Post-weaning Exposure to High-Fat Diet Induces Kidney

Lipotoxicity in Adult Rats.

Cynthia R. Muller1, 2, Ana Paula d. Leite3, Rodrigo Yokota3, Renata O. Pereira3, Anna
Laura V. Americo1, Nilberto R. Nascimento4, Fabiana S. Evangelista6, Vera Farah5,
Manasses C. Fonteles4, Patricia Fiorino5*

1
University of São Paulo, Brazil, 2University of California, San Diego, United States,
3
Federal University of São Paulo, Brazil, 4State University of Ceará, Brazil, 5Mackenzie
Presbyterian University, Brazil, 6School of Arts, Science Humanities, University of São
Paulo, Brazil
Submitted to Journal:

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Frontiers in Nutrition

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Specialty Section:

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Lipid and Fatty Acid Research

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ISSN:
2296-861X

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Article type:
Original Research Article

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Received on:

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22 Dec 2018

Accepted on:
15 Apr 2019

Provisional PDF published on:

15 Apr 2019

Frontiers website link:

www.frontiersin.org

Citation:
Muller CR, Leite AD, Yokota R, Pereira RO, Americo AV, Nascimento NR, Evangelista FS, Farah V,
Fonteles MC and Fiorino P(2019) Post-weaning Exposure to High-Fat Diet Induces Kidney Lipotoxicity
in Adult Rats.. Front. Nutr. 6:60. doi:10.3389/fnut.2019.00060

Copyright statement:
© 2019 Muller, Leite, Yokota, Pereira, Americo, Nascimento, Evangelista, Farah, Fonteles and Fiorino.
This is an open-access article distributed under the terms of the Creative Commons Attribution
License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the
original author(s) or licensor are credited and that the original publication in this journal is cited, in
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This Provisional PDF corresponds to the article as it appeared upon acceptance, after peer-review. Fully formatted PDF
and full text (HTML) versions will be made available soon.

Frontiers in Nutrition | www.frontiersin.org

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 1 Post-weaning Exposure to High-Fat Diet Induces Kidney Lipid Accumulation and
2 Function Impairment in Adult Rats.

3 Muller CR1,5; Leite APO1,2; Yokota R 2; Pereira RO 1,3; Americo ALV1,5; Nascimento

4 NRF4; Evangelista FS6; Farah V1,3; Fonteles MC1,4&; Fiorino P1&*.

5 &- Equal contribution to the paper

6 1 Renal, Cardiovascular and Metabolic Physiopharmacology Laboratory, Health and

7 Biological Science Center, Mackenzie Presbyterian University, São Paulo, Brazil

8 2 Renal Division, Federal University of São Paulo, Brazil.

9 3 Translational Medicine Division, Federal University of São Paulo, Brazil.

10 4 Superior Institute of Biomedical Sciences, Ceara State University, Fortaleza, Ceara,

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Brazil.

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5 Experimental Pathophysiology Dept., Faculty of Medicine, University of São Paulo,

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Brazil

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6 School of Arts, Science and Humanities, University of Sao Paulo, Brazil.

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Correspondence:

Patricia Fiorino, PhD.

18 patriciafiorino@mackenzie.br

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25 Abstract
26 Aim: We investigated the kidney morphofunctional consequences of high-fat diet intake
27 since post-weaning in adult rats. Main Methods: Male Wistar rats were divided into two
28 groups: ND (normal diet; n=10) and HD (high-fat diet; n=10). The high-fat diet was
29 introduced post-weaned and animals were followed for 8 weeks. Key Findings: HD group
30 did not change body weight gain even though food consumption has decreased with no
31 changes in caloric consumption. The HD group showed glucose intolerance and insulin
32 resistance. The glomerular filtration rate was decreased in vivo (ND: 2.8±1.01; HD:
33 1.1±0.14 ml/min) and in the isolated perfusion method (34% of decrease). Renal
34 histological analysis showed a retraction in glomeruli and an increase in kidney lipid
35 deposition (ND: 1.5±0.17 HD: 5.9±0.06%). Furthermore, the high-fat diet consumption
36 increased the pro-inflammatory cytokines IL-6 (ND: 1276±203; HD: 1982±47
37 pg/mL/mg) and IL-1b (ND: 97±12 HD: 133±5 pg/mL/mg) without changing anti-
38 inflammatory cytokine IL-10. Significance: Our study provides evidence that high-fat
39 diet consumption leads to renal lipid accumulation, increases inflammatory cytokines,
40 induces glomeruli retraction and renal dysfunction. These damages observed in the
41 kidney could be associated with an increased risk to advanced CKD in adulthood
42 suggesting that reduction of high-fat ingestion during an early period of life can prevent
43 metabolic disturbances and renal lipotoxicity.

