Renal effects of excessive fructose intake in Spontaneously Diabetic Torii-Leprfa (SDT fatty) rats

Abstract

Excessive fructose intake has been reported to increase the risks of obesity, diabetes and kidney diseases, including diabetic nephropathy. We investigated the effects of a high-fructose diet on nephropathy in both male and female Spontaneously Diabetic Torii-Lepr^fa (SDT fatty) rats, a model for obese type 2 diabetes. 11-week-old male and female Sprague-Dawley (SD) and SDT fatty rats that developed diabetes were each divided into 2 groups receiving either a high-fructose diet (60% fructose) or a basal diet. The diets were fed ad libitum for 7 weeks. Body weights, food consumption, clinical chemistry, urinalysis, kidney weights and histopathology of the kidneys were evaluated. In the SDT fatty rats, fructose intake increased the urinary excretion of calcium and inorganic phosphate in both sexes. Histopathological examination revealed that fructose intake worsened nephropathy (mineralization, inflammatory cell infiltration, and increased mesangial matrix in the glomeruli) in the female SDT fatty rats. In conclusion, a 7-week high-fructose diet induced several renal changes in SDT fatty rats, including urinary electrolyte imbalances and associated mineral deposition in the kidney, suggestive of urinary stone formation. It suggests that the SDT fatty rat is a useful model to investigate type 2 diabetes and diabetic nephropathy and that excessive fructose intake can be a risk factor for the development and progression of urinary stone formation and diabetes- related kidney injury.

INTRODUCTION

Fructose is a simple carbohydrate that is present in natural foods such as fruits, vegetables and honey. In recent years, high-fructose corn syrup (HFCS) has been extensively used as a sweetener in carbonated beverages, sweetened drinks and sweet foods. Due to increased consumption of HFCS, fructose intake has risen over the past several decades (Jung et al., 2022).

Recent analyses have shown that excessive fructose intake increases the risk of obesity, diabetes, nonalcoholic fatty liver disease, cardiovascular disease, and kidney disease (Jung et al., 2022; Bray, 2010; Shoham et al., 2008). Given the epidemiological relationship between fructose and diabetes, there are reports suggesting that fructose intake may play a direct or indirect role in the development of diabetic nephropathy and chronic kidney disease (Shoham et al., 2008). Furthermore, epidemiological studies have reported that dietary fructose intake increases the risk of kidney stones and has been shown to cause associated kidney damage (Taylor and Curhan, 2008).

