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Effect Of Elevated Extracellular Chloride Ions Biology Essay

This study is principally concerned with the effect of elevated extracellular chloride ions and hydrogen ions, which are expected to occur in patients using saline-based fluids as their intravenous therapy, on constrictor tone of the isolated porcine renal arteries. This introduction starts with a brief outline of the intravenous therapy and those commonly employed intravenous fluids. A number of evidences for the occurrence of hyperchloraemic acidosis in Saline-utilizing patients have been included. Then, those adverse effects associated with hyperchloraemic acidosis have also been described. Finally, a brief background of the study have been outlined, and followed by several specific aims in order to investigate whether there are benefits to use Normal Saline in the intravenous therapy.

Intravenous therapy (IV therapy), involves the direct injection of fluids into a vein. It is also referred to as an intravenous drip because this administration retains a drip chamber, which prevents gas bubbles entering in a vascular system and allows flow rate estimation. It is generally used for different purposes, such as replacing fluids and electrolytes or nutrition, transfusing blood and blood products, and delivering medications etc. It is considered a faster-acting therapy compared with other forms of administration of medication because the IV therapy can supply the entire body with fluids or medicines more efficiently.

1.1.1 Two major categories of intravenous fluids

Normally, there are two major intravenous fluids, which are colloids and crystalloids.

Colloids consist of larger insoluble molecules, such as gelatin and blood. As they are too large to pass through the capillary membrane, they remain in the intravascular space and thereby increase the osmotic pressure in such intravascular space. As a result, more fluids are moved from the interstitial and intracellular space to the intravascular space. Therefore, colloids can also be referred to as volume expanders. However, the short half-lives of these costly fluids make them not in a widespread used.

Crystalloids are made up of aqueous solutions of mineral salts or other water-soluble molecules. As there are no any high molecular weight solutes, the crystalloids can quickly diffuse across the capillary wall into the tissue, rather than stay in the intravascular space for a long period of time. Generally, there are few crystalloids, which are commonly used as intravenous fluids. They include Normal Saline (NS), Ringer’s lactate (RL) (also called Hartmann’s solution) and 5% Dextrose in Water (D5W).

1.2 Normal Saline (NS) solution

The Normal Saline is 0.9% sodium chloride solution. Usually, it is given in intraoperative, resuscitation, and maintenance therapy. The composition of these saline-based fluids suggests that they are non-physiological. [5] There are three reasons. Firstly, the normal blood level concentration of chloride ions is about 95-105mMl-1 while the level of chloride ions in Normal Saline is 154mMl-1. [3] Secondly, the saline-based fluids do not contain potassium, calcium, glucose, and magnesium, but all of these electrolytes are present in normal plasma. Thirdly, such fluids cannot maintain the plasma pH within normal limits because of their lack of bicarbonate of bicarbonate precursor buffer. [5]

The idea of using saline for treatment was first discovered by Dr William Brooke O’Shaughnessy in 1831. He successfully treated the severely dehydrated cholera patients by injecting the highly-oxygenate salts. Afterwards, this method was further developed and the composition of Normal Saline has been clarified. [1]

Though Normal Saline is believed to be a safe intravenous fluid for at least 50 years, the incidence of the associated hyperchloraemic acidosis has been increasingly concerned. [2]

1.3 What is hyperchloraemic acidosis?

-anion gap

1.3.1 Evidences of the hyperchloraemia and associated acidosis in saline-based patients

There are numbers of findings supporting the correlation between the Normal saline solution and hyperchloraemic acidosis.

Nicholas J. Wilkes et al. (2001) studied randomized elderly surgical patients given a more physiologically balanced electrolyte formulation, such as Hartmann’s solution and Hextend, or saline-based fluids. By measuring the biochemical indices and acid-base balance, they found that the saline-based group showed a larger increase in the postoperative chloride levels than the balanced fluid group (9.8 vs 3.3 mmol/L, P=0.0001). Also, two-thirds of the saline group developed postoperative hyperchloraemic acidosis (p=0.0001), but none in the balanced fluid group. [4]

Scheingraber et al. (1999) studied two groups of gynecologic surgical patients who randomly received saline or lactated Ringer’s solution. The pH, arterial carbon dioxide, potassium, chloride, lactate, and total protein were assessed in the intervals of 30-minute They discovered that those patients, who were infused with 30mlkg-1h-1 saline solution during anesthesia and surgery, had the metabolic acidosis with hyperchloraemia and a coexistent decrease in the strong ion difference while these were not found in those patients with the lactated Ringer’s solution. [6]

Waters JH et al. (1999) examined twelve patients undergoing prolonged surgeries which lasted for at least 4 hours. The plasma volume, arterial blood gas parameter, serum electrolytes and urine electrolytes of those patients were measured pre- and postoperatively. The researchers found that there was a significant correlation between the volume of saline infused and the change in base excess (r2=0.86, p<0.0001), whereas there was no correlation with the lactated Ringer’s solution. Also, they even noted a stronger association between the total amount of chloride injected and the change in base excess (r2=0.93, p<0.0001). [7]

Afterward, Waters JH et al. (2001) enrolled sixty-six patients who were undergoing open aortic aneurysm repair. Lactated Ringer’s solution or Normal saline were randomly given to those patients in a double-blinded condition. At the end of the assessment, they concluded that hyperchloraemic acidosis was developed in the Normal saline patients, and more bicarbonate therapy (30±62ml in Normal saline group vs 4±16ml in Lactated Ringer’s group) was also given to them. Moreover, those patients with Normal saline received larger amount of platelet transfusion (478±302ml in Normal saline group vs 223±24ml in Lactated Ringer’s group) and significant more blood product (p=0.02). [8]

Mustafa I. et al. (2002) studied numbers of surgical patients who underwent elective coronary artery bypass grafting (CABG). They were either given hypertonic lactate solution or hypertonic saline solution. At the end of such study, the researchers found that saline solution induced a significant decrease in arterial pH and bicarbonate while lactate solution increased those two parameters. [9]

1.4 Clinical implications

Nowadays, the exact physiological risks of hyperchloraemic acidosis are not yet confirmed. [5] However, current trend shows that the hyperchloraemic acidosis may be clinically relevant. Based on various studies, several clinical problems have been shown relevance to the hyperchloraemic acidosis. These clinical issues include impaired renal function, influenced renal vasculature, poor gastric perfusion, impaired coagulation and increased release of inflammatory markers etc. [2] As a result, all of these post-operative complications increase patients’ hospital stay time.

