A novel in vivo method to quantify slit diaphragm protein abundance in murine proteinuric kidney disease
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A novel in vivo method to quantify slit diaphragm protein abundance in murine proteinuric kidney disease. / Haase, Raphael; Potthoff, Sebastian Alexander; Meyer-Schwesinger, Catherine; Frosch, Clara; Wiech, Thorsten; Panzer, Ulf; Königshausen, Eva; Stegbauer, Johannes; Sellin, Lorenz; Rump, Lars Christian; Quack, Ivo; Woznowski, Magdalena.
In: PLOS ONE, Vol. 12, No. 6, 2017, p. e0179217.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - A novel in vivo method to quantify slit diaphragm protein abundance in murine proteinuric kidney disease
AU - Haase, Raphael
AU - Potthoff, Sebastian Alexander
AU - Meyer-Schwesinger, Catherine
AU - Frosch, Clara
AU - Wiech, Thorsten
AU - Panzer, Ulf
AU - Königshausen, Eva
AU - Stegbauer, Johannes
AU - Sellin, Lorenz
AU - Rump, Lars Christian
AU - Quack, Ivo
AU - Woznowski, Magdalena
PY - 2017
Y1 - 2017
N2 - Injury of the glomerular filter causes proteinuria by disrupting the sensitive interplay of the glomerular protein network. To date, studies of the expression and trafficking of glomerular proteins have been mostly limited to in vitro or histologic studies. Here, we report a novel in vivo biotinylation assay that allows the quantification of surface expression of glomerular proteins in mice. Kidneys were perfused in situ with biotin before harvest. Afterwards glomeruli were isolated and lyzed. The protein of interest was separated by immunoprecipitation and the amount of surface-expressed protein was quantified by Western blot analysis with streptavidin staining. As proof-of-concept, we examined the presence of nephrin in the slit diaphragm in two well-established murine models of proteinuric kidney disease: nephrotoxic nephritis and adriamycin nephropathy. In proteinuric animals, significantly less nephrin was detected in the slit diaphragm. When proteinuria decreased once again during the course of disease, the amount of surface nephrin returned to the baseline. Our present results suggest that our assay is a valuable tool to study the glomerular filter in proteinuric kidney diseases. Note that the assay is not limited to proteins expressed in the slit diaphragm, and all surface proteins that are accessible to biotin perfusion and immunoprecipitation qualify for this analysis.
AB - Injury of the glomerular filter causes proteinuria by disrupting the sensitive interplay of the glomerular protein network. To date, studies of the expression and trafficking of glomerular proteins have been mostly limited to in vitro or histologic studies. Here, we report a novel in vivo biotinylation assay that allows the quantification of surface expression of glomerular proteins in mice. Kidneys were perfused in situ with biotin before harvest. Afterwards glomeruli were isolated and lyzed. The protein of interest was separated by immunoprecipitation and the amount of surface-expressed protein was quantified by Western blot analysis with streptavidin staining. As proof-of-concept, we examined the presence of nephrin in the slit diaphragm in two well-established murine models of proteinuric kidney disease: nephrotoxic nephritis and adriamycin nephropathy. In proteinuric animals, significantly less nephrin was detected in the slit diaphragm. When proteinuria decreased once again during the course of disease, the amount of surface nephrin returned to the baseline. Our present results suggest that our assay is a valuable tool to study the glomerular filter in proteinuric kidney diseases. Note that the assay is not limited to proteins expressed in the slit diaphragm, and all surface proteins that are accessible to biotin perfusion and immunoprecipitation qualify for this analysis.
KW - Journal Article
U2 - 10.1371/journal.pone.0179217
DO - 10.1371/journal.pone.0179217
M3 - SCORING: Journal article
C2 - 28604827
VL - 12
SP - e0179217
JO - PLOS ONE
JF - PLOS ONE
SN - 1932-6203
IS - 6
ER -