In experimental animal studies using normal rats, high-fructose diet has been shown to cause dyslipidemia, obesity (Bocarsly et al., 2010), insulin resistance (Tobey et al., 1982; Sleder et al., 1980), hepatic steatosis (Botezelli et al., 2012), and elevated blood pressure (Hwang et al., 1987). Several studies have investigated the renal effects of excessive dietary fructose intake in both normal and diabetic rat models. In normal male Sprague-Dawley (SD) rats, a 60% fructose diet for 6–8 weeks increased kidney weight and induced tubulointerstitial lesions (tubular dilatation and degeneration, tubular epithelial hyperplasia, interstitial fibrosis, and macrophage infiltration), accompanied by hemodynamic alterations (elevated blood and mean arterial pressure, increased glomerular capillary pressure, and a reduced ultrafiltration coefficient) and vascular wall thickening (Nakayama et al., 2010; Sánchez-Lozada et al., 2007). In juvenile Wistar rats given 20% fructose in drinking water for 13–14 weeks, early sex-dependent renal effects have been reported: glomerular filtration rate declines in males, whereas females show more pronounced changes in electrolyte excretion (reduced Na/K and increased Mg excretion), with increased macrophage infiltration and reduced renal endothelial nitric oxide synthase (eNOS) expression (Monteiro et al., 2023). In streptozotocin (STZ)-induced diabetic Wistar Furth rats (male only), a 40% fructose diet for 12 weeks increased renal H₂O₂ in the absence of microalbuminuria or histological injury (Bell et al., 2000). In STZ-diabetic Wistar rats (male only) fed fructose for 6 weeks, increased serum creatinine, renal injury biomarkers, oxidative stress markers, inflammatory mediators, as well as tubular degeneration, epithelial hypertrophy, glomerulosclerosis, and interstitial fibrosis were observed (Oraby et al., 2019). In male Spontaneously Diabetic Torii (SDT) rats (a non-obese spontaneous type 2 diabetes model), a 60% fructose diet for 4 weeks increased urinary uric acid, plasma creatinine, renal injury biomarkers, and kidney weight, and induced hyaline casts, tubular dilatation and regeneration, mineralization, fibrosis, and macrophage infiltration (Toyoda et al., 2018). In summary, all of the studies consistently observed renal injury, as evidenced by changes in renal hemodynamics, alterations in renal functional parameters, increases in renal injury biomarkers, increases in kidney weight, and histopathological lesions. SDT rats are a non-obese spontaneous type 2 diabetes model known to develop glycosuria, hyperglycemia, and hypoinsulinemia (Shinohara et al., 2000). Spontaneously Diabetic Torii-Lepr^fa (SDT fatty) rats are an obese spontaneous type 2 diabetes model created by introducing the leptin receptor mutation (Lepr^fa) of Zucker fatty rats into SDT rats. SDT fatty rats are known to develop leptin resistance, hyperphagia, obesity, and hyperglycemia in males from 5 weeks of age and in females from 8 weeks of age. Furthermore, 100% of males and 73% of females develop diabetes (glycosuria and hyperglycemia) at 16 and 32 weeks of age, respectively, accompanied by hyperinsulinemia, insulin resistance and significant changes in lipid parameters (Masuyama et al., 2005). In humans, the pathophysiology of diabetes varies among individuals, and many diabetic patients have been found to have metabolic syndrome diseases such as obesity. In addition, diabetic kidney disease (diabetic nephropathy) is a major complication of type 2 diabetes, and both type 2 diabetes and obesity are well-established risk factors for chronic kidney disease (Alicic et al., 2017; Hsu et al., 2006). Therefore, SDT fatty rats are considered to be an appropriate animal model for evaluating type 2 diabetes and diabetic nephropathy, as they reflect the complex pathophysiology of diabetic patients.

To our knowledge, as described above, fructose-induced renal injury has been reported in normal rat strains, induced diabetes models and non-obese spontaneous diabetes models; however, its effects in an obese spontaneous model of type 2 diabetes and the possibility of sex-specific susceptibility, have not been investigated (Nakayama et al., 2010; Sánchez-Lozada et al., 2007; Monteiro et al., 2023; Bell et al., 2000; Oraby et al., 2019; Toyoda et al., 2018). In this study, we evaluated the renal effects of a high-fructose diet in male and female SDT fatty rats, a spontaneous type 2 diabetes model with obesity, in which diabetes was confirmed by the presence of glycosuria and hyperglycemia. SDT fatty rats were fed a high-fructose diet for 7 weeks and the renal effects were assessed by measuring renal function parameters and performing histopathological examination of the kidneys. In addition, potential sex-specific differences were also investigated.

MATERIALS AND METHODS

Diet

The basal diet, D10012M (AIN-93M blended diet) and a high-fructose diet, D19082801 (60% fructose diet) were purchased from Research Diets, Inc. (New Jersey, USA). The composition of the D10012M and D19082801 diets is shown in Table 1.

Table 1. The composition of the D10012M and D19082801 diets.

Animals

Ten male and 10 female SDT fatty (SDT.Cg-Lepr^fa/JttJcl) rats (5 weeks of age, n=5/group/sex) and 10 male and 10 female Sprague Dawley (Jcl:SD) rats (5 weeks of age, n=5/group/sex) were purchased from CLEA Japan, Inc. (Tokyo, Japan). SD rats were used as normal control animals as SD rats are considered to be appropriate normal control animals for studies of SDT fatty rats because SDT fatty rats were established from SDT rats, which represent an inbred strain of SD rats. All the rats were allowed free access to the basal diet (D10012M) ad libitum before the experiments. At 12 weeks of age, when SDT fatty rats were confirmed to have glycosuria (≥ 1000 mg/day) and hyperglycemia (≥ 300 mg/dL), the animals of each strain were divided into 2 groups and then assigned to either a high-fructose (D19082801) or the basal (D10012M) diet group. There were 4 groups of each sex with 5 animals each for the high-fructose diet fed-SD rats (HF-SD) group, the basal diet fed-SD rats (C-SD) group, the high-fructose diet fed-SDT fatty rats (HF-SDT fatty) group and the basal diet fed-SDT fatty rats (C-SDT fatty) group. Two male animals in the basal diet fed-SDT fatty rats (C-SDT fatty) group were found dead during the study. The cause of death for these animals was unknown. Therefore, baseline data at 11 weeks of age (pre-treatment) were available for five animals (n=5), whereas only three (n=3) remained at 18 weeks of age (week 7 of the experimental period). During the experiments, the rats were allowed free access to their assigned diets. Tap water was available for drinking ad libitum. The animals were housed individually in plastic cages with bedding kept in an air-conditioned room with a 12-hr light-dark cycle (lighting from 7:00 a.m. to 7:00 p.m.) at a temperature of 23 ± 1°C, a relative humidity of 55 ± 5% and a ventilation rate of about 15 times per hour. All the animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the Yokohama Research Center, Central Pharmaceutical Research Institute, Japan Tobacco Inc. This study was conducted in accordance with the Act on Welfare and Management of Animals (Law No. 105 issued in Japan on October 1, 1973).

