Single-molecule labeling for studying trafficking of renal transporters

The ability to detect and track single molecules presents the advantage of visualizing the complex behavior of transmembrane proteins with a time and space resolution that would otherwise be lost with traditional labeling and biochemical techniques. Development of new imaging probes has provided a robust method to study their trafficking and surface dynamics. This mini-review focuses on the current technology available for single-molecule labeling of transmembrane proteins, their advantages, and limitations. We also discuss the application of these techniques to the study of renal transporter trafficking in light of recent research

Exaggerated salt-sensitive hypertension in the ALMS1 (alstrom syndrome 1) knockout rat

We recently found that a protein named ALMS1 (Alstrom syndrome 1) is expressed in the kidney thick ascending limb (TAL) where it mediates endocytosis of the renal Na/K/2Cl cotransporter termed NKCC2. To study the role of ALMS1 in renal physiology we generated ALMS1 knockout (KO) rats in a Dahl salt-sensitive genetic background via zinc-finger nuclease targeting. We previously found that the amount of NKCC2 in the apical surface is higher in the TALs from ALMS1 KO rats compared to WT- Salt-Sensitive (SS) rats. In order to determine the role of NKCC2 on blood pressure (BP) we utilized both noninvasive tail cuff measurements and invasive radio-telemetry to study the effects of dietary sodium (.22% Na chow and 4% Na chow) on the systolic blood pressure (SBP) of the ALMS1 KO rats and WT-SS rats. We hypothesized that deletion of the ALMS1 gene will increase SBP and enhance salt-sensitivity of BP, in part due to higher NKCC2-mediated Na reabsorption. First, we used tail cuff measurements to obtain the SBP in both groups of rats. We found that with normal Na intake (0.22% Na chow), the ALMS1 KO rats had a higher SBP than the WT-SS rats (KO: 136±3 and WT-SS: 125±3 mmHg, p=0.0461). To further explore the salt-sensitive response of these rats we utilized radio-telemetry monitoring for 4 weeks. We found the ALMS1 KO rats to have a higher baseline SBP on .22% Na chow compared to the WT-SS rats (KO: 145±2 and WT-SS: 134±1 mmHg, p=0.0009). After 2 weeks of high Na intake (4% Na chow) the SBP in ALMS1 KO rats increased to 181±1 mmHg, a 35±3 mmHg increase, whereas the SBP increased to 159±2 mmHg in WT-SS rats, a 25±1 mmHg increase (p\u3c0.01 vs ALMS1 KO). Thus, the SBP was higher in ALMS1 KO rats fed a high salt diet (p=0.0001). We then explored the involvement of NKCC2 in the hypertension observed in ALMS1 KO rats. A daily dose of the loop diuretic bumetanide (3mg/kg), an NKCC2 inhibitor, decreased the SBP in both groups (KO: -23±4 and WT: -30±6 mmHg, p=0.40). After 7 days of treatment with bumetanide, the SBP was normalized only in the WT-SS rats while the SBP remained elevated in the ALMS1 KO rats (KO: 131±3 and WT: 115±4 mmHg, p\u3c0.025). Therefore, we conclude that the ALMS1 KO rats have a higher salt-sensitivity of BP than the WT- (Dahl) SS rats and this in part is mediated by enhanced renal sodium reabsorption in the TAL. Furthermore, our data suggest that additional mechanisms are likely involved in the hypertension observed in ALMS1 KO rats fed a high salt diet

Real-time monitoring of NKCC2 endocytosis by total internal reflection fluorescence (TIRF) microscopy

