Final Report Summary - NOVEL KIDNEY MODEL (zebrafish model to study human hypertension)
The proposal was designed to develop a novel model for studying how WNK and SPAK/OSR1 kinases regulate the thiazide-sensitive sodium chloride transporter (Slc12A3; NCC) in the nephron. We largely succeeded in accomplishing this goal. We had developed a novel antibody directed toward the zebrafish homologue of the thiazide-sensitive NaCl cotransporter, Slc12a3. We showed that this antibody worked well by both immunofluorescence and western blot, with clear identification of the late distal tubule in zebrafish pronephros. We also confirmed that Slc12a3 is activated by phosphorylation along its amino terminus, just like mammalian SLC12A3 is. We had shown that knocking down Slc12a3 led to developmental abnormalities in the fish. Here, we confirmed that knocking down WNK kinase led to premature mortality and misshapenness. We showed that this effect was specific, as these defects could be rescued by with WNK4 RNA. Placing zebrafish in brackish water, resembling normal pondwater in some situations, led to changes in the expression of Slc12a3. These experiments were planned to be continued with S. Seyfried, then at the Max Delbrück Center, and in S. Bachmann’s laboratory to complete them. During the course of the year, however, Dr. Seyfried was moving to another institution, slowing progress on this project. Nevertheless, we expect to publish our results in the near future.
We then became aware of a paper (PA 465:1409-21/2013) showing some of the enviseaged work including distal transport protein function. We therefore decided to slightly modify the goals and develop tools that can be used in both zebrafish and other models, to study NCC regulation. So we then continued in parallel with a mouse study to see how the thiazide-sensitive Na-Cl cotransporter (NCC, SLC12A3) is regulated by dietary potassium intake and how this affects blood pressure. The mouse experiments were conducted under the authorization number G0 190/12 of the Berlin Senate. During the past 12 months, we have made substantial progress towards achieving these aims. We have observed that activation of NCC leads WNK4 and SPAK to associate in characteristic punctal structures in the distal convoluted tubule in mice. The nature of these structures is currently undefined. We have been working to identify the nature of these puncta, using immunofluorescence, immunogold, and immunoprecipitation. We have made substantial progress in their identification, but at this time, the results have not been finalized. We will work assiduously during the next 6 months to complete their identification and publish the results. We are furthermore working to develop a light activated SPAK, so that we can use the cryo electron microscopy expertise of the Bachmann laboratory to show how activation of this kinase actually causes activation of NCC, and whether this process occurs predominantly within the cytoplasm or at the plasma membrane. These studies are still ongoing as well.
We then became aware of a paper (PA 465:1409-21/2013) showing some of the enviseaged work including distal transport protein function. We therefore decided to slightly modify the goals and develop tools that can be used in both zebrafish and other models, to study NCC regulation. So we then continued in parallel with a mouse study to see how the thiazide-sensitive Na-Cl cotransporter (NCC, SLC12A3) is regulated by dietary potassium intake and how this affects blood pressure. The mouse experiments were conducted under the authorization number G0 190/12 of the Berlin Senate. During the past 12 months, we have made substantial progress towards achieving these aims. We have observed that activation of NCC leads WNK4 and SPAK to associate in characteristic punctal structures in the distal convoluted tubule in mice. The nature of these structures is currently undefined. We have been working to identify the nature of these puncta, using immunofluorescence, immunogold, and immunoprecipitation. We have made substantial progress in their identification, but at this time, the results have not been finalized. We will work assiduously during the next 6 months to complete their identification and publish the results. We are furthermore working to develop a light activated SPAK, so that we can use the cryo electron microscopy expertise of the Bachmann laboratory to show how activation of this kinase actually causes activation of NCC, and whether this process occurs predominantly within the cytoplasm or at the plasma membrane. These studies are still ongoing as well.