PKD Public Seminar

Abstract 1:

WHOLE GENOME SEQUENCING (WGS) AS A NEW VALIDATED DIAGNOSTIC TEST FOR ADPKD

ADPKD is the most common monogenic renal disease. There are many benefits of genetic diagnosis, including early diagnosis, family planning, cascade testing, living-donor selection and predict disease severity.  However, diagnostic sequencing is challenged because 6 pseudogenes share 97% sequence homology with PKD1 and confound standard sequencing techniques. WGS has been shown to overcome this pseudogene homology, but had not been validated as a diagnostic test. In order to validate WGS, we studied 42 unrelated patients with an ADPKD phenotype. Thirty patients first underwent sequencing by long-range PCR/Sanger sequencing/MLPA of PKD1 and PKD2 in the Mayo Clinic. Blinded WGS was then performed after PCR-free library preparation in the Garvan Institute. Concurrently, 12 patients were first sequenced via WGS and then blinded standard sequencing of PKD1 and PKD2. With WGS, the same results as standard sequencing were obtained in 40/42 patients. In 2 patients, WGS did not detect mosaic variants, however filtering was confined to 50:50 variant calls. WGS defined the breakpoints of 2 multi-exon deletions, which had not been possible with prior methods. There were no false positive or false negative results. Optimization of variant filtration was a crucial component for the successful analysis. WGS provides the basis of a new diagnostic test for ADPKD and this has now been translated to a diagnostic test. WGS avoids laborious sample preparation and overcomes pseudogene homology and unlike targeted sequencing, WGS allows scope for broadened genomic analysis if no PKD1 or PKD2 variants are identified.


Abstract 2:

ELUCIDATION OF THE CENTRAL ORIGINS OF ENHANCED VASOPRESSIN RELEASE IN A RAT MODEL OF POLYCYSTIC KIDNEY DISEASE

Conor F Underwood, Jacqueline K Phillips & Cara M Hildreth

Department of Biomedical Science, Faculty of Medicine and Health Sciences, Macquarie University, Sydney NSW 2109 Australia;

Greater circulating vasopressin drives disease progression in polycystic kidney disease (PKD) via renal V2 receptors. It is unknown whether activation of vascular V1 receptors contribute to hypertension in PKD, and what population/s of hypothalamic neurons are responsible for elevating vasopressin secretion. We addressed this knowledge gap using the Lewis Polycystic Kidney (LPK) rat model of PKD. Experiments were performed in urethane-anaesthetised rats prepared to measure systolic blood pressure (SBP) and administer drugs intravenously and into discrete hypothalamic nuclei. Acute intravenous administration of a V1 antagonist reduced SBP in LPK (206±9 vs. 180±13 mmHg, n=6; P<0.001), but not in control animals (134±6 vs. 134±6 mmHg, n=7; P>0.05), suggesting that LPK display vasopressor levels of vasopressin. To determine the hypothalamic source of enhanced vasopressin release in LPK, we microinjected muscimol, a GABA-A receptor agonist, bilaterally into either the paraventricular (PVN) or supraoptic (SON) nucleus in order to silence neuronal activity. Though we observed a far greater depressor response to PVN muscimol in the LPK compared to control animals (-43±4 vs. -18±3 mmHg, n=13; P<0.0001), this response was unaffected by intravenous administration of a V1 antagonist (P>0.05). This suggests that PVN neurons maintain hypertension in LPK but the mechanism is independent of vasopressin release. In contrast, SON microinjection of muscimol produced an increase in SBP in LPK (21±7 mmHg, n=5; P<0.01), but no change in controls (P>0.05), indicating that GABA-A receptors may be excitatory in vasopressin-secreting SON neurons in LPK. Together, our data indicate that enhanced vasopressin release may contribute to hypertension in LPK rats and raise the possibility that enhanced vasopressin release may relate to an inhibitory-to-excitatory switch in GABA-A receptor function in SON neurons.

Greater circulating vasopressin levels are commonly described in patients with autosomal dominant polycystic kidney disease (PKD). Activation of renal vasopressin type 2 receptors contributes substantially to disease progression. Furthermore, activation of vascular type 1 (V1) receptors has been suggested to contribute to the hypertension observed in this disease (Torres. Semin Nephrol. 2013; 28(3): 306-317), though this has not been directly tested. It is also unknown what population of vasopressin-secreting hypothalamic neurons are responsible for the exaggerated vasopressin release in PKD .

