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Renal Nephron Segment‐Specific Gene Expression using Adeno‐Associated Viral (AAV) Vectors
Author(s) -
Konkalmatt Prasad,
Asico Larry D,
Santiago Cuevas,
Armando Ines,
Jose Pedro A
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.1034.5
Subject(s) - distal convoluted tubule , nephron , kidney , transgene , microbiology and biotechnology , biology , promoter , gene expression , convoluted tubule , tubule , gene , endocrinology , genetics
AAV vectors provide sustained long‐term gene expression with minimal immunological consequences. Systemic administration of AAV9 vector transduces efficiently various organs, such as the liver, heart, lung and skeletal muscle but provides very low transduction in the kidney. We have reported that the administration of AAV9 vector by retrograde ureteral infusion provided efficient gene transfer to renal cells including proximal and distal tubule cells, and podocytes with minimum off‐target gene expression. In studies on the genetics of renal tubule function, it is important to restrict the expression of the transgene to the desired cell type within the kidney, so that the physiological parameters measured will represent the function of the transgene expressed in that specific renal cell type. We hypothesized that use of nephron segment‐specific promoters sodium‐glucose cotransporter‐2 (SGLT2), Na + ‐K + ‐Cl − cotransporter‐2 (NKCC2), E‐cadherin (ECAD), and kidney‐specific cadherin (KSPC) promoters will provide preferential gene expression in the proximal tubule, thick ascending limb of Henle (TALH), distal convoluted tubule, collecting duct, and entire nephron, respectively. We have constructed, and validated in appropriate cell lines, AAV vector encoding EGFP gene under the control of nephron segment‐specific promoters SGLT2, NKCC2, ECAD, or KSPC. AAV9 vector with SGLT2 promoter driving the expression of EGFP provided robust expression in mouse primary renal proximal tubule cells but not renal collecting duct cells. KSPC promoter provided AAV9‐mediated expression in primary mouse renal proximal tubule as well as renal collecting duct cells. NKCC2 promoter provided weak expression in primary mouse renal proximal tubule and collecting duct cells. Specificity and distribution of gene expression in the mouse kidney from each promoter was determined by immunofluorescence staining using antibody against the marker proteins for proximal tubule, TALH, distal convoluted tubule, and collecting duct cells. Preliminary results, using SGLT2, a proximal tubule‐specific promoter, showed expression of EGFP only in proximal tubules of the mouse kidney. These results demonstrate that AAV9 vectors carrying nephron segment‐specific promoters can provide expression in their respective segment of the nephron.