Inhibition of sodium‐glucose cotransporter 2 ameliorates renal injury in a novel medaka model of nonalcoholic steatohepatitis‐related kidney disease

The effect of sodium‐glucose cotransporter 2 inhibitor (SGLT2I) on nonalcoholic steatohepatitis (NASH) has been reported, but there are few studies on its effect on NASH‐related renal injury. In this study, we examined the effect of SGLT2I using a novel medaka fish model of NASH‐related kidney disease, which was developed by feeding the d‐rR/Tokyo strain a high‐fat diet. SGLT2I was administered by dissolving it in water of the feeding tank. SGLT2I ameliorates macrophage accumulation and oxidative stress and maintained mitochondrial function in the kidney. The results demonstrate the effect of SGLT2I on NASH‐related renal injury and the usefulness of this novel animal model for research into NASH‐related complications.

The effect of sodium-glucose cotransporter 2 inhibitor (SGLT2I) on nonalcoholic steatohepatitis (NASH) has been reported, but there are few studies on its effect on NASH-related renal injury. In this study, we examined the effect of SGLT2I using a novel medaka fish model of NASHrelated kidney disease, which was developed by feeding the d-rR/Tokyo strain a high-fat diet. SGLT2I was administered by dissolving it in water of the feeding tank. SGLT2I ameliorates macrophage accumulation and oxidative stress and maintained mitochondrial function in the kidney. The results demonstrate the effect of SGLT2I on NASH-related renal injury and the usefulness of this novel animal model for research into NASHrelated complications.
HFD-fed medaka exhibited enlarged glomeruli, dilated glomerular capillaries, and expanded mesangium, changes comparable to changes observed in humans with metabolic syndrome-related glomerulopathy [26]. To date, only a few studies demonstrated the beneficial effect of SGLT2Is on chronic renal injury in NASH animal models and elucidated the underlying mechanisms [27][28][29][30][31]. The mechanisms include the renal inflammation, fibrosis, ER stress, apoptosis and lipid accumulation due to the increased renal expression of reactive oxygen species related to oxidative stress [11,28] and TGF-b1, type IV collagen, and fibronectin [11]. Therefore, we examined whether NASH-related renal injury could be ameliorates by the SGLT2I Tofo and assessed renal changes, inflammation, oxidative stress, and renal mitochondrial damage in the medaka model of NASH. We found that Tofo ameliorates the accumulation of macrophages and oxidative stress and maintained mitochondrial function in the renal tubules of medaka fish in the HFD-induced renal injury model. These results demonstrate the beneficial effect of an SGLT2I in NASH-related renal injury and further reveal the utility of the medaka model of NASH in examining these effects and evaluation of other potential therapeutic compounds for NASH-related complications.

Animals and diets
All animal experiments were conducted in full compliance with the regulations of the Institutional Animal Care and Use Committee at Niigata University (Niigata, Japan) that approved the study protocol. All animals received humane care according to the criteria outlined in the 'Guide for the Care and Use of Laboratory Animals' by the National Academy of Sciences and published by the National Institute of Health. The d-rR/Tokyo strain of medaka fish (strain ID: MT837) was supplied by NBRP Medaka (https://shigen. nig.ac.jp/medaka/). All animals were 4 months old and maintained in plastic tanks containing 2 L tap water in plastic tanks under fluorescent light from 8 AM to 8 PM. The water temperature was maintained at 25 AE 1°C. The medaka NASH model was developed by feeding the fish an HFD described in a previous study [25,32]. Briefly, each tank was supplied with a control diet (Hikari Labo M-450; Kyorin, Hyogo, Japan) or HFD (HFD32; CLEA Japan, Tokyo, Japan), at a rate of 20 mgÁday À1 per fish to be consumed within 14 h.

Tofogliflozin administration
Tofogliflozin (Kowa Co. Ltd., Tokyo, Japan) was prepared at a final concentration of 0.5 mgÁL À1 in the tank containing the fish to be treated, as described previously [25]; this concentration is the C max of 500 ngÁmL À1 in humans treated with a standard Tofo dose of 20 mg. Briefly, Tofo was dissolved in dimethyl sulfoxide (Nacalai Tesque, Kyoto, Japan) to a concentration of 100 mgÁmL À1 and then added to the water of the plastic tank at a final concentration of 0.5 mgÁL À1 . The same amount of dimethyl sulfoxide was administrated to the tank of the HFD group as vehicle control.

Biochemical analyses
Blood samples were collected from the animals following a 12-h fasting period for all instances, as previously reported [25]. Fish were kept on ice for 1 min; thereafter, they were bled by cutting the ventral portion of the tail fin. Blood was collected in a heparinized microcapillary tube (VC-H075H; Terumo, Tokyo, Japan) and centrifuged at 1200 g for 12 min at 22°C. Blood glucose levels were analyzed using a glucometer (Glucocard G Black, GT-1830; Arkray, Kyoto, Japan).