Body weights & Food consumption

The animals of each group were fed their assigned diets for 7 weeks (from 11 to 18 weeks of age). Body weights and food consumption were measured at least twice a week and total food consumption during the experimental period was calculated.

Urinalysis

Urinalysis was conducted in 11 weeks of age (pre-treatment) and 18 weeks of age (week 7 of the experimental period). The animals were placed in metabolism cages to collect the urine samples. After measurement of the urine volume, the urine samples were used for dipstick analysis of pH (N-Multistix SG-L and CLINITEK Advantus Urine Chemistry Analyzer, Siemens Healthcare Diagnostics K.K., Tokyo, Japan). Urinary concentrations of total protein (TP), albumin (ALB), glucose (GLU), uric acid (UA), calcium (Ca), inorganic phosphorus (IP), creatinine (CRN), were measured with a TBA-120FR automated analyzer (TOSHIBA Corporation, Tokyo, Japan) using standard reagents by the pyrogallol red method for TP, the turbidimetric immunoassay method for ALB, the HX-G-6-PDH method for GLU, the Uricase-F-DAOS method for UA, the OCPC method for Ca, and the PNP-XDH method for IP (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) and using standard reagents by the CR-SOX-POD-CRN method for CRN (Canon Medical Systems Corporation, Tochigi, Japan). The individual urinary excretion of each parameter was also calculated based on the urinary concentrations in each urine sample and the relevant urine volume.

Clinical Chemistry

Blood samples were taken from all the animals at 11 weeks of age (pre-treatment) and 18 weeks of age (week 7 of the experimental period). Approximately 900 µL blood samples were collected from the subclavian vein into lithium heparin-treated syringes. The blood samples were centrifuged at 9000 rev·min^–1 for 15 minutes to obtain the plasma. At the end of the experimental period, blood samples were collected from the abdominal aorta of all the animals under isoflurane anesthesia. The blood samples were collected into heparinized tubes and were centrifuged at 3000 rev·min^–1 4°C for 30 min to obtain the plasma. Plasma concentrations of GLU, triglycerides (TGL), phospholipid (PL), non-esterified fatty acid (NEFA), urea nitrogen (UN), Ca, IP, acetoacetic acid (AcAc), 3-hydroxybutyric acid (OHBA), total ketone bodies (TKB), total cholesterol (T-CHO), CRN, Ca and IP were measured with a TBA-120FR automated analyzer using standard regents by the HX-G-6-PDH method for GLU, the GPO-HMMPS method and glycerol blanking method for TGL, the CO-DAOS method for PL, the ACS•ACOD method for NEFA, the Urease-GLDH method for UN, the OCPC method for Ca, and the PNP-XDH method for IP (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan), using standard regents by the 3-HBDH method for AcAc, OHBA and TKB (KAINOS Laboratories Inc., Tokyo, Japan), using standard regents by the COD method for T-CHO, the CR-SOX-POD-CRN method for CRN (Canon Medical Systems Corporation, Tochigi, Japan). Plasma insulin levels were measured by using an ELISA kit (Morinaga Ultra Sensitive Mouse/Rat Insulin ELISA Kit, Morinaga BioScience, Inc., Kanagawa, Japan) according to the manufacturer’s instructions.

