Portal Venous Flow Is Increased by Jejunal but Not Colonic Hydrogen Sulfide in a Nitric Oxide-Dependent Fashion in Rats

Abstract

Hydrogen sulfide (H₂S) is a recently discerned endogenous signaling molecule that modulates the vascular system. Endogenous hydrogen sulfide has been shown to dilate both the mesenteric and portal vasculature. Gut microbiome, via sulfur reducing bacteria, is another source of H₂S production within the gut lumen; this source of H₂S is primarily produced and detoxified in the colon under physiologic conditions. Nitric oxide (NO), a major endogenous vasodilator in the portal circulation, participates in H₂S-induced vasodilation in some vascular beds. We hypothesize that jejunal but not colonic H₂S increases portal vein flow in a NO-dependent fashion. To evaluate the effects of luminal H₂S, venous blood flow, portal venous pressure, and systemic venous pressure were measured in rats after administration of either vehicle or an H₂S donor (NaHS) into the jejunum or the colon. We found that portal venous pressure and systemic pressure did not change and were similar between the three study groups. However, portal venous blood flow significantly increased following jejunal administration of NaHS but not in response to colonic NaHS or vehicle administration. To test the contribution of NO production to this response, another group of animals was treated with either an NO synthase inhibitor (N-Ω-nitro-l-arginine, L-NNA) or saline prior to jejunal NaHS infusion. After L-NNA pretreatment, NaHS caused a significant fall rather than increase in portal venous flow compared to saline pretreatment. These data demonstrate that H₂S within the small intestine significantly increases portal venous blood flow in a NO-dependent fashion.

Introduction

H₂S is a recently described gaseous signaling molecule that plays an important role in the physiology of many organs including those of the cardiovascular system where it has a significant impact on health and disease [1]. Endogenously produced H₂S has been shown to regulate both mesenteric [2] and portal [3] hemodynamics. Conflicting reports have shown H₂S causing either vasodilation [2] or vasoconstriction [4] of the mesenteric vasculature. Other studies also showed that H₂S can either increase [4] or decrease [5] systemic blood pressure in subjects with liver disease. Thus, the mechanism of action of H₂S on blood vessels is not fully understood, especially in the setting of liver disease. Previous studies of physiological responses to H₂S have mainly relied on the use of donor molecules, such as sodium hydrosulfide (NaHS) or sodium sulfide (Na₂S). NaHS in aqueous solutions disassociates into HS⁻ and Na⁺ that equilibrate with H⁺ ions in the solution to form H₂S [6]. In the gut lumen, unbound H₂S can diffuse through cell membranes into surrounding tissue to enter the portal circulation [7].

The intestine is a major source of exogenously produced H₂S. The normal gut microbiota contains multiple H₂S-producing genera, collectively called sulfate-reducing bacteria or SRB [8], that produce H₂S from hydrogen, a by-product of fermentation. In a healthy gut, SRB-generated H₂S is found primarily in the colon where it is largely inactivated by a detoxification system in the epithelium. Measurements of H₂S in the colonic lumen of rats have found concentrations of up to 1000 parts per million, far in excess of the “safe” exposure level of 20 parts per million for 10 min allowed by OSHA [9]. In a healthy colon, bacteria-produced H₂S is detoxified by several mechanisms including oxidation by rhodanese of colonocytes to thiosulfate and subsequent catalysis of thiosulfate and cyanide to produce thiocyanate [9, 10]. However, the capacity for detoxification of H₂S by the small intestine is 1/8^th that of the large intestine [11, 12]. With this regional difference, the effect of H₂S on mesenteric and portal blood flow may depend greatly on the site and concentration of production of this gas.

A deficiency of nitric oxide has been associated with intrahepatic vasoconstriction in liver diseases. Specifically, portal hypertension has been found to correlate with reduced activity of nitric oxide synthase (NOS) in the sinusoidal endothelial cells of the liver [13]. By reducing the production of nitric oxide, NOS inhibitor, and L-NNA, [14] has demonstrated that the vasodilatory effects of H₂S depend in part on the NO pathway [1].