1.4.1 Impaired renal function

Bennett-Guerrero et al. (2001) examined 200 cardiac surgical patients who received one of the following fluids: 5% albumin in a normal saline (NS)-like vehicle, 6% hetastarch in a NS-based vehicle, 6% hetastarch in a balanced-salt vehicle and lactated Ringer’s solution, in a randomized blinded fashion. The levels of serum creatinine, urine output and creatinine clearance of those patients were measured. The researchers suggested that there was a significant deterioration of these indices in the patients with saline-based solution, compared with the balanced salt solution group. [10]

Natiuresis might become much less in those with saline solution due to the high level of chloride. [4] Also, less diuresis was found in those with saline solution, compared with the Hartmann’s and 5% dextrose solutions. [11] [12]

1.4.2 Influenced renal vasculature

Wilcox CS (1983) has firstly shown that hyperchloraemia can lead to renal vasoconstriction. It is because he found that excess plasma chloride can reduce lower renal blood flow and glomerular filtration rate, thus in turn decrease the urine volume. [13]

Thereafter, Kotchen TA et al. (1987) studied the effect of alterations of sodium chloride cotransported in the thick ascending limb of Henle (TALH) of kidney on renin release. They found that increased chloride delivery to the loop might inhibit renin release, which could lead to renal vasoconstriction. It is because renin is part of the body’s renin-angiotensin system (RAS) and mediates the arterial vasoconstriction. [14]

Furthermore, Reid et al. (2003) studied nine young men who were given either 2L 0.9% saline infusion or Hartmann’s solution in a double-blind trial. The result showed that less urine (450ml vs 1000ml) with lower sodium content (73mmol vs 122mmol) was voided in those with saline solution, compared with the Hartmann’s solution group, despite the former had higher sodium content. [15]

1.4.3 Poor gastric perfusion

The study conducted by Wilkes NJ et al. (2001) also examined the gastric perfusion of two surgical patients with either saline solution or balanced fluid. Gastric tonometry was used to predict the gastric perfusion outcome after surgery. [16] They discovered that better gastric mucosal perfusion was seen in the balanced fluid group, rather than the saline group. It is because the gastric tonometry showed a significantly greater increase in Pg-aCO2 from start to end of the operation in saline group (1.7±0.5 kPa, end of operation; range, 0 to 6.3 kPa), compared with the balanced fluid group (0.9±1.1 kPa, end of operation; range, -0.6 to 2.2 kPa; p=0.0394). [4]

Also, the poor gastric perfusion may be a potential risk factor for post-operative nausea and vomiting. [17] This finding was further supported the study of Wilkes NJ et al. (2001), who found that more events of post-operative nausea and vomiting and a more frequent use of rescue antiemetic were notified in the saline group. [4]

1.4.4 Impaired coagulation

The optimal pH and electrolyte levels are essential for coagulation system to function effectively. [5] Several studies have presented differences in bleeding and coagulation in those with saline solution and balanced electrolyte crystalloids or colloids. All of these have shown increased incidence of bleeding and more derangement of coagulation function in the saline solution group. [18] [19]

In the study of Scheingraber et al. (1999), a trend of more blood loss in the saline group than the lactated Ringer’s solution was shown. [6] Also, Gan TJ et al. (1999) investigated 120 patients, who received either ‘Hextend’ (6% hetastarch in a suspension like Hartmann’s) or the identical 6% hetastarch in saline, in randomized placebo-controlled study. The patients with ‘Hextend’ had a less blood lost (956ml less, P=0.02), compared with the saline-based group. [18]

1.4.5 Increased release of inflammatory markers

Those with saline-based solution might result in an increased release of inflammatory markers. By supporting this, Kellum JA et al. (2006) conducted a study using twenty adult male rats, which were randomly infused with either 0.1N HCl to reduce the standard base excess (SBE) (comparable to the environment of hyperchloraemic acidosis occurred with saline solution) or similar volume of lactated Ringer’s solution. As a result, they found that those rats with severe acidosis presented greater increase in the cytokines, including tumor necrosis factor (TNF), interleukin (IL)-6 and IL-10. [20]

1.5 Renal artery

Basically, most of the vessels are lined with the thin layer of smooth and flat endothelial cells. The arteries’ endothelial lining is surrounded by thick wall which is made up of smooth muscle and connective tissue. [21] Smooth muscle is an involuntary and unstriated muscle. Compared with skeletal muscle cells, the smooth muscle cells are spindle in shape, with a single nucleus, and are relatively smaller in size. [22]

It has been showed that the membrane potentials of pig renal artery was approximately -67mV, whereas vein was -47mV. [23][24][25] There are numerous studies supported that the Na-K pump might partly contribute to maintain the resting membrane potential in renal arteries, which was also observed in the pig mesenteric arteries. [26][27]

Moreover, effect of catecholamine on the responses of vascular smooth muscle would be extremely different with location and species. [25][28][29] Normally, noradrenaline, a catecholamine, would depolarize the membrane of vascular smooth cells and slightly reduce the membrane resistance. [30]

1.6 Background of the study

Due to the extensive administration of Normal saline in the IV therapy, its safety issue has been increasingly concerned. As mentioned above, hyperchloraemic acidosis may be a predictable consequence of the Normal saline administration. Any problems associated with Normal saline may be attributed to its unique composition. Therefore, based on current evidences on the hyperchloraemic acidosis and its relevant clinical implications, further studies can be conducted in order to better understand the effects of such hyperchloraemic acidosis and develop a more favorable intravenous fluid.

