Defining regional anatomical variation in cellular composition, transcriptome and epigenome in the human kidney

The kidneys play a vital role in cleaning the blood and controlling the body's water balance. Unfortunately, their function may be affected by a number of common diseases including infection, diabetes, damage by reduced blood supply and high blood pressure or the toxic effects of some drugs. Kidney failure can be treated with dialysis or transplant, but both (particularly dialysis) are associated with reduced life expectancy.

The individual building blocks of the kidneys are called cells, and each kidney has hundreds of millions of cells that work in groups that have a similar function. The interaction of individual cells, and groups of cells, is required for normal organ function.
Among the different cell types that reside in the kidney are immune cells, the body's defence system cells. These sentinels stand guard and alert the body to local problems by starting an inflammatory response. They also play an important role in tissue healing and repair. Because of their job of excreting waste and controlling water balance, the kidneys make an unusual and difficult environment for immune cells; some parts of the kidney have a very high salt concentration, so the cells are living in a salt marsh and this can affect their function. At present, we don't fully understand the different types of cells that make up the kidney, how many different of immune cells live there, how they are positioned relative to each other, or whether the extreme environment of some parts of the kidney changes how the cells behave. Answering these questions will help us to determine how cell function goes wrong in kidney diseases and will therefore allow the development of better treatments.

Our aim is to make a cell map of the human kidney.

We will take two experimental approaches:
The first is to take a piece of kidney, mash it up to break up the cell groups so that we can analyse individual cells. Different cells have different functions and identification marks because of differences in which parts of their genetic material (called genes) are translated into proteins. Protein are the molecules that mark a cell and allow it to function. Every cell contains tens of thousands of proteins, giving it a unique signature, but these are hard to measure in a single cell. However, we can measure the molecule that acts as an intermediate between genes and proteins, this is called messenger RNA or mRNA. In the past 10 years, technology has advanced such that we can measure thousands of mRNA molecules in a single cell, this is what we plan to do with the mashed kidney samples that we will take from 6 different regions across the human kidney.

Our second experimental approach is to take a piece of kidney and keep it intact so that the relationship of the cells in space is maintained. We will then make slices of the piece of kidney so that they are thin enough to be seen under a microscope. We will use the mRNA information from our first experiment to identify a small number of markers that can be visualised by the microscope using probes that are fluorescently labeled. This will allow us to investigate the position of cells relative to each other and how different cells in both the adult and developing kidney provide support or a 'niche' for immune cells.

Making a complete map of cells in the normal kidney will provide a critical reference atlas that will help researchers in the future to understand which cells become abnormal in different kidney diseases and how cell interactions are disrupted in disease. This will provide invaluable information to help identify new treatment targets.

The kidneys contain a network of cells, including resident immune cells, which interact to carry out organ function, defence and repair. The homeostatic role of the kidney generates extreme anatomical variation in tissue environment (eg, oxygenation and sodium concentration), which can impact cell function. A comprehensive understanding of the cellular networks in different regions of the normal human kidney is currently lacking, and will provide an important reference dataset from which to investigate disease-associated perturbations. This project aims to address this need by:

1. Generating single-cell molecular profiles across the entire cross-section of the adult human kidney. We will perform droplet based RNA sequencing (10x Genomics platform) on single cell suspensions obtained from n=6 dissociated tissue samples (1cm3) taken from adjacent anatomical areas of n=3 adult human kidneys, spanning from the outer cortex to the upper ureter. We will profile unselected kidney cells and isolated immune cells from each anatomical region in parallel, and will use citeSEQ antibodies to enhance immune cell characterisation.

2. Characterising the epigenetic landscape of immune cells residing in different anatomical regions of the kidney. We will use the 10x scATAC seq platform to assess the chromatin landscape in immune cells obtained from cortex, medulla and pelvis of n=3 adult kidneys, Together these data will allow us to assess how different environment cues affect the potential for transcriptional activity.

3. Ascertaining the precise spatial relationships of transcriptionally characterized cells within different anatomical regions of the fetal and adult kidney. We will use RNA Scope and imaging mass cytometry to determine how kidney epithelium, endothelium and fibroblasts co-localise with immune cells. We will determine whether the survival niche for resident immune cells differs in the developing fetal kidney compared with adulthood.

Who might benefit from this research?
1. Human Cell Atlas
2. Academic beneficiaries
3. Translational/ Pharma beneficiaries
4. Patients, economic and societal benefits

How might they benefit from this research?
1. Human Cell Atlas project - As described above, our data will make a substantial contribution to the human kidney atlas.

2. Academic beneficiaries. The data, tissue processing and storage protocols and analysis platforms we will generate will be of interest to human immunologists, mononuclear phagocyte biologists, kidney researchers, computational biologists and academic clinicians, informing their research, and providing validation and comparator datasets and tools to enable more efficient use of their own data and resources. This will ultimately enable academics to derive the maximum utility from their own datasets and experiments, synergizing efforts in the field.

3. Translational and industry beneficiaries - The reference dataset of normal human kidney cell types generated by this project can be used to allow diseases-associated genes, proteins and pathways to be mapped onto specific cell types providing important potential insights into aetiopathogenesis and potentially identifying novel therapeutic targets in a number of kidney diseases. We will ensure dissemination of this information to colleagues in industry via peer-reviewed publication and by presentations at joint meetings with pharma, such as the Cambridge Varsity Meeting with GlaxoSmithKline. The project will be of particular relevance to those developing therapies for :
a. Autoimmune diseases involving the kidneys.
b. Glomerulonephritides, including IgA nephropathy, membranous GN, diabetic nephropathy.
c. Acute kidney injury (AKI), which most commonly occurs due to hypotension, ischaemia or nephrotoxins, resulting in acute tubular necrosis (ATN). ATN is also the commonest cause of delayed graft function in kidney transplantation.
d. Chronic kidney disease (CKD). CKD represents represents the common end-phenotype of a number of processes and is characterized by fibrosis and tubular atrophy. There are currently no specific treatments.

4. Patients, economic and societal benefits
The identification of new therapeutic strategies in kidney disease would have a significant health-economic benefits, as renal replacement therapy (dialysis and transplantation) is very costly. For example, ATN associated with AKI affects 10% of hospital inpatients and round 10-15% of these patients will require at least one session of dialysis costing £150 per session, and together costing the NHS tens of millions of pounds per annum. Furthermore, some patients with CKD progress to end-stage renal failure, requiring dialysis or a transplant to sustain life. Provision of chronic dialysis requires substantial resource, 2% of total NHS budget, and in the US, $25 billion, representing 27% of the total Medicare budget.
Amelioration of AKI in renal transplantation would also achieve a significant cost saving; more than half of kidneys obtained from deceased cardiac death donors develop ATN, necessitating the patient to receive one or more sessions of dialysis with its attendant risks and costs. Having treatments for AKI and CKD would also expand the number of organs that could be used for transplantation, with the ultimate effect of reducing the number of patients on dialysis. The avoidance of dialysis not only has economic benefits, but also has societal benefits, since many patients receiving dialysis are unable to work due to the time constraints imposed by the requirement for thrice weekly dialysis sessions. Transplantation restores patient independence and frequently facilitates a return to work.
Therefore datasets that will potentially shed light on new treatment strategies for these conditions would have substantial societal and economic benefits, as well as benefits for individual patient's quality of life.