The Single-Cell Transcriptomic Landscape of Early Human Diabetic Nephropathy Research Paper

Background of Paper and Hypothesis of Paper

Diabetic neuropathy is one of the leading causes of kidney failure or end-stage renal disease. However, the available literature and research studies have revealed little early cellular or genetic changes that precede this condition. Before this study, bulk RNA sequencing (RNA-seq) had been used to understand gene transcriptional changes that occur in human diabetic glomeruli. However, this form of sequencing is limited, as it does not quantify gene expression in individual cells. Bulk RNA-seq is only capable of measuring averaged and integrated gene expression in many cells. Additionally, the available laboratory techniques, such as measuring urine protein or serum creatinine levels, are not sensitive enough to identify the early signs of diabetic kidney disease. Consequently, it was difficult to identify cellular changes that occur in the early stages of kidney failure in diabetic patients, which hindered efforts to intervene appropriately.

However, advancement in isolation techniques, such as single-cell RNA sequencing (scRNA-seq), has enabled researchers to study transcriptional changes in single cells. According to Wilson et al. (2019), scRNA-seq “quantifies gene expression in individual cells, and unlike bulk RNA-seq, it can interrogate transcriptional states and signaling pathways in multiple cell types simultaneously” (p. 19619). Therefore, scRNA-seq solves the problems associated with the non-specificity and sensitivity of bulk RNA-seq and other laboratory isolation techniques. Based on this understanding, the authors of this article wanted to use scRNA-seq to identify gene expression changes that occur in the early stages of kidney failure among diabetics. The findings of this study would help significantly in the early detection of diabetic neuropathy using biomarkers for disease progression for timely and appropriate intervention. The authors hypothesized that using scRNA-seq to isolate and study single cells in the kidney cortex in patients at early diabetic neuropathy would show gene expression patterns and signaling pathways changes that occur as the body adapts to hyperglycemia, which is elevated blood sugar levels.

Explanation of all Datasets

The first data set involved changes in signaling networks in the diabetic glomerulus. In this case, diabetic mesangial cells had “increased expression of CCN1 and SLIT3” (Wilson et al. 2019, p. 19620). SLIT3 gene regulates cell migration while CCN1 gene is induced by the growth factor, and it modulates tissue repair. The second dataset was on the infiltration of immune cells in diabetic neuropathy whereby 347 leukocytes were identified. This elevated number of leukocytes was eight times more in diabetics as compared to controls. The first and second datasets support the overall hypothesis that cellular changes occur in the early stages of diabetic neuropathy.

The third dataset was on changes in gene expression in the thick ascending limb (TAL) and the proximal convoluted tubule (PCT). The TAL had “3,788 cells and was enriched for regulation of sodium ion trans-membrane transporter activity, regulation of potassium ion, and cell junction assembly” (Wilson et al. 2019, p. 19621). On the other hand, PCT had 6,518 cells, all with advanced capabilities to regulate IL-8 and angiogenesis (Wilson et al. 2019). The last dataset was on gene expression changes that occur due to diabetes to enhance the secretion of potassium in principal cells and late distal convoluted tubule (DCT2). In this case, the LDCT had 1,652 cells to enrich the mediation of ion transport, specifically K⁺ and Ca²⁺. These findings demonstrate the up-regulation of diabetes-induced pro-angiogenic pathways and genes as the diabetic glomerulus adapts to hyperglycemia, which supports the overall hypothesis of the article. The technique used in this study is scRNA-seq whereby single and multiple cells are analyzed to identify genetic changes that occur in early end-stage renal disease.

Summary and Future Directions

The paper concludes that scRNA-seq is a useful tool that could be used to identify cellular and genetic changes that occur in kidney glomeruli of diabetics. The sample consisted of 3 diabetics after undergoing nephrectomy for renal mass and three non-diabetic controls. scRNA-seq of the glomeruli from the samples indicated that different parts of the kidney had increased up-regulation of certain genes and pathways that prepare it for the elevated blood sugar levels, which are typical in diabetics. Overall, the genetic changes that were identified in the sampled glomeruli of diabetics were associated with increased K⁺ secretion and decreased Mg²⁺ and Ca²⁺. Other cells showed angiogenic signatures, especially in diabetics, which is a clear indication of early signs of disease progression. In simple terms, diabetic glomeruli studied in this article had an increased number of cells and gene expression associated changes that occur in the early stages of diabetic neuropathy.

One of the problems with this paper is the small number of samples used for the study. A sample of 3 glomeruli is not large enough to generalize the findings. Therefore, in the future, this study could be repeated using a large number of glomeruli to enhance the generalizability of the findings. In addition, given that diabetes is the underlying cause of diabetic neuropathy, future studies could use scRNA-seq techniques and study early cellular changes that occur in the pancreatic islets of Langerhans where insulin is produced as a way of detecting diabetes before it progresses.

Reference

Wilson PC, Wu H, Kirita Y, Uchimura K, Ledru N, Rennke HG,… & Humphreys BD. 2019. The single-cell transcriptomic landscape of early human diabetic nephropathy. Proceedings of the National Academy of Sciences, 116(39): 19619-19625.