To support the development of new therapies for autosomal dominant polycystic kidney disease, we offer a kidney-specific PKD1 knockout mouse model. The age-dependent nature of cyst formation allows for tailoring of the model, enabling researchers to choose the most appropriate study design to fit their drug development pipeline.
Autosomal Dominant Polycystic Kidney Disease (ADPKD)
Autosomal dominant polycystic kidney disease (ADPKD) is the most common single-gene disorder and the most prevalent (4 to 10:10 000) progressive kidney disorder. The kidneys of ADPKD patients are affected by fluid-filled cyst formation and growth, ultimately resulting in kidney failure. However, the disease course of ADPKD patients tends to be highly variable with the age of onset ranging from early childhood to>80 years. Reports show that 50% of the patient population develops end-stage kidney failure by the time they reach 60 years of age (1).
Currently, the gold standard and only approved drug for ADPKD treatment is Tolvaptan. However, Tolvaptan induces serious side effects and does not allow long-term treatment. This limits the treatment of ADPKD to disease management strategies, highlighting the need for novel drugs targeting cystogenesis, that would slow down and/or halt further disease progression. Addressing this currently unmet medical need requires novel interventions to be tested in both a cost-effective and timely manner, which demands well-characterized, translational animal models of ADPKD.
ADPKD Pathophysiology and Relevant Targets
The main genes mutated in ADPKD patients are PKD1 or PKD2, with 85% of patients carrying the PKD1 gene mutation. A two-hit hypothesis has been proposed to explain the focal nature of cyst development, in which a germline mutation (first hit) in one of the two alleles together with a somatic mutation knocking out the second allele (second hit) lead to non-functional polycystin (PC) proteins. Although other genes have given mechanistic insights into the disease pathophysiology, the PKD1 gene is the most studied gene with respect to ADPKD. Moreover, the PKD1 protein interacts with the PKD2 protein, which is mutated in 15% of the ADPKD patient population (1). Therefore, models affecting the PKD1 gene represent an attractive tool for researchers involved in the search for novel therapeutics targeting ADPKD.
The gene product of PKD1 is PC1, which modulates several signaling pathways together with PC2, encoded by the PKD2 gene. Mutations in the PKD1 gene cause changes in PC1 expression, which interferes with several intracellular signaling pathways that are in control of cell proliferation, fluid secretion, and ciliary function, amongst others (Figure 1). The resulting dysregulation of cell proliferation and fluid secretion in the kidney leads to cyst development (1). The complex network of signaling pathways that are dysregulated in cystic kidneys provides many potential targets for therapeutic interventions (Figure 1).
FIGURE 1. Overview of autosomal dominant polycystic kidney disease (ADPKD) pathophysiology and main targets of potential treatments. Polycystin-1 and polycystin-2 (PC1 and PC2) are expressed in different subcellular locations (apical and basolateral membranes) where they are involved in the regulation of 1. cell proliferation 2. fluid secretion 3. ciliary function 4. cell-cell adhesion and 5. cell-matrix interactions (depicted as green boxes). Dysfunction in PC1 and PC2 leads to abnormal ciliary function and a decrease in intracellular calcium concentration which results in cAMP generation and mTOR activation, affecting cell proliferation and cyst development. The candidate drug targets (red boxes) include mTOR inhibitors (everolimus, rapamycin, metformin, curcumin), PC2 agonists, V2R antagonists (tolvaptan), somatostatin analogs, miRNA inhibitors, nrf2 activators, HDAC and CDKs inhibitors (roscovitine), EGFR inhibitors, CTFR modulators, and TNF-alpha inhibitors. Tolvaptan is a selective arginine V2R antagonist and decreases renal cAMP levels. TNF-alpha inhibitors target inflammation with a positive effect on pro-inflammatory markers found in ADPKD patients’ urine and renal cyst fluid. RNA-targeted therapies such as miRNA inhibitors, RNA-mediated interference, anti-sense oligonucleotides, and non-coding RNAs, can be used to regulate the expression of target mRNAs implicated in ADPKD and therefore their protein product. HDACs modulate gene expression by removing acetyl groups from histones and regulate a diverse array of intracellular pathways by acting on nonhistone proteins (e.g., cell cycle progression inhibition, downregulation of cAMP…). CFTR modulators act on CFTR, which is a chloride ion channel facilitating the transtubular chloride secretion to the cysts. Lastly, metabolic approaches are also an attractive way to target ADPKD, given that ADPKD cysts show dysregulated metabolism with a high rate of glycolysis and a low rate of mitochondrial oxidative phosphorylation.
