PKR inhibition by C16 ameliorates Tg26 mice kidney phenotype.

(A-D) Shown are the following: plasma creatinine (mg/dL), urinary albumin-to-creatinine ratio (mg/g creatinine), urinary albumin-to-creatinine ratio (mg/g creatinine) of 6 and 12 weeks of age, urinary NGAL-to-creatinine ratio (ng/g creatinine). (E-G) Representative PAS staining images of WT, Tg26 and C16 treated Tg26 kidney. (H, I) Quantitative analysis of glomeruli for segmental scarring and global sclerosis. (J-L) Representative Picrosirius Red-staining images of WT, Tg26 and C16 treated Tg26 kidney. (M) Quantitative analysis of Picrosirius Red-staining area. (One-way ANOVA (A, B, D), t-test (H, I, M); *, P<0.05; **, P<0.01; scale bars are 50 μm)

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) UMAP plot of single-nuclear RNA-seq data from 8 samples, 56,976 nuclei, showing 23 clusters. (D) Shown is a dot plot of 23 marker genes, each characteristic for the cluster. (E) Shown is the ratio of nuclei grouped to each cluster in each sample.

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 WT. (B) Shown is the enrichment plot of oxidative phosphorylation pathway based on bulk mRNA-seq comparing C16 treated Tg26 and Tg26. (C) Heatmap of expressed genes in oxidative phosphorylation pathway (n=123), based on data from bulk mRNA-seq. (D) Western blot to identify mitochondrial subunits CI through CIV and VDAC. (E) Dot plot showing expression of oxidative phosphorylation pathway genes in PT-Mito, PT-S1, PT-Inj, Endo, and Fibroblast cluster by snRNA-seq.

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 genes showed signals inside nuclei of WT mice. (Scale bars are 50 μm) (D) Shown is the activation Z-score of the oxidative phosphorylation pathway by pathway analysis of each cluster comparing Tg26 vs WT mice. (E) Trajectory analysis results including PT-S1, PT-Mito, PT-Inj from WT and Tg26 mice. (F) Ceruloplasmin (Cp) expression in the trajectory analysis plot. (G) PKR downstream pathway mapping by IPA comparing Tg26 vs WT by bulk mRNA-seq data. (H) Activation Z-score of PKR pathway by IPA in each cluster comparing Tg26 vs WT.

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 downregulation, quantified as log2-fold change, p-value; and adjusted p-value by false discovery rate are shown. Solid line indicates known positive regulation, dotted line indicates known negative regulation. (C) Representative immunoblotting of phospho-STAT3, STAT3 and β-ACTIN. (D) Quantitative results of phospho-STAT3/STAT3 by immunoblotting. (one-way ANOVA; *, P<0.05) (E-G) Phospho-STAT3 immunostaining of mouse kidneys is shown (Scale bars are 50 μm). Arrows indicate p-Stat3 detection in injured tubular cells. (H) STAT3 activation Z-score by upstream regulator analysis comparing Tg26 vs WT in each cluster by snRNA-seq.

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 WT, Tg26, and C16 Tg26 kidney tissue. (B) Maximum respiration rate was calculated by OCR measurements of glomeruli. (C) Spare respiratory capacity was calculated by OCR measurements of glomeruli. (D) Shown are oxygen consumption rate (OCR) measurements during cell mitochondrial stress testing, using extracted proximal tubules from WT, Tg26, and C16 Tg26 kidney tissue. (E) Maximum respiration rate was calculated by OCR measurements of isolated proximal tubules. (F) Spare respiratory capacity was calculated by OCR measurements of isolated proximal tubules. (One-way ANOVA; *, P<0.05; **, P<0.01)

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 μm) (E) Podocount analysis showed podocyte loss in Tg26 and was rescued by C16. (One-way ANOVA; *, P<0.05; ***, P<0.001) (F) Trajectory analysis of podocytes by snRNA-seq data from WT, Tg26 glom samples. (G) Trajectory map showing nef expression. (H) Pathway analysis results by IPA comparing Tg26 vs WT using glomerular snRNA-seq data from podocyte cluster. (I) Dot plot showing podocyte marker genes and representative differentially expressed genes in podocytes by glomerular snRNA-seq. (J) Dot plot comparing expression of representative genes in glomerular Tg26 podocytes between nef-positive and nef-negative podocytes.

Cell-cell interaction analysis shows activated ligand-receptor interaction: PDGF-D-PDGFR-B pathway in Tg26.

(A-C) Shown are dot plots depicting results from cell-cell interaction analysis of WT, Tg26, C16 Tg26 snRNA-seq data. (D) Violin plots showing Pdgfd expression levels in the PT-Inj cluster. (E, F) PDGF-D immunostaining of mouse kidneys is shown. (Scale bars are 50 μm)