Consequently, the main objective of this study is that “Do hyperchloraemia and its associated acidosis lead to any vascular effect in kidney?” Thus the results of this study can become parts of the evidences to demonstrate the physiological effects of the Normal saline-induced hyperchloraemic acidosis.

This study is a vitro study. Porcine renal arteries were used because it is believed that such arteries would act in a similar physiological manner to human arteries. Moreover, additional results, which were from the same type of study but using arteries from mesenteries instead of kidney, have been used for comparison and analysis.

1.7 Aims

2. Methods

2.1 Tissue Preparation

Before starting the experiments, all tissues, which were the porcine renal arteries, should be prepared in a systematic way in order to avoid any undesired factors to influence the results. Basically, porcine kidneys, which were obtained from the freshly killed pigs, were placed in a plastic container of Krebs-Henseleit solution and transported to the laboratory on ice. Then, crude dissection of the kidneys should be finished as soon as possible on the day of arrival. This crude dissection involved the removal of a 3-5 cm long renal artery from each kidney. Each set of dissected artery was put in a separated vial, which was filled with the control Krebs-Henseleit solution (described below), and stored overnight at 4℃ for the next day experiment.

On the following day, the arteries were left at room temperature for about 30 minutes before starting the fine dissection. This fine dissection involved the removal of fat and connecting tissues surrounding the arteries and was carried out in a Petri dish filled with the control Krebs-Henseleit solution. Finally, each artery was cut into segments of 6mm long.

2.2 Krebs-Henseleit solution Preparation

In this study, three types of Krebs-Henseleit solution were needed to be prepared. They were control Krebs-Henseleit solution (with pH 7.4), modified with sodium gluconate Krebs-Henseleit solution (with pH 7.4) and modified with sodium gluconate Krebs-solution (with pH 7.2). For these three solution, after dissolving the corresponding chemcials in distilled water, they had been gassed with 95% O2 : 5% CO2 for ten minutes.

2.2.1 Control Krebs-Henseleit solution (with pH 7.4)

Composition of control Krebs-Henseleit solution (with pH 7.4):

Chemical

Grams/L in solution

mM in solution

NaCl

6.896

118

KCl

0.358

4.8

MgSO4·7H2O

0.296

1.2

CaCl2·2H2O

0.191

1.3

NaHCO3

2.1

25

KH2PO4

0.163

1.2

Glucose

2.1

11.7

2.2.2 Modified with sodium gluconate Krebs-Henseleit solution (with pH 7.4)

Composition of modified with sodium gluconate Krebs-Henseleit solution (with pH 7.4):

Chemical

Grams/L in solution

mM in solution

NaCl

5.726

98

KCl

0.358

4.8

MgSO4·7H2O

0.296

1.2

CaCl2·2H2O

0.191

1.3

NaHCO3

2.1

25

KH2PO4

0.163

1.2

Glucose

2.1

11.7

C6H11NaO7

4.36

20

For this solution, 20mM of sodium chloride (NaCl) was replaced by 20mM sodium gluconate (C6H11NaO7). In the other words, 20mM chloride ion had been reduced. As the normal blood level concentrations of chloride ions is within the range of 98 to 108 mmol/L, the control Krebs-Henseleit solution can be regarded as hyperchloraemic solution while this modified with sodium gluconate Krebs-Henseleit solution does not.

2.2.3 Modified with sodium gluconate Krebs-Henseleit solution (with pH 7.2)

This slightly acidic solution was produced by adding a Krebs-Henseleit solution without sodium bicarbonate and calcium chloride to the modified with sodium gluconate Krebs-Henseleit solution.

Composition of Krebs-Henseleit solution without sodium bicarbonate (NaHCO3) and calcium bicarbonate (CaCl2·2H2O):

Chemical

Grams/L in solution

mM in solution

NaCl

5.726

98

KCl

0.358

4.8

MgSO4·7H2O

0.296

1.2

KH2PO4

0.163

1.2

Glucose

2.1

11.7

C6H11NaO7

9.814

45

The deducted sodium ions had been replaced by additional sodium gluconate. Also, by preventing precipitation, the calcium chloride had been excluded.

By producing a curve of relationship between pH and mixture of Krebs-Henseleit solution without sodium bicarbonate and calcium chloride and the modified with sodium gluconate Krebs-Henseleit solution, it was found that the following composition gave the pH 7.2 in 1L solution:

Krebs-Henseleit solution without sodium bicarbonate and calcium chloride/mL

Krebs-Henseleit solution modified with sodium gluconate/mL

450

550

2.3 Experimental set-up

Each segment of arteries was mounted by two different stainless steel wire supports. One of them has a rectangular shape wire attached to an L-shaped glass rod. The other support has a triangular shape wire attached to a cotton thread. Therefore, lumen of each artery segment was passed through by the wires of those two supports. Then, a small hook on each wire was closed in order to fix the position of each artery segment. It had been made careful checks of the two wires were not crossed within the lumen of arteries and the two small hooks of each set of supports were in opposite direction. A diagram of this set-up was described in Figure 1.

Figure 1.

Thereupon the glass rod support was clamped on a stand. Hence, the tissue was suspended in an organ bath and the cotton thread of the support could be linked to a transducer loosely.

