The transcriptome of early compensatory kidney growth reveals cell and time specific responses

Summary Following kidney removal, the remaining kidney enlarges and increases its function. The mechanism and signals driving this compensatory kidney hypertrophy and the enlargement of its constituent kidney cells remains elusive. RNA-seq studies in mice undergoing hypertrophy 24, 48, and 72 h following nephrectomy were undertaken to understand the early transcriptional changes. This revealed substantial enhancement of cholesterol biosynthesis pathways, increases in mitochondrial gene expression and cell cycle perturbations. Single nuclei RNA-seq delineated cell specific changes at 24 h post nephrectomy and showed that sterol binding protein 2 (SREBP2) activity increases in medullary thick ascending limb cells in keeping with promotion of cholesterol synthesis. Cultured renal tubular cells were examined for insulin-like growth factor-1 (IGF-1) stimulated hypertrophy and SREBP2 was found to be required for increase in cell size. This work describes the early cell specific growth pathways mediating cellular and kidney hypertrophy with an intriguing role for cholesterol synthesis.

after the surgical procedures, the remaining kidney tissue was collected and RNA was extracted.Quantitative PCR (QPCR) analysis was performed to evaluate the relative gene expression levels.The significance levels were denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.Non-significant differences were represented as ns (not significant

Figure S1 :Figure S2 :
Figure S1: Protein expression analysis of rpS6 P-S240/244 and total rpS6 in mice (C57BL6) following a unilateral nephrectomy, related to Figure 1.Mice (C57BL6) were subjected to either sham surgery or left unilateral nephrectomy (Unx).(A) Two weeks following a left kidney nephrectomy the remanent kidney (labelled U) was photographed to demonstrate relative whole kidney size change when compared to control sham operation (labelled S).(B)At 24 hours post-surgery, the remaining kidney was harvested (4-5 mice per group).Whole tissue lysates were prepared and separated by SDS-PAGE.Subsequently, the proteins were transferred to membranes for immunoblotting.The membranes were probed with specific antibodies against rpS6 phosphorylated at S240/244 (rpS6 P-S240/244) and total rpS6.β-tubulin (55 kDa) was used as a loading control for normalization.The data indicates increases in rpS6 phosphorylation following unilateral nephrectomy, confirming early mTORC pathway signalling.

Figure S3 :Figure S4 :
Figure S3: Untargeted Mass Spectrometric analysis of kidney tissue lysates 24 hours post surgery, related to Figure 2. Sham surgery (n=6) was compared to nephrectomy (n=6).(A) Volcano plot showing the top 10 upregulated and top 10 downregulated protein at 24h. (B) Venn diagram showing the overlap in differentially expressed mRNAs and protein.Note that mRNAs were measure for all proteins detected but not vice versa.(C) Venn diagram showing the overlap in relatively upregulated protein and mRNAs in common.(D) Bar graph showing pathway enrichment of MsigDB Hallmark from Enrichr using significant (adjusted P-value <0.05) shared upregulated protein and RNA, vertical line indicates adjusted P-value of 0.05.

Figure S5 :Figure S6 :Figure S7 :Figure S8 :Figure S9 :
Figure S5: snRNA-seq batch analysis and overlapping gene expression analysis between bulk RNAseq and snRNA-seq, related to Figure 5. A) PCA of snRNA-SEQ batches 1 and 2 with corrected PCA embeddings using Harmony package in R B) Umap cluster analysis shows comparable clustering of cell types between batches.C) shows percentage overlap of genes upregulated and down regulated in sn-RNA-Seq (in grey) and bulk in black.

Table S1 :
Top 20 differentially expressed genes at 24 hours ranked by adjusted P-value with a

Table S2 :
Top 20 differentially expressed genes at 24 hours ranked by adjusted P-value with a negative l ld ≤-1, related to Figure1.

Table S3 :
Top 20 differentially expressed genes at 48 hours ranked by adjusted P-value with a

Table S4 :
Top 20 differentially expressed genes at 48 hours ranked by adjusted P-value with a negative l ld ≤-1, related to Figure1.

Table S5 :
Top 20 differentially expressed genes at 72 hours ranked by adjusted P-value with a

Table S6 :
Top differentially expressed genes at 72 hours ranked by adjusted P-value with a negative l ld ≤-1, related to Figure 1.

Table S7 :
Additional Key Resource Oligonucleotides, related to Star Methods.