Torres, Vicente E.

Introduction Maintaining fluid intake sufficient to reduce arginine vasopressin (AVP) secretion has been hypothesised to slow kidney cyst growth in autosomal dominant polycystic kidney disease (ADPKD). However, evidence to support this as a clinical practice recommendation is of poor quality. The aim of the present study is to determine the long-term efficacy and safety of prescribed water intake to prevent the progression of height-adjusted total kidney volume (ht-TKV) in patients with chronic kidney disease (stages 1–3) due to ADPKD. Methods and analysis A multicentre, prospective, parallel-group, open-label, randomised controlled trial will be conducted. Patients with ADPKD (n=180; age ≤65 years, estimated glomerular filtration rate (eGFR) ≥30 mL/min/1.73 m 2 ) will be randomised (1:1) to either the control (standard treatment+usual fluid intake) or intervention (standard treatment+prescribed fluid intake) group. Participants in the intervention arm will be prescribed an individualised daily fluid intake to reduce urine osmolality to ≤270 mOsmol/kg, and supported with structured clinic and telephonic dietetic review, self-monitoring of urine-specific gravity, short message service text reminders and internet-based tools. All participants will have 6-monthly follow-up visits, and ht-TKV will be measured by MRI at 0, 18 and 36 months. The primary end point is the annual rate of change in ht-TKV as determined by serial renal MRI in control vs intervention groups, from baseline to 3 years. The secondary end points are differences between the two groups in systemic AVP activity, renal disease (eGFR, blood pressure, renal pain), patient adherence, acceptability and safety. Ethics and dissemination The trial was approved by the Human Research Ethics Committee, Western Sydney Local Health District. The results will inform clinicians, patients and policy-makers regarding the long-term safety, efficacy and feasibility of prescribed fluid intake as an approach to reduce kidney cyst growth in patients with ADPKD. Trial registration number ANZCTR12614001216606.

Significance Statement The only treatment approved for PKD inhibits production of cAMP, the main PKA activator. It is only partially effective, likely because side effects restrict dosing and because other sources of cAMP and mechanisms of cAMP-independent PKA activation exist. Which PKA isozyme(s) promotes PKD is uncertain and selective PKA inhibitors usable in vivo have not been available. Experiments in a mouse model show PKA-I is the main PKA isozyme promoting cystogenesis and that constitutive PKA-I downregulation and a novel, highly selective PKA inhibitor ameliorate PKD. The dose of PKA inhibitor used had no detectable adverse effects. This information provides a strong rationale for a strategy that may be more effective, or substantially increase the efficacy of the currently approved treatment. Background Upregulation of cAMP-dependent and cAMP-independent PKA signaling is thought to promote cystogenesis in polycystic kidney disease (PKD). PKA-I regulatory subunit RI α is increased in kidneys of orthologous mouse models. Kidney-specific knockout of RI α upregulates PKA activity, induces cystic disease in wild-type mice, and aggravates it in Pkd1 RC/RC mice. Methods PKA-I activation or inhibition was compared with EPAC activation or PKA-II inhibition using Pkd1 RC/RC metanephric organ cultures. The effect of constitutive PKA (preferentially PKA-I) downregulation in vivo was ascertained by kidney-specific expression of a dominant negative RIαB allele in Pkd1 RC/RC mice obtained by crossing Prkar1α R1αB/WT , Pkd1RC/RC , and Pkhd1- Cre mice (C57BL/6 background). The effect of pharmacologic PKA inhibition using a novel, selective PRKACA inhibitor (BLU2864) was tested in mIMCD3 3D cultures, metanephric organ cultures, and Pkd1RC/RC mice on a C57BL/6 × 129S6/Sv F1 background. Mice were sacrificed at 16 weeks of age. Results PKA-I activation promoted and inhibition prevented ex vivo P-Ser133 CREB expression and cystogenesis. EPAC activation or PKA-II inhibition had no or only minor effects. BLU2864 inhibited in vitro mIMCD3 cystogenesis and ex vivo P-Ser133 CREB expression and cystogenesis. Genetic downregulation of PKA activity and BLU2864 directly and/or indirectly inhibited many pro-proliferative pathways and were both protective in vivo . BLU2864 had no detectable on- or off-target adverse effects. Conclusions PKA-I is the main PKA isozyme promoting cystogenesis. Direct PKA inhibition may be an effective strategy to treat PKD and other conditions where PKA signaling is upregulated. By acting directly on PKA, the inhibition may be more effective than or substantially increase the efficacy of treatments that only affect PKA activity by lowering cAMP.

