Uremia is a systemic chemical toxemia with repercussions on different organs and systems. Chronic renal failure patients demonstrate several endocrine dysfunctions, such as disturbance of thyroid hormone metabolism and are different from patients with the euthyroid sick syndrome. In the later, the conversion of T4
is reduced, but the generation of reverse T3
) from T4
is increased. In uremic patients, rT3
is typically normal [32
]. In addition, Lim et al
. showed thyroid hormone resistance in hemodialysis patients with significantly reduced peripheral tissue sensitivity to thyroid hormone [17
]. Recent data from our laboratory indicate that in order to maintain the euthyroid state showed that uremia increases T3
influx across erythrocyte's membrane [35
]. Taken together, these findings suggest that CRF affects thyroid function in multiple ways. The molecular mechanisms involved and the role of thyroid hormone receptor in this dysfunction, however, are not fully understood.
In the present study we observed that uremic plasma impaired the ability of TRβ1 and VDR heterodimers (TRβ1-RXRα and VDR-RXRα) to bind to DNA (DR-4 and DR-3 respectively), whereas that the ability of PPARγ-RXRα to bind to DNA (DR-1) was not altered. Interestingly, there was no correlation between the inhibitory activity and the plasma levels of urea, creatinine, parathyroid and thyroid hormone of the patients enrolled in this study. However, the small number of patients precludes any definitive conclusions.
To investigate whether these findings were secondary to the presence of uremic dialyzable toxins, we compared the effect of uremic plasma collected before and after hemodialysis, on TRβ1-RXRα or VDR-RXRα binding to DNA. Our results showed that the inhibitory effect of uremic plasma was significantly reduced by hemodialysis, suggesting that dialyzable toxins were in fact involved. We did not identify which toxin is responsible for this effect, but our results suggest the presence of thermo-resistant molecule(s). Further analyses of these dialyzable toxins are currently being conducted to identify and characterize the molecules responsible for this inhibitory effect.
The mechanisms responsible for our findings are not clear. In uremic syndrome the reduced clearance of many toxins plays a key role in this pathogenesis. Although VDR degradation has been suggested in renal failure [36
], the uremic inhibition of TRβ1-RXRα binding to DNA could not be explained by proteolytic activity of uremic plasma since our SDS-PAGE did not show any uremic plasma-dependent degradation of TRβ1.
Our results are in agreement with other studies, which have shown that uremic toxins are involved in VD3
resistance observed in patients with chronic renal failure [37
]. Uremic ultrafiltrates derived from hemo or peritoneal dialyzed patients have been shown to inhibit the interaction of VDR with DNA [27
]. Further studies showed that the VDR complex formation on different types of VDREs can be reduced by uremic solutions collected from patients after hemo or peritoneal dialysis [28
]. Our results allow us to speculate on the possibility of a common inhibitory mechanism involving the same uremic toxin(s) inhibiting both TR-RXR-DR-4 and VDR-RXR-DR-3 complex formation.
To evaluate whether uremic toxins also affect other members of the nuclear receptor family, we studied the effect of uremic plasma on PPARγ-RXRα binding to DR-1. Contrary to what we observed with TRβ1 and VDR, pre-incubation of PPARγ with uremic plasma did not influence PPARγ-RXRα binding to DNA. This result suggests that the inhibition of protein-DNA complex caused by uremic plasma occurs only with some nuclear receptors. Taken together, our results indicate that uremic toxins exert their inhibitory effect by acting specifically on TRβ1 and VDR heterodimers.
The molecular mechanism involved in this phenomenon is not clear. The fact that PPARγ-RXRα heterodimer was not affected by uremic plasma suggests that these toxins do not interact directly with RXRα. Another possible model to explain the effects of the toxins from uremic plasma on the binding of TR to DNA would be a direct action on DNA that would block its interaction with TRβ1 and VDR heterodimers. However, even though we used the DR-1 in PPARγ assay, in contrast to DR-4 (TRE) and DR-3 (VDRE), this hypothesis is not strongly supported by the results from this study, as PPARγ-RXRα heterodimers bind normally to DNA in the presence of uremic plasma. Another alternative that can not be excluded is an inhibitory effect of the uremic toxin on the surface of TR and VDR DNA binding domain (DBD), disrupting its ability to binding to DNA. Patel et al.
attributed to the formation of Schiff bases between "reactive aldehydes" and lysine residues of the DBD of the VDR to explain the inhibitory effect of the uremic ultrafiltrate on the binding of VDR to DNA [26
]. Nevertheless, in another study, point mutagenesis of different lysine residues in the DBD could not confirm this idea [28
]. In addition, we should consider that the uremic toxins can interact with TR and VDR, causing structural conformational changes on these receptors, consequently, impairing heterodimers formation.
We attempted to demonstrate the physiological relevance of these results by examining the effect of uremic toxins on T3 transcriptional activation. Our results showed that uremic ultrafiltrate collected before hemodialysis inhibited T3-induced transcriptional activation, confirming the in vitro findings. Conversely, in the presence of ultrafiltrate collected after hemodialysis, the transcriptional activation induced by T3 was similar to the control group treated with ultrafiltrate collected from normal individuals. Therefore, we hypothesize that dialyzable toxins are responsible for the resistance to T3 action documented in CRF patients.
In summary, uremic toxins circulating in the plasma of CRF patients selectively reduced the binding of TRβ1-RXRα to DNA and impaired the TRβ1 transcriptional activation mediated by T3. Moreover, hemodialysis partially corrected this inhibitory effect, suggesting the presence of a dialyzable toxin. Since TRβ1 functions as a heterodimer with RXRα, these findings might explain some features of hypothyroidism and thyroid hormone resistance commonly found in CRF patients. Future studies are necessary to identify the toxins and further characterize the mechanisms involved in resistance to T3 action in CRF patients.