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I Kid-ney you not! Single cell sequencing is great for research in kidney disease!

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I Kid-ney you not! Single cell sequencing is great for research in kidney disease!

Your kidneys are an amazing, underappreciated organ! These complicated bean-shaped organs, sit either side of your spine and perform vital tasks to keep us healthy. At roughly the size of your fists, they maintain fluid, electrolyte, and metabolite balance within the body, in addition to, regulating blood pressure and maintaining red blood cell homeostasis. The functional part of the human kidney constitutes around a million filtering units called nephrons. Each nephron consists of a capillary networks called the glomerulus, which filters the blood, and renal tubules, to return nutrients, fluids and other substances to the blood and eliminates excess waste as urine. To perform this highly convoluted process, the kidney contains nearly 20 specialized epithelial cells. Following size-selective filtering from the glomerulus, the proximal convoluted tubule reabsorbs more than 70% of the primary filtrate, which includes ions, water, and other nutrients, and removes toxins to adjust the filtrate pH. Filtrate then passes through the loop of Henle to concentrate the urine, and the distal convoluted tubule to regulate sodium reabsorption. Lastly, the collecting duct determines the final concentration of the urine by regulating the reabsorption of water and solutes from the filtrate. With such a diverse and complicated system, various kidney diseases can develop, such as acute kidney injury, kidney stones, infection, cancer, and the most common, chronic kidney disease (CKD). Examining the development of the cellular diversity is essential to understand kidney homeostasis, disease, and regeneration.

World Kidney Day is a global campaign to raise awareness about the importance of your kidneys. It is a collaborative initiative of the International Society of Nephrology (ISN) and the International Federation of Kidney Foundations – World Kidney Alliance (IFKF-WKA). Their mission is to promote kidney health worldwide and reduce the frequency and impact of kidney diseases and the associated health problems. To fulfil this, the campaign emphasizes key risk factors for CKD such as diabetes and high blood pressure and encourages regular screenings for CKD in patients with diabetes and hypertension. They also promote preventative behaviors and provide educational opportunities for medical professionals in detecting and reducing the risk of CKD. In addition to this, the campaign encourages transplantation as the optimal option for kidney failure and supports organ donations as a life-saving initiative. CKD affects more than 10% of the global population, amounting to more than 800 million individuals, for which there is no cure.

Figure 1. Our mascot Simon promoting world kidney day and promoting kidney health.

Understanding the cellular composition and role of each cell type in the kidney is important to decipher the complexity of this organ. Previous studies have relied on microscopy and fluorescence-activated cell sorting (FACS); however, these methods provide a low resolution on cell types, status, and heterogeneity (1). Single cell sequencing has accelerated our ability to gain a comprehensive measurement of the cellular state to delineate cell type specific gene expression patterns associated with various kidney diseases. As previously stated, CKD is an epidemic, with limited treatment options. Factors such as diabetes, hypertension and immune injury can lead to loss of glomerular function, which is a major cause of CKD. Repair mechanisms to restore function and how it leads to chronic fibrosis is understudied due to the difficulty to isolate glomerular cells. A recent study purified glomeruli and used single cell sequencing to characterize the cell types in four different common types of kidney injury (immune, metabolic, genetic, and toxic) in mice (2). Approximately 75,000 glomerular cells were sequenced which provided a detailed transcriptional insight of all cell types in the glomerulus and highlighted cell-type specific and injury-type specific responses.


