Gut alterations in a chronic kidney disease rat model with diet-induced vascular calcification

Abstract

Intestinal disorders and vascular calcification (VC) are often associated with chronic kidney disease (CKD). While gut barrier alterations have been reported in CKD (such as abnormal intestinal permeability, bacterial overgrowth, and inflammation), it is not clear if vascular calcification influences these alterations. To investigate whether the bidirectional relationships between VC and gut dysfunction could be mediated by increased inflammation and uremic toxin generation, we used the SNx-VC model of uremic vascular calcification (rats undergoing subtotal 5/6th nephrectomy and fed a procalcifying high-phosphate and vitamin D diet). We confirmed the presence of CKD and VC by von Kossa staining and observed increased gut-origin uremic toxin, indoxyl sulfate (IS), in SNx-VC animals compared to controls. In SNx-VC rats, we observed decreased mucus production (Alcian blue, Mucin 2 staining) in the colon and ileum which was correlated with the level of calcification. There was no change in inflammation markers or tight junction protein expression. We assessed intestinal levels in the NOD-like receptor family pyrin domain containing 6 (NLRP6) protein, known to regulate mucus secretion, and found no change in the colon or ileum. Nlrp6 mRNA was, however, decreased in the colon of SNx-VC rats, along with other mRNA (Ly96, Sod1), while Tlr2 was increased compared to controls. Our observations of low mucus, low Nlrp6 mRNA, and high IS in SNx-VC rats confirm a link between gut barrier alterations and uremic VC. This suggests that alterations in the mucus layer could favor the generation of gut-origin uremic toxins and promote VC in CKD. Thus, improving the gut mucus barrier function in the context of uremic VC could be considered as a possible therapeutic strategy in CKD patients.

Keywords: NLRP6; chronic kidney disease; gut; mucus; vascular calcification.

Fig. 1

Determination of plasma concentrations of gut‐related uremic toxins in the different groups of rats: the level of indoxyl sulfate increased in the plasma of SNx rats with or without VC, while the level of p‐cresyl sulfate remained statistically unchanged. (A) Plasma concentration of IS and (B) Plasma concentration of PCS measured by ESI/MS. Data are presented as mean ± SEM, and each point represents data from an individual rat. P‐value from Tukey post hoc tests after ANOVA are shown: *P < 0.05; **P < 0.01.

Fig. 2

Colon and ileum tissues show no clear infiltration of immune cells in SNx rats with or without VC compared with controls. (A) Representative hematoxylin/eosin‐stained colon and ileum sections. The arrow indicates the presence of an accumulation of immune cells and highlights cellular infiltration. Original magnification ×200. (B) Immune cell infiltration score was quantified using hematoxylin/eosin staining according to Erben et al. [28]. (C) Representative CD68 immunostaining of colon and ileum sections showing CD68 positive cells (arrow). Original magnification ×200. (D) CD68‐positive cells quantification (percentage of tissue stained) by immunohistochemistry (IHC). Original magnification ×200. Data are presented as mean ± SEM, and each point represents data from an individual rat. P‐value < 0.1 from Tukey post hoc tests after ANOVA is shown.

Fig. 3

Mucus content and Mucin 2 level decreased in SNx rats with vascular calcification compared to control rats in colon and ileum tissues. (A) Representative alcian blue staining of colon and ileum sections. Original magnification ×200. (B) Mucus content quantification using alcian blue staining. (C) Representative Mucin 2 immunostaining in colon and ileum sections. Original magnification ×200. (D) Mucin 2 protein level by IHC. Arrows indicate goblet cells. Data are presented as mean ± SEM, and each point represents data from an individual rat. P‐value from Tukey post hoc tests after ANOVA are shown: *P < 0.05; **P < 0.01.

Fig. 4

The level of NLRP6 protein is not altered by CKD in the colon and ileum. (A) Representative images of NLRP6 staining in colon and ileum. Arrows show mucosal tissue. Magnification ×200. (B) NLRP6 protein level by IHC. Data are presented as mean ± SEM, and each point represents data from an individual rat.

Fig. 5

Occludin and Claudin 1 protein levels are unchanged in colon tissue of SNx rats compared to control rats. (A) Representative image of Occludin and β‐actin expressions in colon tissue by western blot. (B) Quantification of Occludin protein expression by western blot (expressed as a ratio to β‐actin expression). (C) Representative image of Claudin1 and β‐actin expressions in colon tissue by western blot. (D) Quantification of Claudin1 protein expression by western blot (expressed as a ratio to β‐actin expression). Data are presented as mean ± SEM, and each point represents data from an individual rat.