Organ Weights

At the end of the experimental period, the final body weights were recorded. All the animals were euthanized by exsanguination from the abdominal aorta under isoflurane anesthesia. The kidneys from the animals were weighed and the relative weight to the final body weight was calculated.

Histopathology & Histopathological Grading

The kidneys collected from the animals were fixed in 10% neutral buffered formalin and prepared for histopathological examination by embedding in paraffin wax, sectioning and staining with hematoxylin and eosin (HE). Additional sections of the kidneys stained with von Kossa were also prepared. The slides were evaluated microscopically. Histopathological findings were semi-quantitatively graded by a toxicologic pathologist certified by the Japanese Society of Toxicologic Pathology (JSTP) according to lesion severity, as commonly applied in general toxicology studies. The grading scale was defined as follows: –: No abnormal changes, ±: Very slight, +: Slight, 2+: Moderate, 3+: Severe. This grading reflects the relative severity of lesions, not an objective score or count, and is intended to facilitate qualitative comparisons among groups. The grading criteria followed internal standard operating procedures based on international guidelines (e.g., International Harmonization of Nomenclature and Diagnostic Criteria (INHAND)), and the same pathologist performed all evaluations to ensure consistency.

Statistical analysis

The mean values and standard deviations in each group were calculated for the body weights, food consumption, clinical chemistry, urinalysis and organ weights.

For each animal, values at 11 weeks of age (initial) and at 18 weeks of age (final) and the change from baseline (Δ = final − initial) were analyzed. For the male C-SDT fatty group, Δ values were calculated based on the initial values of the 3 animals that survived until the final evaluation. Three pre-specified pairwise comparisons were evaluated: (i) control SD vs. SDT fatty rats receiving basal diet; (ii) within SD rats, basal vs. high-fructose diet; and (iii) within SDT fatty rats, basal vs. high-fructose diet. For each endpoint (initial, final, and Δ), between-group differences were tested using either Student’s two-sample t-test (assuming equal variances) or Welch’s t-test (unequal variances), conditional on an F-test for equality of variances. Endpoints were treated as separate multiplicity families. Within each endpoint, multiplicity across the 3 planned comparisons was controlled using a Bonferroni correction by adjusting the P values. P values were multiplied by 3 and reported as Bonferroni-adjusted (P_adj). A two-sided P_adj < 0.05 was considered statistically significant.

RESULTS

Body weights, food consumption, glucose-related parameters and lipid parameters

The data for body weights, food consumptions and the summary of the glucose-related parameters (plasma and urinary GLU, plasma insulin levels), lipid parameters (plasma TGL, PL, T-CHO, NEFA, TKB levels), and urinary UA levels are shown in Table 2.

Table 2. The data for body weights, food consumptions and the summary of the glucose-related parameters and lipid parameters.

1. Comparison between the C-SD and C-SDT fatty groups

Total body weight gain and final body weights were lower in male C-SDT fatty rats than in male C-SD rats. In contrast, female C-SDT fatty rats showed higher total body weight gain as well as higher initial and final body weights than female C-SD rats. Total food consumption was higher in the C-SDT fatty groups than in the C-SD groups for both sexes. Both initial and final values of plasma and urinary GLU were higher in the C-SDT fatty groups than in the C-SD groups for both sexes. Although not statistically significant, urinary GLU means were 0.4–2.3 mg/day in C-SD groups versus 5.2×10³–19.7×10³ mg/day in C-SDT fatty groups, indicating marked separation between groups. With respect to the plasma insulin levels, both initial and final values in females were higher in the C-SDT fatty groups than in the C-SD groups. In males, the initial values tended to be higher in the C-SDT fatty groups than in the C-SD groups (not statistically significant), whereas the final values were lower in the C-SDT fatty groups than in the C-SD groups. Plasma PL, T-CHO and NEFA levels were higher in the C-SDT fatty groups than in the C-SD groups for both sexes. As for the plasma TKB levels, the initial values in males and final values in females were higher in C-SDT fatty rats than in C-SD rats. Both the initial and final values for urinary UA excretion for in females were higher C-SDT fatty rats than in C-SD rat.

2. Comparison between the C-SD and HF-SD groups

There was no apparent effect of fructose on the body weights and total food consumption in SD rats of either sex. There was no apparent effect of fructose on the glucose-related parameters, lipid parameters, or urinary UA excretion between the C-SD and HF-SD groups. Although plasma PL levels in male HF-SD rats showed a statistically significant difference in Δ values, the final PL values did not differ between groups, suggesting that the observed change reflects a baseline imbalance rather than a treatment effect.

