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 5

Podocyte morphometric analysis of P3h2ΔPod and P3h2fl/fl mice.

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Podocyte morphometric analysis of P3h2ΔPod and P3h2fl/fl mice. (A) Repre...
(A) Representative immunofluorescence images of WT and KO mouse kidney tissue stained for SYNPO, DACH1, and DAPI. The images were used for the measurement of podocyte number, podocyte density, glomerular volume, and average podocyte volume (average podocyte volume = total podocyte volume/podocyte number [TPV/PN]). Scale bar: 10 μm. (B) Podocyte numbers for WT and KO mice. The number of podocytes was significantly decreased in KO mice glomeruli, indicating podocyte loss. (C) Podocyte density for WT and KO mice. In KO mouse glomeruli, the podocyte density was significantly decreased compared with that of WT glomeruli, again indicating podocyte loss. (D) Glomerular volume for WT and KO mice. There was no significant difference in glomerular volumes between WT and KO mice. (E) Average podocyte volume for WT and KO mice. A significant increase in the average podocyte volume was observed in KO mouse glomeruli, indicating podocyte hypertrophy. (F) Podocyte hypertrophy evaluation for P3h2ΔPod and P3h2fl/fl mice using immunofluorescence staining for ribosomal (Rb) p-S6, SYNPO, and DAPI. Representative immunofluorescence images show Rb p-S6 (green) in podocytes from both WT and KO mice (white arrows). Scale bars: 20 μm; inset zoom, ×5. (G) Quantification of immunofluorescence images showed a significant increase in podocyte hypertrophy in KO glomeruli when compared with WT glomeruli. (H) PEC activation analysis of P3h2ΔPod and P3h2fl/fl mice via immunofluorescence staining for CD44, SYNPO, and DAPI. Representative immunofluorescence images for KO mice show CD44 (green) signal in PECs (white arrow), indicating PEC activation. Representative immunofluorescence images for WT mice show no CD44+ signal in PECs (white arrow). Scale bars: 20 μm. Inset zoom, ×5. (I) Quantification of immunofluorescence images indicated a significant increase in the percentage of glomeruli with activated PECs in KO mice when compared with those of WT mice. n = 6. Data show the mean ± SD. *P < 0.05, by unpaired, 2-tailed Student’s t test. Graphs in B–E show the median ± IQR (n ≥6); *P < 0.05, **P < 0.01, and ****P < 0.0001, by Mann-Whitney U test. Graphs in G and I show the mean ± SD (n = 6).