The apical Na-K-2Cl cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb (TAL). The amount of NKCC2 at the apical membrane of TAL cells is determined by exocytic delivery, recycling, and endocytosis. Surface biotinylation allows measurement of NKCC2 endocytosis, but it has low time resolution and does not allow imaging of the dynamic process of endocytosis. We hypothesized that total internal reflection fluorescence (TIRF) microscopy imaging of labeled NKCC2 would allow monitoring of NKCC2 endocytosis in polarized Madin-Darby canine kidney (MDCK) and TAL cells. Thus we generated a NKCC2 construct containing a biotin acceptor domain (BAD) sequence between the transmembrane domains 5 and 6. Once expressed in polarized MDCK or TAL cells, surface NKCC2 was specifically biotinylated by exogenous biotin ligase (BirA). We also demonstrate that expression of a secretory form of BirA in TAL cells induces metabolic biotinylation of NKCC2. Labeling biotinylated surface NKCC2 with fluorescent streptavidin showed that most apical NKCC2 was located within small discrete domains or clusters referred to as puncta on the TIRF field. NKCC2 puncta were observed to disappear from the TIRF field, indicating an endocytic event which led to a decrease in the number of surface puncta at a rate of 1.18 ± 0.16%/min in MDCK cells, and a rate 1.09 ± 0.08%/min in TAL cells (n = 5). Treating cells with a cholesterol-chelating agent (methyl-β-cyclodextrin) completely blocked NKCC2 endocytosis. We conclude that TIRF microscopy of labeled NKCC2 allows the dynamic imaging of individual endocytic events at the apical membrane of TAL cells

Deletion of ALMS1 (alstrom syndrome 1) enhances salt-sensitive hypertension, and induces insulin resistance and obesity in rats

The Na/K/2Cl cotransporter NKCC2 mediates NaCl absorption by the Thick Ascending Limb (TAL). Increased NKCC2 activity and apical trafficking are associated with salt sensitive hypertension in rodents and humans. A 150 amino acid region in the carboxyl terminus of NKCC2 (C-NKCC2) was shown to be important for apical targeting of NKCC2. We hypothesized that proteins which bind to C-NKCC2 play a role in regulating NKCC2 trafficking and activity. Using a targeted proteomics approach to screen TAL proteins that bind C-NKCC2, we identified Alstrom syndrome 1 (ALMS1) as an interacting partner. The ALMS1 gene has been linked to hypertension and renal function in Genome Wide Association Studies. However its function in the kidney is not known. To study the role of ALMS1 we obtained ALMS1 KO rats in collaboration with the Genome Editing Rat Resource Consortium at MCW. We found that in TALs from ALMS1 KO rats, surface NKCC2 was higher due to lower rates of NKCC2 endocytosis. Thus, we hypothesized that deletion of ALMS1 would increase NKCC2-mediated NaCl reabsorption, and induce salt sensitive hypertension. To study NKCC2-mediated Na transport in vivo, we measured bumetanide-induced natriuresis and diuresis. ALMS1 KO rats exhibited higher bumetanide-induced natriuresis (ALMS: 1292 ± 65 vs WT: 564 ± 31 μmoles/8 hr, p\u3c0.01, n=5) and diuresis (ALMS1: 3.1 ± 0.32 vs WT: 1.6 ± 0.13 ml/8 hr, p\u3c0.05, n=5), indicative of higher TAL NaCl reabsorption. On a normal salt diet (0.22% Na in chow) and tap water, ALMS1 KO had a higher systolic blood pressure and were hypertensive (ALMS1: 147 ± 4 vs WT: 127 ± 5 mmHg, p\u3c 0.02, n=6). Upon addition of 0.5% NaCl in drinking water for 6 days, the systolic blood pressure in ALMS1 KO rats increased in ALMS1 KO rats but not in WT rats (ΔSBP ALMS1: 12.8 ± 5 vs ΔSBP WT: 4.3 ± 7.2 mmHg, p\u3c0.05, n=6). The hypertension and salt sensitivity appears to be independent of the renin angiotensin system because plasma renin activity was lower in ALMS1 KO rats compared to WT rats (ALMS1: 1.3 ± 0.3 vs WT: 2 ± 0.3 ng/ml/hr, p\u3c0.05, n=6). However, we also found that at 3 months of age ALMS1 KO rats developed obesity (Body weight ALMS1: 437.2 ± 8 vs WT: 359.5 ± 5 g, p\u3c0.01, n=5) and insulin resistance evidenced by elevated plasma insulin levels (ALMS1: 13 ± 5 vs WT: 1.8 ± 1.2 ng/ml, p\u3c0.05, n=6) and glucose levels after fasting (ALMS1: 108 ± 20 vs WT: 61 ± 5 mg/dl, p\u3c0.05, n=6). Combined, these data indicate that ALMS1 KO rats are hypertensive and more salt sensitive than WT controls. The mechanism causing salt-sensitive hypertension in ALMS1 KO rats may in part be due to higher TAL NaCl reabsorption and higher NKCC2 activity. However, it is possible that insulin resistance and obesity that develops in these ALMS1 KO rats may play a role in the salt sensitivity. Thus, ALMS1 is not only important for renal function but may also be involved in the regulation of glucose homeostasis and metabolism in rats

