Role Of Nuclear Factor Kappa B In Polycystic Kidney Disease

Abstract

Polycystic kidney diseases (PKDs) are a group of disorders resulting in the formation of multiple renal cysts, and are characterised by renal interstitial inflammation, cell proliferation and apoptosis. The nuclear factor (NF)-κB system regulates the transcription of genes involved in inflammation, growth and apoptosis, but it is unknown whether NF-κB has a role in the pathophysiology of PKD. Hence, the aim of this thesis was to determine the endogenous expression and activity of NF-κB in PKD, and to begin to elucidate its role in this disease. Chapter One. Chapter One demonstrates that PKD is a complex disease involving several pathophysiological processes (particularly proliferation and inflammation) and multiple dysfunctional signalling pathways. The chapter discusses the current and emerging therapies and argues that new translational therapies should focus on targeting ‘master regulators’ that simultaneously control several pathways of PKD. The chapter then provides evidence supporting the case for NF-κB as one such key gene regulator that may be involved in PKD, and introduces the main hypothesis that NF-κB expression and activity are upregulated in PKD, and may mediate cyst expansion in this disease. Chapter Two. Chapter Two presents the methodology and preliminary results of two methods that were attempted but not achieved due to technical limitations. These include the 3D collagen cyst model, which aimed to provide information on the effect of NF-κB inhibition on cyst expansion, and the electrophoretic mobility shift assay (EMSA), which aimed to characterise the DNA binding activity of NF-κB proteins in renal tissue. Chapter Three. Chapter Three examined the endogenous renal expression of NF-κB proteins in Lewis Polycystic Kidney rats (LPK, a genetic orthologue of human nephronophthisis-9) compared to healthy Lewis controls from postnatal weeks 3 to 20. Progressive kidney enlargement in LPK rats was accompanied by increased renal cell proliferation and interstitial monocyte accumulation (peaking at weeks 3 and 10 respectively), and progressive interstitial fibrosis. Rel/NF-κB proteins (phosphorylated-p105, p65, p50, c-Rel and RelB) were expressed in cystic epithelial cells (CECs) of LPK kidneys as early as postnatal week 3 and were present until late-stage disease at week 20. From weeks 10 to 20, nuclear p65, p50, RelB and cytoplasmic IκBα protein levels were upregulated in LPK compared to Lewis kidneys. NF-κB proteins were consistently expressed in CECs of human autosomal and recessive PKD (ADPKD and ARPKD). This study indicated that several NF-κB proteins are consistently expressed in CECs in human and experimental PKD, and may be a constitutive and early pathological feature of cystic renal diseases. Chapter Four. Chapter Four tested the hypothesis that an inhibitor of NF-κB, pyrrolidine dithiocarbamate (PDTC), reduces the progression of PKD in vivo. Chronic administration of PDTC to LPK rats from postnatal weeks 4 until 11 was associated with a slower increase in total kidney volume (TKV) compared to vehicle treatment. By serial magnetic resonance imaging (MRI) at weeks 5 and 10, the relative within-rat increase in TKV was 1.3-fold greater, while the increase in cyst volume was 1.4-fold greater, in LPK rats treated with vehicle compared to the higher-dose PDTC group. By week 11 in LPK rats, PDTC had attenuated kidney weight to body weight ratio by 25%, and decreased proteinuria by 66%, but did not improve renal dysfunction. PDTC did not alter histological interstitial inflammation and fibrosis, or renal cell proliferation in LPK rats. The phosphorylated form of the NF-κB p105 subunit was increased in CECs of LPK rats, but was not altered by PDTC. Moreover, PDTC did not significantly alter nuclear expression of the p50 subunit or NF-κB (p65)-DNA binding. In conclusion, PDTC reduced renal cystic enlargement and proteinuria without decreasing inflammation or NF-κB activity in LPK rats. Chapter Five. This chapter expanded upon the findings of Chapter Three by examining the mechanism by which PDTC reduces renal cyst growth, in vitro. Chapter Five tested the hypothesis that PDTC reduces the proliferation of immortalised ADPKD CECs in an NF-κB-dependent manner. Serum-induced proliferation was similar between normal human kidney cortical (HK-2) cells and ADPKD cells over 72 hours. PDTC decreased proliferation in both ADPKD and HK-2 cells, but these anti-proliferative effects were delayed in ADPKD cells compared to HK-2 cells. Basal NF-κB-dependent luciferase reporter activity was lower in ADPKD cells compared to HK-2 cells. Classical NF-κB stimulants, lipopolysaccharide (LPS) and tumour necrosis factor (TNF)-α, increased NF-κB luciferase activity in HK-2 cells, whereas in PKD cell lines, NF-κB activity was only induced by TNFα. However, neither stimulant altered proliferation in any cell line. PDTC reduced TNFα-stimulated NF-κB activity in HK-2 cells only. In conclusion, this study indicated that PDTC had anti-proliferative properties in ADPKD cells but did not consistently alter NF-κB activation, suggesting that other signalling pathways are likely to be involved in its ability to attenuate renal cyst growth in vivo. Chapter Six. Given the efficacy of the mammalian target of rapamycin complex 1 (TORC1) inhibitor, sirolimus, in slowing the rate of TKV growth in previous pre-clinical models of PKD, Chapter Six investigated whether sirolimus can also modulate NF-κB activity in PKD when administered at different stages of disease. Male LPK rats and Lewis controls were administered either vehicle or sirolimus, during early-stage (postnatal weeks 3 to 10) or late-stage disease (weeks 10 to 20). In early-stage disease, sirolimus completely prevented the increase in MRI-assessed TKV but only partially reduced the percentage cyst area (by 19%), and did not affect the decline in endogenous creatinine clearance (CrCl). Rats treated in late-stage disease only had a partial reduction in TKV, with no change in percentage cyst area and marginal improvement in CrCl. Sirolimus reduced TORC1 but did not alter the expression of markers of TORC2 activation or the expression of NF-κB-dependent genes (CCL2 and TNFα). Late but not early sirolimus treatment increased p65 NF-κB DNA binding activity in Lewis and LPK kidneys. Therefore, the time at which sirolimus was initiated, determined the drug’s efficacy in attenuating cystic growth. However, sirolimus did not alter cystic microarchitecture, significantly improve renal function, or decrease NF-κB expression and activity in LPK rats. These data suggest that additional approaches to normalise cellular dedifferentiation and inflammation are required to completely arrest the progression of PKD. Chapter Seven. Chapter Seven highlights the novel findings of this thesis, which were namely, the first-time localisation of several NF-κB proteins to the CECs in human and experimental PKD; demonstration of a decrease in TKV and cyst volume with PDTC in an animal model of PKD in vivo; and the identification of non-canonical NF-κB signalling in PKD. It puts forward the potential implications of this thesis, such as the capacity of dithiocarbamates to provide renoprotective effects in cystic renal disease. It also underlines the lack of demonstration of a functional relationship between NF-κB and cyst growth by this work, and proposes recommendations to experimentally investigate this concept in future in vitro studies (e.g. a 3D cyst model) as well as in vivo studies (particularly the use of NF-κB gene deletion in an orthologous model of ADPKD).