The Journal of Clinical Investigation: Video abstract: A personal perspective on new research in the JCIM
Fouad Chebib, M.D., Research Chair and Consultant, Nephrology and Hypertension, Mayo Clinic in Florida: I am Fouad Chebib, a nephrologist and physician-scientist at Mayo Clinic in Florida. In autosomal dominant polycystic kidney disease, tolvaptan can slow disease progression by blocking vasopressin V2 receptor signaling. But for many patients, the same mechanism creates a daily burden: very high urine volume, thirst and waking repeatedly at night to urinate. Our study began with a practical question: Can we reduce the aquaretic burden of tolvaptan without losing its disease-modifying benefit?
Normally, water reabsorption in the collecting duct is controlled by vasopressin. Vasopressin activates the V2 receptor, increases cyclic AMP, activates protein kinase A, and promotes apical accumulation of aquaporin-2. Aquaporin-2 is the final regulated water gate of the collecting duct. When it reaches the apical membrane, water can be reabsorbed. When it is removed, water is lost into the urine.
But collecting duct cells do not listen only to vasopressin. They integrate hormonal, paracrine and metabolic signals. Urate was an intriguing candidate. Humans have relatively high urate levels, urate handling is linked to water balance, and the urate transporters GLUT9b and ABCG2 are present in the collecting duct principal cells. So we asked whether soluble intracellular urate might directly regulate aquaporin-2 trafficking.
In polarized collecting duct cells expressing aquaporin-2, apical urate moved aquaporin-2 to the apical membrane, similar to dDAVP. But the mechanism was different. When V2 receptor expression was reduced or when the receptor was blocked with tolvaptan, dDAVP no longer moved aquaporin-2. Urate still did. The response was concentration dependent, appeared at physiologically relevant urinary urate concentrations, and was specific to soluble urate itself.
Mohamad Hadla, Ph.D., Research Associate, Mayo Clinic in Florida: The next question was how urate enters and stays inside the cell. GLUT9b acted as the apical urate entry pathway. When GLUT9 was knocked down, urate could no longer move aquaporin-2 to the surface. ABCG2 acted in the opposite direction, as an apical urate efflux pathway. When ABCG2 was knocked down or inhibited, intracellular urate increased and aquaporin-2 accumulated at the apical membrane. Probenecid reproduced this effect, and the response still required GLUT9. This defined a transport-coupled axis: GLUT9b brings urate in, ABCG2 moves urate out, and the intracellular urate level determines aquaporin-2 localization.
The surprising finding was that urate did not activate the classic cyclic AMP and protein kinase A pathway. Instead, urate reduced Ser256 phosphorylation, lowered PKA activity and decreased intracellular cyclic AMP. Aquaporin-2 still moved to the apical membrane. We then found that this depended on phosphodiesterase 4, also called PDE4. Blocking PDE4 prevented the urate effect, while activating PDE4 mimicked it. As cyclic AMP was degraded, AMP increased, the AMP-to-ATP ratio rose, and AMPK was activated. Urate was not turning on vasopressin signaling. It was creating a distinct PDE4 and AMPK signaling route to aquaporin-2.
AMPK was required for this pathway. AMPK inhibition or AMPK alpha knockdown abolished the urate response, while AMPK activation was sufficient to move aquaporin-2 apically. The trafficking route was also different. Urate required ongoing endocytosis and redirected aquaporin-2 through Rab7 positive late endosomes and Rab11 positive recycling endosomes, with small evidence of lysosomal or exosomal targeting. The model is that intracellular urate, through AMPK, improves recycling of preexisting aquaporin-2 pools back to the apical membrane.
Dr. Chebib: We then tested whether this pathway mattered in vivo. In wild-type mice, probenecid reduced tolvaptan-associated water intake and urine output in a dose-responsive manner. In Pkd1RC/RC ADPKD mice, adding probenecid lowered daily water intake and urine output while preserving the benefit of tolvaptan on kidney weight-to-body weight ratio. Probenecid also restored apical aquaporin-2 expression suppressed by tolvaptan. Importantly, this antiaquaretic effect persisted in a mouse model designed to better approximate human urate metabolism.
Finally, in the phase 2 open label SereNDIpity trial, 17 participants with ADPKD on stable tolvaptan received oral probenecid. After at least seven days, mean 24-hour urine volume decreased by about 30%. Nighttime voiding decreased from nearly four episodes per night to less than one. Patient-reported aquaresis symptoms improved, and morning urine osmolality increased. These clinical results are early and hypothesis generating, but they closely align with the cell biology and mouse data.
Together, these findings identify intracellular urate as a physiologic regulator of collecting duct water handling. By linking GLUT9b and ABCG2 transport to PDE4, AMP kinase and aquaporin-2 recycling, this work reveals a vasopressin-independent pathway for renal water reabsorption. For ADPKD, the larger implication is that aquaresis may be modulated separately from cyst growth suppression. The next step is to develop more selective and durable ways to target this pathway safely for patients.
I want to close by expressing my sincere gratitude to the coauthors, collaborators, research teams, study coordinators, laboratory members and clinical partners who made this work possible. This study required close integration of molecular nephrology, transport physiology, animal modeling and clinical investigation, and it reflects the commitment and creativity of many people across disciplines. Most importantly, I want to thank the patients with ADPKD and their families, especially those who participated in the SereNDIpity trial. Their willingness to contribute to research while living with the daily burden of this disease and its treatments is what allows us to move the field forward toward better, more tolerated therapies.