{"id":28495,"date":"2019-09-25T11:10:46","date_gmt":"2019-09-25T11:10:46","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=28495"},"modified":"2020-04-22T10:11:53","modified_gmt":"2020-04-22T10:11:53","slug":"decreased-renal-function-induced-by-high-fat-diet-in-wistar-rat-the-role-of-plasma-angiotensin-converting-enzyme-2-ace2","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol12no3\/decreased-renal-function-induced-by-high-fat-diet-in-wistar-rat-the-role-of-plasma-angiotensin-converting-enzyme-2-ace2\/","title":{"rendered":"Decreased Renal Function Induced by High-Fat Diet in Wistar Rat: The Role of Plasma Angiotensin Converting Enzyme 2 (ACE2)"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

In recent years, HFD has increasingly been seen as a significant risk factor that can lead to disease.\u00a0However, the mechanism of HFD that has a negative impact on\u00a0health is still difficult to understand.\u00a0Current research related to HFD focuses on the impact of HFD on medical conditions, the underlying mechanisms and the development of therapeutic strategies1<\/sup>.<\/p>\n

HFD is a risk factor for kidney disorders2<\/sup>. In HFD induced obesity it causes damage to kidney structure, inflammation3<\/sup>,<\/sup>4<\/sup> and oxidative stress5<\/sup>. Decreased renal function is characterized by increased urinary albumin excretion or albuminuria6<\/sup>,<\/sup>7<\/sup>, increased plasma creatinine levels3<\/sup> and lower creatinine clearance8<\/sup>. However research on the effects of HFD on renal function with biomarkers of cysC levels was very small and showe different findings. HFD causes an increase in serum cysC levels which implies glomerular and proximal tubular changes7<\/sup>. Crinigan et al<\/em> (2015) found that although HFD increased serum cysC levels, it was not statistically significant9<\/sup>.<\/p>\n

CysC\u00a0is a marker\u00a0for measuring Glomerolus Filtration Rate (GFR)10<\/sup>,<\/sup>11<\/sup> and\u00a0more appropriately used to diagnose kidney damage with a decrease in GFR compared to creatinine clearance10<\/sup>.\u00a0CysC\u00a0is a low molecular weight protein (13kD) which\u00a0is an\u00a0endogenous cysteine\u00a0proteinase inhibitors produced by all nucleating cells in the human body at a fairly constant level.\u00a0CysC is\u00a0filtered freely by the glomerulus and is not secreted\u00a0and is almost completely absorbed back in the proximal tubule11<\/sup>.\u00a0This characteristic makes\u00a0cysC\u00a0useful for detecting\u00a0early stage renal dysfunction.\u00a0CysC\u00a0not influenced by\u00a0age, gender, muscle mass and ethnicity12<\/sup>.<\/p>\n

One of the pathophysiological mechanisms that also plays a central role in the development of kidney disease is the activation of RAS13<\/sup>. RAS plays a key role in controlling kidney function14<\/sup>,<\/sup>15<\/sup>. One of the integral components of the RAS is ACE216<\/sup>.<\/p>\n

ACE2 is a monocarboxypeptidase that degrades angiotensin (Ang) II to Ang (1-7)13<\/sup> and convert angiotensin (Ang) I to Ang (1-9)17<\/sup>. Imbalance between increasing levels of Ang II and ACE2\/Ang (1-7)\/Mas receptor axis contribute to kidney injury13<\/sup>. ACE2 is widely expressed in the kidneys especially in proximal tubular epithelial cells14<\/sup> and visceral glomerolus18<\/sup>. ACE2 protein and mRNA levels have been shown to change in diabetic kidney disease, hypertensive kidney disease and various injurious renal models14<\/sup>.<\/p>\n

Li\u00a0et al<\/em>\u00a0(2015) examined the effect of\u00a0giving HFD to\u00a0various\u00a0RAS\u00a0components. However, they did not\u00a0examine the effect on\u00a0ACE2 which is also one of the components of the RAS19<\/sup>. The findings is encouraging research on the effects of HFD to the activation of\u00a0RAS components through the role of ACE2. Very\u00a0few\u00a0data is available about the\u00a0effects of giving HFD to\u00a0plasma ACE2\u00a0levels.\u00a0Not even found data related to the relationship between plasma ACE2 levels with\u00a0serum cysC levels\u00a0in animals\u00a0induced\u00a0by HFD.\u00a0We hypothesize that\u00a0administration of\u00a0HFD\u00a0can cause a decline in early stage renal function through the role of ACE2.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Diets<\/strong><\/p>\n

Preparation of animal diet was conducted in Animal Food and Nutrition Division,\u00a0Faculty of Animal Husbandry, Hasanuddin University, Makassar, Indonesia.\u00a0The animal diet was arranged\u00a0with\u00a0normal diet (ND) composition consisting of 3.1% fat, 16.1% protein, 3.9% fiber and 5.1% ash\/ mineral.\u00a0Composition of\u00a0the\u00a0high fat diet (HFD)\u00a0consisted of 21.4% fat, 17.5% protein, 50% carbohydrate, 3.5% fiber\u00a0and\u00a04.1% ash\/mineral20<\/sup> (table 1).\u00a0To get rations\/feed according to the composition that has been prepared, analysis of the feed samples using the proximate method was carried out.\u00a0The analysis was done twice to get accurate results (table 2).<\/p>\n

