The Effect of High Salt Diet in Renal Fibrosis Through CHOP Protein Stimulated Apoptosis in Rat Model

І. Background: Prolonged excessive salt intake is an important risk factor for development of renal brosis. In the onset of renal tubular destruction, KIM-1 appears in urine. CHOP is an important apoptosis stimulator protein. The aim of present study was to investigate the effect of high salt diet in development of renal brosis through apoptosis. ІІ. Methods and results: The 25 male Wistar rats were divided randomly into ve groups and treated with 0%, 0.5%, 1%, 1.2%, 1.5% of NaCl dissolved in distilled water for 8 weeks. For conrmation of renal tubular destruction, the urinary KIM-1 was measured. The slides of renal tissue were prepared and stained with Hematoxylin and Eosin and Masson´s Trichrome for brosis detection. To investigate the role of CHOP protein in development of renal tubulointerstitial destruction, the relative gene expression of CHOP in renal tissue was analyzed using qRT-PCR method.There was no signicant differences in urea, creatinine and total protein concentration of rats received different concentrations of NaCl compared to the control group. Urinary KIM-1 and mRNA level of CHOP was found to be increased signicantly in rats treated with 1.5% NaCl compared to the control group. Mild renal brosis was observed in the same group too. III. Conclusion: Excessive salt intake can lead to brosis through increasing the expression of apoptotic CHOP gene in renal tissue. KIM-1 can be detectable in urine long before the development of renal brosis. 1.2% w/v NaCl in distilled water as drinking water Group5: 1.5% w/v NaCl in distilled water as drinking water Treatment was continued for 8 weeks. At the end of the treatment; 24 hour urine with the aid of metabolic cage was collected. The collected urine stored at -80 °C for measuring urinary KIM-1, creatinine, urea and total protein concentration. Animals were anesthetized by diethyl ether and then; kidney samples were separated. The part of kidney samples were immediately frozen in liquid nitrogen and transferred into -80˚C freezer for qRT-PCR assay; and the other part was xed with 10% formalin for histopathology examination. The blood samples were taken from the cardiac puncture; serum was separated and stored at -20˚C for measuring creatinine and urea concentration.


Introduction
Chronic kidney disease (CKD) is a global threat to public health and if not treated in time, it will lead to renal failure (RF). In the early stages of CKD, there are usually no clinical symptoms and the disease does not appear unless there is a signi cant reduction in renal function. The total number of adults affected with CKD is 220 million men and 270 million women worldwide [1,2]. CKD is de ned as tubulointerstitial destruction with a glomerular ltration rate (GFR) of less than 60 ml / min / 1.73 m 2 for at least 3 months [3,4].Prolonged excessive salt intake has been identi ed as a risk factor for the development of renal brosis and CKD [5]. Excessive salt intake increases the osmotic pressure inside the renal tubulointerstitial cells. High osmotic pressure inside the nucleus; destroys the chromatin structure, which will alter the expression of genes; like reducing the expression of genes involved in DNA repair. Additionally, high level of unfolded proteins in the cytoplasm of renal tubulointerstitial cells results in osmotic stress in the endoplasmic reticulum (ER) [6]. In other words, high osmotic pressure, causes DNA damage, disruption of DNA repair systems and ER osmotic stress. Due to destruction of DNA structure, the cell remains in the G 2 phase of the cell cycle and cannot enter mitosis [7][8][9].
It has been demonstrated that, inhibition of ER osmotic stress in the salt-sensitive rats, prevented from developing renal tissue brosis. It means that; prolonged excessive salt intake may cause in ammation and brosis in kidneys through ER osmotic stress [10]. Some in vitro studies indicated that ER osmotic stress increased the expression of the pre-apoptotic molecule CHOP (C/EBP Homologous Protein GADD 153), which promoted apoptosis through inhibiting the anti-apoptotic molecule BCL-2. It seems that, CHOP is one of the key proteins in stimulating apoptosis [11][12][13]. Recent in vivo studies con rmed that rats with a defect in the CHOP gene did not develop in ammation and brosis in kidneys [7,11].
CHOP is a 29 kDa protein consisting of 169 amino acids in humans and 168 amino acids in rodents.
BCL-2 is an important inhibitor protein in apoptosis pathway. CHOP binds to BCL-2 and in this way; stimulates apoptosis [14,15]. In apoptotic tissue, the in ammatory process will lead to brosis [7,12,16] . Kidney Injury Molecule 1 (KIM-1) is a 90 kDa transmembrane protein found in the membrane of renal tubular cells. The outer domain of KIM-1 separates from the membrane and enters the lumen of the renal tubules during apoptosis [17]. Urinary KIM-1 is identi ed as a diagnostic marker for renal tissue destruction [1].
In an attempt to gain further insights into the effects of prolonged excessive salt intake on renal function; this study was carried out in rat model. We hypothesized that high salt diet could a) increase relative gene expression of CHOP in renal tissue , b) increase urinary KIM-1, c) develop renal tissue brosis. Our study focused on the association between renal tissue brosis and urinary diagnostic markers and could be applied as a strategy to prevent from developing progressive CKD and RF.

