment of reproducible animal models is essential for elucidating the mechanisms underlying CKD-associated anemia and for developing novel treatment strategies.

The present study employed an Nx-based rat model to reproduce CRF and its associated anemia. The Nx model induces progressive renal injury through surgical reduction of nephron mass without nephrotoxic drug administration, thereby mimicking the gradual deterioration observed in clinical CKD.¹⁶⁾ Serial blood sampling did not significantly influence renal function, supporting the internal validity of the experimental design. It should also be noted that time-matched terminal Sham cohorts at weeks 3 and 6 were not included for renal functional assessments to minimize animal use, which represents a limitation of the present study. Furthermore, co-administration of TAC, a calcineurin inhibitor known to promote renal fibrosis, exacerbated renal injury and was associated with a more pronounced decline in Hct. TAC administration for two weeks after Nx surgery reproduced a degree of anemia and fibrosis comparable to that observed at later stages in Nx alone, indicating that TAC can accelerate pathological progression within this established CKD model.

The Nx model reproduced progressive renal impairment characterized by elevated plasma creatinine, BUN, and urinary albumin excretion, accompanied by declining Hct values. Notably, plasma EPO levels failed to exhibit a compensatory increase in response to anemia. This finding is consistent with previous reports describing defective hypoxia-sensing mechanisms in CKD-associated anemia.¹⁷⁾ Impaired activation of the HIF signaling pathway has been implicated in insufficient EPO synthesis in diseased kidneys.¹⁵⁾ In line with these observations, our immunohistochemical analysis demonstrated reduced HIF-2α expression in the renal interstitium of Nx rats at 9 weeks after surgery. Although the present study did not establish a direct causal link, the attenuation of HIF-2α expression may partly explain the blunted EPO response observed in advanced renal injury.

Iron-related parameters also showed alterations. Despite relatively preserved TSAT values, decreased plasma iron and UIBC suggested disturbances in iron handling.

These findings resemble clinical observations in CKD patients, in whom functional iron deficiency has been associated with hepcidin elevation and subsequent sequestration of iron within macrophages and hepatocytes.^18,19) Thus, the Nx model recapitulates multiple components of renal anemia, including impaired EPO synthesis and dysregulated iron metabolism.

Although TAC is commonly used as an immunosuppressive agent following organ transplantation, its nephrotoxic properties—manifested as tubular atrophy, arteriolopathy, and interstitial fibrosis—are well documented in both clinical and experimental settings.^20–22) In the present study, TAC administration further exacerbated renal injury and fibrosis, as evidenced by increased interstitial collagen deposition, α-SMA expression, and deterioration of renal function parameters. Interestingly, plasma EPO levels in TAC-treated Nx rats were not proportionally suppressed despite more severe anemia. Rather than indicating absolute EPO deficiency, this pattern may reflect a relative inadequacy of erythropoietic response under aggravated renal injury. Similar phenomena have been reported in advanced CKD, where inflammation and oxidative stress contribute to reduced erythroid responsiveness despite detectable circulating EPO levels.^23,24) However, because direct markers of erythropoietic activity (e.g., reticulocyte counts) were not evaluated in this study, these interpretations should be considered exploratory.

The complementary use of PSR and MT staining enabled both quantitative assessment of total collagen deposition and structural evaluation of interstitial remodeling. Progressive fibrosis observed in Nx rats was markedly enhanced by TAC treatment, supporting the concept that fibrotic remodeling contributes to deterioration of renal function and may influence EPO-producing (REP) cell activity within the renal interstitium. Nevertheless, correlation analyses between fibrosis severity and anemia parameters were not performed in the present study and warrant further investigation.

Furthermore, Western blot analysis showed upregulation of α-SMA, a well-established marker of myofibroblast activation, supporting enhanced interstitial fibrogenesis in Nx rats, particularly following TAC administration. These findings are consistent with the observed histological progression of interstitial fibrosis and deterioration of renal function. Although direct assessment of REP cell function was not performed, progressive interstitial remodeling may influence oxygen-sensing mechanisms and erythropoietic regulation within the diseased kidney.

Taken together, the present study establishes a reproducible experimental system that reflects key features of advanced CKD-associated anemia, including progressive renal fibrosis and an insufficient erythropoietic response. Compared with Nx alone, the addition of TAC accelerates pathological progression and facilitates the induction of advanced renal injury within a shorter experimental period. This model may therefore serve as a practical preclinical platform for investigating mechanisms of renal anemia and for evaluating therapeutic strategies targeting fibrosis, iron metabolism, or impaired erythropoietic regulation.

CONCLUSION

In conclusion, the present study established a reproducible rat model of renal anemia based on Nx, which recapitulates key features of CKD, including progressive renal dysfunction, interstitial fibrosis, and an insufficient erythropoietic response. Reduced plasma EPO levels and attenuation of renal HIF-2α expression were observed in association with advancing renal injury. Furthermore, co-administration of TAC exacerbated renal fibrosis and functional decline and was associated with more pronounced anemia. Although circulating EPO levels were not completely suppressed in TAC-treated Nx rats, Hct remained reduced, suggesting a relative inadequacy of erythropoietic response under aggravated renal injury. The combination of Nx and TAC provides an experimental framework that facilitates the induction of advanced renal pathology within a shorter timeframe and may serve as a practical preclinical platform for investigating the mechanisms of CKD-associated anemia and for evaluating therapeutic strategies targeting fibrosis, iron metabolism, or impaired erythropoietic regulation.

Acknowledgments

The authors would like to express their sincere gratitude to Mrs. Miho Sumita for her dedicated care and technical assistance in animal handling throughout this study. The authors also thank the undergraduate members of our laboratory for their support in data collection and daily maintenance of the experimental animals.

DECLARATIONS

Funding

This work was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant Numbers: JP23K14399 to Y.N. and JP22K06736 to S.M.).

Author Contributions

Y.N. and S.M. contributed to the conceptualization and design of the study. S.M. supervised the experimental procedures. S.K. and Y.N. performed the animal experiments and data analyses. Y.N. and S.M. drafted the manuscript, and all authors reviewed and approved the final version.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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