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. 2018 Apr;29(4):1128-1140.
doi: 10.1681/ASN.2017070752. Epub 2018 Jan 15.

Nephronectin Regulates Mesangial Cell Adhesion and Behavior in Glomeruli

Susan E Zimmerman  1 , Chitkale Hiremath  1 , Jun Tsunezumi  1 , Zhufeng Yang  1 , Bronwyn Finney  1 , Denise K Marciano  2
Affiliations

Nephronectin Regulates Mesangial Cell Adhesion and Behavior in Glomeruli

Susan E Zimmerman et al. J Am Soc Nephrol. 2018 Apr.

Abstract

A critical aspect of kidney function occurs at the glomerulus, the capillary network that filters the blood. The glomerular basement membrane (GBM) is a key component of filtration, yet our understanding of GBM interactions with mesangial cells, specialized pericytes that provide structural stability to glomeruli, is limited. We investigated the role of nephronectin (Npnt), a GBM component and known ligand of α8β1 integrin. Immunolocalization and in situ hybridization studies in kidneys of adult mice revealed that nephronectin is produced by podocytes and deposited into the GBM. Conditional deletion of Npnt from nephron progenitors caused a pronounced increase in mesangial cell number and mesangial sclerosis. Nephronectin colocalized with α8β1 integrin to novel, specialized adhesion structures that occurred at sites of mesangial cell protrusion at the base of the capillary loops. Absence of nephronectin disrupted these adhesion structures, leading to mislocalization of α8β1. Podocyte-specific deletion of Npnt also led to mesangial sclerosis in mice. These results demonstrate a novel role for nephronectin and α8β1 integrin in a newly described adhesion complex and begin to uncover the molecular interactions between the GBM and mesangial cells, which govern mesangial cell behavior and may have a role in pathologic states.

Keywords: cell biology and structure; cell-matrix-interactions; glomerular basement membrane; mesangial cells; nephronectin.

