Background PKHD1 is the main genetic cause of autosomal recessive polycystic kidney disease (ARPKD), a hereditary hepato-renal fibrocystic disorder which is the most important cause of end-stage renal disease during early childhood. ARPKD can also present in adulthood with milder phenotypes. In this study, we describe a 24-year-old woman with atypical polycystic kidney, no family history of renal disease and no obvious extra-renal manifestations who was referred for genetic investigation. Methods We used a combination of next generation sequencing, Sanger sequencing and RNA and microscopy studies performed on urine-derived renal epithelial cells (URECs) to provide a genetic diagnosis of ARPKD. Results A next generation sequencing panel of cystic ciliopathy genes allowed the identification of two heterozygous sequence changes in PKHD1 (c.6900C > T; p.(Asn2300=) and c.7964A > C; p.(His2655Pro)). The pathogenicity of the synonymous PKHD1 variant is not clear and requires RNA studies, which cannot be carried out efficiently on RNA extracted from proband blood, due to the low expression levels of PKHD1 in lymphocytes. Using URECs as a source of kidney-specific RNA, we show that PKHD1 is alternatively spliced around exon 43, both in control and proband URECs. The variant p.(Asn2300=) shifts the expression ratio in favour of a shorter, out-of-frame transcript. To further study the phenotypic consequence of these variants, we investigated the ciliary phenotype of patient URECs, which were abnormally elongated and presented multiple blebs along the axoneme. Conclusions We confirm the power of URECs as a tool for functional studies on candidate variants in inherited renal disease, especially when the expression of the gene of interest is restricted to the kidney and we describe, for the first time, ciliary abnormalities in ARPKD patient cells.

Osteosarcoma (OS) accounts for 60% of all global bone cancer diagnoses. Intravenous administration of Doxorubicin Hydrochloride (DOXO) is the current form of OS treatment, however, systemic delivery has been linked to the onset of DOXO induced cardiomyopathy. Biomaterials including calcium phosphate cements (CPCs) and nanoparticles (NPs) have been tested as localized drug delivery scaffolds for OS cells. However, the tumor microenvironment is critical in cancer progression, with mesenchymal stem cells (MSCs) thought to promote OS metastasis and drug resistance. The extent of MSC assisted survival of OS cells in response to DOXO delivered by CPCs is unknown. In this study, we aimed at investigating the effect of DOXO release from a new formulation of calcium phosphate-based bone cement on the viability of OS cells cocultured with hMSC in vitro. NPs made of PLGA were loaded with DOXO and incorporated in the formulated bone cement to achieve local drug release. The inclusion of PLGA-DOXO NPs into CPCs was also proven to increase the levels of cytotoxicity of U2OS cells in mono-and coculture after 24 and 72 h. Our results demonstrate that a more effective localized DOXO delivery can be achieved via the use of CPCs loaded with PLGA-DOXO NPs compared to CPCs loaded with DOXO, by an observed reduction in metabolic activity of U2OS cells in indirect coculture with hMSCs. The presence of hMSCs offer a degree of DOXO resistance in U2OS cells cultured on PLGA-DOXO NP bone cements. The consideration of the tumor microenvironment via the indirect inclusion of hMSCs in this study can act as a starting point for future direct coculture and in vivo investigations.

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