Effects of excessive sodium chloride loading in the spontaneously diabetic torii (SDT) fatty rats, a preclinical model of type 2 diabetes mellitus

Abstract

Type 2 diabetes mellitus represents an international health concern with its growing number of patients worldwide. At the same time, excessive salt consumption is also seen as a major cause of diseases such as hypertension and may expedite renal complications in diabetic patients. In this study, we investigated the effects of excessive sodium chloride supplementation on the kidney of the Spontaneously Diabetic Torii-Lepr^fa (SDT fatty) rat, an obese type 2 diabetes model. Male and female SDT fatty rats and normal Sprague-Dawley (SD) rats at 5 weeks of age were loaded with 0.3% sodium chloride (NaCl) in drinking water for 13 weeks. Blood serum and urinary parameters were observed throughout the experiment and kidney samples were examined in histopathological and genetical analyses. Significant changes on the body weight, blood pressure, urine volume, creatinine clearance, blood urea nitrogen (BUN), relative gene expressions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), monocyte chemotactic protein-1 (MCP-1) and transforming growth factor-β (TGF-β) were observed in the salt-loaded male SDT fatty rats. Urinary L-type fatty acid-binding protein (L-FABP) and albumin levels were higher observed in the salt-loaded male SDT fatty rats throughout the period, but urinary albumin levels in the female SDT fatty rats remain unchanged. In the kidney, slight Armani-Ebstein changes, tubular degeneration, hyaline cast, and inflammatory cell infiltration were observed in female SDT fatty rats while the levels of some changes were higher in the salt-loaded group. The kidney of the salt-loaded male SDT fatty rats demonstrated a higher degree of lesions compared to the female group and the male unloaded group. Histopathological changes in salt-loaded SDT fatty rats show that excessive salt consumption may act as a diabetic pathology exacerbation factor, but the pathology may be influenced by gender difference. Urinary L-FABP levels may act as a useful biomarker to detect slight tubular damages in the kidney. Excessive salt loading was shown to exacerbate the renal injury in SDT fatty rats.

INTRODUCTION

Type 2 diabetes mellitus (T2DM) had become an international health concern with its growing number of patients globally, especially in developing countries (Shaw et al., 2010; Whiting et al., 2011). At the same time, excessive salt consumption is also seen as a major cause of diseases such as hypertension and may expedite renal complications in diabetic patients. Countries with the highest daily salt consumption include Kazakhstan (15.2 g), Uzbekistan (14.3 g), and Mauritius (13.8 g), and East Asian and Southeast Asian countries like Korea (13.2 g), Japan (12.4 g), China (12.3 g), Thailand (13.5 g), and Singapore (13.1 g) were also among the top 20 countries with the highest consumption of salt in 2010 (Powles et al., 2013). The relationship between salt intake and deaths from cardiovascular diseases was reported at a daily intake of more than 2 g per day, claiming that 80% of the deaths occurred in low-income or middle-income countries (Mozaffarian et al., 2014). The World Health Organization (WHO) strongly recommended reducing dietary salt intake to reduce the number of deaths from hypertension, cardiovascular disease, and stroke. However, some scientists still advocate the possibility of increased risk of cardiovascular disease morbidity and mortality at extremes of low salt intake (Ha, 2014).

In this study, we investigated the effects of excessive salt supplementation on the kidney of the Spontaneously Diabetic Torii-Lepr^fa (SDT fatty) rat, an obese type 2 diabetes model. The SDT fatty rat was established by introducing the fa gene of the Zucker fatty rat into the original SDT rat genome (Masuyama et al., 2005). The female SDT fatty rats show hyperphagia, leading to overt obesity, hyperglycemia, and hyperlipidemia at a young age (Ishii et al., 2010). The early incidence of diabetes leads to early diabetic complications compared to other T2DM model animals (Katsuda et al., 2014b; Matsui et al., 2008). Female SDT fatty rats were reported to develop diabetes mellitus at 6 weeks of age, following renal and cataract complications from 16 weeks of age (Masuyama et al., 2005). These complications were caused by hyperglycemia and pancreatic function weakness, according to a previous study by Ishii et al. (2010).

Physiological changes induced by salt intake in the SDT fatty rats were previously reported by Ohta et al. and Katsuda et al., both in 2014, with a dosage of 1% salt water (Ohta et al., 2014; Katsuda et al., 2014a). The results included a rise in the systolic blood pressure (SBP) and higher severity of pathological findings in the kidney, and lethal bodies were observed in the salt-loaded male SDT fatty rats. As tubulointerstitial damage of the kidney is reported to contribute to renal dysfunctions in Diabetic Kidney Disease (DKD) and End-Stage Renal Disease (ESRD), it is important to study the tubulointerstitial damage in this study (Fioretto and Mauer, 2007; Pourghasem et al., 2015). The urinary Liver-type Fatty Acid Binding Protein (L-FABP) was introduced as a novel biomarker of tubulointerstitial damage as it accurately reflects the degree of oxidative stress, peritubular blood flow, and tubulointerstitial damage (Kamijo-Ikemori et al., 2006; Kamijo-Ikemori et al., 2013). In SDT fatty rats, the urinary L-FABP is well correlated with decreased renal blood flow and oxidative stress status (Tanabe et al., 2019). It was widely used as a tubular marker to detect early diabetic nephropathy in clinical practice (Araki et al., 2013; Nielsen et al., 2010). On the other hand, interleukin-17A (IL-17A) was reported to regulate renal sodium transporters and renal injury in Angiotensin II-induced hypertension (Norlander et al., 2016), and an induction of pathogenic Th17 cells autoimmune disease driven by sodium chloride (NaCl) (Kleinewietfeld et al., 2013).

