STUDYING THE EFFECT OF TWO TYPES OF SALT IN PROGRESSION OF CHRONIC RENAL FAILURE IN RATS

1
STUDYING THE EFFECT OF TWO TYPES OF SALT
IN PROGRESSION OF CHRONIC RENAL
FAILURE IN RATS
Esraa M. Awad-Allah ; Ahmed A. Ameen and Alaa O. Aboraya
Nutrition and Food Science Department, Faculty of Home Economics, Helwan
University, Cairo, Egypt.
Key Words: chronic kidney disease, rock salt, refined salt, rats.
ABSTRACT
Salt ingestion has been linked to kidney disease via hemodynamic
(increased introglomerular pressure) and non-hemodynamic mechanisms
(increased oxidative stress), independent of blood pressure. The aim of
the present study was to compare the effect of rock salt (Halite) to refined
salt in different concentrations on chronic renal failure rats. Thirty adult
male Sprague-Dawley rats which weighing (170+5g) were divided
randomly into two main groups as follow: the first group (-ve control= 5
rats) was fed on basal diet. The second group (25 rats) were fed on 14 %
casein diet containing 0.7% adenine for 4 weeks to induce chronic kidney
disease (CKD), then divided into 5 subgroups from group 2 to group 6.
Subgroup 2 (+ve control) fed on basal diet. Subgroup 3 and 4 fed on
basal diet supplemented with 4% and 8% of refined salt, respectively.
Subgroup 5 and 6 fed on basal diet supplemented with 4% and 8% of
rock salt, respectively. At the end of the experimental period (4 weeks),
rats were scarified and serum was collected to determine kidney
functions. The results showed that serum concentrations of creatinine,
urea, uric acid, sodium and potassium were significantly elevated
(P<0.05) by adenine administration (positive control) compared to
negative control, in contrast, serum total protein, albumin and globulin
were significantly reduced (P<0.05). Also, it was indicated that rock salt
administration recorded the best changes for these parameters in CKD
rats compared to refined salt. It be concluded that restriction of sodium
intake is an important preventive and therapeutic measure in patients
with chronic renal diseases or at risk of renal damage such as
hypertensive. So, it be recommended to use rock salt instead of refined
salt and further studies are required to elucidate beneficial effect and the
mechanism of how rock salt attenuates blood pressure.
INTRODUCTION
Chronic kidney disease (CKD) is an important, widespread
clinical problem which has multiple etiologies. Control of blood pressure,
cholesterol, and glucose are important strategies to slow progression of
CKD towards end-stage renal disease (ESRD) (Walker et al., 1989).
The common sources of salt for consumers can be classified into
refined (table) salt, sea salt, rock salt, and processed salt (Lee et al.,
Egypt. J. of Appl. Sci., 35 (11) 2020 117-126
2
2007). The relationship between salt and blood pressure has been
discussed in medical literature for decades and it has been shown that
increased salt intake contributes to the prevalence of hypertension and
proteinuria (Swift et al., 2005).
Less intensely studied are the direct nephrotoxic effects of sodium
chloride. Salt ingestion has been linked to kidney disease via
hemodynamic (e.g. increased introglomerular pressure) and nonhemodynamic
mechanisms (e.g. increased oxidative stress), independent
of blood pressure (Weir and Fink, 2005).
Although there is growing awareness by the general public of the
health benefits of rock salt, there is little information on whether
consumption of rock salt can have a direct effect on blood pressure and
CKD (Chanumuang, 2010).
The aim of the present study was to compare the effect of rock
salt (Halite) to refined salt in different concentrations on chronic renal
failure rats.
MATERIALS AND METHODS
MATERIALS:
Rock salt and refined salt in the form of powder, were purchased
from El-Gomhoriya Pharm., Cairo, Egypt. Adult male Sprague-Dawley
rats (n= 30) which weighing (170+5g) were purchased from Farm of
experimental animals in Helwan, Egypt. Adenine, casein, all vitamins,
minerals, cellulose, choline and starch were obtained from El-Gomhoria
Company, Cairo, Egypt.
METHODS:
1- Induction of CKD in rats:
Rats were inducted with chronic renal failure by using the method
described by (Yokozawa et al., 1986) by feeding rats on 14 % casein diet
containing 0.7% adenine for 4 weeks.
2- Preparation of salts:
Salts were grinded, then the powder was mixed with basal diet in
different levels.