The transducer was connected via a Mac Lab Bridge Amplifier to the Maclab Data Acquistion system. Any detected change on tension was converted into electrical signal and recorded in grams using software called Mac Lab Chart 3.5 running on a Macintosh computer. On the beginning of each experiment day, each transducer had been calibrated using a 10g weight.

The organ bath was loaded with 15mL control Krebs-Henseleit solution, which was continuously gassed with 95% O2 : 5% CO2 and warmed at 37℃. It should be ensured that the whole tissue was immersed in the control Krebs-Henseleit solution and the glass rod support did not touch the bottom of each organ bath.

As there were four sets of organ bath set in the apparatus, they could be run concurrently for each experiment day and two paired results would be collected. It is because two segments of tissues, which were from the same branch of artery of the same kidney, were used in a paired organ bath. Thus one organ bath acted as a control while the other acted as a tested sample.

Subsequently, the four segments of tissues were left to equilibrate and stabilize for about 45 minutes. Afterward around 10g tension was applied to each segment by raising the position of each transducer manually. Then, each of them was left again for another 45 minutes to admit relaxation to a stable baseline.

2.4 Stimulation with potassium chloride (KCl)

After all segments of arteries had reached the steady level of resting tension, each tissue was exposed to 60mM KCl. Normally, their responses would reach the peak and remain constant. This usually took about 10 to 20 minutes. Then, all of the organ baths were washed with control Krebs-Henseleit solution and the tissues were allowed to relax to baseline levels. This would take approximately 20 to 30 minutes. Afterwards, the above procedure was repeated once or twice.

These stimulations with KCl were required on each experiment day and before any experimental procedures. The purpose of this was to examine the viability and reproducibility of such tissues. It is necessary that responses produced by each corresponding tissue were similar. If there was any tension which went down to below 3.0g, the tissue was required to be re-applied tension manually to about 6g before any further procedure. If the tissue remained nearly or absolutely unresponsive after the three stimulations with KCl, such tissue could be regarded as incompetent for the experiment.

2.5 Studying the effect of hyperchloraemia (‘excess chloride ions’)

For the following experiment 1 to 5, before starting any experimental procedure in each experiment or after finishing any washing out procedures, one preparation of each paired organ bath was replaced with control Krebs-Henseleit solution while the other preparation was replaced with modified sodium gluconate Krebs-Henseleit solution. Then, all of the tissues were left to equilibrate for about 20 minutes.

2.5.1 Experiment 1: Investigating effect of hyperchloraemia on the response of porcine renal artery (PRA) to KCl CRC (cumulative concentration response curve to 60mM)

For this experiment, cumulatively increasing concentration of KCl was added to each organ bath. The starting concentration added was 6mM and left for about 5 minutes. For each following addition, another 6mM KCl was added and left for approximately 5 to 10 minutes to allow the response to become leveled off. Then, further addition should be continued until the cumulative concentration had reached to 60mM. Those tissues were left for at least 10 minutes after the final addition. When the responses had become leveled off, all the organ baths were washed with control Krebs-Henseleit solution. Afterward, those tissues were required to be left for at least 20 minutes to allow their tensions returning to baseline level.

2.5.2 Experiment 2: Investigating effect of hyperchloraemia on the response of PRA to NaCl CRC (up to 60mM)

For this experiment, all procedures were almost the same as experiment 1 (2.5.1). The only difference was the added chemical, which should be NaCl, rather than KCl.

2.5.3 Experiment 3: Investigating effect of hyperchloraemia on the response of PRA to 60mM NaCl

60mM NaCl was added at a time into each organ bath, and then the tissues were left for about 10 to 20 minutes to allow any response. Thereupon all organ baths were washed out with control Krebs-Henseleit solution and waited for the tension to return to baseline.

2.5.4 Experiment 4: Investigating effect of hyperchloraemia on the response of PRA to Noradrenaline CRC (up to 30µM)

Cumulatively increasing concentration of Noradrenaline was added to each organ bath. The first concentration added was 10mM. If no response occurred within 10 minutes, further addition of Noradrenaline was performed. When the response had reached a peak and gone down, next concentration of Noradrenaline was added. The addition of Noradrenaline was continued until the cumulative concentration had come to 30µM. When all tensions became leveled off after the final additions, those organ bath were washed out as before.

2.5.5 Experiment 5: Investigating effect of hyperchloraemia on PRA tone to Substance P and A23187 after pre-contraction with U46619

Firstly, cumulatively increasing concentration of U46619, starting from concentration of 1nM, were added to each organ bath until the contractions had reached approximately 40% to 70% of the responses to 60mM KCl. Relative longer waiting time was required between each U46619 addition. Once the contraction had achieved the desired tension, 10nM Substance P was added. The tissues were left for 10 to 20 minutes to allow responses. After the tensions had returned to baseline, 1µM A23187 was added to each organ bath. Again they were left for responses and washed out as described before.

2.6 Experiment 6: Investigating effects of gluconate ions and chloride ions on porcine renal artery tone after pre-contraction with U46619 under non-hyperchloraemic condition

For this experiment, both preparations in each paired organ bath were filled with control Krebs-Henseleit solution. Then, U46619 was added to increase the contraction tone as described above. Once the tensions had reached desired tones, cumulatively increasing concentration of sodium gluconate was added into one preparation of each paired organ bath while the cumulatively increasing concentration of sodium chloride was added to the other preparation. The starting concentration of both sodium gluconate and sodium chloride was 6mM. About 5 to 10 minutes waiting time was allowed between each addition. The addition was stopped when the cumulative concentration had reached to 60mM. Then, all organ baths were washed out as above once the tensions had leveled off.

