Circulating proteins linked to apoptosis processes and fast development of end-stage kidney disease in diabetes
Katsuhito Ihara, Eiichiro Satake, Parker C. Wilson, Bozena Krolewski, Hiroki Kobayashi, Zaipul I. Md Dom, Joseph Ricca, Jonathan Wilson, Jonathan M. Dreyfuss, Monika A. Niewczas, Alessandro Doria, Robert G. Nelson, Marcus G. Pezzolesi, Benjamin D. Humphreys, Kevin Duffin, Andrzej S. Krolewski
Katsuhito Ihara, Eiichiro Satake, Parker C. Wilson, Bozena Krolewski, Hiroki Kobayashi, Zaipul I. Md Dom, Joseph Ricca, Jonathan Wilson, Jonathan M. Dreyfuss, Monika A. Niewczas, Alessandro Doria, Robert G. Nelson, Marcus G. Pezzolesi, Benjamin D. Humphreys, Kevin Duffin, Andrzej S. Krolewski
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Research Article Nephrology

Circulating proteins linked to apoptosis processes and fast development of end-stage kidney disease in diabetes

Abstract

Many circulating proteins are associated with risk of ESKD, but their source and the biological pathways/disease processes they represent are unclear. Using OLINK proteomics platform, concentrations of 455 proteins were measured in plasma specimens obtained at baseline from 399 individuals with diabetes. Elevated concentrations of 46 circulating proteins were associated (P < 1 × 10–5) with development of ESKD (n = 143) during 7–15 years of follow-up. Twenty of these proteins enriched apoptosis/TNF receptor signaling pathways. A subset of 20 proteins (5–7 proteins), summarized as an apoptosis score, together with clinical variables accurately predicted risk of ESKD. Expression of genes encoding the 46 proteins in peripheral WBCs showed no difference between cells from individuals who did or did not develop ESKD. In contrast, plasma concentration of many of the 46 proteins differed by this outcome. In single-nucleus RNA-Seq analysis of kidney biopsies, the majority of genes encoding for the 20 apoptosis/TNF receptor proteins were overexpressed in injured versus healthy proximal tubule cells. Expression of these 20 genes also correlated with the overall index of apoptosis in these cells. Elevated levels of circulating proteins flagging apoptotic processes/TNF receptor signaling pathways — and likely originating from kidney cells, including injured/apoptotic proximal tubular cells — preceded the development of ESKD.

Authors

Katsuhito Ihara, Eiichiro Satake, Parker C. Wilson, Bozena Krolewski, Hiroki Kobayashi, Zaipul I. Md Dom, Joseph Ricca, Jonathan Wilson, Jonathan M. Dreyfuss, Monika A. Niewczas, Alessandro Doria, Robert G. Nelson, Marcus G. Pezzolesi, Benjamin D. Humphreys, Kevin Duffin, Andrzej S. Krolewski

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Figure 6

snRNA-Seq analysis and enrichment of apoptosis pathways and TNF receptor proteins in injured (PT_VCAM1) and control proximal tubule cells.

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snRNA-Seq analysis and enrichment of apoptosis pathways and TNF receptor...
Reanalyzed data from previous publication (19–21). The gene names for the proteins in the Figure are listed in Supplemental Table 1. (A) Cell-specific expression of gene encoding for 20 apoptosis/TNF receptor proteins and 26 other proteins among kidney cell types. Cell-specific genes were computed using the Seurat FindMarkers function comparing the indicated cell type to all other cell types using a Wilcoxon rank sum test with an adjusted P < 0.05 threshold. The intersection between cell-specific genes and the 20 apoptosis/TNF receptor proteins and 26 other proteins is displayed as a bar plot for each cell type. (B) Differential expression of genes encoding for 46 circulating ESKD risk–associated proteins in PT_VCAM1 versus control proximal tubule cells is shown. The y axis corresponds to a Benjamini-Hochberg adjusted P value and only 14 genes that meet the adjusted P value threshold (P < 0.05) are displayed. Blue circles indicate 11 apoptosis/TNF receptor proteins, and red circle indicate 3 other proteins. For this analysis, we analyzed 9,901 proximal tubule cells and 1,577 PT_VCAM1 cells. (C) Pearson correlation between an aggregate measure of Hallmark 205 apoptosis genes (apoptosis index) and genes encoding the 46 circulating ESKD risk–associated proteins was computed for the PT_VCAM1 cell state. Asterisks indicate genes that are significantly correlated with apoptosis index using an unadjusted P < 0.05. Genes that are filled gray were not detected in the snRNA-Seq dataset in the PT_VCAM1 cell state. Names in red indicate apoptosis/TNF receptor proteins. (D) Pearson correlation between an aggregate measure of hallmark 205 apoptosis genes (apoptosis index) and genes encoding the 46 circulating ESKD risk–associated proteins following imputation. See Supplemental Figures 1 and 2. (E) Escape hypothesis. Healthy proximal tubules express markers like SLC34A1 that mediate normal homeostatic functions. When proximal tubules are injured, they begin to express markers like VCAM1, CD24, and CD133 (21). The injured cells overexpressed genes encoding for the apoptosis/TNF receptor proteins, which are released into circulation. A subset of injured proximal tubule cells ultimately progress to apoptosis.