The hydraulic equivalent of Ohm’s law (resistance x flow = pressure) illustrates the relationship between blood flow and resistance to vessel hydraulic pressure. In the conventional portal blood circulation model, hepatic sinusoids (intrahepatic vasculature), the mesenteric vascular bed, and portal vein circulation all are incorporated to define the sites of resistance [13]. In this model, portal vein flow combined with continuous vascular resistance in hepatic sinusoids defines portal pressure, and any variation of either of these variables will ultimately alter the pressure. Thus, in this conventional model, increased resistance to vascular flow (as seen in hepatic fibrosis) is the main cause of increased pressure, known as portal hypertension. In fibrosis and cirrhosis, increased portal pressure due to a fixed increase in resistance is the presumed cause of sequalae of liver disease such as variceal bleeding, hepatorenal syndrome and ascites [13]. However, under Ohm’s law, increased flow can also increase pressure independent of changes in resistance. While portal resistance has been well studied, humoral regulators of portal flow are not well understood. In this study, we tested the hypothesis that portal vein flow can be increased by absorption of jejunal, but not colonic, H₂S in a nitric oxide-dependent fashion.

Methods

Experimental Overview

We measured portal venous blood flow, portal venous pressure, and systemic venous pressure before (baseline) and after administration of an H₂S donor, NaHS, into the jejunum (jejunal NaHS) or colon (colonic NaHS). Saline was administered as a vehicle control. To test the role of NO, L-NNA or saline was administered as pretreatment to the jejunal NaHS group.

Animals

All procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the University of New Mexico. All protocols conformed to the National Institutes of Health guidelines for animal use in research. Male Sprague–Dawley rats (325–375 g, Harlan Laboratories, n = 8/group) were housed (2 animals per cage) with environmental enrichment under conditions of 12-h light/12-h dark cycle, environmental temperature of 20–23 °C, and food and drink ad libidum. Animals were given at least 2-day acclimation prior to any experimentation. Body weight was recorded prior to experimentation. All protocols were performed under isoflurane gas anesthesia after an overnight, 12-h fast with ad libidum water intake during the fast. Stomach, duodenum, jejunum, and colon were identified during laparotomy for administration of NaHS or vehicle. The site of jejunal infusion was distal to the Ligament of Treitz, ~ 10 cm from the gastric outlet. The site of colonic infusion was the descending colon. For the L-NNA pretreatment group, animals were administered L-NNA (10 mg/kg, i.v.) as a bolus before baseline recordings and before administering NaHS. I.V. saline was infused as control. Animals were euthanized at the end of the experiment.

Surgical Approach

Animals were anesthetized under inhaled isoflurane (5% in oxygen for induction and 2% in oxygen for maintenance) and instrumented through a midline incision. A perivascular ultrasonic probe (Transonic, Ithaca, NY) was placed around the portal vein to measure blood flow; recorded as mL/min and reported as percent change from baseline. A fluid-filled PE20 catheter was glued into the portal vein to measure portal venous pressure, and a second fluid-filled PE20 tubing was tunneled through the femoral vein into the inferior vena cava to measure systemic venous pressure. All variables were monitored until flow and pressure values were stabilized following surgical manipulation and then recorded for 10 min as the baseline data.

Effects of H₂S Infusion

After recording baseline values for 10 min, a loading bolus of NaHS in saline (16.8 mg/mL/kg) was administered followed by continuous infusion (0.56 mg/mL/kg/min) for 20 min. Control animals were administered equi-volume saline using the same approach. Flow and pressure were recorded continuously throughout the 20-min infusion. Measurements are represented as percent change from baseline mean ± SEM in 60-second segments for 20-min total. The end-point measurement (at 20 min) was used for statistical analysis.