In the present study, we investigated the physiological features of the kidney in male and female SDT fatty rats loaded with a lower dose of NaCl (i.e., 0.3% salt supplementation) to study the effects of lower salt intake levels in T2DM individuals and to evaluate the use of newly introduced biomarkers such as the L-FABP and IL-17A in SDT fatty rats.

MATERIALS AND METHODS

Animals

Male and female Sprague Dawley (SD) rats and SDT fatty rats from CLEA Japan Inc. (Tokyo, Japan) were used in this study. The rats were housed in a climate-controlled room with a temperature of 23 ± 3°C, relative humidity of 55 ± 15%, and a 12 hr/12 hr light-dark cycle in the animal institute of Japan Tobacco Inc. Basal diet (CRF-1; Oriental Yeast Co., Ltd, Tokyo, Japan) and water or 0.3% NaCl solution were provided ad libitum for 12 weeks, from 5 to 17 weeks of age. All animal procedures and protocols complied with the guidelines for animal experimentation set by the Ethics Committee for Animal Use at Biological/Pharmaceutical Research Laboratories, Japan Tobacco Inc.

Measurement of biological parameters

Body weights, blood chemistry parameters were periodically evaluated from 5 to 17 weeks of age. SBP and heart rate in the conscious non-fasted state were measured at 12 weeks of age by the indirect tail-cuff method using a Softron BP-98A indirect blood pressure meter (Softron Co. Ltd., Tokyo, Japan). The blood pressure was measured between 13:00 and 16:00 hr. Five measurements were made for each rat and then averaged. Blood samples were collected from the tail vein in a non-fasting state. Blood glucose, triglyceride (TG), total cholesterol (TCHO), creatinine levels, and blood urea nitrogen (BUN) levels were measured using commercial kits (Roche Diagnostics, Basel, Switzerland) and an automatic analyzer (Hitachi Ltd., Tokyo, Japan) as biochemical parameters. Urinalysis including urinary volume, urinary albumin excretion (UAE), and creatinine clearance (Ccr) was performed at 9, 13, and 17 weeks of age. Urine samples were collected by placing the animals in metabolic cages with water for 6 hr. Urinary albumin level was measured with a rat-albumin enzyme immunoassay (ELISA) kit (AKRAL-120, Shibayagi, Gunma, Japan). Urinary L-FABP level was measured using a Rat L-FABP ELISA Kit (CMIC Holdings Inc., Tokyo, Japan). Urinary creatinine levels were measured using QuantiChrom^TM Creatinine Assay Kit (DICT-500, BioAssay Systems, Hayward, CA, USA). Ccr was calculated by dividing the urinary creatinine level by the serum creatinine level and body weight.

Tissue sampling

Necropsy was performed at 17 weeks of age. All animals were sacrificed by exsanguination under isoflurane anesthesia. The kidneys were immediately sampled, weighed, and fixed in 4% paraformaldehyde or snap-frozen at −180°C in liquid nitrogen for subsequent histopathological and molecular analysis, respectively.

Total RNA isolation and reverse transcription

Total RNA was isolated from frozen renal cortex samples using Sepasol-RNA I Super G (Nacalai Tesque Inc., Kyoto, Japan) according to the manufacturer’s protocol. Total RNA was quantified with spectrophotometry using Nanodrop 2000c (Thermo Fischer Scientific, Kanagawa, Japan) at an absorbance of 260 nm absorbance ratio. The value ranged from 1.9 to 2 for all samples. Complementary DNA (cDNA) was prepared from 2 μg total RNA by reverse transcription using 2 μL of Prime Script^TM RT Master Mix (Takara Bio Inc., Shiga, Japan). The resulting cDNA was stored at −30°C.

qRT-PCR

The expression of inflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β], monocyte chemotactic protein-1 [MCP-1]), interleukin-17A [IL-17A] and IL-17 receptor A [IL-17RA]), fibrosis-related factors (transforming growth factor-β [TGF-β], α-smooth muscle actin [α-SMA], collagen-I [col-I], collagen-IV [col-IV]), Renin-Angiotensin System (RAS) related factors (angiotensin-converting enzyme [ACE], ACE II, angiotensin II receptor type 1 [AT1R], angiotensin 1-7 receptor [MAS1]) was assessed with GAPDH as the internal reference. The primers used are shown in Table 1. Real-time quantitative PCR (qRT-PCR) was performed using Thermal Cycler Dice Real-Time System II (Takara Bio Inc.). Each 25-μL reaction contained 2 μL cDNA, 1 μL gene-specific forward and reverse primers (10 μM), 8.5 μL dH₂O, and 12.5 μL SYBR Premix Ex Taq (Takara Bio Inc.). The cycling conditions were as follows: initial template denaturation for 30 sec at 95°C, followed by 40 cycles of denaturation at 95°C for 5 sec, annealing at 60°C for 30 sec, and dissociation at 95°C for 15 sec, 60°C for 30 sec, and 95°C for 15 sec. Fluorescence detection was performed during each cycle at 60°C and 95°C to identify the positive samples. The fold changes in gene expression relative to the levels obtained in SD rats, which were set equal to 1, were analyzed and calculated using the 2^−ΔΔct method.

Table 1. Primers used for the real-time quantitative PCR (qRT-PCR).

