Introduction
The increase of chronic kidney disease (CKD) patients becomes the serious problem for humans because CKD elevates cardiovascular events and causes end-stage kidney disease [
1]. Various treatments are used to delay the progression of CKD. However, only these treatments were not sufficient. As one of the pathogenic factors for progression, an unfavorable effect of uremic toxins recently attracts attention. Indoxyl sulfate (IS) and
p-cresyl sulfate (PCS) are produced in the intestine by the bacteria from tryptophan or tyrosine, respectively, absorbed from the colon, metabolized into sulfur-conjugated substances, and excreted in the urine from the kidney [
2]. Once accumulated by renal impairment, these substances cause the tissue damages through oxidative stress [
3,
4]. Another uremic solute, indole acetic acid (IAA) is also shown to damage endothelial cells and increase cardiovascular events in CKD [
5]. Charcoal adsorbent AST-120 (AST) is considered to be one of the options to delay the progression of CKD in Asia. It was designed to bind to uremic toxins in the colon and protect against the entry of IS precursors and reduced the plasma IS, thereby slowing the progression of CKD [
6,
7]. Although the recent large clinical trial failed renal protective effects [
8], several small sized clinical studies have shown its efficacy against the renal impairment [
9,
10]. Besides, it was reported that the tight junction in the colon was disrupted in CKD, which was reversed by AST [
11]. This effect is supposed to be beneficial to the maintenance of gut barrier function and inhibit the systemic inflammation evoked by the intrusion of various toxins from leaky gut [
10]. In this way, the organ interrelationship between gut and kidney has been gaining the scientific interest, coined as ‘the intestinal-renal syndrome’ [
12]. The modulation of this relationship can be a plausible strategy against CKD. We previously demonstrated that
Lactobacillus (
Lact) was decreased in CKD rats and that the supplementation of
Lact improved the intestinal barrier, systemic inflammation and the kidney function [
13]. However, the effects of AST on microbiota have not been reported thus far.
In this study, we demonstrated that AST elevates the population of Lact in CKD rats, which improved the disruption of intestinal barrier and systemic inflammation through TLR2 and TLR4. The present study provides the novel mechanism of AST through the modulation of gut environment.
Discussion
The present study demonstrated renoprotective effects through gut environment by AST in CKD rats. AST attenuated proteinuria, renal dysfunction, and glomerulosclerosis without affecting SBP.
Our previous paper showed that the renoprotective effects by Lactobacillus improve the decreased tight junction expressions through TLR2 in CKD rats. We explored the mechanisms for these effects by AST and found that AST ameliorated the decreased gut Lactobacillus and the decreased expression of tight junction in Nx through TLR2 and TLR4 pathway which was not observed in the single treatment with Lactobacillus. We also found the reduction of serum uremic toxins, which might contribute to the amelioration of renal damages. These systemic and renal favorable effects by Lactobacillus in combination resulted in the mitigation of renal tissue damages. We also demonstrated that there exist the upper limits of Lactobacillus by the combination experiment with AST and Lactobacillus.
AST is believed to reduce the serum levels of uremic toxins by capturing its precursors in the intestine and blocking the entry of these substances. In the present study, we have shown the increase in fecal excretion of indole,
p-cresol, and phenol by AST and the reduction in serum IS, PCS and IAA. In addition, we proposed that the amelioration of intestinal barrier disruption contributed to the blockade of the entry of these toxic molecules. We showed the decrease in the expressions of intestinal tight junction in Nx, which were ameliorated by AST as was already demonstrated previously [
11]. One of the important mechanisms for the restoration of tight junctions is the deprivation of uremic toxin such as indole in the intestinal lumen. As we previously reported that in vitro studies, indole downregulates the expressions of tight junctions with a concentration-dependent manner [
13]. Recently, some papers focused on the protective effect of indole on the intestinal barrier. The difference among the papers might be caused by the models (the model of colitis or the model or CKD) and the amount of uremic toxins. However, the CKD condition, in which numerous and abundant uremic toxins accumulate in the intestine, at least, provokes the decrease of tight junction by the concentration.
Uremic toxins have been shown to activate aryl hydrocarbon receptor (AhR) [
24‐
28] or pregnane X receptor (PXR) and the activation of PXR by indole derivatives regulated intestinal barrier expression through TLR4 [
29]. TLR4 can be activated by circulating LPS derived from bacteria in the intestine [
30]. Though LPS in the intestine is not absorbed by AST, the absorption of uremic toxins which reduced the activation of AhR or PXR leads to the decreased expression of TLR4 (Fig.
3b). These effects cumulated in the elevation of the intestinal barrier (Fig.