Effects of NOS Inhibition (L-NNA Pretreatment)

To test the role of NO in this response, L-NNA dissolved in saline (10 mg/kg body weight) or saline as control (1 mL/kg body weight) was infused into the vena cava prior to the administration of jejunal H₂S. After pretreatment with L-NNA or saline, baseline recordings were obtained. (Note pretreatment had no effect on pressure or flow.) After baseline recordings were obtained for 10 min following pretreatment, a loading bolus of NaHS, (16.8 mg/mL/kg) was administered followed by continuous infusion (0.56 mg/kg/min) for 20 min into the jejunum. Two control groups were administered saline as i.v. bolus pretreatment. After saline treatment, one control group was administered a loading bolus of NaHS (16.8 mg/mL/kg) followed by continuous infusion (0.56 mg/mL/kg/min) for 20 min into jejunum [saline/NaHS group]. In the second control group, after saline pretreatment, equi-volume saline was administered into the jejunum using the same approach [saline/vehicle group]. Flow and pressure were recorded continuously throughout the 20-min infusion. Measurements are represented as percent change from baseline with mean ± SEM for 60-second segments reported. The end-point measurement (at 20 min) was used for statistical analysis.

Statistics Analysis

There were no significant differences in baseline measures between groups. Baseline data over 10 min was averaged and set as the reference point. To account for inter-animal variability at baseline, percent change from baseline was used to compare responses between groups. Within-group changes in portal and systemic pressure were compared by repeated-measures two-way analysis of variance (RM-ANOVA) to evaluate the effects of H₂S on portal and systemic venous pressures. Two-way RM-ANOVA was also used to compare portal blood flow between and within the groups.

Results

Weight at time of surgery was not different between groups. At baseline, there were no differences in portal vein flow, portal vein pressure, and systemic venous pressure between groups (Table 1).

Table 1.

Baseline characteristics groups testing the effects of H₂S showing similar starting hemodynamics of portal and systemic values along with similar weights in all three groups (n = 8/group)

	Weight	Baseline portal flow	Baseline portal pressure	Baseline systemic pressure
Jejunal infusion NaHs	367 ± 5.6	21.1 ± 5	6.2 ± 0.8	3.8 ± .9
Colonic infusion NaHs	359 ± 7.7	20.2 ± 2.8	7.2 ± 1.6	3.1 ± .6
Vehicle	3611 ± 9.6	17.8 ± 3.2	5.8 ± 0.9	3.9 ± 1.1
	NS	NS	NS	NS

NS no statistical difference for each group

Systemic and portal measurements were monitored after completion of 20-min infusion. Portal venous measurements that changed during the experiment slowly returned to baseline in varied timeframes, occasionally taking up to an hour. No changes were noted in pressure and flow measurements during the experiment, and all measurements remained at baseline after stopping infusion.

Effects of H₂S Infusion

Portal vein pressure after jejunal NaHS infusion (5.2 ± 5.2%) was not different at the end of infusion (20 min) when compared to colonic NaHS infusion (7.0 ± 5.1%) or jejunal vehicle infusion (2.8 ± 1.9%).

Systemic venous pressure after jejunal NaHS infusion (12.3 ± 12.7%) was not different at the end of infusion (20 min) compared to colonic NaHS infusion (−4.7 ± 5.3%) or jejunal vehicle infusion (22.4 ± 26%) (data not shown).

Portal venous flow increased significantly after jejunal NaHS infusion (25.8 ± 8.4%, p < 0.005) at the end of infusion (20 min) when compared to colonic NaHS infusion (− 1.9 ± 5.2%) and jejunal vehicle infusion (1.1 ± 2.9%) (Fig. 1).

Fig. 1.

Portal vein blood flow during 20-min infusion period shown as percent change from baseline (average of 7 min prior to start of infusion). Portal blood flow after infusion of jejunal NaHS significantly increased compared to colonic NaHS infusion. Data show mean ± SEM. “+” represents statistically significant difference between jejunal NaHS infusion and other groups. Data were compared using two-way repeated-measures ANOVA with time and treatment as the dependent variables

Effects of NOS Inhibition

Baseline portal vein pressure and flow and baseline systemic venous pressure were not different between groups. The slow intravenous infusion of L-NNA or saline as pretreatment also did not change these parameters from baseline (Table 2).

Table 2.