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44

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45 Keywords: Post-weaning, High-fat diet, kidney lipid deposition, lipotoxicity,

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46 inflammation

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49 1. Introduction
50
51 High-fat diets are becoming increasingly common in many countries and they contribute
52 to the development of chronic noncommunicable diseases (NCDs), such as obesity,
53 hypertension, and chronic kidney disease (CKD) (de Castro Engler, R et al., 2016). NCDs
54 kill 41 million people each year, 71% of the world’s total deaths (WHO, 2017b). It is
55 recommended that fats account for 20 to 35% of total energy intake (Aranceta and Pérez-
56 Rodrigo, 2012), but daily total fat consumption accounts for 50% of total energy intake
57 in some countries (Elmadfa and Kornsteiner, 2009).
58
59 High-fat diets, in general, are associated with metabolic disorders, and the type of dietary
60 fat is a determinant risk factor since saturated fats are more linked to a positive fat balance
61 and visceral adipose tissue accumulation than to other types of fat (Coelho et al., 2011).
62 Saturated fat intake is also more associated with increased serum LDL and total
63 cholesterol than the consumption of other fatty acids (Mensink R.P., 2016). The World
64 Health Organization recommends a reduction in saturated fat consumption as one of the
65 worldwide strategies to reduce mortality from chronic NCDs (WHO, 2017a).
66
67 Children are important targets for food and beverage companies that use aggressive

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68 advertising strategies to generate a preference for diets with high levels of fat. As a result,

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69 the consumption of high-fat diets typically starts early in life, especially in developed and

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70 developing countries (Mallarino et al., 2013). High-fat diet habits in childhood can predict

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71 the development of several diseases in adulthood, such as obesity, hypertension,

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72 metabolic syndrome, and CKD (Lobstein et al., 2015; Reynolds et al., 2015). Our group
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demonstrated that high-fat diet ingestion since the early period of life increases white
visceral adipose tissue and induces cardiometabolic damage in adult rats (Fiorino et al.,
2016). Morenga and colleagues (2017) showed that a reduction of saturated fat intake was
associated with significant reductions in LDL and total cholesterol and arterial blood
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pressure of children and adolescents aged 2 to 19 years old (Te Morenga and Montez,
2017).

High-fat diet affects the energy balance (Oosterman et al., 2015) leading to lipid
accumulation in ectopic sites and in intracellular compartments (Guebre-Egziabher et al.,
2013; Landeiro and Quarantini, 2011). The renal ectopic accumulation of lipids associated
with insulin resistance has been correlated with a progressive decline in renal function
84 (Guebre-Egziabher et al., 2013; Jiang et al., 2005; Kume et al., 2007). The deleterious
85 effects exerted by lipids on cells and tissues is called lipotoxicity (Escasany et al., 2019;
86 Martins and Mas, 2015).
87 Many studies have demonstrated the link between altered lipid metabolism and the
88 development of kidney injury in mice fed a high-fat diet (Guebre-Egziabher et al., 2013;
89 Kume et al., 2007; Muller et al., 2018; Wahba and Mak, 2007). The literature reports an
90 important association between renal lipid accumulation and increased renal pro-
91 inflammatory mediators, such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor
92 necrosis factor alpha (TNFα) (Martins and Mas, 2015). Furthermore, excessive renal lipid
93 deposition can lead to renal tubular cell injury (Nosadini and Tonolo, 2011),
94 tubulointerstitial fibrosis (Takabatake et al., 2017), podocytes damage, mesangial
95 sclerosis (Abrass, 2004), and structural glomeruli alterations (Keane, 2000; Zhou et al.,
96 2016). Renal lipotoxicity is also strongly associated with the development of proteinuria,
97 glomerulonephritis, and CKD (Bobulescu, 2010). However, the role of renal lipid
 98 accumulation that could lead to kidney damage in a high-fat diet has not been completely
99 understood.
100
101 Although the pathological consequences of a high-fat diet on the kidneys are well
102 documented, the repercussions in renal morphology and function as a result of a high-fat
103 diet from an early age are not clear. Thus, the aim of this study was to investigate the
104 effects of high-fat diet intake from weaning on the morphology and renal function of adult
105 rats. We hypothesized that the intra-renal lipid accumulation induced by high-fat diet can
106 be associated with damages in the renal morphology and function, leading to a higher risk
107 of developing CKD in adulthood.
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110 2. Materials and Methods
111
112 2.1 General Procedures:
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114 Experiments were performed in male Wistar rats post-weaned (21 days old), weighing
115 between 50-60g. The animals were randomly assigned to two groups and followed for
116 eight weeks: standard normal diet (ND, n=10) and high-fat diet (HD, n=10). The high-fat

