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.
View: Text | PDF
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

×

Figure 2

Generation and characterization of P3h2ΔPod mice by urinalysis.

Options: View larger image (or click on image) Download as PowerPoint
Generation and characterization of P3h2ΔPod mice by urinalysis. (A) Sche...
(A) Schematic strategy for the generation of P3h2ΔPod mice. (B) KO confirmation of the P3h2ΔPod mice. qPCR P3h2 mRNA expression analysis was performed in sorted podocytes from WT and KO mice. P3h2 mRNA levels were reduced by 99% in P3h2ΔPod mice compare with levels in P3h2fl/fl mice. (C) Immunofluorescence staining of WT and KO kidney tissues for P3H2, NPHS1, and DAPI. There was no detectable P3H2 in the P3h2ΔPod mouse podocytes. Scale bar: 10 μm; inset zoom, ×5. (D) Body weights of P3h2ΔPod and P3h2fl/fl mice were measured starting at 5 weeks until 48 weeks of age. There was no significant difference at any time point in body weights between the WT and KO mice (E) UACR of P3h2ΔPod and P3h2fl/fl mice. KO mice started to present with albuminuria at 36 weeks of age, and this had increased at 48 weeks of age. (F) Serum urea measurements for P3h2ΔPod and P3h2fl/fl mice. No significant increase was observed in the KO mice. (G) Serum cystatin C measurement for P3h2ΔPod and P3h2fl/fl mice. No significant increase was observed in the KO mice. (H) Hematuria was detected in spot urine of P3h2ΔPod mice. Representative images of urine from mice of each genotype. There were significantly more and dysmorphic RBCs in KO urine than WT urine. Scale bar: 50 μm: inset zoom, ×5. (I) Quantification of urinary RBCs under a light microscope. n ≥ 3. Graphs show the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by unpaired, 2-tailed Student’s t test.