Real-time monitoring of NKCC2 endocytosis by total internal reflection fluorescence (TIRF) microscopy

The apical Na-K-2Cl cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb (TAL). The amount of NKCC2 at the apical membrane of TAL cells is determined by exocytic delivery, recycling, and endocytosis. Surface biotinylation allows measurement of NKCC2 endocytosis, but it has low time resolution and does not allow imaging of the dynamic process of endocytosis. We hypothesized that total internal reflection fluorescence (TIRF) microscopy imaging of labeled NKCC2 would allow monitoring of NKCC2 endocytosis in polarized Madin-Darby canine kidney (MDCK) and TAL cells. Thus we generated a NKCC2 construct containing a biotin acceptor domain (BAD) sequence between the transmembrane domains 5 and 6. Once expressed in polarized MDCK or TAL cells, surface NKCC2 was specifically biotinylated by exogenous biotin ligase (BirA). We also demonstrate that expression of a secretory form of BirA in TAL cells induces metabolic biotinylation of NKCC2. Labeling biotinylated surface NKCC2 with fluorescent streptavidin showed that most apical NKCC2 was located within small discrete domains or clusters referred to as puncta on the TIRF field. NKCC2 puncta were observed to disappear from the TIRF field, indicating an endocytic event which led to a decrease in the number of surface puncta at a rate of 1.18 ± 0.16%/min in MDCK cells, and a rate 1.09 ± 0.08%/min in TAL cells (n = 5). Treating cells with a cholesterol-chelating agent (methyl-β-cyclodextrin) completely blocked NKCC2 endocytosis. We conclude that TIRF microscopy of labeled NKCC2 allows the dynamic imaging of individual endocytic events at the apical membrane of TAL cells

Role of Alström syndrome 1 in the regulation of blood pressure and renal function

Elevated blood pressure (BP) and renal dysfunction are complex traits representing major global health problems. Single nucleotide polymorphisms identified by genome-wide association studies have identified the Alström syndrome 1 (ALMS1) gene locus to render susceptibility for renal dysfunction, hypertension, and chronic kidney disease (CKD). Mutations in the ALMS1 gene in humans causes Alström syndrome, characterized by progressive metabolic alterations including hypertension and CKD. Despite compelling genetic evidence, the underlying biological mechanism by which mutations in the ALMS1 gene lead to the above-mentioned pathophysiology is not understood. We modeled this effect in a KO rat model and showed that ALMS1 genetic deletion leads to hypertension. We demonstrate that the link between ALMS1 and hypertension involves the activation of the renal Na+/K+/2Cl- cotransporter NKCC2, mediated by regulation of its endocytosis. Our findings establish a link between the genetic susceptibility to hypertension, CKD, and the expression of ALMS1 through its role in a salt-reabsorbing tubular segment of the kidney. These data point to ALMS1 as a potentially novel gene involved in BP and renal function regulation

Rare pathogenic variants in WNK3 cause X-linked intellectual disability

Purpose: WNK3 kinase (PRKWNK3) has been implicated in the development and function of the brain via its regulation of the cation-chloride cotransporters, but the role of WNK3 in human development is unknown. Method: We ascertained exome or genome sequences of individuals with rare familial or sporadic forms of intellectual disability (ID). Results: We identified a total of 6 different maternally-inherited, hemizygous, 3 loss-of-function or 3 pathogenic missense variants (p.Pro204Arg, p.Leu300Ser, p.Glu607Val) in WNK3 in 14 male individuals from 6 unrelated families. Affected individuals had ID with variable presence of epilepsy and structural brain defects. WNK3 variants cosegregated with the disease in 3 different families with multiple affected individuals. This included 1 large family previously diagnosed with X-linked Prieto syndrome. WNK3 pathogenic missense variants localize to the catalytic domain and impede the inhibitory phosphorylation of the neuronal-specific chloride cotransporter KCC2 at threonine 1007, a site critically regulated during the development of synaptic inhibition. Conclusion: Pathogenic WNK3 variants cause a rare form of human X-linked ID with variable epilepsy and structural brain abnormalities and implicate impaired phospho-regulation of KCC2 as a pathogenic mechanism.</p