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117 diet produced contained 30% of fat, 23% of carbohydrates and 19% of proteins. The fat

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118 in HD is composed mainly of saturated fat. The standard diet contained 3% of fat, 55%

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119 of carbohydrate and 22% of proteins (Nuvilab®, Paraná, Brazil). Caloric densities of a

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120 high-fat diet and standard diet were respectively 381 kcal/100g and 257 kcal/100g.

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121 Animals were maintained in the Central Animal Facility at the Mackenzie Presbyterian
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University (Sao Paulo) under the same housing conditions (12-h light/12-h dark cycle,
temperature 23±2°C) with free access to tap water and food ad libitum. All procedures
were performed in accordance with the Guide for the Care and Use of Laboratory Animals
published by the US National Institutes of Health (n. 85-23, revised in 1996) and
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approved by the Ethics Committee of Mackenzie Presbyterian University (Protocol:
063/02/2010). Body weight was measured weekly at the same time of the day using a
digital balance (TOLEDO, model 9094c/4). Body weight gain was calculated as the
difference between the body weight measured at the beginning and at the end of the
protocol.

2.2 Glucose Tolerance Test (GTT):

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134 GTT was performed after 8-week protocol. After an 8-h fast, glucose (1.5 g/kg body
135 weight) was injected as a bolus intraperitoneally. Blood glucose concentration was
136 determined by using a glucometer (AccuChek Advantage Roche Diagnostics®). Blood
137 samples were taken from a cut made on the tip of the tail at 0, 15, 30, 60, and 90 minutes
138 after glucose administration. The area under the curve (AUC) was calculated using
139 GraphPad Prism 5 (GraphPad Software Inc, San Diego, CA, EUA).
140
141 2.3 Insulin Tolerance Test (ITT):
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143 72 h after the GTT test, a similar procedure was performed for ITT. Briefly, after a 4-h
144 fast, rats were anesthetized with Pentobarbital (50 mg/kg body weight, i.p.) and an insulin
145 load (0.75 U/kg body weight) was injected as a bolus in the caudal vein. Blood glucose
146 levels were determined from a cut made on the tip of the tail at 0, 4, 8, 12, and 16 minutes
147 after insulin administration. Constant rate for blood glucose disappearance (Kitt) was
148 calculated using the formula 0.693/t1/2, and the blood glucose half-time (t1/2) was
149 calculated from the slope of the least squares regression of the blood glucose
150 concentration during the linear phase of decline (Bonora et al., 1989; Fiorino et al., 2016).
151
152 2.4 Kidney function evaluation:
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154 In the 7th week of the protocol, the animals were housed individually in metabolic cages
155 (Tecniplast, Buguggiate, VA, Italy). Urine samples were collected during the 24-h period
156 and used to determine urine creatinine. A blood sample (500 µL) was also collected at
157 the end of the 24h-period. Urinary and serum creatinine levels were quantified using a
158 colorimetric method (LABTEST Biochemical Kit, Brazil). The creatinine clearance was
159 used to estimate the Glomerular Filtration Rate (GFR) and was calculated using the
160 following formula: [(Urine (Creatinine) X Urine Vol) / Serum (Creatinine)].
161
162 2.5 Isolated perfused kidney method:
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164 At the end of the 8 weeks, rats were anesthetized with sodium pentobarbital (50 mg/kg
165 body weight i.p.) and the right renal artery was cannulated through the mesenteric artery,
166 without blood flow interruption, and placed into the perfusion system (Fonteles et al.,

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167 1983), isolating the kidney from endocrine and neural interference. The perfusate was a

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168 modified Krebs–Henseleit solution with the following composition (in mM): 147 Na+, 5

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169 K+, 2.5 Ca2+, 2.0 Mg2+, 110 Cl−, 25 HCO3−, 1 SO42−, and 1 PO43-. This was dialyzed for

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170 48 h after the addition of six grams of bovine serum albumin (BSA) (Fonteles et al., 1983).