Table 1: The Composition of Animal Diet<\/strong><\/p>\n\n\n\n\n\n\n
<\/td>\n<\/td>\n<\/td>\nHFD<\/strong><\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/td>\nND<\/strong><\/td>\n<\/td>\n<\/td>\n<\/tr>\n
Composition<\/strong><\/td>\nCH (%)<\/strong><\/td>\nProtein (%)<\/strong><\/td>\nFat (%)<\/strong><\/td>\nFiber<\/strong><\/p>\n

(%)<\/strong><\/td>\n

Ash<\/strong><\/p>\n

(%)<\/strong><\/td>\n

CH (%)<\/strong><\/td>\nProtein (%)<\/strong><\/td>\nFat<\/strong><\/p>\n

(%)<\/strong><\/td>\n

Fiber (%)<\/strong><\/td>\nAsh<\/strong><\/p>\n

(%)<\/strong><\/td>\n<\/tr>\n

Corn<\/strong><\/p>\n

Bran<\/strong><\/p>\n

MBM<\/strong><\/p>\n

Premix<\/strong><\/p>\n

Fish Flour<\/strong><\/p>\n

Soybean meal<\/strong><\/p>\n

Tallow<\/strong><\/p>\n

Vegetable oil<\/strong><\/td>\n

38,11<\/p>\n

6,32<\/p>\n

 <\/p>\n

 <\/p>\n

 <\/p>\n

5,76<\/p>\n

 <\/td>\n

4,50<\/p>\n

2,29<\/p>\n

1,29<\/p>\n

 <\/p>\n

1,16<\/p>\n

7,54<\/p>\n

0,41<\/td>\n

2,02<\/p>\n

1,87<\/p>\n

0,33<\/p>\n

 <\/p>\n

0,17<\/p>\n

0,53<\/p>\n

16,16<\/p>\n

0,41<\/td>\n

0,74<\/p>\n

1,06<\/p>\n

0,07<\/p>\n

 <\/p>\n

0,08<\/p>\n

0,44<\/p>\n

 <\/td>\n

0,01<\/p>\n

1,28<\/p>\n

 <\/p>\n

 <\/p>\n

0,22<\/p>\n

1,18<\/p>\n

0,02<\/td>\n

2,20<\/p>\n

48,30<\/p>\n

 <\/p>\n

5,40<\/p>\n

6,32<\/p>\n

 <\/td>\n

 <\/p>\n

4,72<\/p>\n

 <\/p>\n

7,07<\/p>\n

2,29<\/p>\n

1,74<\/td>\n

0,09<\/p>\n

0,56<\/p>\n

 <\/p>\n

0,50<\/p>\n

1,87<\/p>\n

0,26<\/td>\n

 <\/p>\n

0,94<\/p>\n

 <\/p>\n

0,41<\/p>\n

1,06<\/p>\n

0,12<\/td>\n

 <\/p>\n

0,20<\/p>\n

 <\/p>\n

1,11<\/p>\n

1,28<\/p>\n

0,32<\/td>\n<\/tr>\n

Total<\/strong><\/td>\n50,19<\/td>\n17,19<\/td>\n21,49<\/td>\n2,39<\/td>\n2,71<\/td>\n62,22<\/td>\n15,82<\/td>\n3,28<\/td>\n2,53<\/td>\n\u00a0\u00a0\u00a0\u00a0 2,91<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

CH: Carbohydrate; MBM: Meat Bone Meal; ND: Normal Diet; HFD: High Fat Diet<\/p>\n

Table 2: Proximate Analysis Results<\/strong><\/p>\n\n\n\n\n\n\n\n\n
No<\/strong><\/td>\nSample<\/strong><\/td>\nComposition<\/strong><\/td>\n<\/tr>\n
Water<\/strong><\/td>\nProtein<\/strong><\/td>\nFat<\/strong><\/td>\nFiber<\/strong><\/td>\nCH<\/strong><\/td>\nAsh<\/strong><\/td>\n<\/tr>\n
1<\/strong><\/td>\nND1<\/strong><\/td>\n13,04<\/td>\n20,41<\/td>\n4,67<\/td>\n3,70<\/td>\n67,54<\/td>\n3,68<\/td>\n<\/tr>\n
2<\/strong><\/td>\nND2<\/strong><\/td>\n13,32<\/td>\n20,52<\/td>\n5,34<\/td>\n4,39<\/td>\n66,51<\/td>\n3,24<\/td>\n<\/tr>\n
3<\/strong><\/td>\nHFD1<\/strong><\/td>\n10,32<\/td>\n19,85<\/td>\n23,53<\/td>\n3,59<\/td>\n49,02<\/td>\n4,00<\/td>\n<\/tr>\n
4<\/strong><\/td>\nHFD2<\/strong><\/td>\n10,35<\/td>\n19,26<\/td>\n24,71<\/td>\n3,81<\/td>\n47,69<\/td>\n4,52<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Except for water, all fractions are expressed in dry matter. ND1 and HFD1: results of 1st<\/sup> analysis; ND2 and HFD2: results of the 2nd<\/sup> analysis. CH: Carbohydrate; ND: Normal Diet; HFD: High Fat Diet.<\/p>\n

Animals<\/strong><\/p>\n

30 male wistar rats aged 10-12 weeks (body weight between 170-220 grams) were maintenance in Molecular Biology and Immunology Laboratory, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia21<\/sup>,<\/sup>22<\/sup>,<\/sup>23<\/sup>. After acclimatization for 2 weeks (in the room with sufficient air circulation, room temperature 28\u00b12\u2070C, humidity 50\u00b110% and room lights arranged in a 12-h light and dark cycle), wistar rats were randomly divided into 5 groups (6 rats\/group). Group I was the group 0 weeks (baseline), group II (ND8) was the controls group given ND for 8 weeks, group III (ND16) was a control group given ND for 16 weeks, group IV (HFD8) was the treatment group given HFD for 8 weeks and group V (HFD 16) was the treatment group given HFD for 16 weeks. All wistar rats have free access to food and drink (ad libitum). Blood samples were taken through intracardiac at week 0 (after acclimatization) for the baseline group, week 8 for the HFD 8 and ND8 groups and week 16 for the HFD16 and ND16 groups for examination of plasma ACE2 levels and serum cysC levels. All of these procedures were carried out at the Laboratory of Molecular Biology and Immunology, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia.<\/p>\n

