The nephron in the kidney is the main site for extensive solute exchange in mammals. The kidneys are highly complex organs and their primary purpose is in homeostasis of small organic solutes and minerals in the body. Most solutes are absorbed from the blood and residual products are excreted as urine. The proximal tubule of the nephron absorbs the majority of the small inorganic solutes, through members of the solute carrier (SLC) transporters in the membranes. The exchange of solutes and the regulation of osmotic pressure and acid/base homeostasis in the nephron is thus of vital importance for animal life.
Already in ancient Greece physicians had noticed small “stones” in the kidneys. Formation of kidney stones is one of the oldest known human disorders. The medical condition is called nephrolithiasis or urolithiasis and is cased by the formation of inorganic crystals usually formed from, calcium, phosphate/oxalate and uric acids (Pak, 1998). A number of diseases have been attributed to the malfunction of anion transporters. Inorganic sulfate is amongst the most abundant anions present in the mammalian plasma (Bolt et al, 2004). Sulfate is involved in activating and detoxifying xenobiotics, neurotransmitters, and bile acids. The sulfate ion is essential for the biosynthesis of glycosaminoglycans, cerebroside sulfate and the aminoacids cysteine and methionine. Undersulfation in the cartilage proteoglycans has been associated with human inherited osteochondrodysplasia disorders (Rossi et al, 1996, Thiele et al, 2004). In mammals, the sulfate homeostasis is regulated primarily by the kidney. The majority of sulfate is reabsorbed in the proximal tubules, only up to 20% is secreted through the urine (BECKER et al, 1960).
To understand and study acid/base balance we identify and characterize both soluble and membrane bound protein involved with pH regulation, we use a bioinformatic approach initially to identify new possible protein targets or interaction partners. We perform a broad cloning strategy into both eukaryote and bacterial expression systems to search for soluble protein. We aim to purify and characterize members of the SLC4 and SLC26 family in vitro. We are developing purification and lipid vesicle reconstitution protocols for the membrane proteins to study their atomic three-dimensional structure. We use X-ray crystallography, small angle X-ray scattering (SAXS) to obtain the structural information. We also make use of a number of biophysical and biochemical which include activity assays and isothermal titration calorimetry (ITC) for protein-protein binding or to test for small molecule interactions. With these methods we hope to move closer to the both structural models and more functional aspects of acid/base homeostasis. The projects regarding NCBE and NBCn1 are carried out in collaboration with Prof. Christian Aalkjaer, Institute of Physiology, Aarhus, Denmark) and Prof. Jeppe Praetorius, Institute of Anatomy, Aarhus, Denmark .
BECKER EL, HEINEMANN HO, IGARASHI K, HODLER JE, & GERSHBERG H (1960) Renal mechanisms for the excretion of inorganic sulfate in man. J Clin Invest 39 : 1909-1913
Bolt MJ, Liu W, Qiao G, Kong J, Zheng W, Krausz T, Cs-Szabo G, Sitrin MD, & Li YC (2004) Critical role of vitamin D in sulfate homeostasis: regulation of the sodium-sulfate cotransporter by 1,25-dihydroxyvitamin D3. Am J Physiol Endocrinol Metab 287 : E744-9
Pak CY (1998) Kidney stones. Lancet 351 : 1797-1801
Rossi A, Bonaventure J, Delezoide AL, Cetta G, & Superti-Furga A (1996) Undersulfation of proteoglycans synthesized by chondrocytes from a patient with achondrogenesis type 1B homozygous for an L483P substitution in the diastrophic dysplasia sulfate transporter. J Biol Chem 271 : 18456-18464
Thiele H, Sakano M, Kitagawa H, Sugahara K, Rajab A, Hohne W, Ritter H, Leschik G, Nurnberg P, & Mundlos S (2004) Loss of chondroitin 6-O-sulfotransferase-1 function results in severe human chondrodysplasia with progressive spinal involvement. Proc Natl Acad Sci U S A 101 : 10155-10160