Loss of the collagen IV modifier prolyl 3-hydroxylase 2 causes thin basement membrane nephropathy
Hande Aypek, … , Tobias B. Huber, Florian Grahammer
Hande Aypek, … , Tobias B. Huber, Florian Grahammer
Published May 2, 2022
Citation Information: J Clin Invest. 2022;132(9):e147253. https://doi.org/10.1172/JCI147253.
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Research Article Nephrology

Loss of the collagen IV modifier prolyl 3-hydroxylase 2 causes thin basement membrane nephropathy

Abstract

The glomerular filtration barrier (GFB) produces primary urine and is composed of a fenestrated endothelium, a glomerular basement membrane (GBM), podocytes, and a slit diaphragm. Impairment of the GFB leads to albuminuria and microhematuria. The GBM is generated via secreted proteins from both endothelial cells and podocytes and is supposed to majorly contribute to filtration selectivity. While genetic mutations or variations of GBM components have been recently proposed to be a common cause of glomerular diseases, pathways modifying and stabilizing the GBM remain incompletely understood. Here, we identified prolyl 3-hydroxylase 2 (P3H2) as a regulator of the GBM in an a cohort of patients with albuminuria. P3H2 hydroxylates the 3′ of prolines in collagen IV subchains in the endoplasmic reticulum. Characterization of a P3h2ΔPod mouse line revealed that the absence of P3H2 protein in podocytes induced a thin basement membrane nephropathy (TBMN) phenotype with a thinner GBM than that in WT mice and the development of microhematuria and microalbuminuria over time. Mechanistically, differential quantitative proteomics of the GBM identified a significant decrease in the abundance of collagen IV subchains and their interaction partners in P3h2ΔPod mice. To our knowledge, P3H2 protein is the first identified GBM modifier, and loss or mutation of P3H2 causes TBMN and focal segmental glomerulosclerosis in mice and humans.

Authors

Hande Aypek, Christoph Krisp, Shun Lu, Shuya Liu, Dominik Kylies, Oliver Kretz, Guochao Wu, Manuela Moritz, Kerstin Amann, Kerstin Benz, Ping Tong, Zheng-mao Hu, Sulaiman M. Alsulaiman, Arif O. Khan, Maik Grohmann, Timo Wagner, Janina Müller-Deile, Hartmut Schlüter, Victor G. Puelles, Carsten Bergmann, Tobias B. Huber, Florian Grahammer

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Figure 6

Relative quantitative proteomics of the GBM of P3h2ΔPod and P3h2fl/fl mice.

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Relative quantitative proteomics of the GBM of P3h2ΔPod and P3h2fl/fl mi...
(A) Coomassie blue staining of enriched GBM and further fractions collected during GBM isolation. The GBM fraction had high-molecular-weight protein bands indicating that the isolated GBM was enriched with ECM proteins. The intracellular protein fraction had protein bands of variable size, showing that during isolation, many intracellular proteins were separated from the enriched GBM. (B) Western blot analysis of the GBM and other fractions for quality control of isolated GBM. ECM proteins were detected in high abundance in the enriched GBM fraction. Intracellular and transmembrane proteins were in low abundance or not detected in the enriched GBM fraction when compared with intracellular and membrane protein fractions. (C) Volcano plot of the relative quantitative GBM proteome. The x axis shows log2 fold changes in the abundance of WT and KO GBM proteins, and the y axis shows the P values for the GBM proteins. The abundance of the main structural GBM proteins was decreased in KO mice, as shown in the left portion of the plot. The vertical dotted line marks the –log(P value) cutoff of 1.3, above which all proteins are considered statistically significant; the dotted horizontal lines indicate the protein abundance difference [log2(KO) – log2(WT)] cutoff of less and –1 or greater than 1. Proteins with a –log(P value) of greater than 1.3 and a difference 1 or less are highlighted in red. (D) Dot plots of the structural proteins in the GBM showing a difference in their abundance. Comparison of the relative abundance values of the GBM proteome of each mouse group shows a significant decrease in the main GBM structural proteins. n = 6. Graphs show the mean ± SD. *P < 0.05 and **P < 0.01, by unpaired, 2-tailed Student’s t test.