Measurement of<\/strong>\u00a0body<\/strong>\u00a0weight, body fat percentage and obesity index<\/strong><\/p>\n

The\u00a0body\u00a0weight\u00a0and length of the naso-anal were measured to obtain the obesity index value.\u00a0The\u00a0obesity\u00a0index\u00a0measured was lee index,\u00a0rohrer\u00a0index,\u00a0and\u00a0TM index.\u00a0Rats were declared obese if Lee index value is >0.3, Rohrer index >30, and TM index >50.\u00a0Body fat percentage is calculated based on TM index24<\/sup>.\u00a0The formula used\u00a0is:<\/p>\n

Rohrer index = {body weight (gram)\/naso-anal length (cm)3<\/sup>}\u00d7103<\/sup><\/p>\n

 <\/p>\n

 <\/p>\n

formula<\/strong><\/p>\n

 <\/p>\n

TM index = body weight (gram)\/naso-anal length (cm)2.383<\/sup>\u00d7103<\/sup><\/p>\n

Body fat percentage\u00a0= 0.581 \u00d7 TM index \u2013 22.03<\/p>\n

Collection of blood samples, Examination of<\/strong>\u00a0<\/strong>Plasma ACE2 Levels<\/strong>\u00a0<\/strong>and<\/strong>\u00a0Serum <\/strong>CysC<\/strong> Levels<\/strong><\/p>\n

Rats were restrained to control head and body movements.\u00a0After intraperitonial anesthesia using ketamine anesthetic agents (100 mg\/kg) and xylasine (10 mg\/kg), blood was taken\u00a0as much as 2\u00a0ml intracardiac using a needle 19-21.\u00a0Blood was\u00a0inserted into the sample tube.\u00a0Blood was centrifuged to obtain plasma\/serum and stored at -80\u00b0C before examination.\u00a0Plasma ACE2 levels were measured by elisa method using\u00a0ACE2 reagents (Rat ACE2\/ACE-2 Elisa Kit LS-F33783, LifeSpan BioScience,\u00a0Inc.).\u00a0Serum\u00a0CysC\u00a0levels were measured by the Elisa method using\u00a0a cystatin C reagent\u00a0(<\/em>Rat CST3\/Cystatin C Elisa Kit LS-F21524, LifeSpan BioScience, Inc.).\u00a0Plasma ACE2 levels\u00a0and\u00a0serum\u00a0CysC were\u00a0read using Elisa Reader 270 (Biomeriaux, France) with a wavelength of 450 nm for 30 minutes in units of ng\/ml. Each the experiment were done in duplicate.<\/p>\n

Statistical Analysis<\/strong><\/p>\n

The data\u00a0obtained was processed using SPSS\u00a0version\u00a024\u00a0for Windows\u00a0then\u00a0analyzed with a significance level of <0.05.\u00a0Before testing the difference hypothesis and correlation, the data normality test was first carried out.\u00a0The difference test\u00a0conducted was a one-way analysis of variance (ANOVA)\u00a0followed by Bonferroni’s multiple comparison\u00a0test.\u00a0Correlation test performed using Pearson\u00a0correlation test.<\/p>\n

Results<\/strong><\/p>\n

Effects of HFD\u00a0on Body Weight, Obesity Index and Body Fat Percentage<\/strong><\/p>\n

As\u00a0expected, there were differences\u00a0in body weight, obesity index and body fat percentage\u00a0between ND and HFD\u00a0groups (table 3).\u00a0The\u00a0body\u00a0weight\u00a0in the\u00a0HFD8 group was higher than of ND8 group\u00a0(p<\u00a00.001), as well as between groups given HFD16 and ND16\u00a0(p<0.001), even between ND8 and ND16 groups (p<0.05).\u00a0Body weight in the HFD8 group was higher than HFD16 group.<\/p>\n

Obesity index measured was Lee index,\u00a0Rohrer\u00a0index and TM index.\u00a0Lee\u00a0index\u00a0in the HFD8 group\u00a0was higher\u00a0than\u00a0ND8 group (p<0.001). Likewise\u00a0between\u00a0HFD\u00a016 group and ND16 group\u00a0(p<0.001).\u00a0The duration of\u00a0administration of HFD did not affect the Lee index as evidenced by not finding differences between groups given HFD\u00a08 and HFD16.\u00a0Interestingly,\u00a0Lee index after administration of\u00a0ND or\u00a0HFD did not show obesity. Rohrer\u00a0index\u00a0and TM index were also higher in the HFD group\u00a0than ND for both between ND8 and HFD8 and between ND16 and HFD16 (respectively, p<0.001 and p<0.001). The interesting thing was also the\u00a0Rohrer\u00a0index\u00a0and TM\u00a0index in all groups showed good value of obesity after given ND or HFD.<\/p>\n

Body fat percentage is calculated based\u00a0on the TM obesity index value\u00a0.\u00a0Body fat\u00a0percentage was\u00a0also higher in the HFD group than ND for both between ND8 and HFD8 and between ND16 and HFD16 (respectively, p<0.001 and p<0.01).<\/p>\n