Gene	Forward primer 5'-3'	Reverse primer 5'-3'
TNF-α	GAA CTC AGC GAG GAC ACC AA	GCC AGT GTA TGA GAG GGA CG
IL-1β	GAC TTC ACC ATG GAA CCC GT	GGA GAC TGC CCA TTC TCG AC
MCP-1	CTT CCT CCA CCA CTA TGC AGG	GAT GCT ACA GGC AGC AAC TG
α-SMA	GTT TGA GAC CTT CAA T	CGA TCT CAC GCT CAG C
TGF-β	CCA TGA CAT GAA CCG ACC CT	CTG CCG TAC ACA GCA GTT CT
col-I	GTA CAT CAG CCC AAA CCC CA	CAG GAT CGG AAC CTT CGC TT
col-IV	CTT CGT TGG CCT CTG TTT GC	TGC ACT GGA TTG CAA AAG GC
ACE	TCC TAT TCC CGC TCA TCT	CCA GCC CTT CTG TAC CAT T
ACE2	GAA TGC GAC CAT CAA GCG	CAA GCC CAG AGC CTA CGA
AT1R	CTG GCA AGC ATC TTA TGT AGT TC	ACA AGC ATT CAC ACC TAA GTA TTC
MAS1	ACT GTC GGG CGG TCA TCA TC	GGT GGA GAA AAG CAA GGA GA
IL-17	GAA GTT GGA CCA CCA CAT GA	TCC CTC TTC AGG ACC AGG AT
IL-17RA	GAC CCA AAC CAC AAG TCC AA	GTC ATC TTC ATC TCC GTG TCC
GAPDH	AGT GCC AGC CTC GTC TCA TA	AAG AGA AGG CAG CCC TGG TA

Abbreviation: TNF-α; tumor necrosis factor-α, IL-1β; interleukin-1β, MCP-1; monocyte chemotactic protein-1, α-SMA; α-smooth muscle actin, TGF-β; transforming growth factor-β, col-I; collagen-I, col-IV; collagen-IV, ACE; angiotensin-converting enzyme, ACE II; angiotensin-converting enzyme II, AT1R; angiotensin II receptor type 1, MAS1; angiotensin 1-7 receptor, IL-17; interleukin-17A, IL-17RA; IL-17 receptor A, GAPDH; Glyceraldehyde-3-phosphate dehydrogenase

Histopathological analysis

After resection, the tissue was paraffin-embedded by standard techniques and thin-sectioned (3 to 5 μM). The deparaffinated samples were stained with hematoxylin and eosin (HE), periodic acid Schiff (PAS), and Sirius red (SR). Immunohistochemistry was performed using ED-1 (Abcam, Tokyo, Japan), α-SMA (Abcam) antibodies, both diluted 100-fold, and desmin (Nichirei Biosciences Inc., Tokyo, Japan) ready-to-use antibodies. Samples were processed with 500 W microwave for 10 min (except desmin samples), cooled at room temperature, then processed with 0.009% H₂O₂ methanol before applying the respective antibodies. Histofine Simple Stain Rat MAX-PO (MULTI) (Nichirei Biosciences Inc.) was used as the secondary antibody. Staining was visualized using DAB Tablet (DAB/4HCl 10 mg/tablet) (Wako Pure Chemical Industries, Osaka, Japan), dissolved in 100 mL Tris buffer solution and 10 μL 30% H₂O₂ for 3 min during each reaction, to produce brown resultants indicating antigen localization. All samples were examined histopathologically or immunohistochemically in a blind manner, and the findings were graded from normal (-) to severe (3+) in accordance with the following criteria. Grade ± was used to represent very weakly positive changes, + was used to represent focal or weakly positive sections, 2+ was used to represent half of the section area positive or moderate changes, and 3+ was used to represent more than half of the whole area positive or severe changes in a tissue section. For the glomerular findings in kidneys, more than one hundred glomeruli per animal were observed and ED-1 positive cells were counted in 100 glomeruli of each sample in a blind manner. Desmin positive areas in the glomeruli were measured in 100 glomeruli of each sample in a blind manner using cellSens imaging software (Olympus, Tokyo, Japan) and calculated to an average positive area of each glomerulus.

Statistical analysis

The results are expressed as the mean ± standard deviation. Statistical analyses were performed using the GraphPad Prism Ver. 6.05 (GraphPad Software, San Diego, CA, USA). The significance of the difference between groups was examined using the one-way analysis of variance (ANOVA) and the Tukey multiple comparison test. P values < 0.05 (two-tailed) were considered significant.

RESULTS

Body weights, biochemical and renal parameters

Changes in body weight, SBP, blood glucose levels, and urinalysis are shown in Fig. 1. The body weights of the female SDT fatty rats were higher than those of the SD group, while salt-loaded SDT fatty rats were significantly lower at 13 and 17 weeks of age (Fig. 1A). Body weights of male SDT fatty rats were comparable to those of the SD groups throughout the experimental period, showing no difference in salt-loaded groups as well (Fig. 1B). The SBP of female SDT fatty rats was significantly higher than those of SD groups but there is no difference in the salt loading (Fig. 1C). Meanwhile, male salt-loaded SDT fatty rats showed higher SBP than the unloaded group, which is comparable to the SD groups (Fig. 1D). The heart rates of both female and male SDT fatty rats did not differ with salt loading, while comparable to those of the SD groups (data not shown). The absolute and relative kidney weights of both female and male SDT fatty rats were higher than those of the SD groups, while showing no difference in salt loading (data not shown). The SDT fatty group showed hyperglycemia compared to the SD group, but blood glucose levels in female salt-loaded SDT fatty rats were lower than the unloaded group (Fig. 1E), while TG and TCHO levels increased in both female and male SDT fatty rats (data not shown). Ccr levels in the SDT fatty group were comparable to the SD groups in both male and female rats, BUN levels were lower in the female salt-loaded SDT fatty rats than then unloaded group, which is comparable to the SD group, while BUN levels were higher in the SDT fatty group compared to the SD group in the male rats (data not shown). The urinary albumin levels of both female and male SDT fatty rats were significantly higher than those of the SD groups, while the level of salt-loaded female SDT fatty rats was comparable to that of the SDT fatty group, male salt-loaded SDT fatty rats showed a higher level of urinary albumin than the unloaded group throughout the experimental period (Fig. 1G and H). The urinary L-FABP levels of both female and male SDT fatty rats were significantly higher than those of the SD groups, while the levels showed a higher trend in the salt-loaded SDT fatty rats in both genders (Fig. 1I and J).