3- Diet composition and experimental animal design:
The basal diet was formulated according to AIN-93M diet
(Reeves et al., 1993). Animals (30 rats) were housed in well conditions
in biological studies lab of Faculty of Home Economics. They were left
for seven days as adaptation period and they were allowed to feed
standard laboratory food and water. After the period of adaptation,
animals were divided into two main groups, as follows: - the first group
(5 rats) was fed on basal diet and served as a negative control group (-
ve), the second group (25 rats) was fed on 14 % casein diet containing
0.7% adenine for 4 weeks. After chronic renal failure rats were divided
as follow:-
118 Egypt. J. of Appl. Sci., 35 (11) 2020
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Subgroup (1): Five Rats with chronic kidney disease (CKD rats)
were fed on basal diet as positive control group
(+Ve).
Subgroup (4): Five CKD Rats were fed on basal diet supplemented
with 4 % Refined salt (Replacement with salt in
basal diet).
Subgroup (5): Five CKD Rats were fed on basal diet supplemented
with 8 % Refined salt (Replacement with salt in
basal diet).
Subgroup (2): Five CKD Rats were fed on basal diet supplemented
with 4 % Rock salt (Replacement with salt in basal
diet).
Subgroup (3): Five CKD Rats were fed on basal diet supplemented
with 8 % Rock salt (Replacement with salt in basal
diet).
At the end of the experimental period (4 weeks), rats were fasted
overnight before scarifying and blood samples were collected from each
rat and were centrifuged at 3000 rpm for 15 min to obtain the serum for
biochemical analysis.
4- Biological evaluation:
Feed intake (FI), body weight gain percentage (BWG %) and feed
efficiency ratio (FER) were determined according to Chapman et al.,
(1959) using the following equation:
BWG% = Final body weight – Initial body weight /
Initial body weight × 100
FER = Weight gain / Feed intake.
5- Chemical Analysis of Rock Salt and Refined Salt:
Chemical analysis of rock salt and refined salt were done in
Agriculture Research Center and determined according to method
describe by Ranganna, (1977).
6- Biochemical analysis of serum:
Serum uric acid was determined in the serum according to
method describe by Milena, (2003). Serum urea nitrogen, creatinine and
total Protein were determined in the serum according to method describe
by Burtis and Ashwood, (1999). Serum potassium and Serum sodium
were determined according to method describe by Hoeflmayr, (1979).
Albumin and globulin were determined in the serum according to method
describe by Young, (2001) and Goldenberg and Drewes, (1971),
respectively. Serum sodium and potassium were determined according to
method describe by Guder, et al., (1982) and Hoeflmayr, (1979).
7- Statistical Analysis:
Results were expressed as the mean standard error ± SE. Data
were statistically analyzed for variance “ANOVA” test at P ≤ (0.05)
Egypt. J. of Appl. Sci., 35 (11) 2020 119
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using SPSS statistical software, version 20 was used for these
calculations (Armitage and Berry, 1987).
RESULTS AND DISCUSSION
Data in Table (1) showed the chemical analysis of of rock salt
and refined salt, it was observed that rock salt contained 87.98% of NaCl
while refined salt contained 99.9% of NaCl.
Lee et al., (2017) reported that rock salt that contains 85.7% NaCl
whereas refined salt contained 99.9% NaCl. In addition to sodium, rock
salt also contains calcium (1.5 mg/g), potassium (2.9 mg/g), magnesium
(3.9 mg/g), and traces amounts of iron, manganese and zinc.
Table 1: Chemical Analysis of Rock Salt and Refined Salt
Sample NaCl%
Rock salt 87.98
Refined salt 99.9
Results recorded in Table (2) showed the effect of rock salt and
refined salt on body weight gain (BWG), feed intake (FI) and feed
efficiency ratio (FER) in CKD rats. It was observed that FI, BWG and
FER significantly decreased in positive control compared to negative
control. Group that fed on refined salt recorded the lower reduction in FI,
BWG and FER than group fed on rock salt. Group that fed on 4% rock
salt recoded the lowest reduction in FI, BWG and FER compared to
positive control.