This finding means\u00a0that administration of HFD increased body weight, body fat percentage and obesity index\u00a0in wistar rats. The longer the HFD administration, the higher the\u00a0Rohrer\u00a0index, TM index and body fat percentage.<\/p>\n

Table 3: The Effect of HFD on Body Weight, Obesity Index and Body Fat Percentage<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
Parameter<\/strong><\/td>\nDiet Groups<\/strong><\/td>\n<\/tr>\n
Baseline<\/strong><\/td>\nND8<\/strong><\/td>\nND16<\/strong><\/td>\nHFD8<\/strong><\/td>\nHFD16<\/strong><\/td>\n<\/tr>\n
Body Weight (gr)<\/strong><\/td>\n185.17 \u00b1 6.75<\/td>\n204 \u00b1 14.77<\/td>\n220.17 \u00b1 5.98d***<\/sup><\/td>\n339.83 \u00b1 14.77a*<\/sup><\/td>\n334.33 \u00b1 10.94b*<\/sup><\/td>\n<\/tr>\n
Lee Index<\/strong><\/td>\n0.22 \u00b1 0.01<\/td>\n0.24 \u00b1 0.00<\/td>\n0.25 \u00b1 0.00<\/td>\n0.30 \u00b1 0.00a*<\/sup><\/td>\n0.30 \u00b1 0.00b*c*<\/sup><\/td>\n<\/tr>\n
Rohrer Index<\/strong><\/td>\n29.10 \u00b1 2.81<\/td>\n34.03 \u00b1 1.44<\/td>\n33.85 \u00b1 1.00<\/td>\n51.58 \u00b1 2.20a*<\/sup><\/td>\n54.21 \u00b1 2.82b*<\/sup><\/td>\n<\/tr>\n
TM Index<\/strong><\/td>\n49.35 \u00b1 4.62<\/td>\n56.67 \u00b1 0.00<\/td>\n58.66 \u00b1 2.80<\/td>\n59.81 \u00b1 5.35a*<\/sup><\/td>\n92.86 \u00b1 6.27b*<\/sup><\/td>\n<\/tr>\n
Body fat percentage (%)<\/strong><\/td>\n6.44 \u00b1 2.59<\/td>\n10.89 \u00b1 2.30<\/td>\n12.03 \u00b1 1.62<\/td>\n29.95 \u00b1 3.09 a*<\/sup><\/td>\n31.07 \u00b1 5.56 b**<\/sup><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Data are presented in the mean \u00b1 SD form (n=6). Differences between groups were analyzed using the one-way ANOVA test followed by Bonferroni’s test. Significant differences are indicated by superscript: a <\/sup>between ND8 and HFD8, b <\/sup>between ND16 and HFD16, c <\/sup>between HFD8 and HFD16 and d <\/sup>between ND8 and ND16. *<\/sup>p<0.001, **<\/sup>p<0.01, ***<\/sup>p<0.05.<\/p>\n

Effects of HFD<\/strong>\u00a0on Plasma ACE2 Levels<\/strong><\/p>\n

Plasma ACE2 levels in the HFD group were higher than in the ND group for both between ND8 and HFD8 and between ND16 and HFD16 (respectively, p<0.001 and p<0.001).\u00a0Plasma ACE2 levels\u00a0in the HFD16 group were higher than HFD8 group (p<0.001).\u00a0The longer the HFD is given, the higher\u00a0the\u00a0plasma ACE2 levels.\u00a0Administration of HFD increases\u00a0plasma ACE2\u00a0levels\u00a0(table 4).<\/p>\n

Effects of HFD<\/strong>\u00a0<\/strong>on<\/strong>\u00a0Serum <\/strong>CysC<\/strong> Levels<\/strong><\/p>\n

Serum\u00a0cysC\u00a0levels in the HFD group were higher than in the ND group between ND8 and HFD8 and between ND16 and HFD16 (respectively, p<0.001 and p<0.001).\u00a0Although statistically it did not show a significant difference, serum cysC levels in the\u00a0HFD16\u00a0group were higher than those of HFD8 group.\u00a0Giving HFD\u00a0increases\u00a0serum cysC levels (table 4).<\/p>\n

Table 4: Effects of HFD on Plasma ACE2 Levels and Serum Cystatin C Levels<\/strong><\/p>\n\n\n\n\n\n\n
Biomarker<\/strong><\/td>\nDiet Groups<\/strong><\/td>\n<\/tr>\n
Baseline<\/strong><\/td>\nND8<\/strong><\/td>\nND16<\/strong><\/td>\nHFD8<\/strong><\/td>\nHFD16<\/strong><\/td>\n<\/tr>\n
Plasma ACE2 Level (ng\/ml)<\/strong><\/td>\n1.63 \u00b1 0.07<\/td>\n1.92 \u00b1 0.07<\/td>\n2.05 \u00b10.06<\/td>\n2.20 \u00b10.06a*<\/sup><\/td>\n2.37 \u00b10.07b*c*<\/sup><\/td>\n<\/tr>\n
Serum Cystatin C Level (ng\/ml)<\/strong><\/td>\n0.99 \u00b1 0.07<\/td>\n1.22 \u00b1 0.08<\/td>\n1.32 \u00b10.06<\/td>\n1.46 \u00b10.07a**<\/sup><\/td>\n1.57 \u00b10.05b*<\/sup><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Data are presented in the mean \u00b1 SD form (n = 6). Differences between groups were analyzed using the one-way ANOVA test followed by Bonferroni’s test. Significant differences are shown by superscript: a <\/sup>between ND8 and HFD8, b <\/sup>between ND16 and HFD16, c <\/sup>between HFD8 and HFD16 and d <\/sup>between ND8 and ND16. *<\/sup>p<0.001, **<\/sup>p<0.01, ***<\/sup>p<0.05.<\/p>\n