Fig. 1

Changes in body weight (BW) (A, B), systolic blood pressure (SBP) (C, D), glucose (E, F), urinary albumin (G, H) and urinary L-type fatty acid-binding protein (L-FABP) (I, J) in in female (A, C, E, G, and I) and male (B, D, F, H, and J) SDT fatty rats and SD rats with or without salt intake. Data are represented as mean ± standard deviation (n = 5). *p < 0.05, **p < 0.01; significantly different from the SD group. ^#p < 0.05, ^##p < 0.01; significantly different from the salt intake group.

qRT-PCR

Changes in the relative mRNA expression of inflammatory cytokines and fibrosis-related factors are shown in Fig. 2. Both female and SDT fatty rats did not show significant differences in the expression levels of inflammatory cytokines (TNF-α, IL-1β, MCP-1) regardless of salt supplementation or comparison with the SD rat groups (Fig. 2A). However, salt-loaded male SDT fatty rats tend to show higher relative expression levels when compared to other groups (Fig. 2A). The relative expression levels of fibrosis-related factors (TGF-β, α-SMA, col-I, col-IV) showed a similar trend to levels of inflammatory cytokines listed above, while salt-loaded male SDT fatty rats showed significant differences in TGF-β, col-IV when compared to the SDT fatty group (Fig. 2B). Changes in the relative mRNA expression of RAS related factors and IL-17 related cytokines between SDT fatty rats and SD rats, or salt loading were not observed in this study (data not shown).

Fig. 2

Relative gene expression of inflammatory cytokines (A) and fibrosis-related factors (B) of male and female SDT fatty rats and SD rats with or without salt intake. Data are represented as mean + standard deviation (n = 5). *p < 0.05; significantly different from the SD group.

Histopathological analysis

Histopathological findings and immunohistochemistry results on the kidney are shown in Tables 2 and 3, and Fig. 3. The findings below were normal in both female and male SD rats and salt-loaded SD rats. Glomerular changes were slight in both female and male SDT fatty rats while the findings were moderately observed in the male salt-loaded group (Tables 2 and 3, Fig. 3). In the tubules, hyaline cast, Armani-Ebstein, tubular regeneration, inflammatory cell infiltration, and fibrosis in the interstitial space were slightly observed in the female SDT fatty group, while moderate in the male SDT fatty group (Tables 2 and 3, Fig. 3). In the salt-loaded SDT fatty rats, the findings were comparable to the unloaded group in the female rats, while male SDT fatty rats developed moderate to severe tubule changes (Tables 2 and 3, Fig. 3). Immunohistochemistry of ED-1 shows that there was no difference between the salt-loaded and unloaded female SDT fatty rats, while the number of positive cells increased in the salt-loaded male SDT fatty rats (Tables 2 and 3). The mean number of macrophages in 100 glomeruli were counted and no ED-1 positive cells were observed in both female and male SD rats. The mean number of ED-1 positive cells were 0.6 ± 1.8 in female and 1.8 ± 1.6 in male SDT fatty groups, while the number increased to 2.4 ± 1.8 in female and 27 ± 50 in male SDT fatty rats loaded with salt (Fig. 4A). The difference between salt-loaded and unloaded group was not significant. ED-1 positive cells were mainly observed in the interstitial site than the glomeruli, this indicates that low concentration salt intake may affect the tubules or interstitial cells. α-SMA positive cells and desmin positive cells were slightly observed in the tubules and glomeruli respectively, and there was almost no difference observed between the salt-loaded and unloaded SDT fatty rats (Tables 2 and 3, Fig. 3). However, the mean desmin positive area/glomerulus counted in 100 glomeruli was around 100-fold higher in the SDT fatty rats compared to SD rats, while the area increased in the salt-loaded SDT fatty rats (Fig. 4B).

Table 2. Histopathological and immunohistochemical findings in kidneys from the female SDT fatty rats and SD rats (n = 5).

