Die nukleare Translokation des Kalziumbindenden-Proteins Calbindin-1 und dessen Rolle bei der Kalziumhomöostase in den distalen Tubuluszellen der Niere.

The nuclear translocation of the calcium binding protein calbindin-1 and its role in the calcium-homeostasis in kidney distal tubule cells Calcium (Ca2+) plays a key role in many physiological processes like hormone secretion, muscle contraction, bone formation, nerve conduction, exocytosis, and (in)activation of enzymes. The body Ca2+ homeostasis is tightly regulated and disturbances in its balance may lead to pathogenesis. Kidney especially renal epithelium as the site of Ca2+ reabsorption plays an important role in the body Ca2+ homeostasis. The Ca2+-reabsorption in the kidney can be paracellular or transcellular. The latest takes place in distal convoluted tubule (DCT) cells and is regulated by PTH and VD3 [1,2], and plays a role in the fine-tuning of whole-body calcium homeostasis. Increase in cytosolic calcium concentration has been associated with cellular dysfunctions in a variety of conditions such as diabetes mellitus, hypertension, hyperparathyroidism and chronic kidney diseases (CKD) [3,4]. In DCT, Ca2+ enters cells across the apical part through the Ca2+ selective channel TRPV5, then binds to calbindin-1 (Calb-1), the major intracellular calcium binding proteins in DCT, and diffuses to the basolateral membrane, where Ca2+ is extruded via the Na/Ca exchanger (Ncx1) or plasma membrane calcium-transporting ATPase 1 (PMCA1) [1]. In this process of transcellular Ca2+ reabsorption, the Calb1 plays a key role Calb-1 as an intracellular Ca2+- buffer and -transit protein from the apical side to the basolateral side without significant modification in the intracellular Ca2+ [1]. Our mice model for chronic kidney disease (Calreticulin heterozygous mice, Calr+/-) suffers from disturbance in Ca2+-homeostasis and cellular calcium toxicity. Histoimmunological investigations of kidney tissues revealed a nuclear translocation of Calb-1 in the DCT-cells from Calr+/- mice. Our preliminary observations shed light on potential still unknown function of Calb-1 in DCT-cells. The aim of the present study is to clear the role, if there is any, of the nuclear translocation of Calb-1 in DCT-cells. The following questions will be addressed: 1. Does Calbindin-1 undergo a nuclear translocation and which conditions are responsible of the cellular compartmental translocation of Calb-1? For this purpose, renal tissue form Calr+/- and Calr+/+ will be stained with antibody against Calb-1 and confocal microscopy analysis will be performed to confirm the nuclear localization of Calb-1 in Calr+/- kidney DCT-cells. Furthermore, established DCT-cell lines (MDCK and MDBK) will be used to investigated the conditions of the nuclear translocation of Calb-1. 2. Does the nuclear translocation of Calb-1 have any impact on its role in Ca2+-buffering and transition in DCT-cells? DCT-cell lines will be subjected to conditions that trigger the Cabl-1 nuclear translocation and the cytosolic free Ca2+ concentration will be investigated using Fura-2 as the indicator of intracellular calcium concentration. Furthermore, similar experiments will be performed under condition of Calb-1 overexpression (plasmid) or down-regulation (siRNA). 3. Which role play the nuclear Calb-1? Does the nuclear Calb-1 plays any role in gene expression regulation?