Background and objectives The progression of polycystic liver disease is not well understood. The purpose of the study is to evaluate the associations of polycystic liver progression with other disease progression variables and classify liver progression on the basis of patient's age, height-adjusted liver cystic volume, and height-adjusted liver volume. Design, setting, participants, & measurements Prospective longitudinal magnetic resonance images from 670 patients with early autosomal dominant polycystic kidney disease for up to 14 years of follow-up were evaluated to measure height-adjusted liver cystic volume and height-adjusted liver volume. Among them, 245 patients with liver cyst volume >50 ml at baseline were included in the longitudinal analysis. Linear mixed models on log-transformed height-adjusted liver cystic volume and height-adjusted liver volume were fitted to approximate mean annual rate of change for each outcome. The association of sex, body mass index, genotype, baseline height-adjusted total kidney volume, and Mayo imaging class was assessed. We calculated height-adjusted liver cystic volume ranges for each specific age and divided them into five classes on the basis of annual percentage increase in height-adjusted liver cystic volume. Results The mean annual growth rate of height-adjusted liver cystic volume was 12% (95% confidence interval, 11.1% to 13.1%; P <0.001), whereas that for height-adjusted liver volume was 2% (95% confidence interval, 1.9% to 2.6%; P <0.001). Women had higher baseline height-adjusted liver cystic volume than men, but men had higher height-adjusted liver cystic volume growth rate than women by 2% (95% confidence interval, 0.4% to 4.5%; P =0.02). Whereas the height-adjusted liver cystic volume growth rate decreased in women after menopause, no decrease was observed in men at any age. Body mass index, genotype, and baseline height-adjusted total kidney volume were not associated with the growth rate of height-adjusted liver cystic volume or height-adjusted liver volume. According to the height-adjusted liver cystic volume growth rate, patients were classified into five classes (number of women, men in each class): A (24, six); B (44, 13); C (43, 48); D (28, 17); and E (13, nine). Conclusions Compared with height-adjusted liver volume, the use of height-adjusted liver cystic volume showed greater separations in volumetric progression of polycystic liver disease. Similar to the Mayo imaging classification for the kidney, the progression of polycystic liver disease may be categorized on the basis of patient’s age and height-adjusted liver cystic volume.

Key Points Asymptomatic pyuria is associated with kidney failure and faster kidney function decline irrespective of the ADPKD gene and cystic growth. The eGFR decline occurred after detection of asymptomatic pyuria without significant changes in the rate of total kidney volume growth. This study supports the use of asymptomatic pyuria as an enriching prognostic biomarker to predict faster disease progression. Background Autosomal dominant polycystic kidney disease (ADPKD) has phenotypic variability only partially explained by established biomarkers that do not readily assess pathologically important factors of inflammation and kidney fibrosis. We evaluated asymptomatic pyuria (AP), a surrogate marker of inflammation, as a biomarker for disease progression. Methods We performed a retrospective cohort study of adult patients with ADPKD. Patients were divided into AP and no pyuria (NP) groups. We evaluated the effect of pyuria on kidney function and kidney volume. Longitudinal models evaluating kidney function and kidney volume rate of change with respect to incidences of AP were created. Results There were 687 included patients (347 AP, 340 NP). The AP group had more women (65% versus 49%). Median ages at kidney failure were 86 and 80 years in the NP and AP groups (log rank, P =0.49), respectively, for patients in Mayo Imaging Class (MIC) 1A–1B as compared with 59 and 55 years for patients in MIC 1C–1D–1E (log rank, P =0.02), respectively. Compared with the NP group, the rate of kidney function (ml/min per 1.73 m 2 per year) decline shifted significantly after detection of AP in the models, including all patients (−1.48; P <0.001), patients in MIC 1A–1B (−1.79; P <0.001), patients in MIC 1C–1D–1E (−1.18; P <0.001), and patients with PKD1 (−1.04; P <0.001). Models evaluating kidney volume rate of growth showed no change after incidence of AP as compared with the NP group. Conclusions AP is associated with kidney failure and faster kidney function decline irrespective of the ADPKD gene, cystic burden, and cystic growth. These results support AP as an enriching prognostic biomarker for the rate of disease progression.