Body Weight, Obesity Index, Body Fat Percentage and Plasma ACE2 Levels<\/strong><\/p>\n

Body weight correlated\u00a0with\u00a0plasma ACE2\u00a0levels\u00a0, direction of positive correlation with very strong correlation strength\u00a0(p<0.001, r=0.867). Obesity index of Lee,\u00a0Rohrer\u00a0and TM also correlated\u00a0with plasma ACE2 levels, direction of positive correlation with very strong correlation strength\u00a0(respectively p<0.001, r=0.882; p<0.001, r=0.866; p<0.001, r=0.870).\u00a0The\u00a0body\u00a0fat percentage\u00a0also correlated\u00a0with\u00a0plasma ACE2\u00a0levels, the direction of the positive correlation with a very strong correlation strength\u00a0(p<0.001, r=0.862).\u00a0The direction of positive correlation means that the higher body weight, obesity index and\u00a0body\u00a0fat percentage,\u00a0the higher the plasma ACE2 levels\u00a0(Figure 1).<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 1: Correlation between Body Weight, Body Fat Percentage and Obesity Index with Plasma ACE2 Levels<\/strong><\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/em><\/strong><\/p>\n

Plasma<\/strong>\u00a0ACE2 <\/strong>Levels<\/strong>\u00a0<\/strong>and<\/strong> Serum CysC<\/strong>\u00a0<\/strong>Levels<\/strong><\/p>\n

Plasma ACE2 levels correlated with serum cysC levels (p<0.001). The direction of correlation was positive with a very strong correlation strength (r=0.918). A\u00a0positive correlation direction means that the higher plasma ACE2 levels, the higher serum cysC levels (Figure\u00a02).<\/p>\n

 <\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 2: Correlation between Plasma ACE2 Levels with Serum Cystatin C Levels<\/strong><\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Discussion<\/strong><\/p>\n

Body weight and body fat percentage after the administration of HFD was higher than ND both for 8 weeks and 16 weeks.\u00a0This finding is consistent with many studies that have been done before.\u00a0There was an increase in adiposity (body weight, fat mass, percentage of fat and adipocyte size) after administration of HFD both for 8 weeks20<\/sup> and for 16 weeks 25<\/sup>,<\/sup>26<\/sup>.\u00a0Significant weight gain occurred during 4 weeks of diet and tended to persist until the end of the study24<\/sup>.\u00a0Recent findings indicate that administration of HFD for 8 weeks adds 169% of adipose retroperitoneal tissue and\u00a0107% of epididymal tissue27<\/sup>. Other recent findings prove that tissue adipose visceral weight increases after 6\u00a0weeks of HFD administration even to 300% weigher after 24 weeks of HFD administration. Administration of HFD\u00a0for 6 weeks is considered\u00a0a short period.\u00a0However, Crinigan\u00a0et al<\/em>\u00a0(2015) reported that administration of HFD short-term (6 weeks) increases visceral adiposity9<\/sup>.<\/p>\n

Increased body weight from several experimental animal studies\u00a0given HFD varied. This is due to differences in research characteristics such as experimental animal clusters, intestinal microbiota conditions25<\/sup>,\u00a0initial body weight, dietary fat composition, method of administration, experimental period and the amount of food intake consumed by experimental animals. Mice\u00a0C57BL\/6\u00a0and wistar rat strains are most widely used in research models of HFD administration.<\/p>\n

The physiological mechanism by which\u00a0a HFD can increase body weight is\u00a0explained in many previous studies. HFD causes hypertriglyceridemia which causes leptin sensitivity. Leptin is a protein secreted by adipocytes, transported across the blood brain barrier and\u00a0work in the central nervous system to regulate food and energy expenditure. Hypertriglyceridemia induced\u00a0with HFD inhibits this mechanism.\u00a0Because of limitations, this study did not measure\u00a0triglyceride\u00a0levels.\u00a0In addition, energy from fat has a greater effect in increasing body weight than energy from non-fat.\u00a0Fat has a very high efficiency in using nutrients compared to protein and carbohydrates. Signs of satiety that are weaker from\u00a0fat\u00a0than carbohydrates\u00a0and proteins also play a role in the\u00a0desire to consume a HFD28<\/sup>.<\/p>\n

Obesity index after administration of HFD is also higher than ND both for 8 weeks and 16 weeks. The longer the diet is given, the higher the obesity index.\u00a0The results of this study are consistent with previous research. Giving a high-fat diet for 8 weeks increased the Lee index, Rohrer\u00a0index, and TM index and body fat percentage.\u00a0The obesity index\u00a0increases\u00a0according to the duration of\u00a0administration of HFD\u00a0and the degree of obesity24<\/sup>. Giving HFD for 12 weeks\u00a0increased\u00a0the lee\u2019s obesity index by\u00a010.45% compared to the control group. Increasing adipocyte mass also increases body weight and obesity index29<\/sup>.\u00a0In contrast, previous studies found that administration of HFD for 3 weeks did not increase obesity index30<\/sup>. This difference may be due to the duration of diet in their study which is too short.\u00a0The macronutrient composition\/percentage of fat in the diet given is not mentioned in their publications.<\/p>\n