Baseline characteristics for pretreatment group showing similar starting characteristics of portal and systemic values along with similar weights in all three groups (n = 8/group)

	Weight	Baseline Portal flow	Baseline portal pressure	Baseline systemic pressure
L-NNA pretreated, jejunal NaHS	374 ± 7.9	22.9 ± 3.7	9.5 ± 2.2	3.8 ± 0.9
Saline pretreated, jejunal NAHS	370 ± 5.4	21.3 ± 5.4	6.2 ± 0.8	3.8 ± .9
Saline pretreated, control	365 ± 10.4	17.9 ± 3.2	5.8 ± 0.9	3.9 ± 1.1
	NS	NS	NS	NS

NS no statistical difference for each group. No change in baseline hemodynamics was noted with pretreatment infusion; characteristics shown are recorded after pretreatment infusion with L-NNA or saline (these characteristics were not altered before pretreatment of L-NNA or saline)

Portal venous pressure after L-NNA pretreatment and jejunal NaHS infusion (5.1 ± 6.1%) was not different at the end of infusion (20 min) compared to saline/NaHS (4.5 ± 5.9%) or saline/vehicle jejunal infusion (4.2 ± 3.2%).

Systemic venous pressure after L-NNA pretreatment and jejunal NaHS infusion (−5.6 ± 25.9%) was not different at the end of infusion (20 min) compared to saline/NaHS (13.5 ± 10.6%) or saline/vehicle jejunal infusion (29.6 ± 23%) (data not shown).

Portal venous flow decreased following jejunal NaHS in the group pretreated with L-NNA (− 15.9 ± 6.1%, p < 0.05) at the end of infusion (20 min) and was significantly lower compared to flow in saline/NaHS (23.7 ± 9.2%) or saline/vehicle groups (0.6 ± 3.5%) (Fig. 2).

Fig. 2.

Portal vein blood flow expressed as % change from baseline after jejunal NaHS or saline in rats pretreated with L-NNA (10 mg/kg I.V.) or vehicle (saline). Pretreatment with IV L-NNA prior to NaHS jejunal infusion significantly lowered portal flow compared to control infusion. Saline IV pretreatment (equi-volume administration) leads to increase in portal flow. Note L-NNA and saline intravenous pretreatment infusions were not shown separately as they did not alter portal flow; changes in flow were only noted after baseline with addition of NaHS. Minute 1 shows initial baseline (0% change) at start of infusion. * represents statistically significant difference between L-NNA infusion group and other groups. Data show mean ± SEM using two-way repeated-measures ANOVA

Discussion

In this study, we found that administration of a H₂S donor increased portal venous flow but not portal venous pressure or systemic venous pressure when delivered into the proximal small bowel but had no effect when administered into the colon. This is the first report of a region-specific effect of gut H₂S on portal venous hemodynamics.

H₂S is generated endogenously by the enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CTH or CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). While endogenously generated H₂S has been shown to regulate portal blood pressure [3], the effects of H₂S in the gut lumen remain unknown [8]. Considering that the concentration of H₂S in the colonic lumen can reach 1000 parts per million (PPM) [15], and that blood or tissue concentrations of H₂S are only ~ 45 microM or 0.045 PPM [8], the gut is a vast reservoir of H₂S that could impact the portal circulation via generation by SRB members of the gut microbiome.

Our novel finding that jejunal but not colonic infusion of NaHS increases portal venous flow demonstrates that H₂S in the gut lumen can profoundly impact portal hemodynamics. Furthermore, the region-specific response to H₂S that may be explained by the presence of a detoxification mechanism in the colon but the detoxification capacity of the small intestine is 1/8th that of the colon [8]. As a result, H₂S generated in the jejunum may have a greater impact on portal venous blood flow. A colon-based detoxification system for H₂S is consistent with the known compartmentalization of the gut microbial community to the distal gut in normal conditions where bacteria concentration of 10¹²/g exists in the colon compared to only 10³/g in the jejunum [16]. Such compartmentalization means that, normally, the colon would need a higher detoxification capacity as the distal gut may be exposed to a much higher amount of bacteria-derived H₂S than the proximal small intestine.