Material And Methods
Research design 8-Weeks old male Wistar rats, body weight of 200-250g, were purchased from Pasteur institute of Iran (IPI). Animals were housed in 12-Hour light/dark period in a stable temperature (21-23°C) and 55%±10% relative humidity. Rats were nourished by standard chow and water ad libitum. 25 animals were divided into 5 groups randomly each as given below. NaCl ACS reagent was dissolved in distilled water and provided for animals as drinking water: Group 1: distilled water as drinking waterGroup 2: 0.5% w/v NaCl in distilled water as drinking waterGroup 3: 1% w/v NaCl in distilled water as drinking waterGroup 4: 1.2% w/v NaCl in distilled water as drinking water Group5: 1.5% w/v NaCl in distilled water as drinking water Treatment was continued for 8 weeks. At the end of the treatment; 24 hour urine with the aid of metabolic cage was collected. The collected urine stored at -80 °C for measuring urinary KIM-1, creatinine, urea and total protein concentration. Animals were anesthetized by diethyl ether and then; kidney samples were separated. The part of kidney samples were immediately frozen in liquid nitrogen and transferred into -80˚C freezer for qRT-PCR assay; and the other part was xed with 10% formalin for histopathology examination. The blood samples were taken from the cardiac puncture; serum was separated and stored at -20˚C for measuring creatinine and urea concentration.
Urea, creatinine, total protein and KIM-1 assay in serum and urine Creatinine concentration was measured in urine and serum using Pars Azmon kit, based on the Jaffa colorimetric method. Urea concentration was tested in urine and serum with the aid of Pars Azmon kit, based on the Kinetic Urease method. Total protein concentration in urine was determined using Grainer kit, based on the Biuret colorimetric method. KIM-1 concentration in urine was measured by Crystalday ELIZA kit.

RNA extraction and qRT-PCR analysis
Total RNA from the excised kidney tissues were isolated using the TRIzol extraction reagent (Invitrogen, 15596026), according to the manufacturer's recommendations. The integrity of mRNA was con rmed by electrophoresis in a denaturing 1% agarose gel. First strand cDNA was synthesized from total RNA with random hexamer primers using the RevertAid H Minus cDNA synthesis kit (Biofact, W2569-100).
Quantitative Real time-PCR of GAPDH (reference gene) and CHOP were carried out using speci c primers are listed in Table 1 The prepared slides are stained with Hematoxylin and Eosin (H and E); and Masson´s Trichrome; which are the speci c staining method for tissue brosis detection. In Masson´s Trichrome staining, the brotic tissue areas are blue and normal tissue areas are red. From each microscopic slide, 5 of them were randomly selected and the ratio of blue to red tissue areas was calculated by Image J software, and the average of these 5 was considered as brosis severity. They were divided into 4 degrees according to the severity of the brosis: grade 0 was interpreted if brosis was not observed and grades 1, 2, 3 were interpreted according to the severity of the brosis.

Statistical Analysis
Data are expressed as means ± SD. Using SPSS version 16 software; the data were statistically computed by one-way ANOVA procedure and subsequently Duncan's test. A P value < 0.05 was considered statistically signi cant.

Blood and urinary biochemical variables
According to data in Table 2 (page 12), there was no statistically signi cant difference in serum and urine urea, creatinine and total protein concentration in groups 2-5 in comparison with normal control group. necrosis, and mild focal brosis. In animals of group 4, hyperemia, hemorrhagic foci, and degenerative changes were observed in the tubules. In some cases; evidence of Bowman's dilatation, brosis, and mild in ammatory cell accumulation was also observed. In animals of group 5, evidence of epithelial cell membrane destruction, hydropic degeneration, glomerular brosis, accumulation of in ammatory cells, decreased glomerular space, coagulation necrosis, brosis foci, degenerative changes in some glomeruli, hyperemia and mild to moderate bleeding were observed. Outstanding features of histopathology in most cases belonged to this group.As shown in Fig.4 and Fig.5 (page 17-18); the brotic score of kidney tissue in group 5 had a signi cant increase compared to normal control group (p-value < 0. 05).
It is noteworthy that the degree of brosis is mild to moderate, which is expected to have no signi cant effect on kidney function.