Kidney tumour development and single cell sequencing

Renal cell carcinomas (RCCs) present diverse histomorphologies, clinical behaviors and immune cell patterns. RCC arises from kidney tubular epithelial cells, where clear cell RCC (ccRCC) is the most common subtype. Delineating cell origin for different subtypes of RCC can enhance understanding of their diverse pathogenesis. Zhang et al employed scRNAseq to build comprehensive benign and malignant renal cell atlases to decipher the cell origin of more than 10 RCC subtypes. The single cell analysis also revealed novel insights into the tumour microenvironment of ccRCC, including the role of tumour epithelia in promoting immune cell infiltration (11). Another study investigated cancer evolution and tumour differentiation to identify cells from which tumours arise, focusing on kidney tumours. The authors utilized a combination of bulk transcriptome analysis with single cell experiments (figure 2). Analysis of single cell mRNA signals in bulk kidney tumour transcriptomes were initially performed, followed by single cell experiments to validate their findings. Childhood and adult kidney tumours were analyzed to discover fetal cellular signals presenting in childhood tumours and the refute of adult tumours dedifferentiating towards a fetal state. They also discovered a close relationship between developmental mesenchymal progenitor cell populations and childhood tumours (12). Together, these studies highlight an important role of scRNA seq to investigate kidney tumours.

Figure 2. Overview of the methodology employed by Young et al, combining bulk transcriptome data and single cell data (Image taken from (12); https://creativecommons.org/licenses/by/4.0/).


Using single cell sequencing to explore immune related kidney diseases

The kidney plays a vital role in maintaining the homeostasis of the immune system by the filtration and excretion of toxins, cytokines, and inflammatory molecules (5). Single cell sequencing technology has revolutionized research into kidney immunology, enabling insights into cell-cell interactions, cellular heterogeneity, and genetic regulation in kidney diseases, to ultimately advance disease diagnosis and the discovery of novel therapeutic targets. Diabetic kidney disease (DKD) is a major cause of end stage renal disease (ESRD) worldwide (6). Several publications have revealed an essential role of immune cells in the pathogenesis of DKD. Fu et al used scRNA-seq and discovered an increase of immune cells in the glomeruli of diabetic mice (7). Macrophage detection was the highest among major immune cells, where a ratio imbalance of macrophage subtypes; M1 and M2, previously associated with tissue damage and repair respectively, is a potential cause of tissue injury in DKD kidneys. Another study from Wilson et al performed scRNA-seq and observed expression of IFN gamma (IFNGR1 and IFNGR2) downstream signaling genes, which have previously been implicated in DKD (8).

IgA neuropathy (IgAN) is a type of primary glomerulonephritis, affecting many individuals worldwide. A study, using Singleron’s GEXSCOPE technology, performed single cell sequencing on IgAN patients (figure 3) and discovered the increased expression of MALAT1, GADD45B, SOX4 and EDIL3 in mesangial cells, highlighting an essential role in the pathogenesis of IgAN (9), which has never been shown before. The authors also revealed genes involved in several inflammatory pathways, including TNF signaling IL-17 signaling and NOD-like receptor signaling were overexpressed in tubule cells in IgAN patients.

Lupus nephritis (LN) is a consequence of systemic lupus erythematosus (SLE) for which the current treatments are toxic and often ineffective. Single cell sequencing of kidney samples from patients with LN and health donors revealed 21 subsets of active leukocytes, in addition to, multiple population of cells demonstrating both pro-inflammatory and inflammation-resolving responses, including myeloid cells, T cells, B cells and natural killer cells. scRNA-seq was also performed with immune cells from urine sample to reveal a high correlation with kidney samples, providing a novel non-invasive method to analyze immune cells in kidneys (10).

Figure 3. Fourteen different cell clusters were identified from 4 IgAN patients and a healthy control (A), where enrichment for each cell cluster was plotted for each subject (B) (Image taken from (9); https://creativecommons.org/licenses/by/4.0/).


Analyzing kidney development with single cell sequencing

Understanding the developmental origin of kidney diseases requires in-depth knowledge of kidney development, which is largely based on mouse models (3). A recent study performed single cell transcriptomics of human fetal kidney since drastic differences in renal embryogenesis between human and mouse had recently been highlighted. The authors discovered 22 different cell types, in additional to known marker genes. Comparison analysis of samples from different developmental stages revealed changes in gene expression in podocytes and epithelial cells lining the outer surface of glomerular capillaries (4).

Altogether, these publications show single cell sequencing as a valuable tool to investigate different aspects of kidney research, ranging from the kidney development, immunology, and tumour differentiation.

If you want to know more about how Singleron technology can complement your research, get in touch at info@singleronbio.com to discuss your project with our single cell experts.