We were the first to research the effect of giving HFD to\u00a0plasma ACE2\u00a0levels\u00a0and we found plasma ACE2 levels in the HFD16 group were higher than in\u00a0the\u00a0HFD8\u00a0group. Administration of HFD increases\u00a0plasma ACE2\u00a0levels. This shows the protective role of ACE231<\/sup>.<\/p>\n

The duration of\u00a0giving a HFD influences the\u00a0dynamic of plasma ACE2 levels\u00a0both short and long term administration.\u00a0Provision of a long-term diet is 8 weeks or more32<\/sup>. The longer the administration of HFD, the\u00a0higher plasma ACE2 levels. We found that\u00a0long-term\u00a0administration of\u00a0HFD\u00a0increased plasma\u00a0ACE2 levels.\u00a0This increase is in response to compensation for renoprotective.<\/p>\n

We also found\u00a0levels\u00a0serum\u00a0cysC\u00a0increased after administration of HFD compared to ND both for 8 and 16 weeks.\u00a0The results of our study support that the administration of HFD causes a decrease in early stage kidney dysfunction seen in increasing\u00a0cysC\u00a0serum levels. This finding is not\u00a0surprising because damage to renal structure and function\u00a0caused by HFD has been known in many previous studies.\u00a0The administration\u00a0of\u00a015 weeks\u00a0of HFD\u00a0in wistar rats caused a\u00a0decrease in kidney function parameters characterized by urinary albumin excretion and increased plasma creatinine levels3<\/sup>.\u00a0Rats given HFD\u00a0for 6 weeks showed lower creatinine clearance8<\/sup>.\u00a0Renal injury in rats\u00a0given\u00a0HFD for 16 weeks occurs due to lipid accumulation, infiltration of macrophages (inflammation) and oxidative stress in the\u00a0nose that causes\u00a0glomerulosclerosis, interstitial fibrosis\u00a0and albuminuria5<\/sup>.\u00a0Changes in kidney function and structure are also observed in C57BL\/6 male mice\u00a0given by HFD\u00a0for 12 weeks showed albuminuria and lipids accumulation\u00a0in the glomeruli and proximal tubules6<\/sup>.\u00a0Changes in structure appear even worse in the renal cortex (glomerulus, tubules, interstitium and blood vessels) due to the administration of longer duration of HFD (18 months)4<\/sup>. A recent study found that giving HFD also causes a decrease in the diameter of the capsule of the tubules and the cell volume of bowman\u2019s capsule27<\/sup>.<\/p>\n

However, research on the effects of HFD on kidney function with biomarkers\u00a0cysC\u00a0levels\u00a0is very little. In accordance with our findings in this study, recent studies in C56BL\/6\u00a0male mice given HFD for 22 weeks proved to\u00a0lead to increased excretion of\u00a0urine albumin and cysC\u00a0levels which implies glomerular and proximal functional changes in tubules.\u00a0Increased\u00a0urinary\u00a0cysC\u00a0excretion was\u00a0accepted as a biomarker for tubular injury7<\/sup>.\u00a0Other\u00a0research\u00a0found different results in which the\u00a0provision of\u00a0short term\u00a0HFD\u00a0for 6 weeks on srague-dawley rats did not cause\u00a0early renal injury.\u00a0CysC\u00a0levels\u00a0in rats given HFD were higher than ND, but not statistically significant.\u00a0Creatinine and urine protein concentrations also\u00a0show no difference.\u00a0The possibility that the duration of HFD administration used\u00a0in this study is not sufficient to cause a decline\u00a0in kidney function9<\/sup>.<\/p>\n

Plasma\u00a0ACE2\u00a0levels\u00a0are positively correlated with\u00a0levels\u00a0cysC\u00a0serum.\u00a0The direction of positive correlation means that the higher\u00a0plasma ACE2\u00a0levels,\u00a0the higher the serum\u00a0cysC\u00a0levels.\u00a0We\u00a0were the first to carry out a study related to the correlation between ACE2 and a decrease in early kidney function disorders using cysC\u00a0biomarker.\u00a0Previous studies have reported\u00a0on the correlation of ACE2 with biomarker of different\u00a0decrease on kidney function.\u00a0The mRNA expression of urine ACE2 gene was positively correlated with kidney function parameters, namely the degree of proteinuria and creatinine serum and negatively correlated with eGFR33<\/sup>. In\u00a0a rat\u00a0model of\u00a0CKD, ACE2 expression decreased significantly.\u00a0Inhibition of ACE2 causes a decrease in\u00a0cortical\u00a0ACE2 activity, reduces 50% of\u00a0FITC-inulin clearance and significantly increases urine albumin excretion34<\/sup>.\u00a0The findings in\u00a0this study\u00a0indicate that\u00a0plasma\u00a0ACE2\u00a0levels\u00a0play\u00a0a\u00a0role in\u00a0decreasing early kidney function, as\u00a0evidenced by an increase in\u00a0serum\u00a0cysC\u00a0levels.<\/p>\n

We also found that body weight, body fat percentage, and obesity index were positively correlated with plasma ACE2 levels with very strong correlation strength. The higher the body weight, the percentage of body fat, and the obesity index, the higher plasma ACE2 levels. Obesity is associated with overly active systemic and local RAS including adipose RAS. This proves that there is a relationship between RAS and obesity with special emphasis on the role of adipose tissue RAS in the pathogenesis of metabolic disorders in obesity35<\/sup>. Possible findings indicate that HFD-induced obesity activates RAS through the role of ACE2.<\/p>\n