3. Comparison between the C-SDT fatty and HF-SDT fatty groups

Fructose intake had no apparent effect on the body weights in either sex of the SDT fatty rats. Total food consumption decreased for both sexes, with a statistically significant reduction observed in females. There was no apparent effect of fructose on the glucose-related parameters, lipid parameters, or urinary UA excretion in either sex of the SDT fatty rats. Although the final values of the urinary UA excretion were significantly higher in male SDT fatty rats fed fructose, the Δ values were negative, suggesting that this finding may not be attributable to fructose intake.

Renal function-related parameters

A summary of the renal function-related parameters in the clinical and urine chemistry (urinary pH, TP, ALB, CRN, Ca, IP and plasma UN, CRN, Ca, IP) is shown in Table 3.

Table 3. The summary of the renal function-related parameters.

1. Comparison between the C-SD and C-SDT fatty groups

Urinary TP and ALB excretion were higher in the C-SDT fatty groups than in the C-SD groups for both sexes. In males, although the between-group differences in final values were not statistically significant, the group means were widely separated: TP 7.81 mg/day in C-SD vs 144 mg/day in C-SDT fatty; ALB 0.31 mg /day in C-SD vs 72 mg /day in C-SDT fatty. Urinary and plasma CRN levels were lower in the C-SDT fatty groups than in the C-SD groups for both sexes. Initial values of plasma UN levels were higher in female C-SDT fatty rats than in female C-SD rats. Urinary Ca excretion was higher in the C-SDT fatty groups than in the C-SD groups for both sexes. Both initial and final values for urinary IP excretion in males and the initial values in females were higher in C-SDT fatty rats than in C-SD rats. The initial plasma IP level values in males and the final values in females were higher in the C-SDT fatty groups than in the C-SD groups.

2. Comparison between the C-SD and HF-SD groups

There was no apparent effect of fructose on urinary TP, ALB and CRN excretion, urinary pH and plasma CRN and UN levels in either sex of the SD rats. Fructose intake had no apparent effect on urinary Ca excretion in SD rats. For urinary IP excretion, fructose intake increased both the final and Δ values in male SD rats. In females, lower baseline IP values resulted in statistically significant Δ values despite no corresponding differences in the final values. Nevertheless, the final mean values were numerically higher in HF-SD fatty rats than in C-SD rats (8.28 mg/day in C-SD vs 11.75 mg/day in HF-SD fatty), suggesting a modest upward trend. For plasma Ca levels in males, significant between-group differences were observed in the initial and Δ values, but not in the final values. Therefore, these differences were not attributed to fructose intake.

3. Comparison between the C-SDT fatty and HF-SDT fatty groups

Fructose intake had no apparent effect on urinary TP and ALB and CRN excretion, and plasma CRN and UN levels in either sex of SDT fatty rats. In male SDT fatty rats, fructose intake decreased Δ values of urinary pH, and final values tended to be lower. Urinary Ca and IP excretion increased with fructose intake in SDT fatty rats of both sexes. In females, although the between-group differences in final urinary Ca values were not statistically significant, the group means were clearly separated (3.63 mg/day in C-SD fatty vs 9.13 mg/day in HF-SDT fatty) and Δ values with fructose were statistically significant. There was no apparent effect of fructose on plasma Ca or IP in either sex of the SDT fatty rats.

Kidney weights and histopathological examination of the kidneys

The data for the kidney weights and histopathological examination of the kidneys are shown in Table 4 and Figs. 1 and 2.

Table 4. Organ weights and histopathological examination of the kidneys.

Fig. 1

Light micrographs of HE-stained kidney sections at lower magnification. (A–D) Male rats: (A) SD rat fed a basal diet, (B) SD rat fed a high-fructose diet, (C) SDT fatty rat fed a basal diet, and (D) SDT fatty rat fed a high-fructose diet. (E–H) Female rats: (E) SD rat fed a basal diet, (F) SD rat fed a high-fructose diet, (G) SDT fatty rat fed a basal diet, and (H) SDT fatty rat fed a high-fructose diet. Scale bar = 200 µm. hyaline cast (*), inflammatory cell infiltration (arrowheads), tubular dilation (★), and degeneration/regeneration of tubules (arrows) were observed in SDT fatty rats.