3a) and the decrease in the systemic inflammation, contributing to the slow progression of CKD.
As another plausible mechanism for the amelioration of intestinal barrier structure, we focused on the changes in microbiota by AST. In uremic conditions, microbes, such as
Clostridiaceae, Enterobacteriaceae and
Verrucomicrobiacea, which produce indole and p-cresol increased, whereas butyrate-producing microbes including
Lact and
Prevotellae decreased as compared with those of healthy subjects [
31]. Our analysis showed the increase in
Bact known as a species of indole producing microbe in Nx. We also demonstrated the restoration of
Lact by AST. We previously demonstrated that the administration with
Lact also reversed the tight junction disruption through TLR2 pathway in vivo and in vitro studies [
13]. The increase of
Lact by AST is also related to the upregulation of tight junction through TLR2 pathway since
Lact is considered to be one of the key regulators to maintain and form the tight junction protein in the gut.
However, the causes why gut microbiota has changed by the AST treatment are still unknown. Recently, Mishima et al. reported that the ClC-2 chloride channel activator lubiprostone in CKD models mice improves the gut environment [
32]. In the paper, they suggested that the retention of uremic toxins, intestinal ischemia, intestinal transit time prolonged by constipation, decreased intestinal fluid secretion, and malnutrition of gut lining with evident atrophy can provoke the shift of gut microbiota. In our case, there are two main causes for the improvement. One cause can be the decrease in the retention of uremic toxins by AST. The other cause can be the improvement from the intestinal ischemia by the improved expression of mucin-2 (the protective layer above the intestinal barrier). As
Lactobacillus was shown to adhere to the mucin layer and survive as an intestinal microbiota. Therefore, AST attenuates the gut microbiota including the elevation of
Lactobacillus.
Moreover, one of the main mechanisms for the renal protective effects by AST can be its anti-inflammatory effects through the reduction of serum IS. It was reported that Nx exhibited low-grade inflammation, as evidenced by increased level of serum IL-6. These effects might result in the impairment of structure and function of the kidney [
33]. It is reported that IS locally induces reactive oxygen species (ROS), which activate the nuclear factor-kappaB (NF-kB) pathway and trigger both oxidative stress, pro-inflammatory cytokine production and renal tissue damages [
34,
35]. Therefore, reduction of IS by AST teleologically mitigates renal damages. Moreover, we demonstrated the reduction in systemic inflammation by AST treatment. The mitigation of tight junction proteins and the restoration of intestinal barrier function would result in the blockade of LPS entry from the intestine and subdued inflammatory state in CKD.
In the previous report, the supplementation of
Lact improved the renal damage and systemic inflammation [
13]. The present data showed that AST also restored the renal impairment and systemic circulation of uremic toxins and systemic inflammation. Therefore, we hypothesized that the combination therapy with the
Lact and AST could retain the additive effect. However, this therapy failed to show further effects. Of note, the fecal concentration of
Lact did not show the significant change between Nx + AST and Nx + AST +
Lact though the elevation of
Lact concentration in Nx + AST +
Lact is predicted. Thus, the population of
Lact did not increase even after the addition of
Lact orally in Nx + AST. This result indicated the upper limit of
Lact residing in the intestinal environment. Similarly, in the previous report, treatment with
Lact did not show dose-dependent effects. Petschow et al. demonstrated that although they administered three different concentrations of
Lact (10
8, 10
9, 10
10 cfu/day) the number of fecal
Lact did not differ among three groups [
36]. Therefore, our data implied that effects by AST including the improvement of the intestinal environment and the alteration of microbiota considered to have the upper limit. This limitation was considered to be defined by the population of
Lact. This also suggests that the effects AST are partly dependent on the alteration of microbiome and are not solely dependent on the serum uremic toxin levels. This mechanism would explain why recent large scale clinical trial in EPIC study failed to show the favorable effects on renal function [
8], while some small clinical trials in Japan have succeeded [
9,
10], since the population of microbiota is to vary among the different races or different food consumptions.
As a limitation, we cannot evaluate all the gut microbiota quantitatively by T-RFLP. Though this method is less difficult and indicates the accurate trends, the resolution is not so high because different DNA fragments sometimes show the same bands because of the restriction enzyme. After T-RFLP methods give us the general trends, confirmatory real-time PCR would be better for more accurate data. Then, this paper mainly focuses on Lactobacillus and Bacteroides, which has some trends with renal function.
In conclusion, AST improved gut environment favorable to Lact which affected the tight junction expressions though TLR pathway with renal protective effects. Our data may help to establish more efficient strategy against CKD with the use of AST in clinical practice.