Conclusions<\/strong><\/p>\n

Administration of HFD\u00a0has been proven to increase body weight, body fat percentage, and obesity index. Administration of HFD increases plasma ACE2\u00a0levels. Administration of HFD causes a decrease in early renal function as evidenced by the increase in serum cysC levels. Plasma\u00a0ACE2\u00a0levels\u00a0play a\u00a0role in the occurrence of an decrease\u00a0in early stage renal function induced by administration of HFD.\u00a0Body weight, body fat percentage, and obesity index\u00a0also play a role in increasing Plasma ACE2 levels.<\/p>\n

Further research is needed on intervention variations in the form of conventional or herbal medicine which can inhibit the decline of early renal function due to HFD administration,\u00a0research on the effects of HFD administration on the cysC gene and other RAS components,\u00a0and research on the administration of HFD with a longer duration.<\/p>\n

Ethical Approval<\/strong><\/p>\n

The research\u00a0was carried out after obtaining recommendations for\u00a0ethical\u00a0approval\u00a0from The Committee on Ethics of Medical Research, Faculty of Medicine, Hasanuddin University (Recommendation number: 366\/H4.8.4.5.31\/PP36-KOMETIK\/2018) date May 14, 2018.\u00a0Procedures for experimental animals were carried out in accordance with the principles of the Declaration of Helsinki.<\/p>\n

Acknowledgments<\/strong><\/p>\n

A higher appreciation to Lembaga Pengelola Dana Pendidikan (LPDP) and all staff from the\u00a0Laboratory of Molecular Biology and Immunology, Faculty of Medicine, University of Hasanuddin, Makassar.<\/p>\n

Conflicts of Interest<\/strong><\/p>\n

The\u00a0authors declare that there is no conflict of interests regarding the publication of this paper.<\/p>\n

Funding Sources<\/strong><\/p>\n

This research was funded by Lembaga Pengelola Dana Pendidikan (LPDP) Indonesia.<\/p>\n

Abbreviations<\/strong><\/p>\n

ACE2: Angitensin Converting Enzyme 2; CH: Carbohydrate; CKD: Chronic Kidney Disease; CysC: Cystatin C; eGFR: Estimated Glomerular Filtration Rate; FITC-Inulin: Fluoresceinyl Isothiocyanate; HFD: High Fat Diet; mRNA: Messenger Ribonucleic Acid; ND: Normal Diet; RAS: Renin Angiotensin System.<\/p>\n