References

  1. 1. Wu H., Humphreys B. D. (2017). The promise of single-cell RNA sequencing for kidney disease investigation. Kidney Int. 92, 1334–1342. doi:10.1016/j.kint.2017.06.03
  2. 2. Chung, Jun-Jae; Goldstein, Leonard; Chen, Ying-Jiun J.; Lee, Jiyeon; Webster, Joshua D.; Roose-Girma, Merone; Paudyal, Sharad C.; Modrusan, Zora; Dey, Anwesha; Shaw, Andrey S. Single-Cell Transcriptome Profiling of the Kidney Glomerulus Identifies Key Cell Types and Reactions to Injury. JASN 31(10):p 2341-2354, October 2020. | DOI: 10.1681/ASN.2020020220
  3. 3. Lindström NO, McMahon JA, Guo J, Tran T, Guo Q, Rutledge E, et al. Conserved and Divergent Features of Human and Mouse Kidney Organogenesis. J Am Soc Nephrol. 2018; ASN.2017080887. pmid:29449453
  4. 4. Hochane M, van den Berg PR, Fan X, Bérenger-Currias N, Adegeest E, et al. (2019) Single-cell transcriptomics reveals gene expression dynamics of human fetal kidney development. PLOS Biology 17(2): e3000152. https://doi.org/10.1371/journal.pbio.3000152
  5. 5. Donnan, M. D., Kenig-Kozlovsky, Y., and Quaggin, S. E. (2021). The lymphatics in kidney health and disease. Nat. Rev. Nephrol. 17, 655–675. doi: 10.1038/s41581-021-00438-y
  6. 6. Thomas, M. C., Brownlee, M., Susztak, K., Sharma, K., Jandeleit-Dahm, K. A., Zoungas, S., et al. (2015). Diabetic kidney disease. Nat. Rev. Dis. Primers 1:15018. doi: 10.1038/nrdp.2015.70
  7. 7. Fu, J., Akat, K. M., Sun, Z., Zhang, W., Schlondorff, D., Liu, Z., et al. (2019). Single-Cell RNA Profiling of Glomerular Cells Shows Dynamic Changes in Experimental Diabetic Kidney Disease. J. Am. Soc. Nephrol. 30, 533–545. doi: 10.1681/asn.2018090896
  8. 8. Wilson, P. C., Wu, H., Kirita, Y., Uchimura, K., Ledru, N., Rennke, H. G., et al. (2019). The single-cell transcriptomic landscape of early human diabetic nephropathy. Proc. Natl. Acad. Sci. U S A 116, 19619–19625. doi: 10.1073/pnas.1908706116
  9. 9. Tang R, Meng T, Lin W, Shen C, Ooi JD, Eggenhuizen PJ, Jin P, Ding X, Chen J, Tang Y, Xiao Z, Ao X, Peng W, Zhou Q, Xiao P, Zhong Y and Xiao X (2021) A Partial Picture of the Single-Cell Transcriptomics of Human IgA Nephropathy. Front. Immunol. 12:645988. doi: 10.3389/fimmu.2021.645988
  10. 10. Arazi, A., Rao, D. A., Berthier, C. C., Davidson, A., Liu, Y., Hoover, P. J., et al. (2019). The immune cell landscape in kidneys of patients with lupus nephritis. Nat. Immunol. 20, 902–914. doi: 10.1038/s41590-019-0398-x
  11. 11. Zhang Y, Narayanan SP, Mannan R, Raskind G, Wang X, et al. (2021). Single-cell analyses of renal cell cancers reveal insights into tumor microenvironment, cell of origin, and therapy response. Proc Natl Acad Sci U S A. 2021 Jun 15;118(24):e2103240118. doi: 10.1073/pnas.2103240118. PMID: 34099557; PMCID: PMC8214680.
  12. 12. Young, M.D., Mitchell, T.J., Custers, L. et al. Single cell derived mRNA signals across human kidney tumors. Nat Commun 12, 3896 (2021). https://doi.org/10.1038/s41467-021-23949-5
A post by Singleron team

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