Recently completed phase 2 clinical trials with bardoxolone methyl have demonstrated a significant improvement in eGFR in patients with CKD and T2D.15,16
However, the precise mechanism responsible for this effect is still being investigated. Consistent with the clinical data for bardoxolone methyl, the present study demonstrated that RTA 405 increased basal GFR and blunted Ang II-induced GFR decline in Sprague Dawley rats. RTA 405 was used in these animal studies as it possesses pharmacologic properties that are similar to bardoxolone methyl, but is better tolerated by Sprague Dawley rats. Based on the similar effect of RTA 405 and bardoxolone methyl in rats and humans, we conducted additional ex vivo
and in vitro
experiments to investigate the mechanism underlying the observed increase in GFR.
Blood pressure, renal blood flow, and mesangial tone are regulators of GFR. Increased blood pressure could lead to increased GFR by raising net filtration pressure. This study shows that treatment of rats with RTA 405 does not increase basal or Ang II-increased blood pressure. Consistent with this result, Pergola et al
. demonstrated that the change in eGFR in CKD patients did not correlate with a change in blood pressure after 52 weeks of treatment with bardoxolone methyl.16
Therefore, RTA 405-induced increase in GFR is unlikely to be due to increase in systemic blood pressure.
The increase in GFR seen with RTA 405 could also result from an increase in RPF. In the present study, despite a significant increase in basal GFR, there was no significant change in basal RPF in Sprague Dawley rats treated with RTA 405, as measured by PAH clearance (). Furthermore, rats treated with RTA 405 maintained a significantly high level of GFR after administration of Ang II without significant changes in RPF. Taken together, these observations do not support changes in renal hemodynamics as a mechanism for the effect of RTA 405 on GFR.
In addition to renal hemodynamics, changes in the surface area and permeability of the glomerular filtration membrane can also alter GFR. A major finding of this study was that RTA 405 significantly inhibits MC contraction. MCs are located within glomerular capillary loops and their contractile properties enable them to alter intraglomerular capillary flow and filtration surface area and thereby GFR. Previous studies demonstrate that MCs are a target for Ang II-mediated regulation of GFR.17,24,25
In the present study, RTA 405 dose-dependently suppressed Ang II-stimulated contraction of human MCs and Ang II-induced decrease in the volume of rat glomeruli. Taken together, these data suggest that inhibition of Ang II-induced MC contraction results in preservation of filtration surface area in whole glomeruli. These in vitro
and ex vivo
findings are consistent with the effect of RTA 405 on GFR observed in intact animals, and further suggest potential contribution from a MC-mediated mechanism for improvement of eGFR in CKD patients treated with bardoxolone methyl.
Based on the conclusion that a MC-mediated mechanism plays a role in the effect of RTA 405 on GFR, we set out to further elucidate the mechanism of action of this class of drugs in cultured MCs. As in vascular smooth muscle cells, the contractility of MCs is tightly controlled by [Ca2+
. Ang II-induced elevation of intracellular Ca2+
is attributed to release from the sarcoplasmic/endoplasmic reticulum, as well as influx from the extracellular compartment.17,33,34 It has recently been demonstrated by our group and others that voltage-operated, receptor-operated and store-operated Ca2+
channels are involved in Ang II-mediated Ca2+
entry and MC contraction.17,35,36
In particular, we have demonstrated the important role of the canonical transient receptor potential 1 (TRPC1) and 6 (TRPC6) in this process in human MCs.17,20
These receptors function as store-operated and/or receptor-operated Ca2+
channels and are structurally and functionally distinct from the L-type voltage-gated calcium channels that are inhibited by calcium channel blockers such as amlodipine.37–39
Although a direct effect of RTA 405 on the TRPC1 and TRPC6 calcium channels can not be formally excluded at this time, it is likely that the inhibition of Ang II-induced Ca2+
influx is related to the potent antioxidant effects of this class.
Numerous reports from studies of vascular smooth muscle cells have demonstrated that ROS activate diverse pathways to increase [Ca2+
and subsequently trigger smooth muscle contraction.40
Likewise, reducing ROS levels in vascular smooth muscle cells has been shown to inhibit Ang II-induced increases in [Ca2+
and cell contraction.29,30
MCs and vascular smooth muscle cells have similar properties and we hypothesized that the mechanisms that occur in smooth muscle cells involving modulation of Ca2+
influx by ROS may also apply to MCs. In this study, we have demonstrated that PEG-catalase abolishes the Ang II-mediated increase in [Ca2+
in mesangial cells, supporting a role for ROS in Ang II-mediated MC contraction. As antioxidants and modulators of inflammation, synthetic triterpenoids upregulate a variety of antioxidant genes through activation of the Keap1/Nrf2 pathway.8,14,32
The present study showed that RTA 405 treatment significantly reduced intracellular ROS levels in cultured MCs, which correlates well with induction of antioxidant Nrf2 target genes in MCs treated with bardoxolone methyl and RTA 405. Thus, similar to the mechanism established in vascular smooth muscle cells, induction of Nrf2 and the corresponding decrease in intracellular ROS levels by RTA 405 in MCs may contribute to inhibition of the Ca2+
response and MC contraction that occurs with RTA 405 treatment.
We recently found that H2
has dual effects on TRPC6 channels.19,20,30,31
On the one hand, H2
acutely activated TRPC6 channels.30,31
On the other hand, chronic exposure to ROS suppressed the synthesis of TRPC6 protein19,20
and thus, decreased the number of functional channels. In the present experimental settings, RTA 405 suppressed Ang II-induced responses in a range of minutes (Ca2+
response) or tens of minutes (contraction). Therefore, the observed RTA 405 effects are consistent with inhibition of acute ROS effects.
In summary, our findings integrated from in vivo
, ex vivo
and in vitro
studies demonstrate that RTA 405 increased GFR at resting state and during Ang II stimulation in Sprague Dawley rats. Our results implicate induction of Nrf2, reduction of intracellular ROS levels, suppression of the glomerular MC Ca2+
response, and suppression of MC contraction as mechanisms contributing to the underlying effect. Inhibition of MC contraction may lead to a relative increase in glomerular capillary surface area resulting in improved GFR. This conclusion is schematically depicted in . Since the activity of the Ang II system and the intracellular levels of ROS in kidneys are substantially elevated in CKD,1
our study provides a cellular and molecular mechanism that likely contributes to the improvement of renal function by bardoxolone methyl in patients with CKD.
The mechanistic pathway for inhibition of Ang II-induced MC contraction by synthetic triterpenoids