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 8

Rescue of the ECM phenotype of P3H2-KO podocyte lines via AAV-CMV-P3H2 infection.

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Rescue of the ECM phenotype of P3H2-KO podocyte lines via AAV-CMV-P3H2 i...
(A) P3H2 expression was determined via Western blotting. P3H2 protein expression was detected in AAV-CMV-P3H2–infected KO cells. (B) Immunofluorescence staining of KO and infected cells. P3H2 localization was observed in infected cells with a 60%–70% infection efficiency. Scale bars: 20 μm. (C) ECM proteomics for WT and KO cells. The x axis shows log2 fold changes in the abundance of WT and KO GBM proteins, and the y axis shows the P values of the GBM proteins. In the volcano plot, downregulation of collagen IV α1 and α2 subchains in the KO ECM was observed when compared with WT. (D) ECM proteomics of AAV-CMV-GFP–infected WT and AAV-CMV-P3H2–infected KO cells. Volcano plot shows that collagen IV α2 was no longer significantly downregulate and that α1 was still significantly downregulated in the ECM of infected KO cells. However, the difference in abundances of collagen IV α1 and α2 subchains were decreased with infection of AAV-CMV-P3H2, indicating that P3H2 reexpression increased the collagen IV α1 and α2 protein abundance and partially rescued the KO ECM phenotype. For C and D, the vertical dotted lines marks the –log(P value) cutoff of 1.3, above which all proteins were 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 of 1 or less are highlighted in red.