High-salt intake during pregnancy may influence the outcome of renal ischemia / reperfusion in in adult offspring

B rie f C om m un ic at io n Introduction High salt diet during pregnancy may alter some physiological functions and organs developments (1-5). Milk content may also change with high salt diet (6), and the offspring from mothers that received high salt diet during pregnancy demonstrated a higher blood pressure (3,7). It is well-known that renin secrets by glomerular cells which are salt sensitive and high salt diet disturb renin secretion in offspring and may develop hypertension (8). Previously we demonstrated that cardiovascular response to angiotensin II in offspring subjected to high salt intake during pregnancy was altered gender dependently (9). Renal ischemia reperfusion (I/R) as a major cause of kidney disorder influences renin-angiotensin system (RAS) (10, 11) which may alter by type of diets during pregnancy (7,12). Objectives This study was designed to find the role of high salt intake during pregnancy on the outcome of renal IR in adult offspring. Materials and Methods Animals Female Wistar rats were subjected to tap water or high-salt water (2%) during mating until delivery. At the 30th days Journal of Renal Injury Prevention, Volume 7, Issue 1, March 2018 http://journalrip.com 9 High-salt intake during pregnancy B rie f C om m un ic at io n of life (day of weaning) the offspring were assigned into eight groups, and they were housed to gain weight in 3-4 months. Groups 1 to 4 called “water” groups included two groups of male (groups 1 and 2) and two groups of female (groups 3 and 4) offspring from mothers who received tap water during mating and pregnancy. Groups 5 to 8 called “salt” groups included two groups of male (groups 5 and 6) and two groups of female (groups 7 and 8) offspring from mothers who received 2% salt in drinking water during mating and pregnancy. Experimental protocol At the day of experiment the adult offspring in groups 1 (male: 227 ± 9 g, n = 8), 3 (female: 169 ± 4 g, n = 6), 5 (male: 260 ± 9 g, n = 7) and 7 (female: 192 ± 8 g, n = 7) were anesthetized with chlorohydrate (450 mg/kg i.p). The kidneys were prepared for vessels clamping, and the vessels were clamped for a period of 45 minutes, and afterwards the rats were subjected to 24 hours reperfusion. These groups were assigned as I/R groups. The same procedures were applied to the animals from group 2 (male: 234 ± 10 g, n = 8), 4 (female: 167 ± 6 g, n = 7), 6 (male: 260 ± 8 g, n = 7) and 8 (female: 180 ± 7 g, n = 5), but the renal vessels were not clamped. These groups were considered as control (sham operated) groups. All the animals were subjected to urine collection during last 6 hours of reperfusion by keeping animals in standard metabolic cages to determine creatinine clearances (ClCr). At the end of reperfusion, blood samples were obtained, and the animals were sacrificed humanly. The kidneys were removed and weighed rapidly. The serum levels of creatinine (Cr), blood urea nitrogen (BUN), and nitrite (by Griess method) were measured. The ClCr was determined by the following ClCr formula. ClCr = UF x UCr/PCr where UF (mL/min), UCr and PCr stand for urine flow, urine Cr level and serum Cr concentration. Ethical issues The research followed the tenets of the Declaration of Helsinki. This project was approved