Fig. 2

Mineral deposition in the kidney. (A–D) von Kossa-stained kidney sections at lower magnification: (A) male SDT fatty rat fed a basal diet; (B) male SDT fatty rat fed a high-fructose diet; (C) female SDT fatty rat fed a basal diet; (D) female SDT fatty rat fed a high-fructose diet. (E) HE-stained kidney sections at higher magnification from a female SDT fatty rat fed a high-fructose diet. (F) von Kossa-stained kidney sections at higher magnification from a female SDT fatty rat fed a high-fructose diet. Compared with the basal diet group in female SDT fatty rats (C), the high-fructose diet group in female SDT fatty rats (D) exhibited more extensive mineralization (arrows, von Kossa-positive areas in brown-black). Scale bar = 1 mm for A–D; 200 µm for E and F.

1. Comparison between the C-SD and C-SDT fatty groups

The kidney weights were higher in the C-SDT fatty groups than in the C-SD groups for both sexes (Table 4). In the histopathological examination of the kidneys, except for basophilia in tubules which is commonly observed spontaneously in SD rats, no changes were observed in either sex of the SD rats (See the C-SD column in Table 4, Fig. 1A and 1E). In SDT fatty rats, mineralization (mineral deposition in the lumen of the renal tubules or in the renal tubular epithelium), hyaline casts, inflammatory cell infiltration, tubular dilation, glycogen accumulation in the distal tubular epithelium, degeneration/regeneration of the tubules, and increased mesangial matrix in the glomeruli were observed in both sexes and inflammation in the renal pelvis was only observed in females (See the C-SDT fatty column in Table 4, Fig. 1C and 1G). Glycogen accumulation in the distal tubules was observed as an Ebstein–Armani lesion, a histological feature commonly seen in diabetic rats (Lau et al., 2013; Hard, 2008). Mineral deposition was primarily observed in the tubular lumen or in the renal tubular epithelium of the medulla and the corticomedullary junction, and to a lesser extent in the cortex (Fig. 2A-D). The formation of large calculi within the tubular lumens caused dilation of the tubules, exfoliation of the tubular epithelium and replacement of the tubules by the calculi (Fig. 2E and 2F). A von Kossa stain was performed to evaluate the nature of the mineralization and the deposits were stained brown-black, suggesting that they were calcium salts (Fig. 2A-D and 2F).

2. Comparison between the C-SD and HF-SD groups

There was no apparent effect of fructose on the kidney weights and histopathology in either sex of the SD rats (See the HF-SD column in Table 4, Fig. 1B and 1F).

3. Comparison between the C-SDT fatty and HF-SDT fatty groups

There was no apparent effect of fructose on the kidney weights in either sex of the SDT fatty rats (Table 4). In female SDT fatty rats, the severity of the mineralization (mineral deposition), inflammatory cell infiltration, and increased mesangial matrix in the glomeruli increased with fructose intake (See the female HF-SDT fatty column in Table 4, Fig. 1G, 1H and 2C-2F), whereas no apparent changes were observed in male SDT fatty rats (See the male HF-SDT fatty column in Table 4, Figure 1C,1D, 2A and 2B). On the other hand, glycogen accumulation in the distal tubular epithelium was reduced by fructose intake in both sexes of the SDT fatty rats (See the HF-SDT fatty column in Table 4).

DISCUSSION

In comparisons between the C-SD and C-SDT fatty groups, C-SDT fatty rats had already developed diabetes (glycosuria and hyperglycemia) and exhibited abnormalities in lipid parameters at baseline (from 11 weeks of age). In male C-SDT fatty groups, both initial and final body weights and the final plasma insulin level were lower than in the C-SD groups. Male SDT fatty rats become obese early in life due to hyperphagia caused by leptin deficiency, leading to hyperinsulinemia from insulin resistance. However, because their insulin-secreting β-cells are fragile, β-cell numbers decline, reducing insulin levels. This reduction in insulin promotes glycosuria, protein catabolism, and lipolysis, eventually leading to weight loss (Masuyama et al., 2005). In addition, changes in renal parameters, kidney weights, and histopathological findings were observed in both male and female SDT fatty rats.

