AB3562P Sigma-AldrichAnti-Na+/K+/Cl- Cotransporter 2 Antibody

The Na-K-2Cl cotransporter 2 (NKCC2) was thought to be kidney specific. Here we show expression in the brain hypothalamo-neurohypophyseal system (HNS), wherein upregulation follows osmotic stress. The HNS controls osmotic stability through the synthesis and release of the neuropeptide hormone, arginine vasopressin (AVP). AVP travels through the bloodstream to the kidney, where it promotes water conservation. Knockdown of HNS NKCC2 elicited profound effects on fluid balance following ingestion of a high-salt solution-rats produced significantly more urine, concomitant with increases in fluid intake and plasma osmolality. Since NKCC2 is the molecular target of the loop diuretics bumetanide and furosemide, we asked about their effects on HNS function following disturbed water balance. Dehydration-evoked GABA-mediated excitation of AVP neurons was reversed by bumetanide, and furosemide blocked AVP release, both in vivo and in hypothalamic explants. Thus, NKCC2-dependent brain mechanisms that regulate osmotic stability are disrupted by loop diuretics in rats.

Uromodulin (Tamm-Horsfall protein) is the most abundant protein excreted in the urine under physiological conditions. It is exclusively produced in the kidney and secreted into the urine via proteolytic cleavage. The involvement of UMOD, the gene that encodes uromodulin, in rare autosomal dominant diseases, and its robust genome-wide association with the risk of chronic kidney disease suggest that the level of uromodulin in urine could represent a critical biomarker for kidney function. The structure of uromodulin is complex, with multiple disulfide bonds and typical domains of extracellular proteins.Thus far, the conditions influencing stability and measurement of uromodulin in human urine have not been systematically investigated, giving inconsistent results. In this study, we used a robust, in-house ELISA to characterize the conditions of sampling and storage necessary to provide a faithful dosage of uromodulin in the urine.The levels of uromodulin in human urine were significantly affected by centrifugation and vortexing, as well as by the conditions and duration of storage.These results validate a simple, low-cost ELISA and document the optimal conditions of processing and storage for measuring uromodulin in human urine.

Hypertension and chronic kidney disease (CKD) are complex traits representing major global health problems. Multiple genome-wide association studies have identified common variants in the promoter of the UMOD gene, which encodes uromodulin, the major protein secreted in normal urine, that cause independent susceptibility to CKD and hypertension. Despite compelling genetic evidence for the association between UMOD risk variants and disease susceptibility in the general population, the underlying biological mechanism is not understood. Here, we demonstrate that UMOD risk variants increased UMOD expression in vitro and in vivo. Uromodulin overexpression in transgenic mice led to salt-sensitive hypertension and to the presence of age-dependent renal lesions similar to those observed in elderly individuals homozygous for UMOD promoter risk variants. The link between uromodulin and hypertension is due to activation of the renal sodium cotransporter NKCC2. We demonstrated the relevance of this mechanism in humans by showing that pharmacological inhibition of NKCC2 was more effective in lowering blood pressure in hypertensive patients who are homozygous for UMOD promoter risk variants than in other hypertensive patients. Our findings link genetic susceptibility to hypertension and CKD to the level of uromodulin expression and uromodulin's effect on salt reabsorption in the kidney. These findings point to uromodulin as a therapeutic target for lowering blood pressure and preserving renal function.

The mechanisms in the retina that generate light responses selective for the direction of image motion remain unresolved. Recent evidence indicates that directionally selective light responses occur first in the retina in the dendrites of an interneuron, i.e., the starburst amacrine cell, and that these responses are highly sensitive to the activity of Na-K-2Cl (NKCC) and K-Cl (KCC), two types of chloride cotransporter that determine whether the neurotransmitter GABA depolarizes or hyperpolarizes neurons, respectively. We show here that selective blockade of the NKCC2 and KCC2 cotransporters located on starburst dendrites consistently hyperpolarized and depolarized the starburst cells, respectively, and greatly reduced or eliminated their directionally selective light responses. By mapping NKCC2 and KCC2 antibody staining on these dendrites, we further show that NKCC2 and KCC2 are preferentially located in the proximal and distal dendritic compartments, respectively. Finally, measurements of the GABA reversal potential in different starburst dendritic compartments indicate that the GABA reversal potential at the distal dendrite is more hyperpolarized than at the proximal dendrite due to KCC2 activity. These results thus demonstrate that the differential distribution of NKCC2 on the proximal dendrites and KCC2 on the distal dendrites of starburst cells results in a GABA-evoked depolarization and hyperpolarization at the NKCC2 and KCC2 compartments, respectively, and underlies the directionally selective light responses of the dendrites. The functional compartmentalization of interneuron dendrites may be an important means by which the nervous system encodes complex information at the subcellular level.