by Ethics Committee of Isfahan University of Medical. Prior to the experiment, the protocols were confirmed to be in accor dance with the guidelines of Animal Ethics Committee of Isfahan University of Medical Sciences (Ethical code #IR.MUI. REC.1394.2.242). Statistical analysis Data are presented as mean ± standard error of the mean (SEM). The comparison between every two groups was performed using student’s independent t test. A value of P ≤ 0.05 was considered as significant. Results The serum levels of BUN and Cr increased by renal I/R, however these parameters in “salt” intake groups increased more than “water” intake groups (Figure 1). The lower ClCr was obtained in “salt” groups of male and female when compared with “water” intake groups by renal I/R (P < 0.05, Figure 1). In general, the level of BUN and Cr were higher and the level of ClCr was lower in “salt” groups when compared with “water” groups after renal I/R, and these parameters were different between genders. The kidney weight (KW) was increased by renal I/R, but the body weight change (∆W) induced by renal I/R was not significantly different between the renal I/R and control groups (Table 1). Finally, the serum nitrite level was increased by I/R in male salt group (P < 0.05) Discussion The major findings of this research demonstrated that salt intake by mothers during pregnancy may influence the Male Figure 1. The kidney function parameters in 8 groups of experiments. I/R stands for ischemia/reperfusion. Significant difference from control group in similar diet (*P < 0.05; # P < 0.2). Line and p value on above indicated difference between the groups. High-salt intake during pregnancy Salt Water Figure 1 : The kidney function parameters in 8 groups of experiments. I/R stands for ischemia/reperfusion. Significant difference from control group in similar diet (*P<0.05; # P<0.2). Line and p value on above indicated difference between the groups. 0 20 40 60 80 100 120 Control I/R B U N (m g/ dL ) * * P=0.15 0.0 0.4 0.8 1.2 1.6 2.0 Control I/R C r ( m g/ dL ) * P=0.026 0.00 0.02 0.04 0.06 0.08 0.10 Control I/R C lC r ( m L/ m in ) # # P=0.005 0 4 8 12 16 20 Control I/R U F (μ L/ m in ) # P=0.06 0 20 40 60 80 100 120 Control I/R B U N (m g/ dL ) * * 0.0 0.4 0.8 1.2 1.6 2.0 Control I/R C r ( m g/ dL ) # * P=0.05 P=0.1 0.00 0.02 0.04 0.06 0.08 0.10 Control I/R C lC r ( m L/ m in ) # P=0.014 P=0.001 0 4 8 12 16 20 Control I/R U F (μ L/ m in ) # P=0.12 Male Female High-salt intake during pregnancy Salt Water Figure 1 : The kidney function parameters in 8 groups of experiments. I/R stands for ischemia/reperfusion. Significant difference from control group in similar diet (*P<0.05; # P<0.2). Line and value on above indicated differen e between the groups. 0 20 40 60 80 100 120 Control I/R B U N (m g/ dL ) * * P=0.15 0.0 0.4 0.8 1.2 1.6 2.0 Control I/R C r ( m g/ dL ) * P=0.026 0.00 0.02 0.04 0.06 0.08 0.10 Control I/R C lC r ( m L/ m in ) # # P=0.005 0 4 8 12 16 20 Control I/R U F (μ L/ m in ) # P=0.06 0 20 40 60 80 100 120 Control I/R B U N (m g/ dL ) * * 0.0 0.4 0.8 1.2 1.6 2.0 Control I/R C r ( m g/ dL ) # * P=0.05 P=0.1 0.00 0.02 0.04 0.06 0.08 0.10 Control I/R C lC r ( m L/ m in ) # P=0.014 P=0.001 0 4 8 12 16 20 Control I/R U F (μ L/ m in ) # P=0.12 Male Female High-salt intake during pregnancy Salt Water Figure 1 : The kidney function parameters in 8 groups of experiments. I/R stands for ischemia/reperfusion. Significant difference from control group in similar diet (*P<0.05; # P<0.2). Line and p value on above indicated difference between the groups. 