Studies of Immunoglobulin Receptors and Human Proximal Tubular Cells Manal Jamal Natto BSc (Hons; K.S.A), MRes (UK) Submitted in fulfilment of the requirement for the degree of Doctor of Philosophy University of Glasgow Division of Cardiovascular & Medical Sciences and FBLS 2007 AAcckknnoowwllleeddggeemmeenn III, II Acknowledgement I I wish to express my sincere gratitude to my supervisor Professor Alan G. Jardine , the one who has guided me into the fascinating world of research. I wish to express my thanks, for his contributions, excellent collaboration, valuable discussions, and unreserved support. I gratefully acknowledge the enthusiastic supervision of Dr. Niall MacFarlane , for his positive and constructive advice and support throughout, and for always being so helpful. This work would not have been possible without the support and the encouragement of my colleagues Dr. Kenneth McDonald and Ms. Helen Millar . Very special thanks also to Dr. Diane Hillyard for her generosity and kindness. In addition, I would like to thank Dr. James McLay (University of Aberdeen), who kindly provided primary cultures of proximal tubular cells. Most importantly, thanks and love to my wonderful parents, Jamal and Najat . They have wished so long for this thesis, waited and supported me throughout these years. To them I dedicate this work. All studies have been supported by grants from the Ministry of Health in Saudi Arabia, and the Saudi Cultural Bureau office in London. Manal Jamal Natto (2007) AAcckknnoowwllleeddggeemmeenn IIIIII, III Acknowledgement II ((((ﺑﺴﺑﺑﺴﺴﺑﺴــــــــﻢﻢ ﺍﷲ ﺍﻟﺮﺣﻤﺍﻟﺮﺣﻤــــــــﻦﻦ ﺍﻟﺮﺣﻴﺍﻟﺮﺣﻴــــــــﻢﻢﻢ)ﻢ))) إـ ـوـ ـ ـــ، وــ أــ ـــــ آــــــن أـــــ . إـ ة ، ع ان اي ، و اء ا . ﻭﺍﻟﺪﻱ ﻭﻭﺍﻟﺪﺗﻲ ﺍﻷﺣﺒﺎﺀ .................. أن ا اس ا.. أ ر او ؛ و ا أ م اة وهت أن أ رد ء . ل ارب، أن ل ات ا ت أ إً وااً . ال ا ا ا أن ر أرد و ءًا ها ا وان . وأً ............ إ آ آن وراء و ا .....ا ً .. .. وإ زآ، ن، س، و إ أ ا ره ا... أهي ة ي اا ها ا اي اوام ؛ وا وراء ا ..... وا وآ؛؛ . ﺍﻟﻤﺤﺒﺔ ﻟﻜﻢ ﺟﻤﻴﻌﺎﹰ ﻣﻨـﺎﻝ ﺟﻤـﺎﻝ ﻧﺘــﻮ AAuuttthhoorrr’’’ss ddeecclllaarrraatttiiioonn, IV Author's declaration I declare that this thesis has been composed by me and is a record of work performed by me, except where stated in the text. The immunohistochemistry of tissue sections in chapter 3 was performed by Dr Barbara Young (Department of Pathology, Western Infirmary, Glasgow). This work has not previously been submitted for a higher degree. Manal J amal Natto (2007) SSuummmmaarrryy, V Summary Glomerulonephritis (GN) is a major cause of progressive chronic renal failure (CRF) and accounts for less than 20% in the UK of patients requiring dialysis and transplantation. There are many forms of GN, of which IgA nephropathy (IgAN) is the most common, affecting up to 2% of the adult population. GN is characterised by irreversible and progressive glomerular and tubulointerstitial fibrosis, reduction in glomerular filtration rate (GFR), and retention of uremic toxins. The rate of progression of GN is increased by various clinical factors including hypertension, and the quantity and specificity of proteinuria. Although albuminuria is most widely studied, higher molecular weight proteins such as immunoglobulins (Ig) are more associated with progression of renal disease. In this thesis I have explored the hypothesis that an interaction between tubular cells and filtered Ig/immuno complex (Ig/IC) may involve specific cellular receptors, and thus provide a novel mechanism for the initiation of tubular injury and fibrosis. Deposition of Ig and IC is seen in renal diseases such as membranous glomerulonephritis, and IgAN. Ig/IC are regarded as responsible for the initiation of glomerular injury in these diseases. However, whether Ig/IC contribute directly to tubular injury is unknown. We previously identified a novel IgA receptor, the Fc α/µ receptor (Fc α/µR), in mesangial cells which may contribute to IgAN. This receptor binds both IgA and IgM but not IgG. I hypothesised that Ig and IC, filtered at the glomerulus, may act on Ig receptors in human proximal tubular epithelial cells (PTEC) and may play a direct pathophysiological role in the development of interstitial fibrosis. Specifically, that filtered IgA may bind to the Fc α/µR in IgAN. The activation of Ig receptors may stimulate tubular cells to alter cell proliferation and extracellular matrix formation (e.g fibronectin (FN)), which are pathophysiological characteristics of tubulointerstitial fibrosis in GN. I studied primary and immortalised human PTEC using qualitative RT-PCR and quantitative real-time PCR (qRT-PCR) for expression of candidate Ig receptors. I investigated the expression of Ig receptors and their regulation by cytokines (e.g. IFN-γ, TGF-β1 and IL-1α) known to be implicated in GN. The receptors studied include