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171 Immediately before starting the perfusion, 100 mg of glucose, 50 mg of urea, and 50 mg
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of inulin was added to the perfusate solution. The pH was then adjusted to 7.4 and the
solution placed in the perfusion system. The perfused rat kidney model followed the
technique previously described by Bowman and Maack and modified by Fonteles and
coworkers (1983) (Bowman and Maack, 1974; Fonteles et al., 1983) by the introduction
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of a silastic membrane oxygenator into the perfusion line. Prior to each experiment, the
system was calibrated for flow and resistance. Each experiment was divided into two
periods of 30 min each, these sample collection periods were further subdivided into equal
intervals of 10 min. During each 10-min period, aliquots of perfusate and urine were
collected to determine creatinine, sodium, and potassium. The perfusion pressure (PP),
renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), and
the percentage of tubular transport of sodium (%TNa+), potassium (%TK+), and chloride
183 (%TCl−) were determined. The percent of proximal and distal tubular sodium, potassium,
184 and chloride transport were calculated using free water and osmolar clearances as
185 described originally by Martinez-Maldonado and Opava Stitzer (Martinez-Maldonado
186 and Opava-Stitzer, 1978).
187
188 2.6 Histological analysis:
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190 The left kidney was used for glomerular injury analysis in H&E stained (Sigma) sections of
191 the kidney (5 μm) embedded in Paraplast. Digital images from thirty glomeruli per animal
192 were obtained using a light microscope (Leica) at 400x magnification. After digitalization,
193 Bowman's capsule area (BCA), glomerular tuft area (GTA) and Bowman’s space area (BSA)
194 were traced and calculated using a computerized morphometric analysis system (Image Pro-
195 Plus 4.1; Media Cybernetics, Silver Spring, MD, USA).
196 Lipid content was measured using quantitative histochemistry of Oil Red O (Sigma-
197 Aldrich) stained kidneys. Tissue sections (8 μm thickness) obtained in a cryostat were
198 examined by light microscopy at 200x magnification and analyzed by a computerized
199 morphometric analysis system (Image Pro-Plus 4.1; Media Cybernetics, Silver Spring,
200 MD, USA). The slides were counterstained with hematoxylin to visualize the nuclei.
201 Lipid accumulation was determined in 12 images per animal based on the percentage of
202 area occupied by lipid droplets. Histological analyses were blinded conducted by RO
203 Pereira.
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205 2.7 Cytokine measurement:
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207 In a subgroup of randomly selected rats (ND: n=6; HD: n=6) cytokines were evaluated in
208 the right kidney approved by the Ethics Committee of Mackenzie Presbyterian University
209 (Protocol: 108/03/2014).
210
211 Measurement of cytokines was performed using the MILLIPLEX™ cytokine panel
212 (Merck Millipore, Billerica, MA), a bead-based immunoassay which allowed the
213 simultaneous quantification of the cytokines IL-1b, IL-6, TNF-α and IL-10 in kidney
214 samples. The results were normalized by kidney total protein.
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216 2.8 Statistical Analyses:

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218 The statistical analysis was performed by using GraphPad Prism 5. The results were

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219 analyzed using the unpaired Student’s t test. The data were reported as mean ± SEM.

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220 The p value for significant differences was set at p ≤ 0.05.