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Figures

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Graphical abstract
Figure 1.
Figure 1.
Nephronectin localizes to glomeruli and renal tubules in mice. (A) Immunolocalization of Npnt in a coronal section of an adult mouse kidney. (Aa) Npnt (NN, green) localizes to glomeruli (arrow) and tubules. The cortex (Cor) and outer medulla (OM) are depicted. At the (Ab) corticomedullary junction and (Ac) outer medulla, Npnt localizes to glomeruli (arrow in Ab), and the basal lamina of proximal tubules marked by apical Lotus Tetragonolubus Lectin (LTL, blue), in the OM but not cortex, indicating it localizes to the S3-segment of proximal tubules. Thick ascending limb tubules are identified by Tamm Horsfall Protein (THP, red). (Ad and Ae) Npnt also localizes to tubules in the (Ad) cortex and (Ae) medulla (Med) that contain Aquaporin 2 (AQ2, red), a marker of collecting ducts. Nephronectin localizes to the GBM in glomeruli (Af and Ag, arrow) adjacent to podocyte foot processes that are marked by podocin (Pod, red). (B) RNA in situ hybridization for Npnt (purple) and control (Ctl, sense probe) in adult kidney shows high expression in glomeruli (arrows) and tubules of the OM, which are likely S3 proximal tubules identified in (A). Staining of glomerular tufts is limited to the outer rims where podocytes reside. (C) Immunolocalization of Npnt in a P0 kidney. (Ca) Low levels of Npnt are found in the primitive nephron tubule, the s-shaped body (SB), including the vascular cleft where the glomerular tuft will develop (arrows). At (Cb) the capillary loop stage and (Cc) in maturing glomeruli, Npnt localizes to the developing GBM. Merge images show NCAM (red), a marker of the developing nephron epithelia, and F-actin (blue) to aid in identification of structures. Scale bars: 50 μm in (Ab and Ac); 20 μm in (Ad and Ae); 10 μm in (Af and C); 5 μm in (Ag). Ctl, control; LTL, Lotus Tetragonolubus Lectin.
Figure 2.
Figure 2.
Absence of nephronectin leads to mesangial expansion. (A) Periodic acid–Schiff–stained kidney sections of 6-week-old mice. A prominent expansion of the mesangium is noted in mutant (Npntf/f; Six2-cre) and heterozygous (Npntf/+; Six2-cre) mice. (B) Masson’s trichrome stain from 6-week-old mice. (C) Jones’ methenamine silver stain from sections of 4-month-old mice. (Cd) Areas of matrix deposition in Npntf/f; Six2-cre mutants are indicated by arrows. (D) Mesangial sclerosis (MS) index in 6-week-old mice. See Concise Methods for details (*P<0.01, **P<0.001, ***P<0.001). (E) Measurements of serum Creatinine (Cr) performed by HPLC in mice at 6 weeks of age (*P<0.03, **P<0.04). (F) Kidney weight shown as a percentage of total body weight at 6 weeks (*P<0.004, **P<0.001). (G) Quantification of glomerular number (Glom) from kidney sections at 6 weeks (P>0.21). (H) Quantification of urinary albumin by ELISA at 6 weeks of age (P>0.19). In (D–F), data were analyzed with a one-way ANOVA and Sidak’s multiple comparisons test was used to adjust P values for multiple comparisons. Scale bars: 50 μm in (A) upper panels, (Ca and Cb); 20 μm in (A) lower panels, (B), (Cb), and (Cd). Ctl, control, (Npntf/f); Het, heterozygous (Npntf/+; Six2-cre); Mut, mutant (Npntf/f; Six2-cre).
Figure 3.
Figure 3.
Absence of Npnt increases mesangial matrix deposition, especially in severely affected glomeruli. (A) Immunofluorescence with laminin in 4-month-old control (Ctl; Npntf/f, Aa) and mutant (Mut; Npntf/f; Six2-cre, Ab and Ac) mice. A severely affected glomerulus is shown in (Ac), which has increased laminin in the mesangial area (arrow). (B) Quantification of laminin, collagen IV (α4), and agrin in the GBM. Peak fluorescence intensity was obtained along lines drawn perpendicularly through the GBM at capillary loops in the rim of the glomerular tuft. These values were normalized to average fluorescence intensity from similar lines through the basal lamina of adjacent control tubules in the same image. Agrin is increased in the GBM of mutant mice (*P=0.003). n=3 mice of each genotype. Analysis with the unpaired t test. (C) Immunofluorescence of agrin, quantified in (3B). (Cc) A severely affected glomerulus has increased agrin in the mesangial area (arrow). (D) Immunofluorescence of perlecan in (Da) control and (Db) mutant mice. (E) Quantification of perlecan in the mesangial matrix. To attempt to normalize for differences in mesangial area, perlecan signal was normalized to PDGFRβ signal within the glomerular tuft using ImageJ (P=0.09). n=3 mice of each genotype. Analysis with the unpaired t test. Scale: 20 μm in (A, C, and D).
Figure 4.
Figure 4.
Absence of Npnt increases mesangial cell number. (A and B) Immunohistochemistry with (A) endothelial marker ERG and (B) podocyte marker WT1 from sections of 6-week-old control (Ctl; Npntf/f) and mutant (Mut; Npntf/f; Six2-cre) mice. Scale bars: 25 μm. (C) Immunofluorescence with mesangial marker α8 integrin, endothelial marker ERG, and nuclear marker DAPI in 6-week-old mice. (D and E) Quantification of (A and B) as the percentage of endothelial cells (ERG+) and podocytes (WT1+) per total glomerular cells is shown; >5000 cells were counted from n=3 mice of each genotype. Analysis with the unpaired t test (*P<0.01 in E). (F) Quantification of the immunofluorescence shown in (C) as the percentage of mesangial cells (α8+, ERG−) per total glomerular cells. Analysis with the unpaired t test (*P<0.03). Scale: 25 μm in (A and B); 10 μm in (C).
Figure 5.
Figure 5.
Npnt localizes with α8 integrin and is required for the formation of unique GBM-mesangial adhesions. (A) Immunofluorescent imaging of α8 and Npnt in an adult mouse glomerulus shows that α8 in mesangial cells and Npnt at the GBM colocalize (arrows) at the lateral aspects of mesangial, tongue-like protrusions contacting the capillary loops (CL). (B) High-magnification images of control (Npntf/f) glomeruli show the lateral enrichment of α8 (Ba and Bd, asterisks) at mesangial protrusions (arrows), which appear to tether the base of capillary loops (CL). Other portions of the mesangial cell (Ba and Bd, arrowheads) have lower α8. CD31 and nephrin indicate the capillary loop and podocytes. (C) High-magnification images of the same structure in mutant (Npntf/f; Six2-cre) glomeruli show little focal α8 enrichment and the absence of mesangial, tongue-like protrusions (Ca and Cd, arrows) at capillary loops (CL). The α8 integrin is more diffuse (arrowheads). (D) Immunoprecipitation (IP) of anti-nephronectin from neonatal kidney lysates. Lanes: (1) Soluble lysate (Sol), the input before IP (2.5%); (2) nonbound lysate (NB), the input after IP (2.5%); (3) eluate from IP with anti-nephronectin antibody (NN); and (4) eluate from IP with preimmune antibody (pre-I). Western blots of α8 integrin and laminin are shown. Nephronectin coimmunoprecipitates α8 integrin. (E) A schematic showing the α8-enriched lateral protrusions of mesangial cells that contact nephronectin at the GBM. These appear to tether the opposing capillary loop. Scale: 4 μm in (A–C).
Figure 6.
Figure 6.
GBM-mesangial adhesions are focal adhesion–like structures. (A) Immunofluorescence with paxillin, α8 integrin, and endomucin (EM, a marker of endothelia) of a capillary loop (CL) and adjoining mesangial cell (M). Paxillin partially colocalizes with α8 integrin at the mesangial pedestal (arrowheads). (B) Immunofluorescence with vinculin, α8 integrin, and endomucin shows that vinculin partially colocalizes with α8 integrin (B, see arrowheads) and is medial to α8 integrin. Scale: 2 μm.
Figure 7.
Figure 7.
Absence of podocyte-derived Npnt leads to mesangial expansion. (A) Periodic acid–Schiff–stained kidney sections of 4-month-old mice. Mesangial expansion is noted in some glomeruli from mutants (Ac and Ad, Npntf/f; Podocin-cre) compared with controls (Aa and Ab, Npntf/f). (Ad) A severely affected glomerulus is shown. (B) Mesangial sclerosis (MS) index in 4-month-old mice. Glomeruli were scored from 0 to 4 (see Concise Methods for details). More than 100 glomeruli were analyzed from each mouse. Analysis was performed in a double-blinded fashion by two independent investigators. Both yielded statistically significant differences between controls and mutants using the unpaired t test (*P<0.01). Ctl, control (Npntf/f); Mut, mutant (Npntf/f; Six2-cre).
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