Regulation of the key molecules of glomerular ultrafiltration in proteinuric models

...................................................................................................................................................................9 INTRODUCTION ...................................................................................................................................................... 11 REVIEW OF THE LITERATURE.......................................................................................................................... 12 1. Glomerulus as a unique structure for filtration of blood.................................................................................... 12 1.1 Development of the glomerular structures................................................................................................... 12 1.2 Endothelial cell layer .................................................................................................................................... 15 1.3 Glomerular basement membrane (GBM) .................................................................................................... 15 1.4 Epithelial cell layer and slit diaphragm (SD) .............................................................................................. 15 1.5 Mesangial cells ............................................................................................................................................. 16 2. Proteinuria............................................................................................................................................................ 17 2.1 Causes of proteinuria .................................................................................................................................... 18 2.2 Congenital nephrotic syndromes.................................................................................................................. 19 2.3 Congenital Nephrotic Syndrome of the Finnish type.................................................................................. 19 2.3.1 Clinical aspects ........................................................................................................................................................20 2.3.2 Pathology of CNF.....................................................................................................................................................20 2.3.3 Diagnosis and treatment ..........................................................................................................................................21 2.3.4 Genetics of CNF .......................................................................................................................................................22 2.4 Minimal change nephropathy....................................................................................................................... 22 3. Experimental models of proteinuria.................................................................................................................... 23 3.1 Puromycin aminonucleoside nephrosis........................................................................................................ 23 3.1.1 Morphological changes in PAN...............................................................................................................................24 3.1.2 Pathogenesis and molecular changes in PAN ........................................................................................................24 3.2 Mercuric chloride (HgCl2) induction ........................................................................................................... 25 3.3 Heymann nephritis........................................................................................................................................ 26 4. Molecular composition of the podocytes............................................................................................................ 27 4.1 Nephrin.......................................................................................................................................................... 27 4.2 CD2AP.......................................................................................................................................................... 28 4.3 Podocin.......................................................................................................................................................... 29 4.4 Alpha-actinin-4 ............................................................................................................................................. 29 4.5 Integrins ........................................................................................................................................................ 30 4.6 Dystroglycans ............................................................................................................................................... 30 5. RNA processing................................................................................................................................................... 32 5.1 Origin and processing of RNA splicing....................................................................................................... 32 5.2 Alternative splicing....................................................................................................................................... 34 AIMS OF THE STUDY ............................................................................................................................................. 39 MATERIALS AND METHODS .............................................................................................................................. 40 1. Animals ................................................................................................................................................................ 40 2. Antibodies ............................................................................................................................................................ 40 3. Primers ................................................................................................................................................................. 41 4. Methods................................................................................................................................................................ 42 4.1 Animal experiments...................................................................................................................................... 42 4.1.1 PAN with modification with HgCl2 and probucol (I-V) ..........................................................................................42 4.1.2 Induction of Heymann nephritis (II, V) ...................................................................................................................42 4.2 Quantitation of urinary albumin (I-V) ......................................................................................................... 42 4.3 Isolation of glomeruli of rat kidneys (I, IV, V) ........................................................................................... 43 4.4 RNA extraction (I-V).................................................................................................................................... 43 4.5 Cloning of rat nephrin cDNA (I).................................................................................................................. 43 4.6 DNase treatment (II, III, IV) ........................................................................................................................ 43 4.7 Semiquantitative RT-PCR (I-V) .................................................................................................................. 43 4.8 Real-time PCR (III) ...................................................................................................................................... 44 4.9 Immunofluorescence microscopy (I, II, IV, V) ........................................................................................... 45