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3. Results

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P 3.1 Body weight and food consumption

No differences in body weight were observed between groups prior to or after the
experimental protocol. In addition to this, no significant difference in body weight gain
was found between groups. Although the food consumption (g/animal/24-h) was reduced
in the HD group, there was no difference in caloric intake (Kcal/animal/24-h) when
compared to the ND group (table 1).
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234 3.2 Glucose metabolism
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236 As shown in table 1, high-fat diet consumption did not change fasting glucose but
237 promoted an increase in the AUC and a decrease in Kitt.
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239 3.3 Renal function
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241 Indices of kidney function are shown in table 2 and figure 1. There was a decrease in the
242 in vivo GFR (ND: 1.8 ± 0,19; HD: 1.1 ± 0.14 ml/min), UF (ND: 9.8 ±0.54 HD: 3.7 ± 0.19
243 mL/24-h) and water intake (ND: 26.9 ±1.45; HD: 16.75 ± 1.35 mL/24-h) in HD group
244 when compared with ND. In addition, the serum creatinine was increased in HD (ND:
245 0.55±0.071; HD: 0.71±0.14 mg/dL).
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247 In the isolated perfused kidney, the RVR was increased in both experimental periods,
248 achieving 47% increase after 60 minutes perfusion, in the HD group (Figure 1A). UF was
249 augmented at 60 minutes (77%) (table 2). The GFR showed a decrease (34%) in the HD
250 group at 60 minutes (figure 1). Furthermore, the RPF decreased in both times, and the PP
251 increased at 60 minutes, in HD. However, the tubular ion transport of Na+, Cl-, K+ have
252 not changed in the HD (table 2).
253
254 3.4 Histological analysis
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256 The morphometric measurements evaluated demonstrate that the glomeruli were retracted
257 in the HD kidneys as shown by the reduction of the Bowman’s capsule area (30%),
258 glomerular tuft area (27%) and Bowman’s space area (49%) (figure 2).
259
260 The kidney lipid deposition showed a 3-fold increase in HD compared to ND (ND: 1.5±
261 0.17 %/area; HD: 5.9 ± 0.06%/area) (figure 3).
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263 3.5 Inflammatory markers
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265 IL-6, a pro-inflammatory cytokine, was significantly increased in HD (ND: 1276 ± 203;

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266 HD: 1982 ±47 pg/mg of protein). In addition, IL1b was also increased (ND: 97 ± 12 HD:

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267 133 ± 5 pg/mg of protein), with no remarkable changes in the TNF-α and IL-10 levels

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268 (figure 4).

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269 3.6 Correlations analysis

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270 We observed a strong negative correlation between intra-renal lipid content and GFR
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(R=-0.84, p=0.0097). Furthermore, there was a positive correlation between intrarenal
lipid content and IL-6 (R=0.79. p=0.02).

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P 4. Discussion

In the present study, we investigated the morphological and functional kidney responses
in adult rats, exposed to a high-fat diet after weaning. Our findings provide evidence that
the consumption of this diet during this critical developmental period induces renal lipid
accumulation, increases pro-inflammatory cytokines content and loss of renal function.
282 After 8 weeks of high-fat diet, the HD group presented no difference in weight gain, and
283 a reduction in food but not at caloric consumption compared to the ND group. These data
284 corroborate our previous demonstration that rats fed a high-fat diet consume less food and
285 have no increase in body weight gain, but have significantly higher adiposity (Fiorino et
286 al., 2016). In addition, this animal model of metabolic syndrome is characterized by
287 increased leptin and triglycerides levels, but lower adiponectin and normal insulin levels
288 (Fiorino et al., 2016). In this context, we decided to investigate the potential renal
289 complications associated with the current model.
290
291 We observed that HD animals did not increase the fasting blood glucose, but presented
292 an increase in AUC and a decreased Kitt. These results demonstrate that these animals
293 developed glucose intolerance and insulin resistance. These results were similar to those
294 of Higa et al. 2014 who demonstrated the same response in rats fed a cafeteria diet. (Higa
295 et al., 2014).
296 The high-fat diet intake induced in vivo changes in renal function, as observed by the
297 decrease in GFR and serum creatinine accumulation (Gomez-Guerrero et al., 2005;
298 Herrera et al., 2011). Moreover, in the Isolated perfused kidney we also observed a
299 decrease in GFR. Interestingly, the animals fed a high-fat diet had an increased intra-renal
300 lipid content. Our study corroborates Muller and coworkers (Muller et al., 2018) which
301 observed a higher kidney lipid content in mice fed a cafeteria diet. Similarly, Bobulescu
302 and coworkers (Bobulescu, 2010) showed in humans, a direct association between body
303 mass index and kidney lipid deposition. Kidney lipid accumulation has been associated
304 with renal function injury and can be a risk factor in CKD (Martins and Mas, 2015).
305 However, little is known about how this pathogenic process occurs in the kidneys,
306 especially when compared to the knowledge base regarding the deleterious effects of
307 lipids on other organs such as the heart, liver, and skeletal muscle (Tsuboi et al., 2017).
308
309 The intrarenal lipid accumulation is correlated to the GFR reduction in the HD group and
310 can be the cause of morphological alteration in the glomeruli observed by a decrease in
311 the Bowman’s capsule, Bowman’s space and glomerular tuft areas demonstrating a
312 glomeruli retraction. In a previous study from our group, Muller and coworkers showed
313 that mice fed a cafeteria diet presented similar morphological kidney damage (Muller et
314 al., 2018). These glomeruli retraction could be at least in part induced by mesangial cell