References<\/strong><\/p>\n

    \n
  1. Duan Y, Zeng L, Zheng C, Song B, Li F, Kong X. Inflammatory links between high fat diets and diseases. Front Immunol<\/em>. 2018;9(2649):1-10.<\/li>\n
  2. Niu H, Li Y, Li H, et al. Matrix metalloproteinase 12 modulates high-fat-diet induced glomerular fibrogenesis and inflammation in a mouse model of obesity. Sci Rep<\/em>. 2016;6(December 2015):1-14.<\/li>\n
  3. Cao J, Inoue K, Sodhi K, Puri N, Peterson SJ, Abraham NG. High-fat diet exacerbates renal dysfunction in SHR: reversal by induction of HO-1\u2013adiponectin axis. Obesity<\/em>. 2017;20(5):945-953.<\/li>\n
  4. Aguila MB, Mandarim-De-Lacerda CA. Effects of chronic high fat diets on renal function and cortical structure in rats. Exp Toxic Pathol<\/em>. 2003;55(2-3):187-195.<\/li>\n
  5. Kume S, Uzu T, Araki S, Sugimoto T, Isshiki K. Role of altered renal lipid metabolism in the development of renal injury induced by a high-fat diet. J Am Soc Nephrol<\/em>. 2007;18(10):2715-2723.<\/li>\n
  6. Deji N, Kume S, Araki S, et al. Structural and functional changes in the kidneys of high-fat diet-induced obese mice. Am J Physiol Ren Physiol<\/em>. 2018;296(1):118-126.<\/li>\n
  7. Park J, Choi B, Ku S-K, et al. Amelioration of high fat diet-induced nephropathy by cilostazol and rosuvastatin. Arch Pharm Res<\/em>. 2017;40(3):391-402.<\/li>\n
  8. Lu J, Bankovic-calic N, Ogborn M, Saboorian MH, Aukema HM. Detrimental effects of a high fat diet in early renal injury are ameliorated by fish oil in han\u202f: SPRD- cy rats. Nutr Interact Toxic<\/em>. 2003;133(1):180-186.<\/li>\n
  9. Crinigan C, Calhoun M, Sweazea KL. Short-term high fat intake does not significantly alter markers of renal function or inflammation in young male sprague-dawley rats. J Nutr Metab<\/em>. 2015:1-9.<\/li>\n
  10. Schanz M, Pannes D, Dippon J, Wasser C, Alscher MD, Kimmel M. The Influence of thyroid function, inflammation, and obesity on risk prediction of acute kidney injury by cystatin c in the emergency department. Kidney Blood Press Res<\/em>. 2016;41(5):604-613.<\/li>\n
  11. Marwyne MNN, Loo CY, Halim AG, Norella K, Sulaiman T, Zaleha MI. Estimation of glomerular filtration rate using serum cystatin C in overweight and obese subjects. Med J Malaysia<\/em>. 2011;66(4):313-317.<\/li>\n
  12. Lafarge JC, Naour N, Cl\u00e9ment K, Guerre-Millo M. Cathepsins and cystatin C in atherosclerosis and obesity. Biochimie<\/em>. 2010;92(11):1580-1586.<\/li>\n
  13. R\u00fcster C, Wolf G. The Role of the Renin-angiotensin-aldosterone system in obesity-related renal diseases. Semin Nephrol<\/em>. 2013;33(1):44-53.<\/li>\n
  14. Soler MJ. ACE2 alterations in kidney disease. 2013;28(1):2687-697.<\/li>\n
  15. Mizuiri S, Ohashi Y. ACE and ACE2 in kidney disease. World J Nephrol<\/em>. 2015;4(1):74-82.<\/li>\n
  16. Tikellis C, Thomas MC. Angiotensin-converting enzyme 2 (ACE2) is a key modulator of the renin angiotensin system in health and disease. Int J Pept<\/em>. 2012:1-8.<\/li>\n
  17. Williams VR, Scholey JW. Angiotensin-converting enzyme 2 and renal disease. Curr Opin Nephrol Hypertens<\/em>. 2018;27(1):35-41.<\/li>\n
  18. Lely A, Hamming I, van Goor H, Navis G. Renal ACE2 expression in human kidney disease. J Pathol<\/em>. 2004;204(5):587-593.<\/li>\n
  19. Li C, Culver SA, Quadri S, et al. High-fat diet amplifies renal renin angiotensin system expression , blood pressure elevation , and renal dysfunction caused by Ceacam1 null deletion. Am J Physiol Endocrinol Metab<\/em>. 2015;309(9):802-810.<\/li>\n
  20. Lozano I, Werf R Van Der, Bietiger W, et al. Disorders in rats\u202f: impact on diabetes risk, hepatic and vascular complications. Nutr Metab (Lond)<\/em>. 2016:1-13.<\/li>\n
  21. Kamelia E, Islam AA, Hatta M, et al. The effect of administration of ethanol extract from musa paradisiaca L. (MPL) fruit on the caspase-3 mRNA gene expression in rat amyloid beta induced, an alzheimer\u2019s disease model, Asian J Pharm and Clin Res<\/em>. 2018; 11(4): 298\u2013302.<\/li>\n
  22. Simanjuntak TB, Hatta M, Tahir AM, Sirait RH, Karo MB, Tambaib T, Junita AR, Analysis of anti-toxoplasma immunoglobulin G and immunoglobulin M antibody levels after intervention with Curcuma Longa extract on early pregnant mice with acute toxoplasmosis.\u00a0 J Global Infect Dis<\/em>. 2019; 11(1): 25-29.<\/li>\n
  23. Kamelia E et al. Evaluation of caspase-3 mRNA gene expression activity in amyloid beta-induced alzheimer\u2019s disease rats \u00a0J Med Science. <\/em>2017;17(3): 117\u2013125.<\/li>\n
  24. Lee S, Kim J, Lee Y, et al. Anti-obesity effect of monascus pilosus mycelial extract in high fat diet-induced obese rats. J Appl Biol Chem<\/em>. 2011;54(3):197-205.<\/li>\n
  25. Cranford TL, Enos RT, Velasguez KT, et al. Role of MCP-1 on inflammatory processes and metabolic dysfunction following high-fat feedings in the FVB\/N strain taryn. Int J Obes<\/em>. 2016;40(5):844-851.<\/li>\n
  26. Chodavarapu H, Chhabra KH, Xia H, Shenoy V, Yue X, Lazartigues E. High-fat diet-induced glucose dysregulation is independent of changes in islet ACE2 in mice. Am J Physiol Regul Integr Comp Physiol<\/em>. 2018;311(51):1223-1233.<\/li>\n
  27. Jos\u00e9 I, Garcia P, C\u00e9zar JS, et al. Effects of high fat diet on kidney lipid content and the Na , K-ATPase activity. Brazilian J Pharm Sci<\/em>. 2018;54(1):1-13.<\/li>\n
  28. Hariri N, Thibault L. High-fat diet-induced obesity in animal models. 2019;23(2):270-299.<\/li>\n
  29. Miao Y, Kang H, Li J, et al. Effect of sheng-jiang powder on multiple-organ inflammatory injury in acute pancreatitis in rats fed a high-fat diet. World J Gastroenterol<\/em>. 2019;25(6):683-695.<\/li>\n
  30. Shabbir F, Khan S, Yousaf MJ, Khan MA, Rajput TA. Comparison of effect of high fat diet induced obesity and subsequent atorvastatin administration on different anthropometric measures in sprague dawley rats. Pak Armed Forces Med J<\/em>. 2016;66(5):699-704.<\/li>\n
  31. Liang B, Li Y, Han Z, et al. ACE2-Ang (1-7) axis is induced in pressure overloaded rat model. Int J Clin Exp Pathol<\/em>. 2015;8(2):1443-1450.<\/li>\n
  32. Xue B, Thunhorst RL, Yu Y, Guo F, Beltz TG, Felder RB. Central renin\u2013angiotensin system activation and inflammation induced by high-fat diet sensitize angiotensin ii \u2013 elicited hypertension. Hypertension<\/em>. 2015;67(1):1-8.<\/li>\n
  33. Wang G, Lai FM, Lai K, Chow K. Urinary mRNA expression of ACE and ACE2 in human type 2 diabetic nephropathy. Diabetologia<\/em>. 2008;51(6):1062-1067.<\/li>\n
  34. Dilauro M, Zimpelmann J, Robertson SJ, Genest D, Burns KD. Effect of ACE2 and angiotensin-(1 \u2013 7) in a mouse model of early chronic kidney disease. Am J Physiol Ren Physiol<\/em>. 2010;298(6):1523-1532.<\/li>\n
  35. Kalupahana NS. The renin-angiotensin system\u202f: a link between obesity, inflammation and insulin resistance. Obes Rev<\/em>. 2011;13(2):136-149.<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

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