Kidney GWAS

We have compiled all Kidney genome wide association data sets (GWAS) and begun deciphering the mechanisms of variants driving changes to renal physiology

Analysis of variants within LD blocks in GWAS of kidney traits and diseases. A) Breakdown of all “kidney” and “renal” GWAS studies in EBI/NHGRI database. B) PolyPhen “Probably Damaging” variants were found in ANXA9 (D166G), PTCRA (V106I), WDR72 (L819F) and the well-studied SH2B3 (W262R), out of the 21 protein coding variants found in LD with kidney traits. C) A total of 138 genes are found within these LD blocks, with most genes having a ubiquitous expression (*). D) Four genes within the LD blocks show kidney specificity as assessed by the kidney TPM (transcripts per million reads) / Average TPM over 37 tissues. E) RegulomeDB ranking for functional impact of 1,291 CEU and 606 YRI variants within LD blocks of kidney-related GWAS studies. Lower scores suggest higher probability for function of the noncoding variant. F) Sample of the SHROOM3 region for roadmap epigenomic core state models identifying three transcriptional start sites for the gene with the LD block (shown below for CEU) found upstream of TSS2 used in kidney.
Analysis of variants within LD blocks in GWAS of kidney traits and diseases. A) Breakdown of all “kidney” and “renal” GWAS studies in EBI/NHGRI database. B) PolyPhen “Probably Damaging” variants were found in ANXA9 (D166G), PTCRA (V106I), WDR72 (L819F) and the well-studied SH2B3 (W262R), out of the 21 protein coding variants found in LD with kidney traits. C) A total of 138 genes are found within these LD blocks, with most genes having a ubiquitous expression (*). D) Four genes within the LD blocks show kidney specificity as assessed by the kidney TPM (transcripts per million reads) / Average TPM over 37 tissues. E) RegulomeDB ranking for functional impact of 1,291 CEU and 606 YRI variants within LD blocks of kidney-related GWAS studies. Lower scores suggest higher probability for function of the noncoding variant. F) Sample of the SHROOM3 region for roadmap epigenomic core state models identifying three transcriptional start sites for the gene with the LD block (shown below for CEU) found upstream of TSS2 used in kidney.

SHROOM3

One example of detailed mechanisms we have discovered was that noncoding variants upstream of a novel transcriptional start site of SHROOM3 regulate gene expression through transcription factor looping. In addition we identified additional rare missense variants in SHROOM3 that associated with Chronic Kidney Disease.