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315 contraction (MCC). It was demonstrated that MCC could be induced by release of

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316 vasoactive hormones such as angiotensin II, that decrease the capillary surface area and

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317 consequently reducing the GFR (Henderson and Barber, 1998; L’Azou et al., 1999;

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318 Pfeilschifter, 1989; Potier M et al., 1998; Rodriguez-Barbero et al., 2000).

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To support this idea, recently our group showed in isolated perfused kidneys obtained
from rats under high fructose diet, another experimental metabolic syndrome model, a
progressive fall in the glomerular filtration rate associated by an increase in the renal
concentrations of angiotensin I and angiotensin II (Yokota et al., 2018). Moreover,
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unpublished data from our laboratory showed that angiotensin II blockade by losartan in
the isolated perfused kidney method determine a higher RVR decrease more in the HD
group than the ND group suggesting that the angiotensin II has an important contribution
to the RVR rise in the HD.

In the present study, the isolated perfused kidney also demonstrated that in HD group
there is an increase in RVR with no changes in tubular sodium and potassium transport
331 suggesting that changes in renal function are associated with glomerular alterations.
332
333 We have also shown that the inflammatory markers were changed by the high-fat diet
334 consumption, as observed by the increase in the kidney pro-inflammatory cytokines IL-6
335 and IL-1b. Interestingly there was no change in the amount of the anti-inflammatory
336 cytokine IL-10. Considering that we did not measure macrophages infiltration in the
337 kidney, we cannot affirm if the source of cytokine production is local or from other
338 tissues, such as adipose tissue. Despite this limitation, it is important to consider that the
339 increased concentration of pro-inflammatory cytokines in the kidney reveals that this
340 tissue is exposed to the deleterious effects typically generated by chronic inflammation,
341 and therefore, may increase the risk of development of lipotoxicity and CKD.
342 Additionally, we observed a positive correlation between kidney lipid accumulation and
343 the IL-6 content in this organ. Saja and coworkers(Saja et al., 2018) have demonstrated
344 that animals with dyslipidemia developed inflammation that played a key role in
345 mediating the deleterious changes in kidney function. Previous experiments have also
346 shown a strong association between renal lipid accumulation and increased renal pro-
347 inflammatory mediators, such as interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor
348 necrosis factor alpha (TNFα) (Martins and Mas, 2015). The renal lipotoxicity is also
349 strongly associated with the development of proteinuria, glomerulonephritis and chronic
350 kidney disease (Bobulescu, 2010). Moreover, Chung et al. 2009 have demonstrated that
351 hypertensive animals fed a high-fat diet have increased the angiotensin II, which is
352 associated with kidney lipid deposition, lipotoxicity, and inflammation (Chung et al.,
353 2010). In this context, our results suggest that the loss of renal function in the HD group
354 can be caused by a lipotoxicity process however more experiments are necessary for the
355 future to support this idea.
356
357 5. Conclusion
358
359 Our study provides evidence that high-fat diet consumption leads to renal lipid
360 accumulation, increases inflammatory cytokines, induces glomeruli retraction and renal
361 dysfunction. These damages observed in the kidney could be associated with an increased
362 risk to advanced CKD in adulthood suggesting that reduction of high-fat ingestion during
363 an early period of life can prevent metabolic disturbances and renal lipotoxicity.
364

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365 Abbreviations

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366

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367 MCC: Mesangial cell contraction.