We also found that in animals pretreated with an NO synthase inhibitor, NaHS infusion decreased rather than increased portal blood flow suggesting H₂S has a vasoconstrictive effect (increased resistance) in the absence of NO generation and a vasodilatory effect (decreased resistance) in the presence of NO production. Because portal pressures remained the same in these animals, vascular resistance was estimated from changes in flow using Ohm’s law. This suggests that the observed changes in portal venous flow after jejunal H₂S administration may be due to decreased portal venous resistance via an NO-dependent mechanism. As L-NNA was administered prior to NaHS infusion and caused no changes in portal blood flow/pressure or systemic pressure; it is unlikely that the impact in Fig. 2 is only driven by L-NNA.

Increased portal venous flow after administration of jejunal NaHS in the presence of unchanged portal venous pressures indicates that portal vascular resistance decreases. The increased portal flow is unlikely to be caused by increased volume in the portal circulation since saline infusion in the jejunum did not alter portal hemodynamics. These findings were observed in healthy animals, without underlying chronic liver disease. It is important to note that the impact of NaHS, or H₂S, in the setting of increased intrahepatic vascular resistance in cirrhosis is likely to be different but has yet to be properly evaluated.

While the impact of NO on portal venous circulation in the setting of portal hypertension has been established for decades [17, 18], its role is primarily discussed in terms of reducing hepatic resistance. The role of hyperdynamic flow as a significant contributor to the development of complications of liver disease has been an area of considerable research interest [19–21]. The hyperdynamic flow observed in cirrhosis is generally explained as an indirect (compensatory) effect of splanchnic vasodilation due to a variety of factors including increases in NO. Our finding that H₂S in the small but not large intestine uniquely increases portal blood flow via an NO-dependent pathway could provide an additional explanation for the hyperdynamic circulation seen in liver disease. Previously, measurement of portal venous pressure was used to predict development of varices and variceal bleeding [22], but increased blood flow could contribute to the observed portal hypertension to explain more directly the finding of hyperdynamic circulation. Although we did not measure the concentration of H₂S in portal or systemic blood, the immediate increase in portal vein flow without a change in portal venous pressure in response to the delivery of NaHS into the jejunum was dramatic and reproducible; this demonstrated the direct and immediate effect of H₂S as the stimulus for the increase.

Previously, Huc et al. [23] reported that administration of high doses of Na₂S, another H₂S donor, into the colon led to a fall in systemic blood pressure and in some animals, death. These severe consequences may be explained by the administration of near-lethal to lethal amounts of H₂S which are known to block aerobic respiration, especially in conditions of low oxygen availability [24]. The NaHS concentration used in our study was selected from preliminary studies of the dose–response to NaHS, modeled after the study by Huc et al. [23] using Na₂S. Three concentrations of NaHS were tested using a logarithmic dose response curve (from 1.7 mg/kg body weight to 53.1 mg/kg body weight) to determine the lowest dose to elicit changes in portal flow without inducing cardiac arrest. We found that NaHS delivered at 16.8 mg/kg consistently increased portal venous flow without causing any systemic effects. Lowering of systemic blood pressure is a well-described effect of systemic H₂S (1). Thus, the lack of an effect on systemic venous pressure with the luminal delivery of H₂S in this study suggests that the administered H₂S was fully detoxified by the liver before reaching the systemic circulation, a situation that expected in healthy animals. We also used a steady infusion rather than administering a bolus injection to better simulate real-world conditions where SRB continuously produce H₂S for several hours after a meal.

A disturbance in the gut microbiome leading to dysbiosis or an abnormal gut microbiome may be induced by a number of disruptors including a high fat diet [25], antibiotic treatment [26], and stress [27]. Such dysbiosis is frequently characterized by small intestinal bacterial overgrowth whereby the microbial community expands from the colon into the small bowel, a change known to be associated with an increase in the population of SRB in the small intestine [28]. In that setting, the small intestine would be expected to be exposed to a greater amount of bacteria-derived H₂S, but inactivation of this gas would be limited allowing the excess H₂S to enter the mesenteric venous circulation and, subsequently, the portal vein and the liver. While a healthy liver could respond to such exposure by detoxifying H₂S via oxidative metabolism [29], a diseased liver might not.