Based on comparisons between the C-SD and HF-SD groups, the apparent effect of fructose intake was an increase in urinary IP excretion in SD rats, but no changes in the other renal parameters and histopathological findings were observed. Based on comparisons between the C-SDT fatty and HF-SDT fatty groups, in SDT fatty rats, food consumption decreased due to fructose intake and fructose intake exacerbated renal changes, including a lower urinary pH in males, increased urinary Ca and IP excretion in both sexes and histopathological changes (mineralization, inflammatory cell infiltration, and increased mesangial matrix in the glomeruli) in females. The exacerbation of the inflammatory cell infiltration and increased mesangial matrix in the glomeruli may be secondary to the nephron obstruction caused by mineral deposition, namely urinary stone formation. Furthermore, the increase in mineral deposition in the SDT fatty rats following fructose intake was considered to be primarily caused by an imbalance in urinary electrolytes, particularly due to the marked increase in urinary excretion of Ca and IP.

Mineral deposition in the kidney was not observed in the SD rats, but it was observed in SDT fatty rats fed either a basal diet or a fructose diet. In the female SDT fatty rats fed a fructose diet, mineral deposition in the kidney tended to be more severe than in those fed a basal diet, as indicated by grades of 2+ in four rats and 3+ in one rat, whereas the basal diet group showed only grades of 1+ in three rats and 2+ in two rats. The von Kossa stain revealed that the mineral deposition in the kidney was composed of calcium salts. The calcium salts that commonly crystallize in the kidney are calcium phosphate and calcium oxalate (Siener, 2021) and calcium oxalate can form on the basis of UA crystals (Kalaiselvi et al., 1999). In this study, fructose intake increased the urinary excretion of Ca and IP in SDT fatty rats and UA levels were elevated at baseline in female SDT fatty rats. These findings suggest that various substances such as calcium phosphate, calcium oxalate and UA became saturated in the urine and formed urinary stones.

Epidemiologically, fructose is known to be a risk factor for urinary stones (Taylor and Curhan, 2008), but the precise mechanism is still not well understood. The causes of urinary stone formation due to fructose intake are considered to be related to increased urinary excretion of Ca, UA and oxalate, and possibly increased insulin resistance, which is associated with lower urine pH (Ng et al., 2018; Nguyen et al., 1995; Abate et al., 2004). In a previous experimental animal study, fructose intake increased the urinary excretion of Ca and IP and induced the deposition of calcium phosphate stones in the kidneys of normal rats (Flisiński et al., 2021). In the report by Flisiński, the increase in urinary Ca excretion was considered to be due to decreased Ca reabsorption caused by proximal tubular injury because increase in the inflammatory markers of the renal tubules was also observed. On the other hand, another study has shown that fructose intake in normal rats decreased the blood levels of 1,25-(OH)₂D₃ (Douard et al., 2013). Since 1,25-(OH)₂D₃ plays a crucial role in Ca reabsorption from the renal tubules, the increase in urinary Ca excretion due to fructose intake is considered to possibly be caused by the reduction of 1,25-(OH)₂D₃. In our study, calcium metabolism-related factors (such as 1,25-(OH)₂D₃) were not investigated. Therefore, the cause of the increase in urinary Ca excretion due to fructose intake remains unknown.

Regarding the relationship between diabetes and urinary stones, it is known that diabetic patients have a higher risk of developing urinary stones (Meydan et al., 2003; Lieske et al., 2006). Insulin resistance is associated with derangements in renal ammonium production, increased urinary acidification, hypocitraturia, and hypercalciuria, all of which can contribute to the development of UA and Ca stones (Abate et al., 2004; Cupisti et al., 2007; Weinberg et al., 2014). It has been reported that diabetic patients with urinary stones excrete significantly greater amounts of urinary oxalate and have a significantly lower urine pH compared to non-diabetic patients. (Eisner et al., 2010).

In our study, mineral deposition in the kidneys was not observed in the SD rats but was observed in the SDT fatty rats fed a basal diet, indicating that mineral deposition is more likely to occur in diabetic animals. Furthermore, fructose intake in the SDT fatty rats increased urinary Ca and IP excretion in both sexes and exacerbated mineral deposition in the kidneys in the females. These results indicate that fructose is a risk factor for urinary stone formation and the development of renal damage and that this risk may be higher in diabetic animals compared to normal animals.