0 20 40 60 80 100 120 Control I/R B U N (m g/ dL ) * * P=0.15 0.0 0.4 0.8 1.2 1.6 2.0 Control I/R C r ( m g/ dL ) * P=0.026 0.00 0.02 0.04 0.06 0.08 0.10 Control I/R C lC r ( m L/ m in ) # # P=0.005 0 4 8 12 16 20 Control I/R U F (μ L/ m in ) # P=0.06 0 20 40 60 80 100 120 Control I/R B U N (m g/ dL ) * * 0.0 0.4 0.8 1.2 1.6 2.0 Control I/R C r ( m g/ dL ) # * P=0.05 P=0.1 0.00 0.02 0.04 0.06 0.08 0.10 Control I/R C lC r ( m L/ m in ) # P=0.014 P=0.001 0 4 8 12 16 20 Control I/R U F (μ L/ m in ) # P=0.12 Male Female High-salt intake during pregnancy Salt Water Figure 1 : The kidney function parameters in 8 groups of experiments. I/R stands for ischemia/reperfusion. Significant difference from control group in similar diet (*P<0.05; # P<0.2). Line and p value on above indicated difference between the groups. 0 20 40 60 80 100 120 Control I/R B U N (m g/ dL ) * * P=0.15 0.0 0.4 0.8 1.2 1.6 2.0 Control I/R C r ( m g/ dL ) * P= .026 0.00 0.02 0.04 0. 6 0.08 0.10 Control I/R C lC r ( m L/ m in ) # # P=0.005 0 4 8 12 16 20 Control I/R U F (μ L/ m in ) # P=0.06 0 20 40 60 80 100 120 Control I/R B U N (m g/ dL ) * * 0.0 0.4 0.8 1.2 1.6 2.0 Control I/R C r ( m g/ dL ) # * P=0.05 P=0.1 0.00 0.02 0.04 0.06 0.08 0.10 Control I/R C lC r ( m L/ m in ) # P=0.014 P=0.001 0 4 8 12 16 20 Control I/R U F (μ L/ m in ) # P=0.12 Male Female High-salt intake during pregnancy Salt Water Figure 1 : The kidney function parameters in 8 groups of experiments. I/R stands for ischemia/reperfusion. Significant difference from control group in similar diet (*P<0.05; # P<0.2). Line and p value on above indicated difference between the groups. 0 20 40 60 80 100 120 Control I/R B U N (m g/ dL ) * * P=0.15 0.0 0.4 0.8 1.2 1.6 2.0 Control I/R C r ( m g/ dL ) * P=0.026 0.00 0.02 0.04 0.06 0.08 0.10 Control I/R C lC r ( m L/ m in ) # # P=0.005 0 4 8 12 16 20 Control I/R U F (μ L/ m in ) # P=0.06 0 20 40 60 80 100 120 Control I/R B U N (m g/ dL ) * * 0.0 0.4 0.8 1.2 1.6 2.0 Control I/R C r ( m g/ dL ) # * P=0.05 P=0.1 0.00 0.02 0.04 0.06 0.08 0.10 Control I/R C lC r ( m L/ m in ) # P=0.014 P=0.001 0 4 8 12 16 20 Control I/R U F (μ L/ m in ) # P=0.12 Male Female High-salt intake during pregnancy Salt Water Figure 1 : The kidney function parameters in 8 groups of experiments. I/R stands for ischemia/reperfusion. Significant difference from control group in similar diet (*P<0.05; # P<0.2). Line and value on above indicated differen e between the groups. 0 20 40 60 80 100 120 Control I/R B U N (m g/ dL ) * * P=0.15 0.0 0.4 0.8 1.2 1.6 2.0 Control I/R C r ( m g/ dL ) * P=0.026 0.00 0.02 0.04 0.06 0.08 0.10 Control I/R C lC r ( m L/ m in ) # # P=0.005 0 4 8 12 16 20 Control I/R U F (μ L/ m in ) # P=0.06 0 20 40 60 80 100 120 Control I/R B U N (m g/ dL ) * * 0.0 0.4 0.8 1.2 1.6 2.0 Control I/R C r ( m g/ dL ) # * P=0.05 P=0.1 0.00 0.02 0.04 0.06 0.08 0.10 Control I/R C lC r ( m L/ m in ) # P=0.014 P=0.001 0 4 8 12 16 20 Control I/R U F (μ L/ m in ) # P=0.12 Male Female High-salt intake during pregnancy Salt Water Figure 1 : The kidney function parameters in 8 groups of experiments. I/R stands for ischemia/reperfusion. Significant difference from control