Patholo gical findings Kidney: Female	Groups
	SDT fatty						SDT fatty + NaCl						SD						SD + NaCl
	-	±	+	++	+++		-	±	+	++	+++		-	±	+	++	+++		-	±	+	++	+++
Glomeruli
Atrophy	5	0	0	0	0		2	0	3	0	0		4	1	0	0	0		4	1	0	0	0
Adhesion	4	1	0	0	0		1	0	4	0	0		1	4	0	0	0		0	5	0	0	0
Hypertrophy	0	0	3	2	0		0	0	3	2	0		1	4	0	0	0		0	5	0	0	0
Mesangial hyperplasia	0	1	4	0	0		0	0	3	2	0		1	4	0	0	0		0	5	0	0	0
Fibrosis	0	4	1	0	0		0	3	2	0	0		3	2	0	0	0		0	4	1	0	0
Renal Tubule
Regeneration	0	0	3	2	0		0	1	4	0	0		4	1	0	0	0		4	1	0	0	0
Tubular dilation	0	0	2	3	0		0	2	3	0	0		4	1	0	0	0		4	1	0	0	0
Armanni-Ebstein lesion	0	1	1	3	0		4	1	0	0	0		5	0	0	0	0		5	0	0	0	0
Hyaline cast	2	0	3	0	0		3	0	2	0	0		5	0	0	0	0		5	0	0	0	0
Tubulointerstitium
Infiltration, inflammatory cell	0	1	3	1	0		1	3	1	0	0		2	3	0	0	0		2	3	0	0	0
Fibrosis	0	0	3	2	0		1	1	3	0	0		1	4	0	0	0		1	4	0	0	0
ED-1 Positivity	2	2	1	0	0		0	3	2	0	0		5	0	0	0	0		5	0	0	0	0
Desmin Positivity	0	1	4	0	0		0	2	3	0	0		5	0	0	0	0		5	0	0	0	0
α-SMA Positivity	0	1	2	2	0		0	0	4	1	0		5	0	0	0	0		5	0	0	0	0

Each of the findings was assigned one of five qualitative grades: normal (-), very slight (±), slight (+), moderate (++), or severe (+++) abnormalities. Each value in the Table (e.g., 0, 1, 2, 3, 4, 5) represents the number of rats with the pathological finding.

Table 3. Histopathological and immunohistochemical findings in kidneys from the male SDT fatty rats and SD rats (n = 5).

Pathological findings Kidney: Male	Groups
	SDT fatty						SDT fatty + NaCl						SD						SD + NaCl
	-	±	+	++	+++		-	±	+	++	+++		-	±	+	++	+++		-	±	+	++	+++
Glomeruli
Atrophy	0	1	4	0	0		0	0	5	0	0		5	0	0	0	0		5	0	0	0	0
Adhesion	2	2	1	0	0		0	2	2	1	0		5	0	0	0	0		5	0	0	0	0
Hypertrophy	0	0	0	5	0		0	0	1	4	0		5	0	0	0	0		4	1	0	0	0
Mesangial hyperplasia	0	0	4	1	0		0	0	1	3	1		3	2	0	0	0		3	2	0	0	0
Fibrosis	0	0	5	0	0		0	0	1	3	1		4	1	0	0	0		3	2	0	0	0
Renal Tubule
Regeneration	0	0	5	0	0		0	1	1	2	1		5	0	0	0	0		4	1	0	0	0
Tubular dilation	0	0	3	2	0		0	0	1	2	2		5	0	0	0	0		5	0	0	0	0
Armanni-Ebstein lesion	0	0	1	4	0		0	0	2	2	3		5	0	0	0	0		5	0	0	0	0
Hyaline cast	1	0	4	0	0		0	0	2	2	1		5	0	0	0	0		5	0	0	0	0
Tubulointerstitium
Infiltration, inflammatory cell	0	1	4	0	0		0	1	4	0	0		4	1	0	0	0		3	2	0	0	0
Fibrosis	0	1	4	0	0		0	0	1	3	1		4	1	0	0	0		3	2	0	0	0
ED-1 Positivity	2	3	0	0	0		0	0	3	1	1		3	2	0	0	0		2	3	0	0	0
Desmin Positivity	0	1	2	2	0		0	0	1	4	0		4	1	0	0	0		2	3	0	0	0
α-SMA Positivity	0	3	2	0	0		0	1	1	2	1		4	1	0	0	0		5	0	0	0	0

Each of the findings was assigned one of five qualitative grades: normal (-), very slight (±), slight (+), moderate (++), or severe (+++) abnormalities. Each value in the Table (e.g., 0, 1, 2, 3, 4, 5) represents the number of rats with the pathological finding.

Fig. 3

Kidney histopathology of male SDT fatty rats and SD rats by salt intake. (A to D): hematoxylin and eosin (HE) staining; arrows, double arrows, and arrowheads indicate obvious hyaline cast, tubular regeneration, and Armani-Ebstein lesion, respectively. (E to H): periodic acid-Schiff (PAS) staining; arrowheads indicate mesangial hyperplasia. (I to L): Sirius red staining; arrows and arrowheads indicate fibrosis (glomeruli) and fibrosis (tubulointerstitium), respectively. (M to P): Immunohistochemistry of desmin. Arrows indicate typical glomeruli that show a positive signal. Bar = 100 μm.

Fig. 4

Average ED-1 positive cells/100 glomerulus (A) and positive area of desmin Immunohistochemistry/glomerulus (B) in the kidney of male and female SDT fatty rats and SD rats with or without salt intake. Data are represented as mean + standard deviation (n = 5). *p < 0.05; significantly different from the SD group.

DISCUSSION

The above results indicate that changes in body weight, SBP, plasma and urinary parameters, and kidney histopathological changes were developed in both female and male SDT fatty rats, with underlying mechanisms manipulated by the inflammatory cytokines and fibrotic factors. After 0.3% salt loading, male SDT fatty rats showed a higher degree of blood pressure and renal changes in plasma, urinary parameters, inflammatory cytokines and fibrotic related factors, and histopathological changes compared to the female rats, which did not demonstrate significant changes except the higher trend in L-FABP and desmin positive areas in the glomeruli.

