Acute pyelonephritis: Increased plasma membrane targeting of renal aquaporin‐2

Abstract

Aim: Aquaporin-2 (AQP2) shuttling between intracellular vesicles and the apical plasma membrane is pivotal in arginine vasopressin-mediated urine concentration and is dysregulated in multiple diseases associated with water balance disorders. Children and adults with acute pyelonephritis have a urinary concentration defect and studies in children revealed increased AQP2 excretion in the urine.This study aimed to analyse AQP2 trafficking in response to acute pyelonephritis.Methods: Immunofluorescence analysis was used to evaluate subcellular localization of AQP2 and AQP2-S256A (mimicking non-phosphorylated AQP2 on serine 256) in cells stimulated with bacterial lysates and of AQP2 and pS256-AQP2 in a mouse model at day 5 of acute pyelonephritis. Western blotting was used to evaluate AQP2 levels and AQP2 phosphorylation on S256 upon incubation with bacterial lysates. Time-lapse imaging was used to measure intracellular cAMP levels in response to incubation with bacterial lysates. Results:In cell cultures, lysates from both uropathogenic and nonpathogenic bacteria-mediated AQP2 plasma membrane targeting and increased AQP2 phosphorylation on serine 256 (pS256) without increasing cAMP levels. Both bacterial lysates induced plasma membrane targeting of AQP2-S256A.Immunofluorescence analysis of renal sections from mice after 5 days of acute pyelonephritis revealed apical plasma membrane targeting of AQP2 and pS256-AQP2 in inner medullary collecting ducts. Conclusion:Uropathogenic bacteria induce AQP2 plasma membrane targeting in vitro and in vivo. cAMP levels were not elevated by the bacterial lysates and AQP2 plasma membrane targeting could occur without S256 phosphorylation. This may explain increased AQP2 excretion in the urine during acute pyelonephritis.

EP1 receptor antagonism mitigates early and late stage renal fibrosis

“…For immunofluorescent labelling of paraffin sections for EP 1 and markers for TAL and CDs, labelling and imaging was carried our as previously described 48 . In brief, 2 µm paraffin sections were deparaffinized overnight in xylene and subsequently rehydrated.…”

“…For immunofluorescent labelling of paraffin sections for EP 1 and markers for TAL and CDs, labelling and imaging was carried our as previously described. 48 In brief, 2 µm paraffin sections were deparaffinized overnight in xylene and subsequently rehydrated. Antigens were retrieved by boiling in TEG buffer (10 mM Tris pH 9, 0.5 mM EGTA) and aldehyde groups quenched by incubation in 50 mM NH4Cl in PBS for 30 minutes.…”

Multiplex immunofluorescence staining of coverslip‐mounted paraffin‐embedded tissue sections

“…Finally, the sections were washed three times in PBS. For imaging, the coverslips were placed in a Chamlide Magnetic Chamber (Live Cell Instrument Co, Republic of Korea) and filled with PBS as for live‐cell imaging (albeit here cell culture media is used instead of PBS) [13].…”

Monomeric α‐synuclein activates the plasma membrane calcium pump

“…The system was controlled by NIS Elements software from Nikon. Imaging was performed similar to previously described (Ernstsen et al , 2022). Images were acquired every second for 8 min in total.…”