Introduction
High salt diet during pregnancy may alter some physiological functions and organs developments (1)(2)(3)(4)(5).Milk content may also change with high salt diet (6), and the offspring from mothers that received high salt diet during pregnancy demonstrated a higher blood pressure (3,7).It is well-known that renin secrets by glomerular cells which are salt sensitive and high salt diet disturb renin secretion in offspring and may develop hypertension (8).Previously we demonstrated that cardiovascular response to angiotensin II in offspring subjected to high salt intake during pregnancy was altered gender dependently (9).Renal ischemia reperfusion (I/R) as a major cause of kidney disorder influences renin-angiotensin system (RAS) (10,11) which may alter by type of diets during pregnancy (7,12).

Objectives
This study was designed to find the role of high salt intake during pregnancy on the outcome of renal IR in adult offspring.

Animals
Female Wistar rats were subjected to tap water or high-salt water (2%) during mating until delivery.At the 30th days of life (day of weaning) the offspring were assigned into eight groups, and they were housed to gain weight in 3-4 months.Groups 1 to 4 called "water" groups included two groups of male (groups 1 and 2) and two groups of female (groups 3 and 4) offspring from mothers who received tap water during mating and pregnancy.Groups 5 to 8 called "salt" groups included two groups of male (groups 5 and 6) and two groups of female (groups 7 and 8) offspring from mothers who received 2% salt in drinking water during mating and pregnancy.

Experimental protocol
At the day of experiment the adult offspring in groups 1 (male: 227 ± 9 g, n = 8), 3 (female: 169 ± 4 g, n = 6), 5 (male: 260 ± 9 g, n = 7) and 7 (female: 192 ± 8 g, n = 7) were anesthetized with chlorohydrate (450 mg/kg i.p).The kidneys were prepared for vessels clamping, and the vessels were clamped for a period of 45 minutes, and afterwards the rats were subjected to 24 hours reperfusion.These groups were assigned as I/R groups.The same procedures were applied to the animals from group 2 (male: 234 ± 10 g, n = 8), 4 (female: 167 ± 6 g, n = 7), 6 (male: 260 ± 8 g, n = 7) and 8 (female: 180 ± 7 g, n = 5), but the renal vessels were not clamped.These groups were considered as control (sham operated) groups.All the animals were subjected to urine collection during last 6 hours of reperfusion by keeping animals in standard metabolic cages to determine creatinine clearances (ClCr).At the end of reperfusion, blood samples were obtained, and the animals were sacrificed humanly.The kidneys were removed and weighed rapidly.The serum levels of creatinine (Cr), blood urea nitrogen (BUN), and nitrite (by Griess method) were measured.The ClCr was determined by the following ClCr formula.ClCr = UF x U Cr /P Cr where UF (mL/min), U Cr and P Cr stand for urine flow, urine Cr level and serum Cr concentration.

Ethical issues
The research followed the tenets of the Declaration of Helsinki.This project was approved by Ethics Committee of Isfahan University of Medical.Prior to the experiment, the protocols were confirmed to be in accor dance with the guidelines of Animal Ethics Committee of Isfahan University of Medical Sciences (Ethical code #IR.MUI.REC.1394.2.242).

Statistical analysis
Data are presented as mean ± standard error of the mean (SEM).The comparison between every two groups was performed using student's independent t test.A value of P ≤ 0.05 was considered as significant.

Results
The serum levels of BUN and Cr increased by renal I/R, however these parameters in "salt" intake groups increased more than "water" intake groups (Figure 1).The lower ClCr was obtained in "salt" groups of male and female when compared with "water" intake groups by renal I/R (P < 0.05, Figure 1).In general, the level of BUN and Cr were higher and the level of ClCr was lower in "salt" groups when compared with "water" groups after renal I/R, and these parameters were different between genders.The kidney weight (KW) was increased by renal I/R, but the body weight change (∆W) induced by renal I/R was not significantly different between the renal I/R and control groups (Table 1).Finally, the serum nitrite level was increased by I/R in male salt group (P < 0.05)

Discussion
The major findings of this research demonstrated that salt intake by mothers during pregnancy may influence the Male Figure 1.The kidney function parameters in 8 groups of experiments.I/R stands for ischemia/reperfusion. Significant difference from control group in similar diet (*P < 0.05; # P < 0.2).Line and p value on above indicated difference between the groups.

High-salt intake during pregnancy
Salt Water

Figure 1 :Figure 1 :Figure 1 :Figure 1 :Figure 1 :Figure 1 :Figure 1 :Figure 1 :Figure 1 :
Figure 1 : The kidney function parameters in 8 groups of experiments.I/R stands for ischemia/reperfusion. Significant difference from control group in similar diet (*P<0.05;# P<0.2).Line and p value on above indicated difference between the groups.