MO436: The Effect of Protein-Restriction on the Tubular Handling of the Uraemic Retention Solute Indoxyl Sulfate
Authors: Lauriola Mara , Dejongh Sander , Farré Ricard , Verbeke Kristin , Meijers Björn 
Published: May 2022

Abstract

BACKGROUND AND AIMS
Chronic kidney disease (CKD) patients present an increased plasma level of protein-bound solutes known as uraemic toxins. Their accumulation is associated with, amongst others, cardiovascular events. The gut microbiome plays an important role in the generation of part of the uraemic toxins. It has been postulated that proximal tubule cells in kidneys are able to sense augmented levels of gut microbial metabolites via receptors and signalling pathways [1]. According to the so-called remote sensing hypothesis, kidney tubular cells respond to uraemic toxins plasma variations modulating the activity of membrane transporters involved in their excretion. Indoxyl sulfate (IS) originates from the microbial fermentation of tryptophan, a diet-derived amino acid. Nonetheless, few studies have investigated whether gut generation, absorption and plasma retention of indolic uraemic toxins and kidney cells’ secretory mechanisms are affected by the protein content in the diet.

METHOD
A total of 18 Sprague–Dawley male rats (Janvier, Le Genest- St Isle, France) 7–8 weeks old (weighing 270–388 g) were induced with CKD utilizing 5/6 nephrectomy and were randomly assigned to a low protein (LP) (n = 10) or a high protein (HP) (n = 8) diet. A sham-operated control group for each diet was used (n = 7 and n = 8 respectively). 24 h-urine was collected after 7 weeks, and euthanasia was carried out after 8 weeks from the induction of the disease (Figure 1). Blood and colon samples were collected. Diffusion chambers were used to assess colon permeability to indole over time (t = 0, 60, 90, 120 min). Indole concentration was quantified by Kovacs assay. Blood creatinine and urea were determined with standard laboratory techniques. Total plasmatic and urinary IS concentrations were measured using LC-MS/MS. The fractional excretion (FE) i.e. the percentage of IS excreted relatively to the kidney filtered load, was calculated to assess IS remote sensing.

RESULTS
Plasmatic levels of IS were significantly higher (P &lt;0.001) in CKD rats compared to sham rats. However, CKD rats on an HP diet showed no difference in plasma IS (P = 0.63) compared to rats on an LP diet. Conversely, 24-h urinary IS was significantly increased in CKD rats on a HP diet (P &lt;0.001). The FE of IS was significantly higher (P = 0.005) in CKD rats on an HP diet (Figure 2) and correlated with 24 h-urinary IS (Spearman r = 0.51, P -value = 0.03) and with protein intake (Spearman r = 0.52, P-value = 0.03) in CKD rats.
Therefore, CKD rodent models’ results suggest higher absorption and/or production of indole at the intestinal level when an HP is administered to CKD rats. Plus, they provide additional proof of the remote sensing theory of indole metabolites. The permeability of colon to indole evaluated ex vivo with the diffusion chamber technique, showed an increased apparent apical-to-basolateral transport of indole (P = 0.049) in CKD rats on an HP diet compared to that of sham rats on the same diet, again suggesting modulatory mechanisms affecting indole metabolites transporters uptake.

CONCLUSION
An HP diet in 5/6 nephrectomized CKD rats leads to increased production of indole in the colon and increased fractional excretion of IS. These results provide an additional proof of mechanisms of remote sensing and signalling of indole-derived uraemic toxins. To conclude, the adoption of a low protein diet remains recommended in chronic kidney disease.