Narrowing the SHROOM3 LD block of CKD GWAS to rs17319721. A) Transcripts of the SHROOM3 gene with Cohesin HiChIP structural looping shown above, ENCODE binding TFs from both rs17319721 and TSS2 shown to the left, and the CRISPR experiments shown below. B) FANTOM 5 CAGE data for TSS2 of SHROOM3 showing 122 datasets using TSS2 as the transcriptional start site. C-D) Reads mapped to SHROOM3 (x-axis) and Nephrin (Y-axis) for from mouse glomeruli RNAseq (C) or mouse single cell (D) RNAseq datasets. Shown below is the mapping of reads near the 5’UTR that starts from TSS2. E) IP of SHROOM3 from human primary podocytes followed by western blots of SHROOM3 identifying two isoforms of SHROOM3. F) Deletion of the entire LD block using CRISPR/Cas9 followed by real time PCR for the SHROOM3 isoform 2. G) Sanger sequencing of  rs17319721 SNP inserted into HEK293T cells in a homozygous manner. H) Expression of SHROOM3 isoform 2 was seen to be elevated following the insertion of rs17319721. SEPT11 and FAM47E are genes that directly flank either side of SHROOM3 and do not have changes in expression when rs17319721 is inserted. I) Imaging of zebrafish dorsal aorta for 70-kDa FITC dextran injected into zebrafish with Shroom3 morpholino (MO) following by human SHROOM3 recovery with the three constructs listed in panel A. J) Quantification of 3 independent experiments in each group from sample panel C. Error bars represent the SEM.
Narrowing the SHROOM3 LD block of CKD GWAS to rs17319721. A) Transcripts of the SHROOM3 gene with Cohesin HiChIP structural looping shown above, ENCODE binding TFs from both rs17319721 and TSS2 shown to the left, and the CRISPR experiments shown below. B) FANTOM 5 CAGE data for TSS2 of SHROOM3 showing 122 datasets using TSS2 as the transcriptional start site. C-D) Reads mapped to SHROOM3 (x-axis) and Nephrin (Y-axis) for from mouse glomeruli RNAseq (C) or mouse single cell (D) RNAseq datasets. Shown below is the mapping of reads near the 5’UTR that starts from TSS2. E) IP of SHROOM3 from human primary podocytes followed by western blots of SHROOM3 identifying two isoforms of SHROOM3. F) Deletion of the entire LD block using CRISPR/Cas9 followed by real time PCR for the SHROOM3 isoform 2. G) Sanger sequencing of rs17319721 SNP inserted into HEK293T cells in a homozygous manner. H) Expression of SHROOM3 isoform 2 was seen to be elevated following the insertion of rs17319721. SEPT11 and FAM47E are genes that directly flank either side of SHROOM3 and do not have changes in expression when rs17319721 is inserted. I) Imaging of zebrafish dorsal aorta for 70-kDa FITC dextran injected into zebrafish with Shroom3 morpholino (MO) following by human SHROOM3 recovery with the three constructs listed in panel A. J) Quantification of 3 independent experiments in each group from sample panel C. Error bars represent the SEM.
Mapping of rare variants for SHROOM3 for functional outcomes. A) Predicted impact score for >1,000 missense variants for SHROOM3 from gnomAD, identifying those of high potential impact including P1244L with a CKD odds ratio (OR) of 7.95. B) Sequence alignment for SHROOM3 for the P1244L variant location. C) Kinase assay with various concentrations of LATS2 recombinant enzyme on 4 different peptides of SHROOM3 showing P1244L to not alter the phosphorylation. D) Crystal structure of wilt type (cyan) with the solved structure of P1244L (red) overlaid for interaction with 14-3-3 (gray).
Mapping of rare variants for SHROOM3 for functional outcomes. A) Predicted impact score for >1,000 missense variants for SHROOM3 from gnomAD, identifying those of high potential impact including P1244L with a CKD odds ratio (OR) of 7.95. B) Sequence alignment for SHROOM3 for the P1244L variant location. C) Kinase assay with various concentrations of LATS2 recombinant enzyme on 4 different peptides of SHROOM3 showing P1244L to not alter the phosphorylation. D) Crystal structure of wilt type (cyan) with the solved structure of P1244L (red) overlaid for interaction with 14-3-3 (gray).

Cultured Podocytes

One of the biggest difficulties of studying genetics of the kidney is the lack of culture techniques for podocytes. Our lab has developed a novel culturing technique that allows us to generate foot processes that network in culture.

Podocyte stained red for actin and blue for the nucleus
Podocyte stained red for actin and blue for the nucleus

Microfluidics of Tubule Cells

Finally we have begun developing complex models of microfludics to test albumin uptake in renal proximal tubule cells

Renal Proximal Tubule Cell microfluidics. A-B) Setup for simple fluidics, allowing for passing over FITC-albumin to observe uptake (B). C) More complex coculture setup for microfluidics. D) HAVCR1 damage response as a result of hypocia. E) Albumin uptake and degradation using ELISA.
Renal Proximal Tubule Cell microfluidics. A-B) Setup for simple fluidics, allowing for passing over FITC-albumin to observe uptake (B). C) More complex coculture setup for microfluidics. D) HAVCR1 damage response as a result of hypocia. E) Albumin uptake and degradation using ELISA.