Regarding sex-specific differences in the effect of fructose, the exacerbation of renal mineral deposition due to fructose intake in SDT fatty rats was observed only in females. However, the reason why the exacerbation of renal mineral deposition occurred only in females remains unclear, because urinary electrolyte imbalances were observed in both sexes. Inflammation of the renal pelvis was observed only in female SDT fatty rats (1 female C-SDT fatty and 1 female HF-SDT fatty rat). Although bacterial infection was not confirmed, such inflammation is often associated with ascending urinary tract infections (Frazier et al., 2012), which are known to contribute to stone formation (Miano et al., 2007). This suggests a potential, though unconfirmed, link between infection and sex-specific renal pathology.

To our knowledge, few studies have examined sex differences in the renal effects of fructose. Monteiro et al. (2023) reported early sex-dependent renal effects in juvenile Wistar rats, with females exhibiting more pronounced alterations in electrolyte excretion. In that study, baseline Ca excretion was higher in females than in males and fructose induced no apparent effect on Ca or IP excretion in normal rats of either sex. In our study, urinary Ca excretion was higher in female normal (SD) rats than in males, with no apparent effect of fructose, consistent with previous findings. In contrast, for an obese spontaneous type 2 diabetes model (SDT fatty rats), fructose increased urinary Ca and IP excretion in both sexes and the final mean values of urinary Ca and IP were numerically higher in males than females (urinary Ca, 25.85 mg/day in male vs 9.13 mg/day in female; urinary IP, 36.54 mg/day in male vs 22.30 mg/day in female), providing new insights into sex-specific responses under diabetic conditions.

In our previous study, we demonstrated that feeding fructose to SDT rats, a non-obese diabetic model, exacerbated renal dysfunction (Toyoda et al., 2018). In SDT rats, injury to the renal tubules due to increased plasma and urinary UA and plasma GLU levels was considered to be the main cause of exacerbation of renal damage. In contrast, based on the results of the present study, urinary electrolyte imbalances and increased urinary stone formation appear to be the main drivers of renal deterioration in SDT fatty rats. These findings suggest that although the overall pattern of renal injury in SDT fatty rats shares similarities with that of SDT rats, a distinct susceptibility pathway may be involved. The genetic difference between SDT rats and SDT fatty rats lies solely in the presence or absence of a leptin receptor mutation which causes differences in phenotypes such as food consumption, lipid parameters and the effects of insulin. In SDT fatty rats hyperphagia may result in increased dietary calcium intake, which could be associated with the increased formation of urinary stones. Furthermore, leptin itself has been reported to have both central and peripheral actions on bone metabolism (Matsunuma and Horiuchi, 2007; Takeda et al., 2002), suggesting that the leptin receptor mutation may be involved in alterations in calcium metabolism. The advantage of the SDT fatty model lies in its characteristic feature of mimicking the pathophysiological conditions of obese type 2 diabetes. In the present study, this disease model demonstrated that fructose primarily exacerbates electrolyte imbalances and promotes stone formation. Therefore, SDT fatty rats serve as a complementary model to SDT rats, enabling mechanistic and interventional studies focused on Ca/IP metabolism and urinary stone formation in the context of obese diabetics.

In conclusion, a 7-week high-fructose diet induced several renal changes in SDT fatty rats, including urinary electrolyte imbalances and associated mineral deposition in the kidney, suggestive of urinary stone formation. It suggests that the SDT fatty rat is a useful model to investigate type 2 diabetes and diabetic nephropathy and that excessive fructose intake can be a risk factor for the development and progression of urinary stone formation and diabetes- related kidney injury.

Funding

No funding was provided for the work.

Conflict of interest

The authors declare that there is no conflict of interest.

Data availability

The data in this study are included in the article/supplementary materials. Contact the corresponding author(s) directly to request the underlying data.

Author contributions

Conceptualization: Kaoru Toyoda

Funding acquisition: Kaoru Toyoda

Investigation: Kaoru Toyoda, Tadakazu Takahashi, Yuki Tanaka, Yusuke Suzuki

Supervision: Kaoru Toyoda

Visualization: Kaoru Toyoda

Writing – original draft: Kaoru Toyoda

Writing – original draft: Kaoru Toyoda, Hideaki Yokoyama, Toshiyuki Shoda, James K. Chambers, Kazuyuki Uchida

Ethical approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

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