Table (2): Effect of Rock Salt and Refined Salt on Feed Intake (FI),
Body Weight Gain% (BWG %) and Feed Efficiency
Ratio (FER) of Chronic Renal Failure Rats
Parameters
Groups
FI (g/d)
BWG%
FER
Negative Control 16.5 21.91±1.44a 0.070±0.004a
Positive Control 15.2 9.11±0.30b 0.030±0.001b
8% Refined Salt 11.55 3.15±0.17d 0.018±0.002d
4% Refined Salt 14 4.50±0.44cd 0.020±0.001cd
8% Rock Salt 13.5 4.18±0.34cd 0.018±0.002d
4% Rock Salt 14.15 6.05±0.43c 0.026±0.003bc
*Mean values are expressed as means ± SE.
*Mean values at the same column with the same superscript letters are not
statistically significant at P<0.05.
Results of BWG were in the same line with Lee et al., (2017),
who found that the final body weight in the group that fed on the high
level of refined salt was significantly lower than all other groups. In the
current study, feed intake of refined salt group was the lowest compared
to the rock salt group. This was due to larger amount of food
consumption by the rock salt groups despite the refined salt diet
120 Egypt. J. of Appl. Sci., 35 (11) 2020
5
containing a higher salt content than the rock salt diet. The fact that rock
salt diets were consumed at a higher rate than the refined salt diets may
be an indication that the chow with rock salt was more palatable to the
rats (Lee et al., 2017).
As showed in Table (3), serum creatinine, urea and uric acid were
elevated significantly (P< 0.05) elevated by adenine administration in
positive control group compared to the negative control group. It was
observed that the group that fed on high level of refined salt (8%)
recorded the most increase in serum creatinine, urea and uric acid levels
compared to all groups. While the rock salt group (4%) recorded the
lower increase in these parameters than other groups that treated with the
different levels of salts compared to positive control group.
Concerning results of kidney functions in were confirmed by
Saad et al., (2018), who showed that adenine administration significantly
increased serum urea, creatinine and uric acid. Salt intake is associated
with the development of impaired kidney function in the general
population, independent of its effects on blood pressure (Sugiura et al.,
2018). Rock salt contains trace amounts of natural minerals such as
MgSO4, CaSO4, CaCl2 and KCl with slightly lower sodium content
compared to refined salt. Although there is growing awareness by the
general public of the health benefits of rock salt, there is little
information on whether consumption of rock salt can have a direct effect
on blood pressure regulation (Chanmuang, 2010).
Table (3): Effect of Rock Salt and Refined Salt on Serum Creatinine,
Urea and Uric Acid of Chronic Renal Failure Rats
Parameters
Groups
Creatinine Urea Uric Acid
mg/dL
Negative Control 0.62±0.05f 23.15±0.43e 2.75±0.75e
Positive Control 0.91±0.09e 30.05±0.29d 3.11±0.04d
8% Refined Salt 1.14±0.04a 35.63±0.31a 5.79±0.10a
4% Refined Salt 0.96±0.06c 33.22±0.30b 4.62±0.80b
8% Rock Salt 0.98±0.05b 35.16±.48a 4.59±0.84b
4% Rock Salt 0.93±0.06d 31.15±0.20c 3.42±0.13c
*Mean values are expressed as means ± SE.
*Mean values at the same column with the same superscript letters are not
statistically significant at P<0.05.
Results in Table (4) showed a significant (P< 0.05) increased in
serum sodium and potassium in positive control by administration of
adenine compared to negative control group. Rats that fed on the high
level of refined salt (8%) recorded the highest elevation in serum Na and
K compared to other treated groups with salts, while rats that fed on 4%
rock salt recorded the lowest elevation in serum Na and K.
Egypt. J. of Appl. Sci., 35 (11) 2020 121
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Results of potassium were in agreement with Kashioulis et al.,
(2018), who reported that plasma concentration of potassium was clearly
elevated in rats that treated with adenine. It is known that dietary
potassium can influence blood pressure, and that it can play a role in
controlling blood pressure in hypertensive patients (Rodrigues et al.,
2014). Suzuki et al., (2007) which reported that salt is essential and
important for maintaining life. Sodium is an essential nutrient and one of
the important cations in the extracellular fluid. Several studies have
suggested that excess salt intake deteriorates a renal function and
increase in blood pressure. In these regards, modulation of salt is of
utmost importance in the fields of hypertension and nephrology
(McMahon et al., 2013). Lee et al., (2017) reported that how the rock
salt diet actually exerts its anti-hypertensive effects is not clear. One
obvious point to consider is the content of minerals other than sodium in
the rock salt.
