No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

Galectin-3 is a β-galactoside-binding lectin implicated in renal collecting duct development and differentiation. Autosomal recessive polycystic kidney disease (ARPKD) affects 1 in 20,000 humans, and is characterised by cyst development from collecting ducts. Galectin-3 retards cystogenesis in at least 2 in vitro models. Hence, I hypothesised that endogenous galectin-3 may reduce cyst formation in vivo, and investigated this in the congenital polycystic kidney mouse (cpk), a well-characterised ARPKD model. Widespread galectin-3 expression was detected in cpk cyst epithelia in a distinct distribution compared to other developmental markers and renal galectins, and also in other cystic mice and human PKD, raising the possibility that galectin-3 may be a common part of a 'cystogenic' pathway. Next, I investigated whether reduced galectin-3 accelerated cyst formation in vivo using cpk and galectin-3 mutants to generate double cpk/galectin-3 mutants. Initial results on a mixed genetic background demonstrated large variability, but still significantly increased cysts in mice lacking galectin-3. I then backcrossed onto a pure 129Sv background but offspring developed unexpected increased mortality and pancreatic cysts, which confounded this experiment. Hence, we imported different galectin-3 mutants to reassess on the C57BL/6j background: cyst formation was less rapid than mixed/129Sv, but significantly more cortical cysts were again observed in galectin-3 null mutants. I detected galectin-3 in the primary cilium and centrosomes both in vivo and in vitro in normal and cystic samples for the first time. At least some of the galectin-3 appears on the outside of the cilia and paclitaxel, a therapy that retards PKD in cpk mice, caused increased extracellular galectin-3, a location where the lectin might interact with cilia. Preliminary experiments were also performed to investigate ciliary function using atomic force microscopy. These data raise the possibility that galectin-3 may act as a 'natural' brake on cystogenesis in cpk mice, perhaps via ciliary roles.

The overall aim of this thesis is ‘to investigate the role of the urothelium in mediating sensory signalling from the bladder’ and provide direct evidence to support the hypothesis that the urothelium and afferent nerve fibres are capable of communicating with each other and via modulation of urothelial mediator release the activity of afferent nerve fibres can be altered . Understanding the influence of the urothelium on afferent nerve sensitivity both during and between distensions of the bladder could reveal future targets for therapeutic intervention for treatment of lower urinary tract disorders, but equally importantly, contribute to our understanding of normal bladder function and the importance of a functioning urothelium in mediating afferent sensitivity. Experiments in this thesis have explored the effect of urothelial signalling on afferent nerve sensitivity by several methods:- 1. Inhibition of urothelial mediator release – see chapter 4 2. Stimulation of urothelial mediator release – see chapter 5 3. Damage to the urothelium and the effect of K+ stimulation – see chapter 5 4. Pharmacological attenuation of urothelial signalling pathways – see chapter 6

Chronic kidney disease (CKD) is characterised by the pathological accumulation of extracellular matrix (ECM) proteins leading to progressive kidney scarring via glomerular and tubular basement membrane expansion. Increased ECM synthesis and deposition, coupled with reduced ECM breakdown contribute to the elevated ECM level in CKD. Previous pre-clinical studies have demonstrated that increased plasmin activity has a beneficial effect in the protein overload model of CKD. As plasmin activation is down regulated by the action of the thrombin activated fibrinolytic inhibitor (TAFI), we tested the hypothesis that inhibition of TAFI might increase plasmin activity & reduce ECM accumulation in an in vitro model of glucose induced ECM expansion. Treatment of NRK52E tubular epithelial cells with increasing concentrations of glucose resulted in a 40% increase in TAFI activity, a 38% reduction in plasmin activity and a subsequent increase in ECM accumulation. In this model system, application of the previously reported TAFI inhibitor UK-396082 [(2S)-5-Amino-2-[(1-n-propyl-1H-imidazol-4-yl)methyl]pentanoic acid] caused a reduction in TAFI activity, increased plasmin activity and induced a parallel decrease in ECM levels. In contrast, RNAi knockdown of plasmin resulted in an increase in ECM levels. An evaluation of serum TAFI and plasmin activity from individuals with CKD indicated a strong correlation between elevated TAFI and reduced plasmin levels with renal impairment. These findings correlated with similar observations made in the rat following sub-total nephrectomy. Pharmacological inhibition of TAFI with UK-396082 reduced glomerular and tubulointerstitial ECM deposition. Renal function, as measured by an increase in creatinine clearance and decrease in proteinuria, and survival improved with TAFI inhibition. Taken together, the data support the importance of the fibrinolytic axis in regulating renal fibrosis and point to a potentially important therapeutic role for TAFI inhibition.