2.7 Studying the effect of acidosis (pH had been decreased from 7.4 to 7.2)

For the following experiment 7 to 12, before starting any experimental procedure in each experiment or after finishing any washing out procedures, one preparation of each paired organ bath was replaced with modified sodium gluconate Krebs-Henseleit solution (with pH 7.4) while the other preparation was replaced with modified sodium gluconate Krebs-Henseleit solution (with pH 7.2). Then, all of the tissues were left to equilibrate for about 20 minutes.

2.7.1 Experiment 7: Investigating effect of acidosis on the response of PRA to KCl CRC (up to 60mM)

All procedures in this experiment were the same as those described in experiment 1 (2.5.1).

2.7.2 Experiment 8: Investigating effect of acidosis on the response of PRA to Noradrenaline CRC (up to 30µM)

Again, all procedures in this experiment were referred to those described in experiment 4 (2.5.4).

2.7.3 Experiment 9: Investigating effect of acidosis on PRA tone to Substance P after pre-contraction with U46619

The procedures were almost the same as in experiment 5 (2.5.5). (The A23187 part in experiment 5 was not included in this experiment.)

2.7.4 Experiment 10: Investigating effect of acidosis on the response of PRA to 60mM NaCl

Again, all procedures were the same as experiment 3 (2.5.3).

2.7.5 Experiment 11: Investigating effect of acidosis on the response of PRA to L-NAME and subsequent 60mM NaCl

100µM L-NAME was added into each organ bath, and left for approximately 10 to 20 minutes. Then, 60mM NaCl was added in each preparation without washing out the L-NAME. All the organ baths were washed out as above after the responses had leveled off.

2.7.6 Experiment 12: Investigating effect of acidosis on the response of PRA to 120mM NaCl and 120mM sodium gluconate

Firstly, 120mM NaCl was added at a time into each preparation, and left for about 10 to 15 minutes. Then, all organ baths were washed out as before. Afterward, 120mM sodium gluconate was added into each preparation, and left for about 10 to 20 minutes. Eventually, all tissues were washed out again as above.

2.8 Data analysis

2.9 Statistical analysis

2.10 Materials

3. Results

3.1 Response to 60mM KCl (before any experimental procedures)

Almost all the tissues used in the experiments responded to 60mM KCl in a reproducible manner. The mean response was 16.5±0.8g (n=66). These tissues were divided into control group and experimental group. Table 4 shows that there is no significant difference in the response to 60mM KCl in both control and experimental groups before performing any experimental procedures (p>0.05).

Experimental Week

Mean response to 60mM KCl/g

P value

Control group

Experimental group

Week 1

12.7

15.3

0.1

Week 2

12.3

15.7

0.1

Week 3

18.6

20.9

0.2

Week 4

14.9

17.3

0.2

Week 5

18.4

20.6

0.2

Table 4. The mean response to 60mM KCl and corresponding p values in both control and experimental groups in the five weeks experiments.

3.2 Response to cumulatively increasing concentrations of KCl

3.2.1 Effect of Hyperchloraemia

In the study of hyperchloraemia, the responses to cumulative increasing concentrations of KCl, within the concentration range of 6mM to 60mM, were examined in control Krebs-Henseleit and modified sodium gluconate solutions, and described in Figure 2.

Figure 2. The responses to cumulatively increasing of KCl expressed as percentage of 60mM KCl response, in the presence of control Krebs-Henseleit solution and modified sodium gluconate Krebs-Henseleit solution. Values are shown as the mean±SEM of n=8 observations in each group.

The mean maximum response in control Krebs-Henseleit solution (Cl- ≈124mM; pH 7.4) was 110.5±4.9% (n=8) of response to 60mM KCl while the mean maximum response in modified sodium gluconate Krebs-Henseleit solution (Cl- ≈104mM; pH 7.4) was 96.0±4.1% (n=8) of response to 60mM KCl.

The mean pEC50 value (concentration of KCl required to induce 50% of the maximum response produced by KCl) of control group (Cl- ≈124mM; pH 7.4) was 1.6±0.03 (n=8), whereas the mean pEC50 value of experimental group (Cl- ≈104mM; pH 7.4) was 1.5±0.08 (n=8). Hence, there is a significant difference in pEC50 values between the two groups (p=0.03).

3.2.2 Effect of acidosis

In the study of acidosis, the responses to cumulative increasing concentrations of KCl, within the concentration range of 6mM to 60mM, were examined in modified sodium gluconate Krebs-Henseleit (with pH 7.4) and modified sodium gluconate solutions (with pH 7.2), and described in Figure 3.

Figure 3. The responses to cumulatively increasing of KCl expressed as percentage of 60mM KCl response, in the presence of modified sodium gluconate Krebs-Henseleit solution with pH 7.4 and modified sodium gluconate Krebs-Henseleit solution with pH 7.2. Values are shown as the mean±SEM of n=7 observations in each group.

The mean maximum response in control group (Cl- ≈104mM; pH 7.4) was 103.2±2.4% (n=7) of response to 60mM KCl while the mean maximum response in experimental group (Cl- ≈104mM; pH 7.2) was 94.9±3.0% (n=7) of response to 60mM KCl.

The mean pEC50 value of control group (Cl- ≈104mM; pH 7.4) was 1.5±0.06 (n=8), whereas the mean pEC50 value of experimental group (Cl- ≈104mM; pH 7.2) was 1.5±0.04 (n=7). Accordingly, there is no significant difference in pEC50 values between the two groups (p=0.86).

3.3 Response to cumulatively increasing concentrations of Noradrenaline

3.3.1 Effect of hyperchloraemia

In the study of hyperchloraemia, the responses to cumulative increasing concentrations of Noradrenaline, within the concentration range of 100nM to 30µM, were investigated in control Krebs-Henseleit and modified sodium gluconate solutions, and described in Figure 4.

