The Urinary Excretion of Uromodulin is Regulated by the Potassium Channel ROMK

Abstract

Uromodulin, the most abundant protein in normal urine, is produced by cells lining the thick ascending limb (TAL) of the loop of Henle. Uromodulin regulates the activity of the potassium channel ROMK in TAL cells. Common variants in KCNJ1, the gene encoding ROMK, are associated with urinary levels of uromodulin in population studies. Here, we investigated the functional link between ROMK and uromodulin in Kcnj1 knock-out mouse models, in primary cultures of mouse TAL (mTAL) cells, and in patients with Bartter syndrome due to KCNJ1 mutations. Both global and kidney-specific Kcnj1 knock-out mice showed reduced urinary levels of uromodulin paralleled by increased levels in the kidney, compared to wild-type controls. Pharmacological inhibition and genetic deletion of ROMK in mTAL cells caused a reduction in apical uromodulin excretion, reflected by cellular accumulation. In contrast, NKCC2 inhibition showed no effect on uromodulin processing. Patients with Bartter syndrome type 2 showed reduced urinary uromodulin levels compared to age and gender matched controls. These results demonstrate that ROMK directly regulates processing and release of uromodulin by TAL cells, independently from NKCC2. They support the functional link between transport activity and uromodulin in the TAL, relevant for blood pressure control and urinary concentrating ability.

SeqStain using fluorescent-DNA conjugated antibodies allows efficient, multiplexed, spatialomic profiling of human and murine tissues

“…Composites of pseudocolored images revealed a predominant presence of proximal tubules (AQP1) consistent with the histology of renal cortex (Figure 8A) (64,65). We were also able to identify the collecting duct (AQP2+ AQP3+ EpCAM+) (Cyto7+ Cyto8+) ( Figure 8A and 8C) (66)(67)(68), Distal convoluted tubules (AQP2-AQP3-EpCAM+),…”

“…Composites of pseudocolored images revealed a predominant presence of proximal tubules (AQP1) consistent with the histology of renal cortex ( Figure 8A ) (64, 65). We were also able to identify the collecting duct (AQP2+ AQP3+ EpCAM+) (Cyto7+ Cyto8+) ( Figure 8A and 8C ) (66–68), Distal convoluted tubules (AQP2-AQP3-EpCAM+), Thick Ascending Loop of Henle’s (EpCAM+ Uromodulin+) and their spatial location with respect to one another ( Figure 8A ) (69). Similarly, in the glomerulus, we were able to discern the three components of the glomerular capillary filter-podocytes, glomerular basement membrane and glomerular endothelial cells (69).…”

SeqStain is an efficient method for multiplexed, spatialomic profiling of human and murine tissues

“…Composites of pseudocolored images revealed a predominant presence of proximal tubules (AQP1) consistent with the histology of renal cortex ( Figure 7B ) ( Nielsen et al, 2002 ; Singh et al, 2019 ). We were also able to identify the collecting duct (AQP2 + AQP3 + EpCAM + Cyto7 + Cyto8 + ) ( Hatta et al, 1987 ; Schiano et al, 2019 ; Skinnider et al, 2005 ), Distal convoluted tubules (AQP2 − AQP3 − EpCAM + Cyto8 + UMOD − ), thick ascending loop of Henle (EpCAM + uromodulin + Na + ,K + -ATPase + AQP2 − AQP3 − ), and their spatial location with respect to one another ( Figures 7B – 7E ) ( Fissell and Miner, 2018 ). Similarly, in the glomerulus we were able to discern the three components of the glomerular capillary filter: podocytes (WT1 + and vimentin + ), glomerular basement membrane (collagen IV + ), and glomerular endothelial cells (CD31 + ) ( Fissell and Miner, 2018 ).…”

Molecular Basis, Diagnostic Challenges and Therapeutic Approaches of Bartter and Gitelman Syndromes: A Primer for Clinicians

“…Due to the strict correlation and functional of the transporters in the TAL, the loss of function of the ROMK channels (BS type 2) could lead to the inactivation of NKCC2 (BS type 1) [ 57 ], which could justify the overlapping phenotype in both types. Furthermore, an interaction of both channels has also been demonstrated in the secretion of uromodulin protein [ 58 , 59 ], which is decreased in patients with type 2 Bartter [ 59 ]. Thus, these results point to a possible role for uromodulin in tubular disorders.…”