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A molecular mechanism explaining albuminuria in kidney disease

Nature Metabolism volume 2, pages 461–474 (2020)Cite this article

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Abstract

Mammalian kidneys constantly filter large amounts of liquid, with almost complete retention of albumin and other macromolecules in the plasma. Breakdown of the three-layered renal filtration barrier results in loss of albumin into urine (albuminuria) across the wall of small renal capillaries, and is a leading cause of chronic kidney disease. However, exactly how the renal filter works and why its permeability is altered in kidney diseases is poorly understood. Here we show that the permeability of the renal filter is modulated through compression of the capillary wall. We collect morphometric data prior to and after onset of albuminuria in a mouse model equivalent to a human genetic disease affecting the renal filtration barrier. Combining quantitative analyses with mathematical modelling, we demonstrate that morphological alterations of the glomerular filtration barrier lead to reduced compressive forces that counteract filtration pressure, thereby resulting in capillary dilatation, and ultimately albuminuria. Our results reveal distinct functions of the different layers of the filtration barrier and expand the molecular understanding of defective renal filtration in chronic kidney disease.

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Fig. 1: PodR231Q/A286V mouse phenotype resembles human genetic glomerular disease.
Fig. 2: STED microscopy allows the visualization of nephrin and podocin in high spatial resolution.
Fig. 3: Morphometric analyses of the SD pattern in STED images reveal altered FP morphology and reduced SD abundance in PodR231Q/A286V mice as compared with controls across five time points.
Fig. 4: Morphological parameters correlate with albuminuria, and mathematical modelling supports the hypothesis of decreased compression of the GBM as the mechanism of albuminuria.
Fig. 5: Ex vivo and in vivo measurements of the capillary diameter at baseline and in response to angiotensin II infusion in control and PodR231Q/A286V mice.
Fig. 6: Validation of our model in human samples.
Fig. 7: Schematic illustration of the present study (clockwise from top left corner).

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Data availability

The data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

We thank M. Brütting, R. Herzog, S. Keller and S. Kayser for excellent technical support. We thank J. Patrakka (KI/AZ Integrated CardioMetabolic Center, Department of Laboratory Medicin,. Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm, Sweden) for providing us with kidney tissue of a tumour nephrectomy. The plasmid pSpCas9(BB)‐2A‐GFP (PX458) was a gift from F. Zhang (Addgene plasmid no. 48138). This work was supported by the Clinical Research Unit (CRU) 329 (KFO 329; A1, A6 and A7) as well as partly by FOR 2743 of the Deutsche Forschungsgemeinschaft (DFG).

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Author notes

  1. These authors jointly supervised this work: Bernhard Schermer, Thomas Benzing.

Authors and Affiliations

  1. Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany

    Linus Butt, David Unnersjö-Jess, Martin Höhne, Julia Binz-Lotter, Dervla Reilly, Robert Hahnfeldt, Markus M. Rinschen, Lena K. Ebert, Matthias J. Hackl, Paul T. Brinkkoetter, Bernhard Schermer & Thomas Benzing

  2. Royal Institute of Technology, Stockholm, Sweden

    David Unnersjö-Jess, Hjalmar Brismar & Hans Blom

  3. Department of Biomedical Engineering, Boston University, Boston, MA, USA

    Aurelie Edwards

  4. Institute of Physiology, University of Regensburg, Regensburg, Germany

    Vera Ziegler, Katharina Fremter & Hayo Castrop

  5. Center for Mass Spectrometry and Metabolomics, The Scripps Research Institute, La Jolla, CA, USA

    Markus M. Rinschen

  6. Department of Medicine IV, Medical Center - University of Freiburg, Faculty of Medicine, and Signalling Research Centres BIOSS and CIBSS, University of Freiburg,, Freiburg, Germany

    Martin Helmstädter & Gerd Walz

  7. Department of Pediatrics and Center for Molecular Medicine, University of Cologne, Faculty of Medicine and University Hospital of Cologne, Cologne, Germany

    Max C. Liebau

  8. MTA-SE Lendület Nephrogenetic Laboratory, 1st Department of Pediatrics, Semmelweis University, Budapest, Hungary

    Kálmán Tory

  9. University Children’s Hospital, Clinic for Pediatrics II, University of Duisburg-Essen, Essen, Germany

    Peter F. Hoyer

  10. Institute of Human Genetics and Center for Molecular Medicine, University of Cologne, Faculty of Medicine and University Hospital of Cologne, Cologne, Germany