Validated, translationally relevant and customizable model to study novel ADPKD treatments
At InnoSer, we offer a uniquely engineered inducible, kidney-specific Cre(lox,lox)–Pkd1 knock-out (KO) model (2). Conditionally disrupting Pkd1 gene expression prevents embryonic lethality of mice that occurs with Pkd1 germline deletion due to a severe cystic phenotype. The inducible feature of this model allows a disruption of PKD1 gene expression at specifically chosen time points, both in developing and adult mice. This not only allows a well-controlled analysis of ADPKD pathogenesis, but also the creation of different ADPKD progression models, thanks to the characteristic age-dependent cyst formation observed in our model.
When conducting preclinical research using our model, there are three time points at which the KO can be induced, referring to the post-natal day (P) at which tamoxifen-induced Pkd1 gene disruption is performed; P10, P18 and P40. As the kidney tissue is found in different proliferative stages in the juvenile and adult mice, differences in susceptibility to cystogenesis are observed in each approach. Consequently, each timing of the Pkd1 gene disruption has a major effect on the severity and progression of the cyst development (Figure 2) (2). Therefore, each study time point presents a unique model with its own benefits and considerations, which we explain below.
FIGURE 2. H&E-stained kidney sections depicting the differences in renal cystic phenotypes between P10 and P18 models. (A-C) Pkd1 knock-out (KO) at post-natal day 10 (P10) shows fast progression to cystogenesis in the outer medulla compared to the other kidney regions. Additionally, this model shows rapid formation of multiple large cysts within a short period. Treatment with previously validated drug against ADPKD, such as Everolimus shows a significant reduction in the cystic phenotype. (D-F) In contrast to the P10 model, the P18 model shows synchronized formation of multiple, smaller cysts throughout the whole kidney region. Treatment with previously validated drug against ADPKD, such as Tolvaptan shows a significant reduction in the cystic phenotype.
P10 study: Perform Fast Lead Compound Screening Studies
P10 studies offer researchers a compound screening platform, highly suitable for quick and robust testing, ideal for pharmacokinetic as well as efficacy and tolerability studies with multiple lead compounds. Moreover, it has been shown that the early onset of ADPKD in neonatal and juvenile patients leads to relatively large cysts, explained by the rapid cyst growth in the developing kidneys, which is also observed in the P10 model.
Disruption of Pkd1 gene expression at P10 leads to the rapid and massive development of cysts in the distal segment of the nephron (Figure 2), since the renal epithelium is still in a proliferative stage (2) (Figure 3). Thanks to its short study nature (in-life phase lasts 4 weeks), P10 studies are the most cost- and time-efficient to perform, in comparison to P18 and/or P40 studies.
FIGURE 3. Differences in renal proliferative state of the different ADPKD disease progression models and the gold-standard treatment response. Histological images show areas of epithelial cell nuclei, positive for the proliferation marker Ki67. (A) The renal tubular epithelium of the P10 model shows significant proliferation around the cysts, indicated by the strong Ki67 expression (shown by the zoomed-in section). (B) Treatment with everolimus reduces the number of proliferative cells around the cysts, indicated by the weak Ki67 staining. (C) In contrast to the P10 model, the P18 model shows a lower number of cells undergoing proliferation around the cysts. Differences in cyst development and growth between the P10 and P18 models are thus likely related to the differences in proliferation. (D) Following treatment with tolvaptan, kidneys from animals of the P18 model showed decreased cell proliferation, indicated by a weak and/or absent Ki67 expression. Scale bars are shown for each respective image.