Statistical analyses
Data were analyzed using one-way or two-way analysis of variance with repeated measures followed by Bonferroni's multiple comparison test. A P value ≤ 0.05 was considered to indicate statistical significance.

Results
Effect of Tofo on blood glucose and kidney size in the medaka model of NASH We first evaluated the effect of Tofo on changes in blood glucose levels over time in chow-fed (chow), HFD-fed (HFD), and HFD-fed and Tofo-treated (HFD + Tofo) groups (Fig. 1A). Over 12 weeks, whereas blood glucose levels did not show significant changes in the chow group over 12 weeks, the HFD group exhibited a significant increase from the control level of 36 (Fig. 1A). The liver tissue showed successful development of significant fatty infiltration in the liver (Fig. 1B) and that Tofo ameliorates the changes (Fig. 1C) as previously reported [25]. The HFD group exhibited enlargement of the renal size from the control level of 737.7 AE 101.3 µm to 1645.5 AE 109.8 µm following 12 weeks of HFD feeding, whereas the HFD + Tofo group showed milder enlargement to 1343.3 AE 92.5 µm (P < 0.01) . *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and NS, no statistical significance. One-way ANOVA followed by Bonferroni's multiple comparison test. (Fig. 1D). Mild and statistically nonsignificant changes in renal size were seen over the 12-week period in the chow group. And the differences between Chow and HFD showed the evidence of successful development of NASH-related kidney disease. These results confirmed that in the HFD-induced NASH model in medaka, Tofo was effective in reducing elevated blood glucose level and renal swelling induced by elevated blood glucose.

Effect of Tofo on glomerular size in the medaka model of NASH
To determine whether glomerular enlargement, a characteristic of HFD-induced renal injury associated with mesangial expansion and glomerular capillary dilatation, was suppressed by Tofo, we assessed time-dependent changes in glomerular size in our medaka fish model of NASH. The HFD group exhibited a significant, time-dependent increase in glomerular size to 140 µm compared with the glomerular size of 60 µm in the chow group over 12 weeks (Fig. 1E-H), and this increase was milder when Tofo was administered (P < 0.05) (Fig. 1I-K). No significant changes in the glomerular size were seen in the chow group, and no difference in expansion of the renal matrix was seen in the HFD and HFD + Tofo groups (Fig. 1L). These results suggested that Tofo slowed down HFD-induced glomerular enlargement in the medaka model of NASH.

Effect of Tofo on oxidative stress in the kidney of the medaka model of NASH
To further elucidate the mechanism underlying the effect of Tofo on HFD-induced renal injury in the medaka model of NASH, we assessed the expression levels of 8-OHdG and GPX1 in the renal tissues using immunohistochemical analyses (Fig. 2). The HFD group showed increases in the percentage of 8-OHdGpositive areas in the renal tissue in a time-dependent manner, from 8.1 AE 0.5% in the control group ( Fig. 2A) Fig. 2E-H). Conversely, the percentage of GPX1-positive areas was significantly decreased from the control level of 6.4 AE 0.6% (Fig. 2I) to 4.4 AE 0.5%, 3.3 AE 0.6%, and 3.1 AE 0.3% (Fig. 2J-L) at 4, 8, and 12 weeks in the HFD group, respectively, and was significantly suppressed to 5.6 AE 0.3%, 5.3 AE 0.4%, and 4.2 AE 0.1% by Tofo administration (Fig. 2M-P). The differences between Chow and HFD (Fig. 2H,P) suggested that the HFD-induced renal injury led to oxidative stress represented by the accumulation of 8-OHdG and the decrease in GPX1-positive staining, both of which were effectively alleviated by Tofo, although the effect is milder in later stage of 12 weeks.

Accumulation of F4/80-positive cells in the kidney of the medaka model of NASH
To examine the HFD-induced inflammation in the kidney, we next determined whether there was macrophage accumulation in the kidneys of medaka fish in the HFD-induced NASH model and whether this could be ameliorates by Tofo. Therefore, we determined the number of cells positively stained with F4/ 80. While no increase with aging in 12 weeks was observed in the chow group (50-60 cells per highpower field) (Fig. 3A) (Fig. 3E-H). The differences between Chow and HFD (Fig. 3H) provided evidence that HFD-induced inflammation and oxidative stress led to an increase in the number of macrophages, which was suppressed by Tofo administration in our animal model although the effect is milder in later stage of 8 and 12 weeks.