In the present study, 0.3% salt load did not influence the blood pressure and heart rate of the female SDT fatty rats, which differs from the study of Katsuda et al., in which the salt load concentration was 1% (Katsuda et al., 2014a). On the other hand, salt-loaded male SDT fatty rats showed an increase in blood pressure from 13 weeks of age (8 weeks of salt loading), which shows that salt sensitivity differs by gender in the SDT fatty rats. Blood glucose levels of the SDT fatty rats were higher compared to the SD groups while showing no changes in salt load in male rats, but a minor decrease of the blood glucose levels in female salt-loaded SDT fatty rats was observed. Although the amount of food intake was not measured in this study, the previous study has shown that there is a trend towards lower food intake and blood glucose in female salt-loaded SDT fatty rats (Katsuda et al., 2014a). This was similar to the present study but the reason behind the reduction of food intake due to salt load is still unknown. TG and TCHO levels of both female and male SDT fatty rats were higher than those of the SD groups but not influenced by salt load, while Ccr levels were not different between SDT fatty rats and the SD rats.

Although there were no significant changes in renal parameters such as Ccr and BUN, the urinary albumin and L-FABP levels of the SDT fatty rats were higher than those of the SD rats showing there are glomerular and tubulointerstitial damages in the kidney of SDT fatty rats. In the salt-loaded groups, male SDT fatty rats showed an increase in both albumin and L-FABP levels. While albumin levels of the female SDT fatty rats did not differ from the unloaded group, it showed a tendency of higher L-FABP levels which indicates that salt-loading may contribute to early tubulointerstitial damage. The difference in the tendency of urinary albumin and L-FABP levels in female and male SDT fatty rats may be explained through salt sensitivity and increased blood pressure in male SDT fatty rats as stated above. In addition, women are more susceptible to non-albuminuric renal impairment, and in the cohort studies, urinary L-FABP has been reported to be more associated with diabetes incidence, especially in women, than albumin (Kubota et al., 2021; Penno et al., 2011). Furthermore, L-FABP was also reported to be useful to identify early renal damages even before albuminuria, which is known as a sensitive biomarker of kidney diseases (Kamijo-Ikemori et al., 2011; Thi et al., 2020). As the progression of renal diseases was known to be strongly related to the tubulointerstitial injury than glomerular injury (Risdon et al., 1968), urinary L-FABP, which is a highly sensitive biomarker for tubulointerstitial damage, is indeed useful to predict the progression of the onset of early DKD or diabetic nephropathy. On the other hand, although IL-17A has been reported to be associated with renal injury (Norlander et al., 2016), no changes in IL-17-related cytokines were observed in this study. Therefore, IL-17A was not found to be useful as a biomarker for renal injury in this model.

The histology findings in the kidney showed slight glomerular and tubular changes in the female SDT fatty rats but were moderate in the male SDT fatty rats. Salt loading did not influence the female rats but in the male SDT ratty rats, the findings in the glomeruli and tubules were higher than the unloaded rats. Increased ED-1 positive cells in the glomeruli and the interstitial space, although not significant, indicate that salt loading potentially affects renal functions by inducing macrophage increase in the kidney. Increased inflammatory cells in the kidney were known to contribute to chronic inflammation and eventually lead to fibrosis in chronic kidney disease (CKD) and ESRD (Pourghasem et al., 2015). Increased positive areas of desmin in the glomeruli also indicate that salt loading in diabetic models may attribute to podocyte injury, which leads to the failure of the glomerular filtration barrier, and finally CKD (Becherucci et al., 2015; Fligny et al., 2011; Reiser and Sever, 2013; Asanuma, 2015). The grades of findings in this study were apparently lower than those in previous studies conducted with 1% salt loading (Ohta et al., 2014; Katsuda et al., 2014a), which means a lower concentration of salt supplementation (0.3%) is essential to slow down the progression of kidney diseases but there is still a minor effect in the kidney. However, as heterogeneity of hypertension or salt sensitivity in human was previously reported (Choi et al., 2015; Weinberger, 1996; Poch et al., 2001), this factor must be considered when evaluating the effects of excessive salt consumption in diabetic patients.

In this study, there was the gender difference in the renal findings observed in salt-loaded SDT fatty rats. One of the factors may be due to the difference between males and females in the findings observed in the original SDT fatty rats. In fact, in male and female SDT fatty rats, common findings such as glomerulosclerosis in the glomeruli, Armanni-Ebstein lesions and tubular dilatation in the renal tubules were observed, but these findings occurred earlier and were severe in male SDT fatty rats (Ishii et al., 2010; Matsui et al., 2008). Similarly, in the results of this study, when compared with male and female SDT fatty rats without salt loading, male had severe findings than female. Clinical studies have also reported that kidney disease progresses more rapidly in men than in women (Neugarten and Golestaneh, 2019). It has been suggested that the antioxidant effect of the female hormone estrogen may contribute to the renal protective effect (Kasimay et al., 2009). On the other hand, when blood pressure was used as an index, women seemed to be more salt-sensitive than men (Wesseling et al., 2011), but in this study, salt-loading did not show any effect on blood pressure in female rats. Considering that 1% salt loading in female SDT fatty rats clearly causes hypertension and renal damage (Katsuda et al., 2014a), the effect on blood pressure may also be involved in the gender difference in renal findings.

From the results of the present study, it is indicated that low concentration salt supplementation may act as an exacerbation factor in hypertension and lowered renal functions, especially tubulointerstitial damage in diabetic patients but the difference of salt sensitivity in gender or race must also be considered. While the damage did not show any changes in albuminuria, other renal parameters and only little changes were found histologically between the salt-loaded and unloaded group, L-FABP proved to be a highly sensitive urinary biomarker to evaluate early stages of tubular damage in DKD.