LPK animals showed greater resting levels of systolic blood pressure (SBP) compared to Lewis controls (** vs. ** mmHg, n=* Lewis vs. n=* LPK; P<0.0001).  To determine whether tonic V1 receptor activation contributes to hypertension in LPK, a selective V1 antagonist (OPC-21268; 3 mg/kg) was administered intravenously in a cohort of animals; systemic V1 antagonism did not affect resting SBP in the Lewis (n=*, P>0.05), but effectively lowered SBP in the LPK (-** mmHg, vs. baseline; P<0.**). This effect was despite an apparently similar sensitivity of the vasculature to vasopressin as evidenced by a similar relative pressor response evoked by an intravenous bolus of vasopressin (** vs. ** % change from baseline, P=**).

Greater circulating vasopressin levels are commonly described in patients with autosomal dominant polycystic kidney disease (PKD) and there is conclusive preclinical and clinical evidence showing that activation of renal V2 receptors is major driver of disease progression. Nevertheless, several questions remain: Does activation of vascular V1 receptors contribute to hypertension in PKD? What population/s of hypothalamic neurons are responsible for elevating vasopressin levels in PKD?


Abstract 3:

TO EXPLORE THE UNDERLYING MECHANISMS OF A NOVEL REGULATOR IN PKD DEVELOPMENT FOR THERAPEUTIC DEVELOPMENT

Wang B.

Department of Anatomy and Developmental Biology, Monash University, Melbourne Australia.

PKD development is characterised with proliferation of kidney epithelial cells and cyst growth with underlying mechanisms including activation of WNT pathway. Inhibition on cyst formation had become a promising treatment for PKD. Any novel regulator on cyst formation and growth will lead to new therapeutic strategy in PKD. Our recent discovery has identified a novel protein, called WISP1, that may play an important role in the formation of kidney cysts and the detrimental scarring of the kidneys that leads to reduced kidney function over time.

Our preliminary data showed the up-regulation of WISP1 in PKD patient kidney and PKD mice model. Deletion of WISP1 inhibit the growth of renal epithelial cells. The WISP1 antibody can reduce the cell proliferation, suggesting a monoclonal antibody based therapy had shed a light on the treatment of cyst formation. More interestingly, knock-down of WISP1 in kidney cells demonstrated an anti-fibrotic function, indicating a broader application of WISP1 in kidney disease treatment.

We will continue to investigate the function of WISP1 in cyst formation using the isolated epithelial cells from a PKD model. Moreover, we will develop a therapeutic antibody that inhibits WISP1 protein production that will be used therapeutically to retard cyst growth and slow or alleviate disease progression, including kidney fibrosis.


Abstract 4:

IS SERUM COPEPTIN A MODIFIABLE BIOMARKER IN AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE (ADPKD)?

ATY Wong1, Mannix C, Zhang J, Harris DCH, Sud K, Lee V, Rangan GK and on behalf of PREVENT-ADPKD collaborators and investigators.

1 Centre for Transplant and Renal Research, Westmead Institute for Medical Research, Sydney Australia

The availability of disease-modifying drugs for the management of autosomal dominant polycystic kidney disease (ADPKD) has accelerated the need to accurately predict renal prognosis and/or treatment response. Arginine vasopressin (AVP) is a critical determinant of postnatal kidney cyst growth in ADPKD. Copeptin (the C-terminal glycoprotein of the precursor AVP peptide) is an accurate surrogate marker of AVP release that is stable and easily measured by immunoassay. Cohort studies show that serum copeptin is correlated with disease severity in ADPKD and predicts future renal events (decline in renal function and increase in total kidney volume, TKV). Our preliminary analysis in an ADPKD cohort (CKD Stages 1 to 3) indicates that serum copeptin is strongly correlated with serum creatinine, suggesting that it may not add value over estimated glomerular filtration rate and TKV as a prognostic biomarker. Alternatively, it has been suggested that copeptin could be a predictive biomarker to select ADPKD patients who are most likely to benefit from AVP-modifying therapies, and prospective data to validate this assumption are required. In this regard, a long-term randomised clinical trial evaluating the effect of prescribed water intake on renal cyst growth may contribute to addressing this hypothesis. In conclusion, although serum copeptin is aligned with the basic pathogenesis of ADPKD, further studies are needed to define if it will contribute to enabling the delivery of personalised care in ADPKD.

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The aims of the Society are to promote and support the study of the kidney and urinary tract in health and disease, and to ensure the highest professional standards for the practice of nephrology in Australia and New Zealand.

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