Uremic Toxic Blood-Brain Barrier Disruption Mediated by AhR Activation Leads to Cognitive Impairment during Experimental Renal Dysfunction

Abstract

Background: Uremic toxicity may play a role in the elevated risk of developing cognitive impairment found among patients with CKD. Some uremic toxins, like indoxyl sulfate, are agonists of the transcription factor aryl hydrocarbon receptor (AhR), which is widely expressed in the central nervous system and which we previously identified as the receptor of indoxyl sulfate in endothelial cells.

Methods: To characterize involvement of uremic toxins in cerebral and neurobehavioral abnormalities in three rat models of CKD, we induced CKD in rats by an adenine-rich diet or by 5/6 nephrectomy; we also used AhR^-/- knockout mice overloaded with indoxyl sulfate in drinking water. We assessed neurologic deficits by neurobehavioral tests and blood-brain barrier disruption by SPECT/CT imaging after injection of ^99mTc-DTPA, an imaging marker of blood-brain barrier permeability.

Results: In CKD rats, we found cognitive impairment in the novel object recognition test, the object location task, and social memory tests and an increase of blood-brain barrier permeability associated with renal dysfunction. We found a significant correlation between ^99mTc-DTPA content in brain and both the discrimination index in the novel object recognition test and indoxyl sulfate concentrations in serum. When we added indoxyl sulfate to the drinking water of rats fed an adenine-rich diet, we found an increase in indoxyl sulfate concentrations in serum associated with a stronger impairment in cognition and a higher permeability of the blood-brain barrier. In addition, non-CKD AhR^-/- knockout mice were protected against indoxyl sulfate-induced blood-brain barrier disruption and cognitive impairment.

Conclusions: AhR activation by indoxyl sulfate, a uremic toxin, leads to blood-brain barrier disruption associated with cognitive impairment in animal models of CKD.

Keywords: chronic kidney disease; dementia; uremia.

Graphical abstract

Figure 1.

Experimental protocol of the study. Description and timing of the experiments conducted in (A) rats and (B) mice. 5/6 Nx, 5/6 nephrectomy; AhR^+/+, wild type; AhR^-/-, Aryl Hydrocarbon Receptor knock-out; BBB, blood-brain barrier; CSF, cerebro spinal fluid.

Figure 2.

ARD induced dose-dependent irreversible tubular lesion and fibrosis; the 0.5% ARD induced uremic toxins accumulation and AhR activation. Biologic and histologic evaluation of control or 0.25% and 0.5% ARD rats. (A) Serum IS over time. (B) Relative AhR activation by the rats’ serum (by percentage of FICZ). (C) Tubular injury score after hematoxylin-eosin staining. (D) Renal histology in optical microscopy after hematoxylin-eosin staining in (D, a and d) control rats, (D, b and e) 0.25% ARD rats, and (D, c and f) 0.5% ARD rats. Original magnification, x20 in D, a–c; x100 in D, d–f. (E) Sirius red staining. (F) Renal histology in optical microscopy after Sirius red staining on full histologic section in (F, a) control rats, (F, b) 0.25% ARD rats, and (F, c) 0.5% ARD rats. **P<0.01; ***P<0.001; ****P<0.0001.

Figure 3.

Renal failure induced neurobehavioral alteration. Neurobehavioral evaluation of control or 0.5% ARD rats. (A) Discrimination index of the NOR test. (B) Correlation between discrimination index of the NOR test and serum IS levels. (C) Discrimination index of the OL test. (D) Sniffing time in social recognition test. *P<0.05; **P<0.01.

Figure 4.

The 5/6 nephrectomy induced comparable outcomes with 0.5% ARD on kidney function, uremic toxins accumulation, AhR activation, neurobehavioral impairment, and BBB permeability in rats. Evaluation of the effects of CKD induced by 0.5% ARD or 5/6 nephrectomy in rats on (A) brain content of 99mTc-DTPA cerebral scintigraphy in percentage of injected activity per gram of brain (% IA/g). (B) Serum indoxyl levels at D28. (C) Relative AhR activation by the rats’ serum (by percentage of FICZ) at D28. (D) Discrimination index of the NOR test. (E) Correlation between discrimination index of the NOR test and brain content in 99mTc-DTPA cerebral scintigraphy expressed as percentage of injected activity per gram of brain (% IA/g). (F) Correlation between serum IS levels and brain content in 99mTc-DTPA cerebral scintigraphy expressed as percentage of injected activity per gram of brain (% IA/g). **P<0.01; ***P<0.001; ****P<0.0001.

Figure 5.

IS overload increased cognitive impairment and BBB disruption in 0.5% ARD rats. Evaluation of ARD rats with IS overload compared with control or 0.5% ARD rats. (A) Serum indoxyl levels at D28. (B) Relative AhR activation by the rats’ serum (by percentage of FICZ) at D28. (C) Discrimination index of the NOR test. (D) Brain content of 99mTc-DTPA cerebral scintigraphy in percentage of injected activity by gram of brain (% IA/g). (E) IS levels in CSF. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Figure 6.

Oral sorbent AST-120 treatment after ARD decreased serum indoxyl sulfate but did not reverse BBB dysfunction in rats. Evaluation of a 4-week treatment with oral sorbent AST-120 after ARD in rats. (A) BBB dysfunction measured by brain content of 99mTc-DTPA cerebral scintigraphy expressed as percentage of injected activity per gram of brain (% IA/g). (B) Serum IS levels. *P<0.05; **P<0.01; ***P<0.001.

Figure 7.

AhR activation was involved in indoxyl sulfate-induced BBB disruption and cognitive impairment in mice. (A) Relative brain content of 99mTc-DTPA cerebral scintigraphy in percentage of injected activity by gram of brain (% IA/g) in wild-type (AhR^+/+) and AhR knockout (AhR^-/-) mice at D7 compared with D0. (B) Serum IS in AhR^+/+ and AhR^-/- mice at D7. (C) Relative AhR activation in vitro by the mice’s serum expressed in percentage of the reference agonist FICZ. (D) Cognitive assessment by discrimination index in the NOR test and the OL test. *P<0.05; **P<0.01.