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES

•  Araki,  S.,  Haneda,  M.,  Koya,  D.,  Sugaya,  T.,  Isshiki,  K.,  Kume,  S.,  Kashiwagi,  A.,  Uzu,  T. and  Maegawa,  H. (2013): Predictive effects of urinary liver-type fatty acid-binding protein for deteriorating renal function and incidence of cardiovascular disease in type 2 diabetic patients without advanced nephropathy. Diabetes Care, 36, 1248-1253.
•  Asanuma,  K. (2015): The role of podocyte injury in chronic kidney disease. Nihon Rinsho Meneki Gakkai Kaishi, 38, 26-36.
•  Becherucci,  F.,  Lazzeri,  E. and  Romagnani,  P. (2015): A road to chronic kidney disease: toward glomerulosclerosis via dendrin. Am. J. Pathol., 185, 2072-2075.
•  Choi,  H.Y.,  Park,  H.C. and  Ha,  S.K. (2015): Salt Sensitivity and Hypertension: A Paradigm Shift from Kidney Malfunction to Vascular Endothelial Dysfunction. Electrolyte Blood Press., 13, 7-16.
•  Fioretto,  P. and  Mauer,  M. (2007): Histopathology of diabetic nephropathy. Semin. Nephrol., 27, 195-207.
•  Fligny,  C.,  Barton,  M. and  Tharaux,  P.-L. (2011): Endothelin and podocyte injury in chronic kidney disease. Contrib. Nephrol., 172, 120-138.
•  Ha,  S.K. (2014): Dietary salt intake and hypertension. Electrolyte Blood Press., 12, 7-18.
•  Ishii,  Y.,  Ohta,  T.,  Sasase,  T.,  Morinaga,  H.,  Ueda,  N.,  Hata,  T.,  Kakutani,  M.,  Miyajima,  K.,  Katsuda,  Y.,  Masuyama,  T.,  Shinohara,  M. and  Matsushita,  M. (2010): Pathophysiological analysis of female Spontaneously Diabetic Torii fatty rats. Exp. Anim., 59, 73-84.
•  Kamijo-Ikemori,  A.,  Sugaya,  T.,  Ichikawa,  D.,  Hoshino,  S.,  Matsui,  K.,  Yokoyama,  T.,  Yasuda,  T.,  Hirata,  K. and  Kimura,  K. (2013): Urinary liver type fatty acid binding protein in diabetic nephropathy. Clin. Chim. Acta, 424, 104-108.
•  Kamijo-Ikemori,  A.,  Sugaya,  T. and  Kimura,  K. (2006): Urinary fatty acid binding protein in renal disease. Clin. Chim. Acta, 374, 1-7.
•  Kamijo-Ikemori,  A.,  Sugaya,  T.,  Yasuda,  T.,  Kawata,  T.,  Ota,  A.,  Tatsunami,  S.,  Kaise,  R.,  Ishimitsu,  T.,  Tanaka,  Y. and  Kimura,  K. (2011): Clinical significance of urinary liver-type fatty acid-binding protein in diabetic nephropathy of type 2 diabetic patients. Diabetes Care, 34, 691-696.
•  Kasimay,  O.,  Sener,  G.,  Cakir,  B.,  Yüksel,  M.,  Cetinel,  S.,  Contuk,  G. and  Yeğen,  B.C. (2009): Estrogen protects against oxidative multiorgan damage in rats with chronic renal failure. Ren. Fail., 31, 711-725.
•  Katsuda,  Y.,  Kemmochi,  Y.,  Maki,  M.,  Sano,  R.,  Ishii,  Y.,  Miyajima,  K.,  Kakimoto,  K. and  Ohta,  T. (2014a): Physiological changes induced by salt intake in female Spontaneously Diabetic Torii-Lepr(fa) (SDT fatty) rat, a novel obese type 2 diabetic model. Anim. Sci. J., 85, 588-594.
•  Katsuda,  Y.,  Ohta,  T.,  Miyajima,  K.,  Kemmochi,  Y.,  Sasase,  T.,  Tong,  B.,  Shinohara,  M. and  Yamada,  T. (2014b): Diabetic complications in obese type 2 diabetic rat models. Exp. Anim., 63, 121-132.
•  Kleinewietfeld,  M.,  Manzel,  A.,  Titze,  J.,  Kvakan,  H.,  Yosef,  N.,  Linker,  R.A.,  Muller,  D.N. and  Hafler,  D.A. (2013): Sodium chloride drives autoimmune disease by the induction of pathogenic TH17 cells. Nature, 496, 518-522.
•  Kubota,  Y.,  Higashiyama,  A.,  Marumo,  M.,  Konishi,  M.,  Yamashita,  Y.,  Okamura,  T.,  Miyamoto,  Y. and  Wakabayashi,  I. (2021): Relationship of urinary liver-type fatty acid-binding protein with cardiovascular risk factors in the Japanese population without chronic kidney disease: sasayama study. BMC Nephrol., 22, 189.
•  Masuyama,  T.,  Katsuda,  Y. and  Shinohara,  M. (2005): A novel model of obesity-related diabetes: introgression of the Lepr(fa) allele of the Zucker fatty rat into nonobese Spontaneously Diabetic Torii (SDT) rats. Exp. Anim., 54, 13-20.
•  Matsui,  K.,  Ohta,  T.,  Oda,  T.,  Sasase,  T.,  Ueda,  N.,  Miyajima,  K.,  Masuyama,  T.,  Shinohara,  M. and  Matsushita,  M. (2008): Diabetes-associated complications in Spontaneously Diabetic Torii fatty rats. Exp. Anim., 57, 111-121.
