Four-jointed knock-out delays renal failure in an ADPKD model with kidney injury.

Autosomal Dominant Polycystic Kidney Disease is characterised by the development of fluid-filled cysts in the kidneys which lead to end-stage renal disease (ESRD). In the majority of cases, the disease is caused by a mutation in the Pkd1 gene. In a previous study, we demonstrated that renal injury can accelerate cyst formation in Pkd1 knock-out (KO) mice. In that study, we found that after injury four-jointed (Fjx1), an upstream regulator of planar cell polarity and the Hippo pathway, was aberrantly expressed in Pkd1 KO mice compared to WT. Therefore, we hypothesised a role for Fjx1 in injury/repair and cyst formation. We generated single and double deletion mice for Pkd1 and Fjx1, and we induced toxic renal injury using the nephrotoxic compound 1,2-dichlorovinyl-cysteine. We confirmed that nephrotoxic injury can accelerate cyst formation in Pkd1 mutant mice. This caused Pkd1 KO mice to reach ESRD significantly faster; unexpectedly, double KO mice survived significantly longer. Cyst formation was comparable in both models, but we found significantly less fibrosis and macrophage infiltration in double KO mice. Taken together, these data suggest that Fjx1 disruption protects the cystic kidneys against kidney failure by reducing inflammation and fibrosis. Moreover, we describe, for the first time, an interesting (yet unidentified) mechanism that partially discriminates cyst growth from fibrogenesis. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The expression of somatostatin receptor 2 decreases during cyst growth in mice with polycystic kidney disease.

IMPACT STATEMENT: Somatostatin (SST) analogs have been shown to halt cyst growth and progression of autosomal dominant polycystic kidney disease by several clinical trials. However, two studies suggest that the effect of the SST analog octreotide on kidney growth during the first year of treatment is reduced in the subsequent follow-ups and the kidney enlargement resumes. This biphasic change in kidney growth during octreotide treatment may be partially explained by alterations in SSTR2 expression. Here, we found that SSTR2 is mainly expressed in distal tubules and collecting ducts in murine kidneys, and the expression of SSTR2 decreases during cyst growth in two PKD mouse models. Our data may thus provide possible explanations for the lack of efficacy in long-term treatment with SST analogs.

Variable Cyst Development in Autosomal Dominant Polycystic Kidney Disease: The Biologic Context.

Patients with autosomal dominant polycystic kidney disease (ADPKD) typically carry a mutation in either the PKD1 or PKD2 gene, which leads to massive cyst formation in both kidneys. However, the large intrafamilial variation in the progression rate of ADPKD suggests involvement of additional factors other than the type of mutation. The identification of these factors will increase our understanding of ADPKD and could ultimately help in the development of a clinically relevant therapy. Our review addresses the mechanisms by which various biologic processes influence cyst formation and cyst growth, thereby explaining an important part of the inter- and intrafamilial variability in ADPKD. Numerous studies from many laboratories provide compelling evidence for the influence on cyst formation by spatiotemporal gene inactivation, the genetic context, the metabolic status, the presence of existing cysts, and whether the kidneys were challenged by renal injury. Collectively, a solid basis is provided for the concept that the probability of cyst formation is determined by functional PKD protein levels and the biologic context. We model these findings in a graphic representation called the cystic probability landscape, providing a robust conceptual understanding of why cells sometimes do or do not form cysts.

Translational research in ADPKD: lessons from animal models.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2, which encode polycystin-1 and polycystin-2, respectively. Rodent models are available to study the pathogenesis of polycystic kidney disease (PKD) and for preclinical testing of potential therapies-either genetically engineered models carrying mutations in Pkd1 or Pkd2 or models of renal cystic disease that do not have mutations in these genes. The models are characterized by age at onset of disease, rate of disease progression, the affected nephron segment, the number of affected nephrons, synchronized or unsynchronized cyst formation and the extent of fibrosis and inflammation. Mouse models have provided valuable mechanistic insights into the pathogenesis of PKD; for example, mutated Pkd1 or Pkd2 cause renal cysts but additional factors are also required, and the rate of cyst formation is increased in the presence of renal injury. Animal studies have also revealed complex genetic and functional interactions among various genes and proteins associated with PKD. Here, we provide an update on the preclinical models commonly used to study the molecular pathogenesis of ADPKD and test potential therapeutic strategies. Progress made in understanding the pathophysiology of human ADPKD through these animal models is also discussed.