A complete understanding of the pathogenesis of diabetic neuropathy continues to be elusive and as a result, progress in developing effective therapies has been disappointing. In particular, there is only limited understanding of why some patients suffer severe chronic pain, whilst others have painless symptoms. Assessment of the peripheral nerves frequently shows no differences between painful and painless diabetic peripheral neuropathy (DPN). There is growing evidence that the nerve damage in DPN is more generalized, involving the entire nervous system including the central nervous system (CNS). The advent of new radiological techniques, such as magnetic resonance spectroscopy (MRS) provides us with non-invasive modalities to study pathophysiological processes in greater detail. In addition, although a clear link between DPN and cardiac autonomic neuropathy (CAN) is recognised, the relationship of autonomic neuropathy with sub-types of DPN is less clear. The development of novel and sensitive measures of CAN, such as spectral analysis of heart rate variability (HRV), may allow the detection of subclinical abnormalities not detected by conventional autonomic function tests (AFT). The principal aim of this thesis was to better understand the nature of the relationship between painful and painless DPN with other parts of the nervous system, namely the CNS and the autonomic nervous system. In the first study the central processing of sensation in people with diabetes was assessed to determine whether central mechanisms have an important role in the perception of pain. In the second study, short-term HRV analysis was used to help define the nature of the relationship between CAN and painful and painless DPN more clearly. A secondary aim was to develop and validate a model incorporating HRV parameters as a sensitive measure of autonomic dysfunction. In the first study, 110 subjects with type 1 diabetes (20 no DPN, 30 subclinical DPN, 30 painful DPN and 30 painless DPN) and 20 healthy volunteers (HV) underwent detailed clinical and neurophysiological assessments (Dyck's NIS(LL)+7 staging criteria). They all underwent proton magnetic resonance spectroscopy of the left thalamic nucleus and somatosensory cortex to measure established markers of neuronal function using long echo time (LET) and neuronal integrity using short echo time (SET) spectroscopic sequences. The results demonstrated significant differences between painful and painless DPN. In the thalamus, at LET, subjects with painless DPN had significantly lower N-acetylaspartate (NAA) compared to other groups (ANOVA p<0.001). No differences were seen at SET. In contrast, in the somatosensory cortex, no inter-group differences were seen at LET, but at SET, the painless DPN group had lower NAA, compared to HV and subjects with diabetes but no DPN, whilst subjects with painful DPN had intermediate levels (ANOVA p<0.001). Various other differences were also seen between painful and painless DPN in other cerebral neurochemicals (particularly myo-inositol and glutamate), despite no differences between the groups in detailed peripheral nerve assessments. These results suggest that astrocyte dysfunction within a hyperglutaminergic state within the thalamus may be a key factor in the development of painful DPN. In a second study, a subset of these patients (20 HV, 20 no DPN, 20 painful DPN and 20 painless DPN) underwent short-term HRV analysis, to assess sympathovagal modulation of the heart rate. Various frequency domain and time domain parameters were assessed. The results showed that despite no differences in conventional AFT, subjects with painful DPN had greater autonomic abnormalities when assessed using HRV analysis, suggesting that it is a more sensitive tool to detect autonomic dysfunction. The greater autonomic dysfunction seen in painful DPN may reflect more predominant small fibre involvement and adds to the growing evidence of its role in the pathophysiology of painful DPN. In the third study, we demonstrated that this method of HRV analysis can be used to develop a sensitive tool to detect early autonomic dysfunction. Using discriminant function analysis, a model was developed which incorporated 8 HRV parameters as well as basic demographic data. It demonstrated a high degree of sensitivity and specificity. From the above studies it can be inferred that changes in neuronal physiology and function may be important in the perception of pain in DPN. They have demonstrated that DPN is a disease that affects the entire nervous system, including the CNS which should trigger a critical rethinking of the disorder.