Dysbiosis is common in patients with fatty liver disease and other chronic liver diseases [30, 31]. Although the concentration of H₂S in the gut in the setting of liver disease is not known, the known bloom in SRB (specifically Desulfovibrio spp, which has been the most studied genus) [32] suggests the intestine is exposed to elevated levels of H₂S in the setting of dysbiosis. Importantly, the effects of dysbiosis on portal circulation are largely unknown [30] and most studies focus on leaky gut with translocation of bacteria and bacterial products to cause disease [33–35]. Patients with chronic liver disease have frequent vascular complications including variceal bleeding, ascites, hepatorenal syndrome, and hepatopulmonary syndrome. Our data suggest H₂S-mediated increases in portal venous flow may contribute to these complications of chronic liver disease in conjunction with portal hypertension caused by abnormal hepatic vascular resistance.

Current understanding of portal hypertension implicates architectural disturbances in the liver as the cause of hemodynamic changes in the portal circulation with sequelae on circulatory hemodynamics driven by splanchnic vasodilation due to NO and other mediators related to microbial translocation [19–22, 36, 37]. In contrast to splanchnic vasodilation due to overproduction of NO, increased hepatic vascular resistance has also been explained by a deficiency in NO. Specifically, it is thought that reduced NO synthase activity leads to increased portal vascular resistance secondary to the fibrosis of the liver parenchyma and vascular occlusions caused by compressive regenerative nodules [13, 20, 36]. These opposing effects suggest reduced NO leads to increased portal venous pressure. As a consequence of this increased portal pressure, splanchnic and systemic arterial beds vasodilate in a compensatory response mediated largely by NO [13].

Our data suggest an alternate explanation whereby absorption of excess gut-derived H₂S leads directly to increased portal venous blood flow. In the setting of chronic liver disease where bacterial overgrowth extends into the small bowel [38], the limited detoxification capacity could be overwhelmed by large amounts of H₂S produced by SRB. The generated H₂S then enters the portal vein to increase blood flow via lower vascular resistance. This increased blood flow could then contribute to dynamic complications in the portal and mesenteric vascular system such as esophageal and gastric varices, independent of static complications such as portal venous pressure increases.

This novel model links portal blood flow to bacteria-derived H₂S in the small but not large intestine and provides a plausible explanation for acute changes in portal hypertension observed in patients with chronic liver disease [39] associated with clinical decompensation and hospital admission. Because increased resistance to blood flow due to liver fibrosis only explains a static rise in portal pressure [40], additional factors such as gut-derived H₂S must also be present to drive the acute, dynamic changes in portal hemodynamics that are known to drive decompensation events such as a variceal bleed.

This initial study shows the regional impact of gut luminal contents on the portal circulation in healthy rats with normal liver function. Future directions should further evaluate the regional impact of gut microbiome contents in the setting of chronic liver disease. As previously discussed, it has been well established that portal pressures increase in cirrhosis/fibrosis and it will be important to evaluate the role of H₂S in disease states. This study used concentrations of NaHS that produce portal changes without systemic impact. The free H₂S concentrations during dysbiosis remain unknown and may reach levels that can impact systemic circulation, which was not tested in this study. Future studies are needed to evaluate the concentrations of H₂S in healthy and diseased states.

As the portal vein delivers the nutrients from the small intestine to the liver, gut-derived H₂S would be expected to have an especially large impact on portal venous hemodynamics. Luminal hydrogen is increased after every meal when microbial fermentation ramps up following delivery of fermentable substrates to the microbiome. In the presence of SRB, these postprandial fluctuations in hydrogen availability could result in large swings in H₂S generation. Our data suggest these variations in H₂S production in the small intestine could drive fluctuating portal blood flow and have clinical consequences as H₂S escapes the limited detoxification system of the small bowel to enter the portal vein.