Significance Statement This study demonstrates for the first time a role of ferroptosis in ADPKD. We show the Pkd1 mutation makes renal epithelial cells prone to ferroptosis through the dysregulation of iron and lipid metabolism. It also suggests the main form of regulated cell death in ADPKD kidneys is ferroptotic but not apoptotic, which helps clarify the controversy over the role of apoptosis in ADPKD. In addition, we found that induction of ferroptosis by erastin promotes cyst growth in Pkd1RC/RC mice, whereas inhibition of ferroptosis by Fer-1 delays cyst growth in rapidly and slowly progressive ADPKD mouse models. These observations suggest management of ferroptosis may be a novel strategy for the treatment of this disease. Background Autosomal dominant polycystic kidney disease (ADPKD), the most common inherited kidney disease, is regulated by different forms of cell death, including apoptosis and autophagy. However, the role in ADPKD of ferroptosis, a recently discovered form of cell death mediated by iron and lipid metabolism, remains elusive. Methods To determine a pathophysiologic role of ferroptosis in ADPKD, we investigated whether the absence of Pkd1 (encoding polycystin-1) affected the expression of key factors involved in the process of ferroptosis, using Western blot and qRT-PCR analysis in Pkd1 mutant renal cells and tissues. We also examined whether treatment with erastin, a ferroptosis inducer, and ferrostain-1, a ferroptosis inhibitor, affected cyst growth in Pkd1 mutant mouse models. Results We found that kidney cells and tissues lacking Pkd1 exhibit extensive metabolic abnormalities, including reduced expression of the system Xc − amino acid antiporter (critical for import of cystine), of iron exporter (ferroportin), and of GPX4 (a key and negative regulator of ferroptosis). The abnormalities also include increased expression of iron importers (TfR1, DMT1) and HO-1, which in turn result in high iron levels, low GSH and GPX4 activity, increased lipid peroxidation, and propensity to ferroptosis. We further found that erastin increased, and ferrostatin-1 inhibited ferroptotic cell death and proliferation of Pkd1 -deficient cells in kidneys from Pkd1 mutant mice. A lipid peroxidation product increased in Pkd1 -deficient cells, 4HNE, promoted the proliferation of survived Pkd1 mutant cells via activation of Akt, S6, Stat3, and Rb during the ferroptotic process, contributing to cyst growth. Conclusion These findings indicate that ferroptosis contributes to ADPKD progression and management of ferroptosis may be a novel strategy for ADPKD treatment.

Abstract Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations of PKD1 or PKD2 genes, is characterized by development and growth of cysts causing progressive kidney enlargement. Reduced resting cytosolic calcium and increased cAMP levels associated with the tonic action of vasopressin are two central biochemical defects in ADPKD. Here we show that co‐targeting two GPCRs, the vasopressin V2 receptor (V2R) and the calcium sensing receptor, using the novel V2R antagonist lixivaptan in combination with the calcimimetic R‐568, reduced cyst progression in two animal models of human PKD. Lixivaptan is expected to have a safer liver profile compared to tolvaptan, the only drug approved to delay PKD progression, based on computational model results and initial clinical evidence. PCK rat and Pkd1 RC/RC mouse littermates were fed without or with lixivaptan (0.5%) and R‐568 (0.025% for rats and 0.04% for mice), alone or in combination, for 7 (rats) or 13 (mice) weeks. In PCK rats, the combined treatment strongly decreased kidney weight, cyst and fibrosis volumes by 20%, 49%, and 73%, respectively, compared to untreated animals. In Pkd1 RC/RC mice, the same parameters were reduced by 20%, 56%, and 69%, respectively. In both cases the combined treatment appeared nominally more effective than the individual drugs used alone. These data point to an intriguing new application for two existing drugs in PKD treatment. The potential for synergy between these two compounds suggested in these animal studies, if confirmed in appropriate clinical investigations, would represent a welcome advancement in the treatment of ADPKD.

