Early Molecular Events Mediating Loss of Aquaporin-2 during Ureteral Obstruction in Rats.

BACKGROUND: Ureteral obstruction is marked by disappearance of the vasopressin-dependent water channel aquaporin-2 (AQP2) in the renal collecting duct and polyuria upon reversal. Most studies of unilateral ureteral obstruction (UUO) models have examined late time points, obscuring the early signals that trigger loss of AQP2. METHODS: We performed RNA-Seq on microdissected rat cortical collecting ducts (CCDs) to identify early signaling pathways after establishment of UUO. RESULTS: Vasopressin V2 receptor (AVPR2) mRNA was decreased 3 hours after UUO, identifying one cause of AQP2 loss. Collecting duct principal cell differentiation markers were lost, including many not regulated by vasopressin. Immediate early genes in CCDs were widely induced 3 hours after UUO, including Myc, Atf3, and Fos (confirmed at the protein level). Simultaneously, expression of NF-κB signaling response genes known to repress Aqp2 increased. RNA-Seq for CCDs at an even earlier time point (30 minutes) showed widespread mRNA loss, indicating a "stunned" profile. Immunocytochemical labeling of markers of mRNA-degrading P-bodies DDX6 and 4E-T indicated an increase in P-body formation within 30 minutes. CONCLUSIONS: Immediately after establishment of UUO, collecting ducts manifest a stunned state with broad disappearance of mRNAs. Within 3 hours, there is upregulation of immediate early and inflammatory genes and disappearance of the V2 vasopressin receptor, resulting in loss of AQP2 (confirmed by lipopolysaccharide administration). The inflammatory response seen rapidly after UUO establishment may be relevant to both UUO-induced polyuria and long-term development of fibrosis in UUO kidneys.

Urinary Extracellular Vesicles for Renal Tubular Transporters Expression in Patients With Gitelman Syndrome.

Background: The utility of urinary extracellular vesicles (uEVs) to faithfully represent the changes of renal tubular protein expression remains unclear. We aimed to evaluate renal tubular sodium (Na+) or potassium (K+) associated transporters expression from uEVs and kidney tissues in patients with Gitelman syndrome (GS) caused by inactivating mutations in SLC12A3. Methods: uEVs were isolated by ultracentrifugation from 10 genetically-confirmed GS patients. Membrane transporters including Na+-hydrogen exchanger 3 (NHE3), Na+/K+/2Cl- cotransporter (NKCC2), NaCl cotransporter (NCC), phosphorylated NCC (p-NCC), epithelial Na+ channel ß (ENaCß), pendrin, renal outer medullary K1 channel (ROMK), and large-conductance, voltage-activated and Ca2+-sensitive K+ channel (Maxi-K) were examined by immunoblotting of uEVs and immunofluorescence of biopsied kidney tissues. Healthy and disease (bulimic patients) controls were also enrolled. Results: Characterization of uEVs was confirmed by nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting. Compared with healthy controls, uEVs from GS patients showed NCC and p-NCC abundance were markedly attenuated but NHE3, ENaCß, and pendrin abundance significantly increased. ROMK and Maxi-K abundance were also significantly accentuated. Immunofluorescence of the representative kidney tissues from GS patients also demonstrated the similar findings to uEVs. uEVs from bulimic patients showed an increased abundance of NCC and p-NCC as well as NHE3, NKCC2, ENaCß, pendrin, ROMK and Maxi-K, akin to that in immunofluorescence of their kidney tissues. Conclusion: uEVs could be a non-invasive tool to diagnose and evaluate renal tubular transporter adaptation in patients with GS and may be applied to other renal tubular diseases.