•  Mozaffarian,  D.,  Fahimi,  S.,  Singh,  G.M.,  Micha,  R.,  Khatibzadeh,  S.,  Engell,  R.E.,  Lim,  S.,  Danaei,  G.,  Ezzati,  M. and  Powles,  J.; Global Burden of Diseases Nutrition and Chronic Diseases Expert Group. (2014): Global sodium consumption and death from cardiovascular causes. N. Engl. J. Med., 371, 624-634.
•  Neugarten,  J. and  Golestaneh,  L. (2019): Influence of Sex on the Progression of Chronic Kidney Disease. Mayo Clin. Proc., 94, 1339-1356.
•  Nielsen,  S.E.,  Sugaya,  T.,  Hovind,  P.,  Baba,  T.,  Parving,  H.-H. and  Rossing,  P. (2010): Urinary liver-type fatty acid-binding protein predicts progression to nephropathy in type 1 diabetic patients. Diabetes Care, 33, 1320-1324.
• Norlander, A.E., Saleh, M.A., Kamat, N.V, Ko, B., Gnecco, J., Zhu, L., Dale, B.L., Iwakura, Y., Hoover, R.S., McDonough, A.A., and Madhur, M.S. (2016): Interleukin-17A Regulates Renal Sodium Transporters and Renal Injury in Angiotensin II-Induced Hypertension. Hypertension (Dallas, Tex. 1979), 68, 167-174.
•  Ohta,  T.,  Katsuda,  Y.,  Maki,  M.,  Shinohara,  M.,  Tong,  B. and  Yamada,  T. (2014): [Effects of salt intake on blood pressure and renal function in Spontaneously Diabetic Torii (SDT) fatty rat]. Hokushinetsu. J. Anim. Sci., 108, 9-14.
•  Penno,  G.,  Solini,  A.,  Bonora,  E.,  Fondelli,  C.,  Orsi,  E.,  Zerbini,  G.,  Trevisan,  R.,  Vedovato,  M.,  Gruden,  G.,  Cavalot,  F.,  Cignarelli,  M.,  Laviola,  L.,  Morano,  S.,  Nicolucci,  A. and  Pugliese,  G.; Renal Insufficiency And Cardiovascular Events (RIACE) Study Group. (2011): Clinical significance of nonalbuminuric renal impairment in type 2 diabetes. J. Hypertens., 29, 1802-1809.
• Poch, E., González, D., Giner, V., Bragulat, E., Coca, A., de La Sierra, A. (2001): Molecular basis of salt sensitivity in human hypertension. Evaluation of renin-angiotensin-aldosterone system gene polymorphisms. Hypertension (Dallas, Tex. 1979), 38, 1204-1209.
•  Pourghasem,  M.,  Shafi,  H. and  Babazadeh,  Z. (2015): Histological changes of kidney in diabetic nephropathy. Caspian J. Intern. Med., 6, 120-127.
•  Powles,  J.,  Fahimi,  S.,  Micha,  R.,  Khatibzadeh,  S.,  Shi,  P.,  Ezzati,  M.,  Engell,  R.E.,  Lim,  S.S.,  Danaei,  G. and  Mozaffarian,  D.; Global Burden of Diseases Nutrition and Chronic Diseases Expert Group (NutriCoDE). (2013): Global, regional and national sodium intakes in 1990 and 2010: a systematic analysis of 24 h urinary sodium excretion and dietary surveys worldwide. BMJ Open, 3, e003733.
•  Reiser,  J. and  Sever,  S. (2013): Podocyte biology and pathogenesis of kidney disease. Annu. Rev. Med., 64, 357-366.
•  Risdon,  R.A.,  Sloper,  J.C. and  De Wardener,  H.E. (1968): Relationship between renal function and histological changes found in renal-biopsy specimens from patients with persistent glomerular nephritis. Lancet, 2, 363-366.
•  Shaw,  J.E.,  Sicree,  R.A. and  Zimmet,  P.Z. (2010): Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res. Clin. Pract., 87, 4-14.
•  Tanabe,  J.,  Ogura,  Y.,  Nakabayashi,  M.,  Nagai,  Y.,  Watanabe,  S.,  Sugaya,  T.,  Ohata,  K.,  Ichikawa,  D.,  Inoue,  K.,  Hoshino,  S.,  Kimura,  K.,  Shibagaki,  Y.,  Ono,  Y. and  Kamijo-Ikemori,  A. (2019): The Possibility of Urinary Liver-Type Fatty Acid-Binding Protein as a Biomarker of Renal Hypoxia in Spontaneously Diabetic Torii Fatty Rats. Kidney Blood Press. Res., 44, 1476-1492.
•  Thi,  T.N.,  Gia,  B.N.,  Thi,  H.L.,  Thi,  T.N. and  Thanh,  H.P. (2020): Evaluation of urinary L-FABP as an early marker for diabetic nephropathy in type 2 diabetic patients. J. Med. Biochem., 39, 224-230.
• Weinberger, M.H. (1996): Salt sensitivity of blood pressure in humans. Hypertension (Dallas, Tex. 1979), 27, 481-490.
•  Wesseling,  S.,  Koeners,  M.P. and  Joles,  J.A. (2011): Salt sensitivity of blood pressure: developmental and sex-related effects. Am. J. Clin. Nutr., 94 (Suppl), 1928S-1932S.
•  Whiting,  D.R.,  Guariguata,  L.,  Weil,  C. and  Shaw,  J. (2011): IDF diabetes atlas: global estimates of the prevalence of diabetes for 2011 and 2030. Diabetes Res. Clin. Pract., 94, 311-321.