Fig 1.
Fold change of Map transcripts in freshly isolated mouse podocytes versus non-podocyte glomerular cells.
Fold change of Map transcripts expression (Affymetrix Mouse Gene 1.0 ST array) of freshly purified mouse podocytes compared to expression non-podocyte glomerular cells. Values > 0 indicate higher expression values in podocyte compared to other glomerular cells (* = significant differential regulation, q-value <0.001)
Fig 2.
MAP1B is highly expressed in kidney podocytes.
Immunofluorescence staining of kidney sections derived from adult human and rat kidney. WT1 served as a marker for podocyte nuclei. Nephrin and synaptopodin served as foot process markers. DAPI was used for staining nuclei. MAP1B is expressed highly specific in podocytes (scale for overview: 50 μm; higher magnification: 25 μm) (A,C). Expression of MAP1B during glomerulogenesis in newborn rat kidney. Glomerular differentiation advances from cortex to medulla (upper right: immature; lower left: mature) (scale for overview: 50 μm; higher magnification: 25 μm) (B). Transmission electron microscopy of kidney sections. Immunolabeling of MAP1B confirms an enrichment in areas with a dense MT cytoskeleton in primary processes and the cell body (primary processes (PP), foot processes (arrowheads), Nucleus (Nu)), glomerular basement membrane (GBM); scale: 200 nm) (D).
Fig 3.
MAP1B is closely associated with the podocyte MT skeleton.
Immunofluorescence microscopy of immortalized podocytes at 33°C (A) and after cellular senescence at 37°C (B) using antibodies for MAP1B and α-Tubulin as well as phalloidin for labeling of the actin cytoskeleton. During differentiation cell-culture podocytes undergo an enormous cell growth which goes along with forming an arborized MT skeleton. MT bundles show absolute co-localization with MAP1B HC (see merge of channels for MAP1B and α-Tubulin, panel far right, scale 50 μm). Semi-quantitative Western Blot analysis of MAP1B in cell culture podocytes at 33°C and 37°C. Lysate of HEK-293T cells served as negative control, lysates of brain and HEK-293T with overexpressed MAP1B served as positive controls. Intracellular concentration of MAP1B increases in response to cellular senescence. Tubulin and CD2AP confirm equal loading concentrations (C). Schematic of our GFP/ mCherry-tagged MAP1B fusion protein: Map1b FL cDNA, flanked by N-terminal GFP and C-terminal RFP has been used for lenti-viral transduction into cell culture podocytes (D). Immunofluorescence microscopy of fixed podocytes, stably expressing GFP/RFP-tagged MAP1B (scale: 50 μm) (E).
Fig 4.
Constitutive MAP1B Knock out (KO) mice do not exhibit a glomerular phenotype.
Immunofluorescence microscopy of kidney sections from constitutive MAP1B KO mice versus control littermates. Autofluorescence of PFA treated tissue is used to display glomerular anatomy. Total depletion of MAP1B HC in podocytes of KO animals as well as the specificity of our antibody for MAP1B expression is confirmed (scale: 50 μm) (A). We did not observe alterations of glomerular morphology using light microscopy of Periodic-acid Schiff (PAS) stained kidney sections of MAP1B KO animals (scale: 50 μm) (B). MAP1B podocyte foot process morphology examination using transmission electron microscopy of MAP1B KO podocytes (Podocyte (Pd), capillary (C), Bowman’s urinary space (B), podocyte foot processes (arrow heads), scale: 10 μm) (C). Proteinuria in the physiological range in both KO and control littermate by measuring urinary albumin/creatinin ratios (n = 7 for KO and control animals, p = 0,205) (D). 9 week old MAP1B KO animals showed a significantly reduced body weight compared to control littermates (n = 7 for KO and control animals, p = 0,010) (E).