    Bodo B. Beck

  11. CECAD, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany

    Bernhard Schermer & Thomas Benzing

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  1. Linus Butt
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Contributions

Conceptualization, B.S. and T.B.; methodology, L.B., L.K.E., M. Höhne, B.S. and T.B.; software, L.B. and M. Höhne; formal analysis, L.B., M. Höhne, A.E., V.Z., K.F. and M.M.R.; investigation, L.B., D.U.-J., M. Höhne, J.B.-L., D.R., R.H., V.Z., K.F. and M. Helmstädter.; resources, M.C.L., K.T, B.B.B. and P.F.H..; writing, original draft, L.B., M. Höhne, B.S. and T.B.; writing, review and editing, L.B., D.U.-J., M. Höhne., A.E., M.M.R., L.K.E., H.C., M.J.H., G.W., P.T.B., M.C.L., B.S. and T.B.; supervision, M. Höhne, H.C., M.J.H., G.W., P.T.B., H. Brismar, H. Blom, B.S. and T.B.; project administration, B.S. and T.B.; funding acquisition, P.T.B., B.S. and T.B.

Corresponding author

Correspondence to Thomas Benzing.

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Extended data

Extended Data Fig. 1 Sanger sequencing of F1 generation mice demonstrates heterozygosity.

a, The upper panel indicates the wildtype sequences on the gDNA and amino acid levels of the region neighboring the R231Q mutation site. The wildtype nucleotide at position c.692 and the wildtype amino acid at position p.231 are highlighted in green. Sequencing of a wildtype mouse aligns with the predicted wildtype sequence. The lower panel indicates the R231Q sequences on the gDNA and amino acid levels of the same region. The point mutation at c.692 and the respective change in the amino acid at position p.231 are highlighted in red. Silent mutations leading to restriction sites are highlighted in orange. Sequencing of a F1 mouse reveals double signals at the respective mutation sites. The black arrow indicates the point mutation and asterisks indicate silent mutation sites. b, The upper panel indicates the wildtype sequences on the gDNA and amino acid levels of the region neighboring the A286V mutation site. The wildtype nucleotide at position c.857 and the wildtype amino acid at position p.286 are highlighted in green. Sequencing of a wildtype mouse align with the predicted wildtype sequence. The lower panel indicates the A286V sequences on the gDNA and amino acid levels of the same region. The point mutation at c.857 and the respective change in the amino acid at position p.286 are highlighted in red. Sequencing of a F1 mouse reveals a double signal at the point mutation site indicated by the black arrow.

Extended Data Fig. 2 Glomerular proteome of wildtype and PodR231Q/A286V mice at 3 weeks of age.

a, Hierarchical clustering of proteins (label-free quantification (LFQ) values) within 3 control and 3 PodR231Q/A286V mice samples based on Euclidian distance. Heat map displays normalized log2 LFQ intensities of all n = 4322 proteins quantified in the samples (red = high intensity, green = low intensity). b, Volcano plot showing logarithmized fold changes of label-free quantification (LFQ) values in control vs. PodR231Q/A286V samples. Log2 ratios of PodR231Q/A286V over control are plotted against the negative logarithmic P value of the two-sided Student’s t-test. Each dot represents a protein (FDR = 0.05, s0 = 0.1). Proteins above the curved line are considered significantly different in terms of abundance. c, The same Volcano plot as in b with podocyte-specific proteins highlighted in red. d, The same Volcano plot as in b with glomerular basement membrane constituents highlighted in green.

Extended Data Fig. 3 Pedigree and clinical history of a patient suffering from PodR229Q/A284V mutations.

a, Schematic drawings of the human podocin protein with the two respective point mutations. Highlighted in yellow and red are the transmembrane domain (TM) and the Prohibitin domain (PHB). The pedigree indicates the mode of inheritance in the patient’s family. The parents as well as the sibling are unaffected carriers of one of the point mutations. b, Measured serum creatinine levels at different ages of the PodR229Q/A284V patient. c, Urinary protein to creatinine ratios at different ages of the PodR229Q/A284V patient. The patient was coincidentally diagnosed with albuminuria at 4 years of age. d, Normalized creatinine clearances at different ages of the PodR229Q/A284V patient shows a decline in renal function in adolescence/early adulthood.