Figure 4. The responses to cumulatively increasing of Noradrenaline expressed as percentage of 60mM KCl response, in the presence of control Krebs-Henseleit solution and modified sodium gluconate Krebs-Henseleit solution. Values are shown as the mean±SEM of n=6 observations in each group.

The mean maximum response in control Krebs-Henseleit solution (Cl- ≈124mM; pH 7.4) was 154.9±6.7% (n=6) of response to 60mM KCl while the mean maximum response in modified sodium gluconate Krebs-Henseleit solution (Cl- ≈104mM; pH 7.4) was 158.6±5.6% (n=6) of response to 60mM KCl.

The mean pEC50 value of control group (Cl- ≈124mM; pH 7.4) was 5.6±0.14 (n=6), whereas the mean pEC50 value of experimental group (Cl- ≈104mM; pH 7.4) was 5.6±0.06 (n=6). Thus, there is no significant difference in pEC50 values between the two groups (p=0.8).

3.3.2 Effect of acidosis

In the study of acidosis, the responses to cumulative increasing concentrations of KCl, within the concentration range of 10nM to 30µM, were investigated in modified sodium gluconate Krebs-Henseleit (with pH 7.4) and modified sodium gluconate solutions (with pH 7.2), and described in Figure 5.

Figure 5. The responses to cumulatively increasing of Noradrenaline expressed as percentage of 60mM KCl response, in the presence of modified sodium gluconate Krebs-Henseleit solution with pH 7.4 and modified sodium gluconate Krebs-Henseleit solution with pH 7.2. Values are shown as the mean±SEM of n=7 observations in each group.

The mean maximum response in control group (Cl- ≈104mM; pH 7.4) was 177.0±12.7% (n=7) of response to 60mM KCl while the mean maximum response in experimental group (Cl- ≈104mM; pH 7.2) was 183.0±21.2% (n=7) of response to 60mM KCl.

The mean pEC50 value of control group (Cl- ≈104mM; pH 7.4) was 5.8±0.10 (n=7), whereas the mean pEC50 value of experimental group (Cl- ≈104mM; pH 7.2) was 5.5±0.13 (n=7). So there is significant difference in pEC50 values between the two groups (p=0.008).

3.4 Response to 60mM NaCl

3.4.1 Effect of hyperchloraemia

In the study of hyperchloraemia, the mean response to 60mM NaCl in control group (Cl- ≈124mM; pH 7.4) was 12.1±2.2% (n=6) of 60mM KCl while in experimental group (Cl- ≈104mM; pH 7.4) was 0.8±0.7% (n=6) of 60mM KCl. Therefore, a significant difference in increase of tension of the control group was observed compared with the experimental group (p=0.002).

3.4.2 Effect of acidosis

In the study of acidosis, the mean response to 60mM NaCl in control group (Cl- ≈104mM; pH 7.4) was 14.6±4.2% (n=4) of 60mM KCl, whereas such mean response of experimental group (Cl- ≈104mM; pH 7.2) was 4.0±1.1% (n=4) of 60mM KCl. However, the difference in such response was insignificant (p=0.09).

3.5 Response to Substance P and A23187 after pre-contraction with U46619

3.5.1 Effect of hyperchloraemia

The U46619 caused a concentration-dependent contraction in the porcine renal artery. The mean response to U46619 (approximately 5nM) in control group (Cl- ≈124mM; pH 7.4) was 48.1±4.2% (n=6) of 60mM KCl. On the other hand, the mean response to U46619 (approximately 5nM) in experimental group (Cl- ≈104mM; pH 7.4) was 52.6±3.0% (n=6) of 60mM KCl. It was observed that a stable and reproducible contraction was produced in each preparation of tissues.

Following the stable contraction, addition of 10nM Substance P elicited a sharp and transient relaxation to the U46619-incduced tone. The mean reduction of U46619-induced tone in control group (Cl- ≈124mM; pH 7.4) was 13.0±5.6% (n=6) of U46619 tone, whereas in experimental group (Cl- ≈104mM; pH 7.4) the mean reduction was 27.7±5.5% (n=6) of U46619 tone. Hence, a significant higher degree of relaxation in experimental group was shown compared to the control group (p=0.02).

Furthermore, subsequent addition of 1uM A23187 after the U46619-induced tone had returned to stable tension caused a relative small relaxation compared to U46619 in both groups. The mean relaxation in control group (Cl- ≈124mM; pH 7.4) was 10.5±4.5% (n=6) of the U46619 tone, while such mean relaxation in experimental group (Cl- ≈104mM; pH 7.4) was 13.8±4.9% (n=6) of the U46619 tone. Obviously, there is no significant difference in response to A23187 between the two groups (p=0.6).

3.5.2 Effect of acidosis

Same as above, U46619 also caused the concentration-dependent contraction in both groups of tissues. The mean response to U46619 (approximately 5nM) in control group (Cl- ≈104mM; pH 7.4) was 51.9±3.7% (n=6) while the mean response of experiment group (Cl- ≈104mM; pH 7.2) was 55.9±5.0% (n=6).

Subsequent addition of 10nM Substance P also produced the sharp and transient relaxation. The mean decrease of U46619-induced tone in control group (Cl- ≈104mM; pH 7.4) was 57.8.1±4.4% (n=6) of the U46619 tone, and such mean decrease of experimental group (Cl- ≈104mM; pH 7.2) was 56.6±6.6% (n=6) of the U46619 tone. But, there is no statistically significant diffidence in response to Substance P between the two groups (p=0.9).

3.6 Response to cumulatively increasing concentration of NaCl under effect of hyperchloraemia

The responses to cumulative increasing concentrations of NaCl, within the concentration range of 6mM to 60mM, were investigated in control Krebs-Henseleit and modified sodium gluconate solutions, and described in Figure 6.