Discussion
The results of the present study indicated that consumption of NaCl in group 5 caused a statistically signi cant increase in the expression of the CHOP gene as well as mild brosis in kidney tissue. Biochemical indicators of renal function such as urea, creatinine in blood and urine, and total protein in urine were not signi cantly different in comparison with normal control group; however, the concentration of KIM-1 increased signi cantly in the urine.
Results of present study indicated that osmotic stress due to prolonged excessive salt consumption increased expression of apoptotic CHOP gene in kidney tissue. In 1998, a study was designed on the mIMCD cell line. The cells were cultured in two isosmotic media (300 mosmol / kg) and hyperosmotic media (300 mosmol / kg + 150 mM NaCl) for 48 hours. Consistent with results of our study; in cells exposed to hyperosmotic media, the expression of GADD45 and GADD153 (CHOP) genes increased signi cantly [21].
Findings of present research demonstrated that urea, creatinine in blood and urine, and total protein in urine of rats received different concentrations of NaCl were not signi cantly different in comparison with normal control group. In the study carried out in 2007, spontaneously hypertensive rats (SHR), were divided into four groups with 0.6% (normal control), 4%, 6%, and 8% NaCl in the diet for 8 weeks. In contrast to results of our research; in animal groups received 6% and 8% NaCl in the diet; serum creatinine concentration, proteinuria and albuminuria increased signi cantly compared to the normal control group. Additionally; statistically signi cant decreased GFR was observed due to mild renal tubular degeneration in the same groups [22].
Based on results of present study, mild renal brosis was observed in rats treated with 1.5% NaCl compared to the control group. According to the study designed in 2011 on SHR rats; in animal groups that received 8% NaCl in the diet for 4 weeks, consistent with results of our study, signi cant glomerular damage and interstitial brosis were observed in comparison with group that received 8% NaCl in the diet with Losartan. It means that modulating osmotic stress with Losartan; could prevent from developing renal tissue brosis [23].
Results of present study demonstrated that, 1.5% NaCl in drinking water for 8 weeks in male Wistar rats, increased the expression of CHOP gene in kidney tissue. In 2015, researchers carried out a study on Sprague Dawley rats; weighing 250 to 300 g. To induce osmotic stress; the animals were deprived of drinking water for 3 days. In the next step; For 7 days, the animals consumed drinking water containing 2% NaCl. In both conditions of water depriving and consumption of water containing 2% NaCl, consistent with ndings of our research, expression of CHOP gene in hypothalamus tissue was signi cantly increased in comparison with the normal control group [10].
In present research we observed mild renal brosis in rats that received 1.5% NaCl in drinking water for 8 weeks. In 2015, the study conducted on mice that had 5/6 kidneys removed. In this study, the animals were divided into three groups and exposed to high-salt (4% NaCl) with Hydralazine, low-salt (0.02% NaCl), and normal (0.4%Nacl) diet for two weeks. At the end of the study, consistent with results of our research, it was found that salt induces stable renal brosis while blood pressure is normalized with Hydralazine. As a result, a prolonged high salt diet causes renal tissue brosis and chronic progressive renal disease independent of blood pressure [24]. According to the study carried out in 2017, Dahl saltsensitive (DSS) rats were divided into two groups with 2% NaCl and 8% NaCl in the diet for 5 weeks. In contrast to results of our research, a statistically considerable kidney tissue brosis was not seen and there was no statistically signi cant difference in serum creatinine and urea levels between the two animal groups. After 15 weeks statistically signi cant difference in serum creatinine and urea levels and notable kidney tissue brosis was observed between two animal groups. Also, at the end of the 15 th week, consistent with results of our study, the expression of the KIM-1 gene in kinney tissue of group received 8% NaCl was signi cantly higher than the group received 2% NaCl [25]. In studies conducted in 2017 and 2018 on male Wistar albino rats; consistent with results of our research, signi cant tubular degeneration and renal tissue brosis were observed in the group received 8% NaCl diet for 8 weeks; compared to the normal control group [26,27].

Conclusion
Osmotic stress due to prolonged excessive salt consumption can result in brosis through increased expression of apoptotic CHOP gene in kidney tissue. At the onset of apoptosis; KIM-1 protein appears in urine. In other words; KIM-1 can be detectable in urine before brosis of signi cant part of kidney tissue and impaired renal function. prior publication: Neither this manuscript nor one with substantially similar content under our authorship has been published or is being considered for publication elsewhere. W certify that all the data collected during the study is presented in this manuscript and no data from the study has been or will be published separately.
Con icting Interest: All contributing authors declare no con icts of interest.