Effect of Tofo on mitochondrial function in renal tubules in the medaka model of NASH
To determine whether HFD led to mitochondrial damage, we assessed the kidney tissue samples in the medaka model of NASH by OPA1 staining. While no change in the percentage of OPA1-stained areas was observed in the chow group, with 6%-7% OPA1-positive areas in the control (Fig. 4A), the HFD group exhibited significant decreases in the percentage of OPA1-positive areas to 5.5 AE 0.6%, 4.2 AE 0.2%, and 3.9 AE 0.3% at 4, 8, and 12 weeks, respectively ( Fig. 4B-D). These decreases were reversed with significant increases in the percentage of OPA1-positive areas to 6.4 AE 0.5%, 6.2 AE 0.3%, and 5.2 AE 0.3% at 4, 8, and 12 weeks, respectively, by Tofo administration (Fig. 4E-H). The differences between Chow and HFD (Fig. 4H) suggested that HFD-induced renal injury was associated with damaged mitochondrial function in the renal tubules represented by OPA1 expression, a key GTPase necessary for mitochondrial fusion and maintenance of mitochondrial homeostasis, which was suppressed by Tofo administration in our animal model although the effect is milder in later stage of 12 weeks.

Discussion
With increasing incidence, NAFLD is becoming the most common chronic liver disease. Disease progression is related to obesity, diabetes, hyperlipidemia, hypertension, and insulin resistance [1][2][3], and chronic renal injury and cardiovascular disease are common complications that aggravate each other and are associated with bad prognosis in patients with NAFLD/NASH [34]. These complications are known to be induced by inflammation and oxidative stress, which aggravate glucotoxicity and lipotoxicity of metabolic syndrome [27,34]. Specifically, NASH-related renal injury shares several features with diabetic kidney disease and is associated with renal changes including glomerular enlargement, focal segmental glomerulosclerosis, and renal tubular damage [35][36][37]. The causative factors underlying these features of chronic renal injury include changes in adipokine levels such as a reduction in adiponectin, which mediates fatty acid metabolism by increasing peroxisome proliferative-activated receptor-a expression and inducing AMP-activated protein kinase phosphorylation [38]. The values represent mean AE SD values (n = 15 for each group). *P < 0.05, **P < 0.01, ***P < 0.001, and NS, no statistical significance. One-way ANOVA followed by Bonferroni's multiple comparison test.
Basic and clinical studies that provide evidence for these mechanisms used renal histological analyses in HFD-induced diabetic nephropathy models and reported inflammation, macrophage activation in glomerular as well as interstitial lesions, accumulation of oxidative stress [39][40][41], and mitochondrial damage in renal tubules [29,37]. However, few studies have investigated the effect of SGLT2Is on NASH-related renal injury in an animal model. Therefore, we have examined the renal inflammation, oxidative stress, and mitochondrial function in a medaka model of HFD-induced renal injury that recapitulates changes in NASH-associated renal injury [26], because of the ease of maintenance of these animals under the same concentrations of chemical compounds dissolved in water and their high fecundity. In this model, we previously reported that Tofo attenuated the fatty and fibrotic changes in the liver [25]. In the current study, we examined the effect of Tofo, a selective SGLT2I, on NASH-related renal injury by assessing macroscopic and microscopic changes in the kidneys, markers of inflammation and oxidative stress, and mitochondrial function in the renal tubules. Comparison of the Chow and HFD groups with regard to the The values represent mean AE SD values (n = 15 for each group). *P < 0.05, ***P < 0.001, ****P < 0.0001, and NS, no statistical significance. One-way ANOVA followed by Bonferroni's multiple comparison test.
levels of blood glucose, sizes of the kidneys and glomerulus (Fig. 1), and levels of 8-OHdG (Fig. 2), GPX1 (Fig. 2), F4/80 (Fig. 3), and OPA1 (Fig. 4) indicated the successful development of NASH-related kidney diseases. Additionally, after demonstrating the effect of Tofo on blood glucose levels in the medaka model of NASH, we showed that Tofo slowed glomerular enlargement that led to renal swelling induced by HFD. However, our results indicated that Tofo did not affect the mesangial expansion, which is consistent with the findings of a previous report [11,28,31]. In addition, in our medaka model of NASH, we showed that these changes associated with Tofo administration were related to the amelioration of macrophage accumulation and oxidative stress in the kidney as well as the maintenance of mitochondrial function in the renal tubules in HFD-induced renal injury in this medaka model of NASH. Our study appeared to have a limitation in the analysis of renal function (including proteinuria and the estimated glomerular filtration rate) because the urinary volume of medaka has been reported to be 1 mLÁh À1 Ákg À1 and urine is difficult to collect appropriately [42]. The comparison of the Chow and HFD + Tofo groups demonstrated that the effect of Tofo became mild with continuous feeding of HFD for 12 weeks, indicating that further analyses regarding the doses and combinations with other antidiabetic drugs, lipid-lowering drugs, etc., will help improve its effectiveness.
In conclusion, we showed that the highly specific SGLT2I Tofo prevented the progression of NASH-related renal injury by reducing oxidative stress and macrophage accumulation and maintaining mitochondrial function in the renal tubules. Additionally, the medaka model might be a useful platform for cost-effective evaluation of the efficacy of therapeutic approaches in NASH-related complications. Therefore, further analyses involving testing of various medicines in the medaka NASH model will help reveal better combinations of medicines for the treatment and prevention of NASH and its complications.