Figure 6. The responses to cumulatively increasing of NaCl expressed as percentage of 60mM KCl response, in the presence of control Krebs-Henseleit solution and modified sodium gluconate Krebs-Henseleit solution. Values are shown as the mean±SEM of n=8 observations in each group. *: Significant difference

The mean maximum response in control group (Cl- ≈124mM; pH 7.4) was 30.0±5.3% (n=8) of response to 60mM KCl while the mean maximum response in experimental group (Cl- ≈104mM; pH 7.4) was 15.9±4.3% (n=8) of response to 60mM KCl.

Moreover, the NaCl, with concentration range from 18mM to 60mM, has shown significant difference in increasing of tension of the control group (Cl- ≈124mM; pH 7.4) compared with the experimental group (Cl- ≈104mM; pH 7.4) (p<0.05).

3.7 Response to gluconate ions and chloride ions after pre-contraction with U46619 under non-hyperchloraemic condition

Firstly, tissues in both groups (gluconate vs chloride groups) responded to the pre-contraction with U46619 in a similar way, i.e. the mean response of gluconate group was 62.8±8.7% (n=5) of 60mM KCl while the mean response of chloride group was 59.6±6.7% (n=6) of 60mM KCl. Those tissues produced stable and reproducible tensions before any addition of gluconate or chloride ions.

Afterward, the responses to the cumulative increasing concentration of sodium gluconate and sodium chloride, range from 6mM to 60mM, were studied in the modified sodium gluconate Krebs-Henseleit solution (Cl- ≈104mM; pH 7.4), and described in Figure 7.

Figure 7. The responses to cumulatively increasing of sodium gluconate and sodium chloride expressed as percentage of U46619-induced tone, under the non-hyperchloraemic condition (modified sodium gluconate Krebs-Henseleit solution). Values are shown as the mean±SEM of n=5 observations in gluconate group and n=6 observations in chloride group. *: Significant difference

The mean maximum response to sodium gluconate was 12.1±8.1% (n=5) of the U46619-induced tone, whereas such response to sodium chloride was 31.0±19.2% (n=6) of the U46619-induced tone. Apparently, the increase in concentration of chloride ions raised the tension more compared to that of gluconate ions.

3.8 Response to 120mM NaCl and 120mM Sodium gluconate under effect of acidosis

The mean response to 120mM NaCl in control group (Cl- ≈104mM; pH 7.4) was 37.5±6.5% (n=6) of 60mM KCl, whereas such mean response of experimental group (Cl- ≈104mM; pH 7.2) was 35.1±14.6% (n=4) of 60mM KCl. Consequently, the difference in such response was insignificant (p=0.38).

Furthermore, the mean response to 120mM sodium gluconate in control group (Cl- ≈104mM; pH 7.4) was 18.4±5.5% (n=6) of 60mM KCl, whereas such mean response of experimental group (Cl- ≈104mM; pH 7.2) was 21.8±9.0% (n=4) of 60mM KCl. Likewise, the difference in such response was insignificant too (p=0.45).

3.9 Response to 100µM L-NAME and subsequent addition of 60mM NaCl under effect of acidosis

The mean response to 100µM L-NAME in control group (Cl- ≈104mM; pH 7.4) was 43.2±5.8% (n=6) of 60mM KCl, and the mean response of experimental group (Cl- ≈104mM; pH 7.2) was 27.9±4.6% (n=6) of 60mM KCl. Thus, there is no significant difference in response to the 100µM L-NAME in both groups (p=0.08).

At a later time, 60mM NaCl was added. The mean response to such 60mM NaCl in control group (Cl- ≈104mM; pH 7.4) was 25.6±4.0% (n=6) of 60mM KCl, and the mean response of experimental group (Cl- ≈104mM; pH 7.2) was 17.3±6.4% (n=6) of 60mM KCl. Similarly, there is no significant difference in response to the subsequent addition of 60mM NaCl between the two groups (p=0.08).

4. Discussion

The present study demonstrates that the hyperchloraemia and acidosis have different vascular effects. Obviously, the excess chloride ions in Krebs-Henseleit solution seems to increase both the K+-induced contraction and NaCl-induced contraction and decrease the Substance P-induced relaxation significantly. On the other hand, the acidosis appears to reduce the Noradrenaline-mediated contraction importantly. More results will be explained in more detail in the following discussion.

4.2 Effect of chloride ion on the vascular smooth cells

4.2.1

A previous study has demonstrated the contraction of renal afferent arterioles induced by depolarization was modulated by extracellular chloride directly. [31] At a later time, a study has also shown the K+-induced contraction of smooth muscle cells in the afferent arterioles of kidney was highly sensitive to chloride. [32] These suggested that chloride might affect the K+-induced contraction of renal artery too.

In this study, it was found that K+-induced contraction of renal artery was significantly greater in the Krebs-Henseleit solution of excess chloride ion (p=0.032), compared with Krebs-Henseleit solution of a normal chloride level as in human plasma. This is consistent with the previous finding in which comparable substitutions of chloride could lead to the inhibition of activation of voltage-gated Ca2+ channel and sequent contraction in response to agonists or K+ depolarization. [33][34] Therefore, it can be concluded that hyperchloraemia does have vasoconstrictor action on the porcine renal artery.

4.2.2

A number of studies have investigated relation of chloride ion to response of noradrenaline-mediated contraction of renal artery. [31][32] Some approaches have supported the concept that norepinephrine-induced vascular smooth muscle contraction is dependent on Cl- current activation. [35] These may suggest that the noradrenaline-mediated contraction could be affected by chloride ion.