Extended Data Fig. 4 Electron micrographs reveal progressive pathological alterations of the podocytes in PodR231Q/A286V mice.

a, Scanning electron microscopy (SEM) of control mice at four different time points shows regularly configured podocytes with typical interdigitating pattern of foot processes. Scale bars correspond to 5 µm. b, SEM of PodR231Q/A286V mice at different time points detects a progressive widening of foot processes and a rarification of the interdigitating pattern. Scale bars correspond to 5 μm. c, Transmission electron microscopy (TEM) of control mice at different time points reveals complete coverage of the glomerular basement membrane (GBM) by the podocyte’s foot processes and cell bodies (CB) of the podocytes that reside within the bowman’s space (BS) (upper panel). Higher magnification in lower panel allows for the visualization of the slit diaphragm (yellow arrows) and the slender foot processes (yellow dotted line). Scale bars correspond to 500 nm (upper panels) and 250 nm (lower panels). d, TEM of PodR231Q/A286V mice at different time points detects podocyte’s cell bodies (CB) directly attached to the glomerular basement membrane (light yellow line), loss of the slit diaphragm (yellow asterisks), and irregular thickening of the glomerular basement membrane at later time points (yellow arrowhead). Higher magnification (lower panel) demonstrates widening of foot processes (yellow dotted line), and ultimately, absence of foot processes (light yellow area). One mouse per age and genotype was imaged. Scale bars correspond to 500 nm (upper panels) and 250 nm (lower panels).

Extended Data Fig. 5 Quantification of morphometric parameters.

a, Workflow of the quantification of foot process (FP) area, perimeter, and circularity. b, Workflow of the quantification of the slit diaphragm (SD) length per area. Data in (a) are shown as mean ± SD. The depicted values in (a) represent measurements from 3 images each of one mouse per genotype c, Workflow of the quantification of the slit diaphragm (SD) grid index. d, Demonstration of the effect of different manual assignment of the capillary surface on the slit diaphragm (SD) length per area and slit diaphragm (SD) grid index values. The difference in the manual assignment does affect the SD length whereas the values of the SD grid index remain very similar. e, Scatter plot of the values of slit diaphragm (SD) grid index against the values of slit diaphragm (SD) length per area of all experimental mice (control and PodR231Q/A286V). Based on Spearman’s rank coefficient (r), there is strong correlation between slit diaphragm (SD) grid index and slit diaphragm (SD) length per area. Each dot represents an individual mouse of which both parameters were determined (n = 43, control and PodR231Q/A286V mice). r, Spearman’s rank coefficient; P-value corresponds to Spearman’s correlation.

Extended Data Fig. 6 Morphological parameters correlate with albuminuria across the entire group of experimental mice (PodR231Q/A286V and control mice).

(a)-(g) Scatter plots of each indicated parameter against the urinary albumin creatinine ratio (ACR) of experimental mice. Across the entire group of experimental mice all parameters with the exception of the foot process (FP) area (p = 0.051) correlate significantly with the ACR. (a)-(b) n = 19 mice, (c)-(g) n = 27 mice. Two-tailed Spearman’s rank correlation coefficient was used to determine statistical significance. r, Spearman’s rank coefficient. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Extended Data Fig. 7 Glomerular filtration rate (GFR) of control (black circles) and PodR231Q/A286V (red triangles) mice at different time points and correlation of the GFR with slit diaphragm (SD) length and urinary albumin creatinine ratio (ACR).

a-b, Measurement of the GFR at the indicated time points. Values are depicted as absolute (a) and normalized to body weight (b). Each dot/triangle represents one mouse (n ≥ 5 mice per genotype and age). c-d, Scatter plots of the normalized GFR against the slit diaphragm (SD) length per area (c) and the ACR of individual experimental mice (d). There is no significant correlation between the GFR and the SD length or the ACR within the groups of control and PodR231Q/A286V mice. Each dot/triangle represents one mouse. Data are presented as mean± SEM. Two-tailed unpaired Student’s t-tests and Spearman rank coefficients were used to determine statistical significance. r, Spearman’s rank coefficient. * p < 0.05, ** p < 0.01.

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Butt, L., Unnersjö-Jess, D., Höhne, M. et al. A molecular mechanism explaining albuminuria in kidney disease. Nat Metab 2, 461–474 (2020). https://doi.org/10.1038/s42255-020-0204-y

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