P18 & P40 studies: Perform Long-term Studies
P18 and P40 studies are typically preferred by researchers who wish to evaluate the efficacy of their compound in a more clinically relevant set-up. It has been reported that individuals with adult-onset ADPKD show cyst formation and growth at a much slower rate compared to juvenile-onset ADPKD patients (3). Knocking out Pkd1 at P18 (in-life phase lasts approx. 18 weeks) and P40 (in-life phase lasts around 27 weeks) leads to a relatively slow cyst development and progression, affecting all segments of the nephron (Figure 2). This resembles the course of the pathophysiology in adult disease onset ADPKD. P18 and P40 studies can additionally provide a platform for researchers looking to perform chronic treatment studies, providing the opportunity to also detect any possible long-term side effects.
Readout Selection: How to Measure Disease Progression
At InnoSer, we use several readouts to assess the disease progression and the effects of novel targeted ADPKD compounds. General pharmacokinetic studies allow the assessment of biodistribution of the compounds of interest, given that kidney-specific compound administration is crucial to prevent systemic toxicity associated with long-term therapy in ADPKD patients. Kidney volume is an important predictor of ADPKD progression in the patient population and is therefore also highly translational in the preclinical testing of novel therapeutics. Leveraging our in vivo capabilities, we perform ultrasound kidney volume measurements to gain translationally relevant longitudinal data. More importantly, the total kidney volume (TKV) is considered a surrogate biomarker for a decline in kidney function in ADPKD patients, approved by EMA and the FDA. The increase in kidney volume is related to a decline in the glomerular filtration rate (GFR), which provides highly relevant information about the progression of the disease. At InnoSer, GFR is assessed transdermally, allowing the collection of longitudinal assessment of renal function. On the other hand, blood creatinine and urea measurements provide more general kidney function parameters and are less expensive to perform than GFR.
In addition to kidney volume and function, cystogenesis, and other associated pathological lesions, as well as the effect of novel treatments on inhibiting or halting the development and/or further growth of cysts can be assessed by routine H&E stains. The analysis of the total area of cysts within the total area of the kidney, referred to as a cystic index, allows us to quantify the process of cystogenesis following routine H&E staining. Finally, specific stainings can be used to assess fibrosis (PSR, Trichrome, alpha-SMA), proliferation (Ki67), and cyst growth (Cyclin D1) to gain additional insight into the therapeutic mechanism of investigational drugs.
Choosing the Right Study Type for Your Research
The most appropriate study type and design ultimately depend on the specific aims of the research question. Each model has its characteristics, such as the age and rate of disease progression, the affected nephron segment, the number of affected nephrons, and the rate of cyst formation and growth. Factors such as the pharmacological properties of the compound being tested, as well as target protein expression in different nephron segments and cystic epithelia, need to be considered.
Consulting with our nephrology study experts will allow you to carry out tailored studies while collecting the most study-appropriate data. We also advise you on the most optimal model selection, taking in your budget and study timelines.
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- Bergmann C, Guay-Woodford LM, Harris PC, Horie S, Peters DJ, Torres VE. Polycystic kidney disease. Nature reviews Disease primers. 2018 Dec 6;4(1):50.
- Lantinga-van Leeuwen IS, Leonhard WN, van der Wal A, Breuning MH, de Heer E, Peters DJ. Kidney-specific inactivation of the Pkd1 gene induces rapid cyst formation in developing kidneys and a slow onset of disease in adult mice. Human molecular genetics. 2007 Dec 15;16(24):3188-96.
- Leonhard WN, Happe H, Peters DJ. Variable cyst development in autosomal dominant polycystic kidney disease: the biologic context. Journal of the American Society of Nephrology. 2016 Dec 1;27(12):3530-8.