Table (4): Effect of Rock Salt and Refined Salt on Serum Sodium
(Na) and Potassium (K) of Chronic Renal Failure Rats
Parameters
Groups
Na K
mg/dL
Negative Control 142.80±0.86e 4.07±0.04e
Positive Control 149.00±1.14d 4.60±0.11d
8% Refined Salt 167.00±1.00a 7.09±0.09a
4% Refined Salt 153.80±0.77c 5.01±0.04c
8% Rock Salt 157.99±0.32b 6.05±0.0b
4% Rock Salt 151.20±0.41cd 4.61±0.17d
*Mean values are expressed as means ± SE.
*Mean values at the same column with the same superscript letters are not
statistically significant at P<0.05.
Data in Table 5 revealed that serum total protein, albumin and
globulin levels were decreased by adenine administration in positive
control group compared with the negative control group. Results also
illustrated that rats treated with 8% refined salt recorded the highest
reduction in serum total protein, albumin and globulin compared with
other treated groups with salts, while rats that fed on 4% rock salt
recorded the lowest reduction in these parameters.
Results of serum total protein and albumin were in agreement
with Saad et al., (2018), who reported that the administration with
adenine significantly decreased serum albumin and total protein. Salt
restriction significantly reduced urinary protein excretion in all studies
that reported proteinuria (McMahon et al., 2015). In fact, a recently
published double-blind controlled randomized trial in patients with CKD
(stages 3 and 4) showed that dietary sodium restriction significantly
122 Egypt. J. of Appl. Sci., 35 (11) 2020
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decreased blood pressure, and consistent reductions in proteinuria and
albuminuria were shown (McMahon et al., 2013 and Hosohata, 2017).
In conclusion, it would appear that restriction of sodium intake is
an important preventive and therapeutic measure in patients with chronic
renal diseases or at risk of renal damage such as hypertensive. It is likely
that the major beneficial effect of rock salt is associated with the mineral
content of the rock salt that is known to be anti-hypertensive such as
potassium, calcium and magnesium. It is also possible that there are as
yet undetermined component(s) of the rock salt that might confer
resistance to hypertension. So, further studies are required to elucidate
the mechanism of how rock salt attenuates blood pressure. Based on our
findings it would also be important to determine if rock salt consumption
would have similar effects on blood pressure in humans.
Table (5): Effect of Rock Salt and Refined Salt on Serum Total
Protein, Albumin and Globulin of Chronic Renal Failure
Rats
Parameters
Groups
Total Protein Albumin Globulin
g/dL
Negative Control 8.84±0.10a 3.65±0.13a 2.45±0.05a
Positive Control 7.58±0.11b 2.99±0.07b 2.00±0.04b
8% Refined Salt 5.95±0.13d 0.98±0.01e 1.04±0.03c
4% Refined Salt 6.71±0.07c 2.04±0.2d 1.89±0.05b
8% Rock Salt 6.11±0.04d 1.97±0.04d 1.17±0.04c
4% Rock Salt 7.03±0.19c 2.28±0.12c 1.99±0.17b
*Mean values are expressed as means ± SE.
*Mean values at the same column with the same superscript letters are not
statistically significant at P<0.05.
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د ا رسة تأثير نوعين من الممح في تطور مرض الفشل الکموي المزمن في الفئ ا رن
إس ا رء مجدي عوض ، أحمد عمي أمين ، آلاء أسامة أبو رية
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لمدة 1 أسابيع لمحث عمى مرض الکمى المزمن ، ثم تم تقسيميا إلى 5 مجموعات فرعية من
Egypt. J. of Appl. Sci., 35 (11) 2020 125
10
المجموعة 5 إلى المجموعة 6. المجم وعة الفرعية 5 )مجموعة ضابطة موجبة( تم تغذيتيا عمى
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مکمل ب 1٪ و 8٪ ممح صخري عمى التوالي في نياية الفترة التجريبية ) 1 أسابيع( تم تشريح
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عمى الأدينين )المجموعة الضابطة الموجبة( مقا رنة بالمجموعة الضابطة السالبة، عمى النقضي
من ذلک انخفض البروتين الکمي في الدم والألبومين والجموبيولين بشکل ممحوظ. کما تم الإشارة
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وعلاجي ميم في المرضى الذين يعانون من أم ا رض الکمى المزمنة أو المعرضين لخطر
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من الممح المکرر وىناک حاجة إلى مزيد من الد ا رسات لتوضيح التأثير المفيد وآلية کيفية تخفيف
الممح الصخري لضغط الدم.
126 Egypt. J. of Appl. Sci., 35 (11) 2020