In this study, it was found that the noradrenaline-mediated contraction of porcine renal artery does not significantly affected by the level of chloride ion in Krebs-Henseleit solution (p=0.8). Apparently it seems not to be consistent with the finding of a study that the norepinephrine-induced contraction is potentiated in the buffer with low chloride concentration. [35] However, such effect of lowing extracellular chloride on norepinephrine-induced contraction is concentration dependent. The peak responses of contraction are only significantly increased when the extracellular chloride level is lowered from 138mM to 41 or 8mM. But, there is no significantly change in response of contraction when the extracellular chloride level is lowered from 138 mM to 106 or 73mM. [35] Therefore, as the difference of chloride level in Krebs-Henseleit solutions in the present study is only 20mM, the result is still concordant with previous findings.

4.2.3

A study investigated the effect of sodium chloride (salt) on vessels and reported some similar results as the present study. Such study found that increased NaCl intake would lead to several indirect effects, such as Na+,K+-ATPase inhibition in vascular smooth muscle. [36] This inhibition could increase blood pressure by producing electrogenic depolarization of vascular smooth muscle cells, and increase calcium influx via the voltage sensitive calcium channels. [37] [38] Also, it could decrease Na+-Ca2+ exchange across plasma membrane of the vascular smooth cells, and then decrease calcium efflux. [39] All of these can increase the contractile activity of vascular smooth muscle.

In the present study, responses to both 60mM NaCl and NaCl CRC (up to 60mM) showed a relatively small increase in tension of renal artery, compared to the contraction seen with KCl. These findings not only support the previous findings that NaCl does have indirect effect on vascular smooth muscle, but also suggest a direct effect on the renal artery. Moreover, there is significantly an increased in contraction of the renal artery in the Krebs-Henseleit solution with excess chloride level. (60mM NaCl: p=0.002; NaCl CRC: p<0.05) These results can further conclude that the vascular effect of excess chloride ion on renal artery.

4.2.4. Response to U46619 and Substance P

In order to assess the response to Substance P, all tissues were firstly pre-contracted by U46619. The U46619 did induce concentration-dependent sustained contraction on all tissues in this experiment. Basically, when the tension of each tissue was increased to approximately 40% to 60% of KCl response by U46619, 10nM Substance P was added. In all tissues, it was found that Substance P did induce the transient relaxation, and then the tension of vessels returned to the pre-contraction level.

U46619

The U46619 is a thromboxane A2 (TxA2) analogue, which is considered as a thromboxane A-receptor (TP) agonist and known as a strong vasoconstrictor. [40] Usually, the U46619 is used, instead of TxA2, to induce a contraction, because TxA2 is unstable. [41]

In fact, U46619-induced contraction is mediated by TP receptor as such contraction is completely blocked by TP receptor antagonist SQ-29548. [42] By binding to the TP receptor, the heterotrimeric G protein (G12/13) is coupled to TP receptor and the guanine nucleotide exchange factor (Rho-GEF) is activated via direct interaction of Gα subunit with GEF. [42] Thereafter, Rho A is activated and Rho-associated kinase is changed from inactive (ROKi) to active form (ROKa), which involves in the Ca2+ sensitization and the entry of extracellular Ca2+. This Ca2+ influx occurs predominately via voltage-gated Ca2+ channels. The Ca2+ activates the calmodulin-dependent MLCK while ROKa inhibits the MLCP. Finally, the level of myosin regulatory light chain phosphorylation is increased and the cross bridge cycling and contraction is activated. [43]

Figure 10 Proposed mechanism of U-46619-induced contraction of rat caudal arterial

smooth muscle

TxA2, thromboxane A2; TPR, thromboxane A2 receptor; SQ-29548, TP receptor antagonist;

G12/13, heterotrimeric G proteins; Rho-GDI, guanine nucleotide dissociation inhibitor; RhoAGDP,

inactive form of RhoA; RhoA-GTP, active form of RhoA; ROKi, inactive form of Rhoassociated

kinase; ROKa, activated form of Rho-associated kinase; Y-27632 and H-1152, ROK

inhibitors; CaM, calmodulin; MLCK, myosin light chain kinase; ML-7, MLCK inhibitor; MLCP,

myosin light chain phosphatase; Pi, inorganic phosphate.

Substance P

Substance P is a neuropeptide. [44] One of its functions is as a vasodilator. [156] The vasodilation induced is via the activation of neurokinin-1 (NK1) receptors located on endothelial cells in arterial vessel, and mediated by nitric oxide (NO) release from the endothelium. [157] The NK1 receptor is a G protein-coupled receptor. [52] It works via a phospholipase C-IP3 second messenger system, and subsequently can directly stimulate the NO production. [53] NO released stimulates the soluble guanylate cyclase which subsequently forms cyclic GMP (cGMP). The cGMP activates protein kinase G, which lead to the myosin light chain phosphatase phosphorylation and so myosin light chain kinase inactivation. Finally, due to the ultimate dephosphorylation of myosin light chain, the smooth muscle relaxes. [47]

However, the relaxation induced by Substance P is transient. There are evidences suggesting that this is due to the internalization [48] [49] [50] and desensitization of NK1 receptor in epithelial cells after binding to Substance P. [51] The desensitization is due to NK-1 receptor phosphorylation, and the internalization or endocytosis and recycling is required for the NK-1 receptor resensitization. [54]

In this experiment, it was demonstrated that chloride ion seems to affect the response of Substance P, whereas the acidosis does not show a significant effect on the Substance P response. The vessels in the Kreb-Henseleit solution with excess chloride level show significantly less relaxation in response to Substance P, compared with Krebs-Henseleit solution with normal chloride level. (P=0.018)

However, few studies suggested that the optimal pH for iNOS was about 7.0. So if the intracellular pH was

Limitations

Future work

SNP

Endothelial

NO

Krebs

Other arteries

Mearsure the ph finish

Conclusion

How NA Work

U46619 work

NA-Cl difference?

H+ acidosis NA work

Acidosis – cl

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