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Defining Cellular Injury in Diabetic Nephropathy by Single Cell RNA Sequencing

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Data Summary

Applicant	Humphreys, Benjamin
E-Mail Address	bhumphre@dom.wustl.edu
Project Title	Defining Cellular Injury in Diabetic Nephropathy by Single Cell RNA Sequencing
CBU ID	17AU3798
External SubContract ID	32307-7
Diabetic Complication	Nephropathy
Funding Program Group	Pilot & Feasibility [PF2017]
Abstract	Despite almost universal implementation within the last 20 years of treatments that were presumed to be reno-protective, diabetes continues to rank as the #1 cause of ESRD. Diabetic nephropathy (DN) is characterized by glomerulopathy, albuminuria and progressive tubulointerstitial fibrosis. Understanding the precise transcriptional changes that occur in single podocytes, tubular epithelium and interstitial cells during diabetic nephropathy may allow us to infer novel cell states and heterogeneity among these cells that will inform our understanding of disease pathogenesis. Single cell RNA-sequencing (scRNA-seq) has a unique advantage in characterizing cell transcriptomes because it can detect them comprehensively on a genomic scale. We have successfully implemented scRNA-seq in human kidney tissue, but we are limited by two critical hurdles. First, our dissociation protocols selectively enrich for proximal tubule epithelia but lack podocyte, fibroblast and endothelial cells, precluding their study. Second, our microfluidic-based scRNA-seq protocol requires very fresh tissue and cannot be performed on archival material. In this application we will attempt to overcome these substantial barriers by performing single nucleus RNA-seq (sNuc-seq) on archival, fresh frozen human kidney tissue of known histology and diagnosis. We believe that the nuclear isolation protocol will free all nuclei within our frozen sample, not just proximal tubule nuclei as with our single cell dissociation experience to date. We have already identified two normal and two diabetic nephropathy kidney samples in collaboration with the Boston Nephrectomy Biobank, led by Sus Waikar MD, MPH. The ability to perfrom sNuc-seq on archival material would also represent an enormous advance and open many new opportunities to study human diabetic nephropathy. We predict that measuring the gene expression repertoire of single nuclei has tremendous power to reveal stochastic gene expression and unappreciated differences in cell states during diabetic nephropathy.
Application PDF	Application Research Plan
Status	Contract Executed
Key Personnel	Kohei Uchimura
Salary Total Costs	41522
Supply Total Costs	24052
Equipment Total Costs	0
Travel/Other Total Costs	0
Direct Costs	65574
Indirect Costs Proposed	34426
Total Costs Proposed	100000
Total Costs Approved	100000
Start Date	11/1/2017
End Date	10/31/2018
IFO Name	Gindhart, Joseph
IFO E-Mail Address	jgindhart@wustl.edu
IACUC/IRB No.	9999
IACUC/IRB Institution	Washington University in St Louis
Entity ID No.	1430653611A1
Report Request Date	11/30/2018
T1D	NO

Type	Count
Invoices	2
Progress Reports	1

Data Submission

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Url	CBU ID	External ID	Institution	Date	Direct	Indirect	Invoice	Balance	PDF
View	17AU3798	32307-7	Washington University in St Louis	7/27/2018	$7,158.16	$3,758.03	$10,916.19	$40.39	View PDF
View	17AU3798	32307-7	Washington University in St Louis	1/23/2019	$58,389.13	$30,654.29	$89,043.42	$40.39	View PDF

Author	Complete Date	Report
Humphreys, Benjamin	11/27/2018	View Progress Report Document

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