Cyst expansion and regression in a mouse model of polycystic kidney disease.

Autosomal-dominant polycystic kidney disease is characterized by progressive cyst formation and fibrosis in the kidneys. Here we describe an orthologous Pkd1(nl,nl) mouse model, with reduced expression of the normal Pkd1 transcript, on a fixed genetic background of equal parts C57Bl/6 and 129Ola/Hsd mice (B6Ola-Pkd1(nl,nl)). In these mice, the first cysts develop from mature proximal tubules around birth. Subsequently, larger cysts become visible at day 7, followed by distal tubule and collecting duct cyst formation, and progressive cystic enlargement to develop into large cystic kidneys within 4 weeks. Interestingly, cyst expansion was followed by renal volume regression due to cyst collapse. This was accompanied by focal formation of fibrotic areas, an increased expression of genes involved in matrix remodeling and subsequently an increase in infiltrating immune cells. After an initial increase in blood urea within the first 4 weeks, renal function remained stable over time and the mice were able to survive up to a year. Also, in kidneys of ADPKD patients collapsed cysts were observed, in addition to massive fibrosis and immune infiltrates. Thus, B6Ola-Pkd1(nl,nl) mice show regression of cysts and renal volume that is not accompanied by a reduction in blood urea levels.

Morphometric analysis of centrosome position in tissues.

Planar cell polarity (PCP) is the polarization of cells within the plane of an epithelial cell layer. PCP is important in many tissues in different processes. In the kidney, it is hypothesized to be important in acquiring and maintaining correct tubular diameter. Aberrant PCP has been shown to be involved in polycystic kidney disease. Therefore, research in this field requires a method to study PCP. As PCP and outward-in signaling via the cilia are interconnected, the position of the centrosome, the base of the cilium can be used as a read-out system for PCP. Here, we provide a method in which the position of the centrosome is measured as read-out for PCP.

Polycystic kidney disease: the complexity of planar cell polarity and signaling during tissue regeneration and cyst formation.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inherited systemic disease with intrarenal cystogenesis as its primary characteristic. A variety of mouse models provided information on the requirement of loss of balanced polycystin levels for initiation of cyst formation, the role of proliferation in cystogenesis and the signaling pathways involved in cyst growth and expansion. Here we will review the involvement of different signaling pathways during renal development, renal epithelial regeneration and cyst formation in ADPKD, focusing on planar cell polarity (PCP) and oriented cell division (OCD). This will be discussed in context of the hypothesis that aberrant PCP signaling causes cyst formation. In addition, the role of the Hippo pathway, which was recently found to be involved in cyst growth and tissue regeneration, and well-known for regulating organ size control, will be reviewed. The fact that Hippo signaling is linked to PCP signaling makes the Hippo pathway a novel cascade in cystogenesis. The newly gained understanding of the complex signaling network involved in cystogenesis and disease progression, not only necessitates refining of the current hypothesis regarding initiation of cystogenesis, but also has implications for therapeutic intervention strategies. This article is part of a Special Issue entitled: Polycystic Kidney Disease.

Altered Hippo signalling in polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive deterioration of renal function and formation of cysts, and is an important cause of end-stage renal disease. Previously we showed that tubular epithelial injury accelerates cyst formation in inducible Pkd1-deletion mice. In these mice, expression of the planar cell polarity (PCP) component Four-jointed (Fjx1) is decreased during epithelial repair, while in control mice Fjx1 expression is increased and may be required during tissue regeneration. In cystic kidneys, however, Fjx1 expression is also increased. Besides a PCP component, Four-jointed is also implicated in the Hippo-signalling pathway. This pathway is involved in organ size control by regulating proliferation and apoptosis. The role of Hippo signalling, together with the opposing expression pattern of Fjx1 during epithelial repair and at cystic stages, triggered us to investigate the activity of the Hippo pathway during these processes. Therefore, we examined its final effector molecule, the transcriptional co-activator Yes-associated protein (YAP) and observed that during tissue repair, YAP expression was not different between Pkd1-deletion mice and controls, ie during tissue regeneration YAP expression was increased and predominantly localized in the cytoplasm but normalized after tissue repair. At a later stage, however, in cystic epithelia and epithelia of dilated tubules, strong nuclear YAP accumulation was observed, accompanied by up-regulation of the YAP transcriptional targets Birc-3, Ctgf, InhbA, and Fjx1. Altered activity of the Hippo pathway was confirmed in renal tissues from human ADPKD and ARPKD patients, as well as in cystic renal tumours. Our data strengthen the concept that during epithelial repair Four-jointed is involved in PCP signalling, while in cystic kidneys it is related to Hippo signalling and cyst growth.

