How preservation of urine can impact kidney disease research
Background:
Each day, 20 people in the UK alone, develop chronic kidney disease, while an estimated 3 million people in the UK are thought to suffer from some form of kidney disease. Kidney disease can occur due to 1) genetics, 2) poorly controlled diabetes, 3) heart disease, and 4) obesity. When the kidneys stop working properly, harmful toxins and excess fluid can build up in the body. People suffering from kidney disease also may suffer from high blood pressure, anaemia and bone and muscle weakness.
Primary ciliopathies are a group of rare genetic conditions, which can affect multiple organ systems including the kidneys (Figure 1). Found in almost all human cells, primary cilia are finger-like protrusions, acting like radio antennae receiving information from the surrounding environment. The primary cilia are also responsible for key cellular signalling mechanisms. Defects in these primary cilia are responsible for primary ciliopathies.
Figure 1. Wheel depicting primary ciliopathies which are known to have a renal phenotype. Other hallmark characteristics of each ciliopathy are also shown, to highlight the broad spectrum and organ-system involvement associated with each primary ciliopathy. Figure generated using BioRender.com
How we currently study primary ciliopathies:
To study primary ciliopathies, we use primary ciliated cells. One of the most frequently used cell lines to do this is fibroblasts, often derived from skin biopsies. Researchers often chose to ship established, confluent fibroblasts live, in flasks full to the brim with growth media. Another method of sample shipment commonly used is cryopreservation. However, delays in transport during long-haul shipments pose a major threat to sample integrity. Evaporation of dry ice, and consequently thawing of samples, results in cells sitting in dimethyl sulphate (DMSO), which is toxic at ambient temperatures.
Another rapidly growing method to study renal ciliopathies focuses on the use of urine. Every day, between 2000 and 7000 cells are shed from the kidneys, into the urine. These cells, known as human urine-derived renal epithelial cells (hURECs) can be isolated to study kidney diseases on a patient-specific basis. We use these hURECs to study several disease aspects, including primary cilia phenotype (how the cilia look and behave), investigating potential genetic drivers of specific diseases, and nephrotoxic drug screening (Figure 2). Currently, as researchers, we must collect, transport, and process urine samples within 4-hours, on ice, to ensure that these cells are viable for expansion. This is not an issue for locally based patients, however, for those patients further afield this 4-hour time frame poses a major hurdle. To complicate matters further, as hURECs are a primary cell line, they tend to be more sensitive to traditional cryopreservation shipment methods; in fact, long-distance collaborating researchers often opt to send hURECs live in plates (increasing contamination risks and likelihood of cell death).
Figure 2. Overview of how human urine-derived renal epithelial cells (hURECs) are currently isolated from whole urine samples, and how these cells are utilised in kidney and ciliopathy-focused research. Figure generated using Biorender.com.
How Atelerix technology can help:
Atelerix offers a solution to this sample shipment problem. Their novel technology specialises in the preservation of biological material at room temperature, removing the need for cell exposure to harmful cryopreservation reagents (like DMSO), eliminating the need for dry ice, and removing the risk of sending live cells. It is hoped that the specialist hydrogel preservation technology developed by Atelerix could be implemented at one of three key time points in the processing of urine samples (Figure 3).
Figure 3. Overview of the urine processing sample, with areas where Atelerix products could be implemented to help overcome issues associated with long-distance samples shipping shown in purple boxes. Areas: 1) after spinning down urine samples, Atelerix products (BeadReady or CytoStor) could be utilised to preserve the whole urinary pellet (which contains hURECs). 2) After urine spinning, and resuspension of the whole urinary pellet, cell suspension could be plated, and the Atelerix WellReady kit used to preserve samples prior to shipment. 3) Following complete urine processing, established hURECs could be shipped using any of Atelerix’s Kits (BeadReady, CytoStor or WellReady). Figure generated using BioRender.com.
Next Steps:
Over a three-month period, the following two aims will be explored to try and establish the impact of Atelerix technologies on renal and primary ciliopathy research.
- Explore the possibilities of using Atelerix technologies to preserve cells during the early stages of hUREC isolation
- Increase the viability of preserved established ciliated cell lines (including primary hURECs and fibroblasts) following preservation at room temperature to mimic shipment periods
Becky Dewhurst
MRC DiMeN DTP iCASE PhD Student
Newcastle University
