Effects of flow and polycystin‐1 dysfunction on ATP release in the inner medullary collecting duct of the kidney

Abstract

After filtration of blood by the kidney, the remaining pro‐urine flows through the renal tubular system with a flow rate that can vary considerably. These variations in pro‐urinary flow can be sensed by mechanosensing proteins on the epithelial cells lining the renal tubules. An important mechanosensing protein is polycystin‐1 (PC1), which is thought to be a key sensor of fluid flow. Mutations in PC1 give rise to autosomal dominant polycystic kidney disease (ADPKD), which is characterized by the formation of fluid‐filled cysts in the kidney. Previous research has highlighted a role for the ATP release channel pannexin‐1 (PANX1) in the increased flow‐induced ATP release in a Pkd1‐/‐ cell model of the distal convoluted tubule. However, it remains unclear if other purinergic signaling components are also involved, especially in the collecting duct.Using the mouse inner medullary collecting duct 3 (mIMCD3) cell line in combination with microfluidic experiments, we show here that a similar flow‐induced ATP release is present in Pkd1‐/‐ mIMCD3 cells under low (0.32 mL/min) or high (1.27 mL/min) flow application for 1 minute compared to wildtype (WT) mIMCD3 cells. This is despite a six‐fold increase in the expression of Panx1 observed in Pkd1‐/‐ mIMCD3 cells. Moreover, application of the specific PANX1‐inhibitor BB‐FCF revealed that PANX1 did not drive the high flow‐induced increased ATP release. Accordingly, intermediate flow application (0.47 mL/min) for 3 hours did not increase Panx1 expression in WT mIMCD3 cells compared to the static condition. Flow did not increase the expression of any of the known purinergic receptors in the collecting duct cells compared to the static condition. We did observe a two‐fold increase in gene expression of the putative ATP release channel connexin‐30.3 (CX30.3) compared to static conditions. Moreover, we also report a three‐fold increase in CX30.3 gene expression in Pkd1‐/‐ mIMCD3 cells compared to WT cells.In conclusion, we show here that flow‐induced purinergic signaling in the collecting duct greatly differs from previously published work on the distal convoluted tubule. Specifically, in the inner medulla of the collecting duct, PANX1 may not be the key flow‐regulated ATP release channel. Instead, CX30.3 may facilitate flow‐induced ATP release in this segment in health and in ADPKD.