Toxic tubular injury in kidneys from Pkd1-deletion mice accelerates cystogenesis accompanied by dysregulated planar cell polarity and canonical Wnt signaling pathways.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by large fluid-filled cysts and progressive deterioration of renal function necessitating renal replacement therapy. Previously, we generated a tamoxifen-inducible, kidney epithelium-specific Pkd1-deletion mouse model and showed that inactivation of the Pkd1 gene induces rapid cyst formation in developing kidneys and a slow onset of disease in adult mice. Therefore, we hypothesized that injury-induced tubular epithelial cell proliferation may accelerate cyst formation in the kidneys of adult Pkd1-deletion mice. Mice were treated with the nephrotoxicant 1,2-dichlorovinyl-cysteine (DCVC) after Pkd1-gene inactivation, which indeed accelerated cyst formation significantly. After the increased proliferation during tissue regeneration, proliferation decreased to basal levels in Pkd1-deletion mice just as in DCVC-treated controls. However, in severe cystic kidneys, 10-14 weeks after injury, proliferation increased again. This biphasic response suggests that unrestricted cell proliferation after injury is not the underlying mechanism for cyst formation. Aberrant planar cell polarity (PCP) signaling and increased canonical Wnt signaling are suggested to be involved in cyst formation. Indeed, we show here that in Pkd1 conditional deletion mice expression of the PCP component Four-jointed (Fjx1) is decreased while its expression is required during tissue regeneration. In addition, we show that altered centrosome position and the activation of canonical Wnt signaling are early effects of Pkd1-gene disruption. This suggests that additional stimuli or events are required to trigger the process of cyst formation. We propose that during tissue repair, the integrity of the newly formed Pkd1-deficient cells is modified rendering them susceptible to subsequent cyst formation.

PAT for vaccines: the first stage of PAT implementation for development of a well-defined whole-cell vaccine against whooping cough disease.

Since variation in process time and process output is commonly accepted to be inevitable for biological processes, application of Process Analytical Technologies (PAT) on these processes is challenging. In this paper the applicability of PAT on the cultivation of Bordetella pertussis bacteria as part of the manufacture of a vaccine against whooping cough disease is investigated. Scrutinizing and eliminating the most prominent sources of variance make the cultivation process step highly reproducible. Furthermore, the use of DNA microarrays allows investigation of how disturbances influence cellular physiology and product quality. Marker genes for product quality were identified, providing the means to quantitatively assess product quality, which is hardly possible using the mandatory animal tests for product quality. The tools and results described in this paper, combined with suitable on line measurements, can make full PAT application for this process step possible. Ultimately, the process can be designed and controlled towards consistent end product quality.

HIV entry inhibitor TAK-779 attenuates atherogenesis in low-density lipoprotein receptor-deficient mice.

OBJECTIVE: HIV combination therapy using protease inhibitors is associated with elevated plasma levels of atherogenic lipoproteins and increased risk for atherosclerosis. We investigated whether the HIV entry inhibitor TAK-779 affects lipoprotein levels and atherogenesis in low-density lipoprotein receptor-deficient mice. TAK-779 is an antagonist for the chemokine receptors CCR5 and CXCR3, which are expressed on leukocytes, especially T-helper 1 cells, and these receptors may be involved in recruitment of these cells to atherosclerotic plaques. METHODS AND RESULTS: TAK-779 treatment of low-density lipoprotein receptor-deficient mice did not elevate the levels of atherogenic lipoproteins, whereas it dramatically reduced atherosclerosis in the aortic root and in the carotid arteries. The number of T cells in the plaque was reduced by 95%, concurrently with a 98% reduction in the relative IFN-gamma area. TAK-779-treated animals showed a decreased percentage of CD4+ and CD8+ T cells in peripheral blood and in mediastinal lymph nodes compared with control-treated animals. CONCLUSIONS: TAK-779 not only suppresses HIV entry via blockade of CCR5 but also attenuates atherosclerotic lesion formation by blocking the influx of T-helper 1 cells into the plaque. TAK-779 treatment may be especially beneficial for young HIV patients as they face lifelong treatment, and this drug impairs atherogenesis.