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368 HD: High-fat diet

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369 ND: Standard normal diet
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Acknowledgments

This study was supported by grants from MackPesquisa (number 059/11) to Fiorino P.
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Américo ALV and Muller CR held a scholarship from Mackenzie PIBIC.

Author Contributions Statement

CM participated in research design, writing of the paper, performance of the research,

and data analysis. AL, RP, AA, participated in performance of research and data analysis
NdN participated in performance of research. FE, VF, PF and MF participated in
381 performance of research and writing of the paper
382
383 Conflict of Interest Statement:
384
385 The authors declare that there are no conflicts of interest
386
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540 Table1. Metabolic parameters at the end of the protocol of 8 weeks.

ND HD
Initial body weight (g) 47 ± 0.9 49 ± 0.8

Final body weight (g) 312 ± 7 295 ± 6.3

Weight gain (g) 265 ± 8 246 ± 6

Food consumption (g/animal/24 h) 26 ± 0.3 21 ± 0.7 *

Caloric consumption (Kcal/animal/24 h) 79 ± 1 79 ± 2.7

Fasting glucose (mg/dL) 108 ± 5 99 ± 6

AUC (mg/dL/min)

Kitt (mg/dL)

o n al 144 ± 6

4 ± 0.2
200 ± 9*

3.3 ± 0.2*

541

542

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AUC = Area Under the Curve; Kitt = Rate constant for glucose disappearance; ND,

Normal Diet; HD, High-Fat Diet. *p≤0.05 versus ND.

543

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550
551 Table 2. Renal functional parameters of Perfusion Pressure, Renal Plasmatic Flow, and

552 tubular transport of Sodium, Potassium, and Chloride at the end of the protocol of 8 weeks

553 of isolated perfused kidney method.

Isolated
perfused
ND HD ND HD
kidney
parameters
30 mins 60 mins
PP 114.7 ± 3.9 130.0 ± 6.7 112 ± 3.5 130 ± 7*
RPF 26 ± 2.1 19.2 ± 1.3* 26 ± 2 20 ± 1.4*

UF 0.14±0.014 0.15±0.03 0.14±0.01 0.23±0.04*

% T Na 83.6 ± 0.9 83.7 ± 3.2 80 ± 3 72 ± 5

%TK

% T Cl
61.3 ± 5.3

89.9 ± 3.7

o n al
69.0 ± 5.6

79.8 ± 4.0
61 ± 5

81 ± 3
65 ± 3

76 ± 4

554

555

o vi si
Perfusion Pressure (PP, mmHg), RPF (Renal Plasmatic Flow, mL.g-1.min-1), UF (Urinary

Flow, mL.g-1.min-1), Percentage of Sodium tubular transport (% T Na+), Percentage of

r
556

557

558
P
Potassium tubular transport (% T K+) and Percentage of Chloride Tubular transport (% T

Cl-). ND, Normal Diet; HD, High-Fat Diet. *p≤0.05 versus ND.

559 Figure 1. Isolated perfused kidney. Renal Vascular Resistance (A; RVR), and Glomerular

560 Filtration Rate (B; GFR) of isolated perfused kidneys from ND, Normal Diet and HD,

561 High-Fat Diet. The results are expressed as mean ± SEM *p≤0.05 versus ND.

562 Figure 2. Glomerular morphological parameters. A: Morphologic parameters of renal

563 histology. B: Representative glomeruli. Lowercase letters show: a. Bowman’s Space; b.

564 glomerular tuft; c. Bowman’s capsule; t: tubules. Magnification: X400. ND, Normal Diet;

565 HD, High-Fat Diet. *p≤0.05 versus ND.

566 Figure 3. Kidney lipid deposition. A: Estimated percentage of lipids in the kidney. B:

567 Lipid deposition in ND and HD, arrows show lipids droplets. Magnification: 400x. ND,

568 Normal Diet; HD, High-Fat Diet. *p≤0.05 versus ND.

569 Figure 4. Inflammatory cytokines. Effects of high-fat diet on cytokines (pg/mg of

570 protein): (A) Interleukin 6 (IL-6), (B) Interleukin 1b (IL-1b), (C) Interleukin 10 (IL-10)

571 and (D) Tumor Necrosis Factor-alpha (TNF-α). ND, Normal Diet; HD, High-Fat Diet.

572 *p≤0.05 versus ND.

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