Polycystic kidney disease (PKD) is the most common monogenic kidney disorder and an important cause of end stage renal disease. Vascular abnormalities are the most important non‐cystic complication and contribute to renal disease progression. Homocysteine (Hcy)‐mediated endothelial dysfunction (ED) and oxidative stress are evident early on, but the underlying mechanisms remain unknown. We hypothesized that abnormalities in Hcy metabolism are responsible for the increased levels, leading to Hcy‐induced ED from early stages of PKD. Methods We investigated the Hcy metabolic pathway (transulfuration/remethylation fig. 1) and downstream effects ‐main sources of renal tubular (RT) and endothelial cell (EC) reactive oxygen species (ROS), NADPH oxidase 4, (NOX4) and mitochondria‐ in 4‐week‐old PCK and Sprague‐Dawley (SD) control rats (n=12 each). Kidneys were harvested, frozen in liquid nitrogen, or preserved in formalin for metabolomics and ex‐vivo studies. Twenty‐four‐hour urine and terminal blood samples were collected for metabolite analysis and chemistries. Plasma folic acid, vitamin B12, and Hcy were determined by ELISA. Hcy pathway metabolites (betaine, dimethylglycine) were determined by 1 HNMR. Endothelial NO (eNOS) and oxidative stress were assessed by double immunofluorescence staining for CD31/eNOS and CD31/NOX4, respectively. RT and EC mitochondrial structure was assessed by electron microscopy. Results Serum creatinine and BUN remained unchanged, yet kidney weight/body weight ratio were increased in PCK. eNOS immunoreactivity was lower in PCK vs SD. Plasma Hcy and urine excretion of methyl donor betaine were elevated in PCK, and Hcy positively correlated with urine betaine. Plasma betaine was similar between the groups, but its tissue concentration was lower in PCK, which may lead to betaine insufficiency, impaired betaine dependent remethylation, and hyperhomocysteinemia. Contrarily, tissue glutathione concentration was higher in PCK vs SD, and plasma folate and vitamin B12 were similar, arguing against a defect in the transsulfuration or folate‐dependent remethylation pathways. RT NOX4 expression and immunoreactivity were increased in PCK compared to SD, in association with renal oxidative stress. PCK had increased number of CD31/NOX4 positive cells in peritubular capillaries, indicating increased EC ROS production. Collecting duct and EC mitochondria were swollen and showed cristae remodeling. Conclusion In PKD, early elevation in Hcy is likely related to betaine insufficiency and impaired betaine‐dependent remethylation may be responsible for the early elevation of Hcy. NOX4 and mitochondria abnormalities may be important contributors to oxidative stress and disease severity. Our data suggests these may not only be early vascular disease markers, but also play an important pathogenic role in vascular complications associated with PKD, and contribute to renal disease progression. Support or Funding Information R25‐DK101405, NIDDK P30 DK090728, NIDDK P30 DK 106912 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Disruption of intracellular calcium likely underlies the upregulation of cyclic AMP (cAMP) signaling and the proliferative and secretory phenotype of the cystic epithelium in ADPKD. However, the mechanisms by which polycystins reduce cytoplasmic calcium remain elusive. Impaired flow‐induced calcium entry through ciliary polycystin complex has been previously proposed as a pathogenic mechanism, but newer evidence suggests the involvement of polycystins in modulation of other mechanosensitive ion channels such as Piezo1, the newly‐identified stretch‐activated non‐selective cation channel. We hypothesized that polycystins, through their physical and functional interaction with Piezo1, modulate various cellular responses to mechanical stimulation. Piezo1 was expressed in both wild type mouse inner medullary collecting duct cells (mIMCD3) cells and PKD2 null cells (~3 fold higher than wild type). Wild type mIMCD3 cells were stably transfected with genetically encoded biosensors, jRGeco1a and Flamindo2, to study spatiotemporal dynamics of calcium and cAMP during live cell imaging, respectively. Piezo1 activation by Yoda1 increased cytoplasmic calcium and simultaneously lowered forskolin‐induced cAMP (Figure 1A). Additionally, exerting physiological fluid shear stress (FSS) (ibidi pump system) led to reduction in cAMP (Figure 1B). Both Yoda1 and FSS lead to an increase in ATP release in mIMCD3 cells (Figure 1C). Furthermore, Yoda1 significantly reduced forskolin‐induced in vitro cystogenesis by 68% in WT and by 48% in PKD2 null mIMCD3 cells (Figure 1D). These results suggest that Piezo1 is involved in renal cystogenesis by controlling FSS‐induced calcium entry. Further studies are required to determine in vivo effect of Piezo1 on renal cystogenesis. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Substantial evidence indicates the importance of elevated cAMP in polycystic kidney disease (PKD). Preclinical and clinical trials have shown the feasibility of therapies (vasopressin V2 receptor antagonists and somatostatin analogs) acting on G protein coupled receptors to inhibit adenylyl cyclase activity and production of cAMP. An additional or alternative approach could be to increase phosphodiesterase (PDE) mediated hydrolysis of cAMP. Of the large PDE superfamily, PDE1 is the only family activated by calcium, which is reduced in PKD cells. To assess the contribution of PDE1A to renal cyst formation, we depleted PDE1A levels using antisense morpholinos in zebrafish. We identified two splice isoforms with alternative starts corresponding to human PDE1A1 and PDE1A5. Two splice‐blocking morpholinos targeting the common hydrolase domain of both isoforms induced renal cysts. Two additional morpholinos, targeting each start, also induced renal cysts with dose dependence and additive effects. Co‐injection of the start 1 morpholino with pkd2 morpholino increased the severity of hydrocephalus, renal cysts, and body curvature, demonstrating functional interaction of PDE1A and PC2. Overall, data implicate PDE1A in PKD pathogenesis and validate further investigation of PDE1A as a therapeutic target in PKD.