the polymeric immunoglobulin receptor (pIgR), the neonatal receptor (FcRn), the Fc α/µR, the classical IgG receptors (Fc γR1, γIIa, γIIb, γIII and the related FcR γ-chain) and the classical IgA receptor (Fc αR). None of the classical IgG receptors were expressed by SSuummmmaarrryy, VI PTEC, nor was the classical IgA receptor. However, the FcRn, pIgR and the Fc α/µR were expressed. Gene expression of these receptors was investigated under cytokine regulation. IFN-γ, IL- 1α and TGF-β1 had no effect on FcRn gene expression. The expression of the pIgR gene was up-regulated by IFN-γ and the Fc α/µR transcript level was up-regulated by IL-1α and IFN-γ but down-regulated by TGF-β1. Expression of Fc α/µR protein by tubular cells was confirmed by western blot analysis by using specific Fc α/µR monoclonal antibody. Immunohistochemistry confirmed the expression of Fcα/µR by PTEC in both normal and diseased kidney and that IgG, IgA and IgM binding varies in parallel with gene expression of related Fc receptors. This binding was associated with increased FN production (assessed by ELISA) and reduced the proliferation of PTEC, demonstrated by [ 3H]- thymidine uptake assay. I screened the ability of PTEC to release cytokines under normal conditions and confirmed this by ProteoPlex cytokine array. Among these cytokines there was no significant effect of Ig/IC on IL-6, IL-8 and GM-CSF released by PTEC, and this was confirmed by specific ELISA for each individual cytokine. The phosphotyrosine/phospho-ERK signalling pathway was unaffected by Ig/IC; however, IgM was able to activate the phospho-ERK pathway. These results suggest that there might be alternative signalling pathways activated by Ig/IC in PTEC. I then studied the effect of calcineurin inhibitor immunosuppressive drugs as a model of tubular toxicity, and statins as immunomodulatory drugs, reputed to protect renal function and known to have immunomodulatory effects. I hypothesised that immunosuppressant agents/statins either alone or in combination with LPS might affect Ig receptors expression, cytokine release, FN production and proliferation by PTEC. My results inferred that immunosuppressive agents alone have a non-inflammatory response on Ig receptor expression. However, in combination with LPS, the study drugs showed a slight inhibitory effect on the expression of the pIgR on PTEC at the highest concentration only. There was no significant effect of immunosuppressant agents alone or in combination with LPS on IL-6, IL8 and GM-CSF released by PTEC. Cyclosporine, FK506 and sirolimus were shown to inhibit both the production of FN and the growth of PTEC at high doses, thereby exerting an antiproliferative effect. SSuummmmaarrryy, VII Both simvastatin and fluvastatin inhibited LPS induced production of IL-6, IL-8 and GM- CSF, FN and proliferation in a dose-dependent manner. 1 micromolar simvastatin/fluvastatin was associated with a 35% reduction in unstimulated FN production, a 51% reduction in proliferation, and reduced production of IL-6 (58%), IL-8 (65%) and GM-CSF (57%). The expression of Ig receptors was increased in a dose- dependent manner in PTEC treated with statins alone or in combination with LPS. In conclusion, the expression of tubular FcRn, pIgR and Fc α/µR by PTEC is regulated by proinflammatory cytokines. The binding of IgA and IgM, possibly through the Fc α/µR, by PTEC may contribute to immune-mediated nephropathy. The FN production and the proliferation suggest that Ig/IC binding may contribute to the pathophysiology of IC- mediated renal disease. The ability of immunosuppressant agents to reduce FN production, proliferation, and pIgR expression by PTEC may offer potential new strategies for the prevention and treatment of nephrotoxicity, proteinuria and chronic allograft nephropathy (CAN). The inhibitory effect of statins alone or in combination with LPS on FN production, proliferation and cytokines released suggest that statin therapy may have potential benefit in interstitial nephritis and fibrosis. On other hand, statins alone or in combination with LPS increased the expression of Ig receptors by PTEC. This thesis shows that the novel human Fc α/µR in PTEC may play an important role in the immune mechanisms involved in the tubular response during renal injury associated with tubular Ig/IC deposits in renal disease. TTaabblllee oofff ccoonnttteenntttss, VIII Table of contents Acknowledgement I ..............................................................................................................II Acknowledgement II............................................................................................................III Author's declaration ...........................................................................................................