PKR activation-induced mitochondrial dysfunction in HIV-transgenic mice with nephropathy
- Kidney Disease Section, Kidney Diseases Branch, NIDDK, NIH, United States
- Department of Pathology, Johns Hopkins Medical Institutions, United States
- Department of Pathology and Anatomical Sciences, Jacobs School of Medicine & Biomedical Sciences, University at Buffalo, United States
- Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, United States
- Polycystic Kidney Disease Section, Kidney Diseases Branch, NIDDK, NIH, United States
- Frederick National Laboratory for Cancer Research, NCI, NIH, United States
- College of Medicine, University of Florida, United States
- Nephrology Endocrinology and Vascular Medicine, Tohoku University Hospital, Japan
Figures
Figure 1
PKR inhibition by C16 ameliorates Tg26 mice kidney phenotype.
(A–C) Shown are the following: plasma creatinine (mg/dl, mean with SD), urinary albumin-to-creatinine ratio (mg/g creatinine, mean with SEM), urinary albumin-to-creatinine ratio (mg/g creatinine) of …
Figure 2 with 2 supplements
Overview of bulk RNA-seq and single-nucleus RNA-seq experiments.
(A) Shown is the workflow of the bulk RNA-seq and single-nucleus RNA-seq experiments. (B) Principal component analysis plot of bulk RNA-seq results. (C) Uniform manifold approximation and projection …
Figure 2—figure supplement 1
Pre-processing of single-nuclear RNA-seq data.
(A) Uniform manifold approximation and projection (UMAP) plot of single-nuclear RNA-seq data before doublet removal from 8 samples, 57,061 cells, showing 27 clusters. (B) UMAP plot showing detected …
Figure 2—figure supplement 2
Additional plots of single-nuclear RNA-seq data.
(A) Uniform manifold approximation and projection (UMAP) plot of single-nuclear RNA-seq data from each 8 samples. (B) Feature plot showing Eif2ak2 expression.
Figure 3 with 1 supplement
Oxidative phosphorylation genes are downregulated in Tg26 mice and downregulation is reversed by PKR inhibition using C16.
(A) Shown is the enrichment plot of oxidative phosphorylation pathway based on bulk mRNA-seq comparing Tg26 and wild-type (WT). (B) Shown is the enrichment plot of oxidative phosphorylation pathway …
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Figure 3—source data 1
Uncropped and labeled gels for Figure 3D.
- https://cdn.elifesciences.org/articles/91260/elife-91260-fig3-data1-v2.zip
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Figure 3—source data 2
Raw unedited gels for Figure 3D.
- https://cdn.elifesciences.org/articles/91260/elife-91260-fig3-data2-v2.zip
Figure 3—figure supplement 1
Additional data about mitochondria and S phase score of podocytes.
(A–D) Quantification of western blot probed against mitochondrial subunits normalized by VDAC (mean with SD). (E and F) Relative expression ratio of Nd1 and 16S divided by Hk2 (mean with SEM). (G) …
Figure 4 with 3 supplements
PT-Mito and PT-Inj cluster characterization.
(A) Shown are dot plots showing the top five marker genes in each of the PT clusters (PT-S1, PT-S1/S2, PT-S2, PT-S2/S3, PT-S3, PT-Inj, PT-Mito). (B, C) In situ hybridization of mt-Co1 and mt-Atp6 …
Figure 4—figure supplement 1
Additional in situ hybridization images.
(A) In situ hybridization images probing dapB (negative control probe) showed no signals. (B) In situ hybridization images probing Ppib (positive control probe) showed strong signals.
Figure 4—figure supplement 2
PT-Mito cluster detection of publicly available human kidney single-nuclear RNA-seq data (GSE131882).
(A) Uniform manifold approximation and projection (UMAP) plot of human kidney single-nuclear RNA-seq data shows 16 clusters. Clusters 1, 4 are proximal tubule (PT) clusters, and cluster 7 is PT-Mito …
Figure 4—figure supplement 3
Single-nuclear RNA-seq data comparison with ischemic reperfusion injury model.
(A) Dot plot showing expression of marker genes reported in new proximal tubular clusters by mouse ischemic reperfusion acute kidney injury model (Kirita.PNAS.2020). (B) Dot plot showing expression …
Figure 5
STAT3 activation downstream of PKR.
(A, B) Shown is mapping of STAT3 regulating genes comparing Tg26 vs WT (Z-score 6.286), Tg26 C16 vs Tg26 (Z-score –3.182) by bulk mRNA-seq. Red color indicates upregulation and green color indicates …
Figure 6
PKR inhibition with C16 reverses mitochondrial dysfunction in Tg26 glomeruli and proximal tubules.
(A) Shown are oxygen consumption rate (OCR) measurements during cell mitochondrial stress testing, using extracted glomeruli from wild-type (WT), Tg26, and C16 Tg26 kidney tissue. (B) Maximum …
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Figure 6—source data 1
Uncropped and labeled gels for Figure 6C.
- https://cdn.elifesciences.org/articles/91260/elife-91260-fig6-data1-v2.zip
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Figure 6—source data 2
Raw unedited gels for Figure 6C.
- https://cdn.elifesciences.org/articles/91260/elife-91260-fig6-data2-v2.zip
Figure 7
HIV-1 gene expression causes Tg26 podocytes dedifferentiation.
(A) Shown is dot plot demonstrating HIV-1 gene expression levels in each cluster detected by snRNA-seq. (B–D) p57 staining of kidney showing podocyte loss and dedifferentiation (scale bars are 50 …
Figure 8
Cell-cell interaction analysis shows activated ligand-receptor interaction: platelet-derived growth factor (PDGF)-D-PDGFR-B pathway in Tg26.
(A–C) Circles plot showing cell-cell interaction weight in each sample at the resolution of each identified cell cluster. (D–F) Shown are dot plots depicting results from cell-cell interaction …
Tables
Table 1
Activation Z-scores from bulk RNA-seq data.
Activation Z-scores of transcription factors by upstream regulator analysis of Ingenuity Pathway Analysis. Triple Z-score was calculated by multiplying three Z-scores in each comparison.
| Upstream regulator | Activation Z-scoreWT C16 vs WT | Activation Z-scoreTg26 vs WT | Activation Z-scoreTg26 C16 vs Tg26 | Triple Z-score |
|---|---|---|---|---|
| STAT3 | –2.183 | 6.286 | –3.182 | 43.66 |
| FOXO1 | –3.618 | 3.991 | –2.697 | 38.94 |
| GLI1 | –2.335 | 5.517 | –2.936 | 37.82 |
| EPAS1 | –2.665 | 3.554 | –3.242 | 30.71 |
| SREBF1 | –3.013 | 3.672 | –2.63 | 29.10 |
| CREB1 | –2.325 | 4.181 | –2.916 | 28.35 |
| ATF4 | –2.606 | 4.445 | –2.028 | 23.49 |
| SMAD2 | –2.207 | 2.791 | –2.335 | 14.38 |
