Integrin beta1-mediated matrix assembly and signaling are critical for the normal development and function of the kidney glomerulus

Abstract

The human kidneys filter 180 l of blood every day via about 2.5 million glomeruli. The three layers of the glomerular filtration apparatus consist of fenestrated endothelium, specialized extracellular matrix known as the glomerular basement membrane (GBM) and the podocyte foot processes with their modified adherens junctions known as the slit diaphragm (SD). In this study we explored the contribution of podocyte beta1 integrin signaling for normal glomerular function. Mice with podocyte specific deletion of integrin beta1 (podocin-Cre beta1-fl/fl mice) are born normal but cannot complete postnatal renal development. They exhibit detectable proteinuria on day 1 and die within a week. The kidneys of podocin-Cre beta1-fl/fl mice exhibit normal glomerular endothelium but show severe GBM defects with multilaminations and splitting including podocyte foot process effacement. The integrin linked kinase (ILK) is a downstream mediator of integrin beta1 activity in epithelial cells. To further explore whether integrin beta1-mediated signaling facilitates proper glomerular filtration, we generated mice deficient of ILK in the podocytes (podocin-Cre ILK-fl/fl mice). These mice develop normally but exhibit postnatal proteinuria at birth and die within 15 weeks of age due to renal failure. Collectively, our studies demonstrate that podocyte beta1 integrin and ILK signaling is critical for postnatal development and function of the glomerular filtration apparatus.

Fig. 1. Podocyte specific expression of Cre-recombinase

(A-B) LacZ staining for ROSA flox mice with (podCRE(+) LacZ flox) or without (podCRE(-)) the podocin-Cre transgene, clearly shows podocyte specific expression of LacZ. (C-D) YFP expression in EYFP Rosa-flox/podocin-Cre mice (podCre (+) YFP flox). Protein expression of YFP was detected with anti-YFP antibody followed by Rhodamine conjugated secondary antibody. Merged images (Rhodamine plus DAPI) are shown. Star in picture (D) indicates glomerulus.

Fig. 2. Podocin-Cre/β1-fl/fl mice exhibit proteinuria with poor prognosis

(A-B) β1 integrin expression in podocyte. Merged images (with podocin) clearly shows a lack of β1 integrin expression in podocytes of the glomerulus in podocin-Cre β1-fl/fl mice (podCRE(+) β1fl/fl). (C-D) α3 integrin expression in glomerulus. In podocin-Cre β1-fl/fl mice, markedly decreased expression of α3 integrin was observed. Representative results from 3 mice in each group are shown. The kidney samples from post-natal day1 (P1) mice are used. (E) Survival curve of podocin-Cre β1-fl/fl mice and control mice. (F) Urinary protein excretion at P1 in podocin-Cre β1-fl/fl mice by SDS-page. (G) Detection of WT1 and podocin protein in the urine by western blot analysis using the same membrane as panel (F). (H-I) Periodic Acid Schiff (PAS) staining of kidney from P1 mice. Arrows in panel (H) indicate defective association of podocyte foot process with the GBM. Representative picture from 5 mice in each group are shown.

Fig. 3. Podocyte slit diaphragm proteins and basement membrane components expression in the podocin-Cre β1-fl/fl mice

Immunofluorescence analysis for (A-B) Nephrin, (C-D) Podocin, (E-J) Collagen typeIV α3-5, (K-N) Laminin β1-2, (O-P) Entactin, (Q-R) CD31. Laminin β1 are localized distinctly along GBM in podocin-Cre β1-fl/fl mice. The representative results of podocin-Cre β1-fl/fl mice (podCRE(+) β1fl/fl) and control (podCRE(-) β1fl/fl) from 3 mice in each group are shown. The kidney samples from P1 mice are used. (S-T) Compensatory upregulation of integrin β3 in podocytes of podocin-Cre β1-fl/fl mice. The representative results from 2 embryos (E 18.5) in each group are shown.

Fig. 4. Ultrastructural analysis of the glomerulus in the podocin-Cre β1-fl/fl mice

(A-B) Transmission electron microscopy (TEM) analysis. Arrows in panel (A) show severe multilamination in podocin-Cre β1-fl/fl mice. All of GBM in the glomerulus of podocin-Cre β1-fl/fl showed multi-lamination in GBM. (C-D) Scanning electron microscope (SEM) analysis. In panel (C), formation of podocyte foot process was severely disrupted in podocin-Cre β1-fl/fl mice. The representative results of podocin-Cre β1-fl/fl mice (podCRE(+) β1fl/fl) and control (podCRE(-) β1fl/fl) from 2 mice in each group are shown. The kidney samples from P1 mice were used here.

Fig. 5. Focal segmental glomerulosclerosis in the kidneys from podocin-CRE ILK-fl/fl mice

(A) Survival curve of podocin-Cre ILK-fl/fl mice (podCRE(+) ILKfl/fl) and control mice (podCRE(-) ILKfl/fl). (B) Gross appearance at 4 weeks of age podocin-Cre ILK-fl/fl mice or control mice. podocin-Cre ILK-fl/fl mice have pale and atrophic kidneys compared to control mice. (C) Urinary albumin excretion at post natal (P1) in podocin-Cre ILK-fl/fl mice. (D) Progressive urinary albumin excretion in podocin-Cre ILK-fl/fl mice. (E) Detection of WT1 and podocin protein in urine by western blot analysis on same membrane as panel (D). (F) Progressive focal segmental glomerulosclerosis in podocin-Cre ILK-fl/fl mice. At 4 weeks of age, progressive FSGS with interstitial alteration was developed in podocin-Cre ILK-fl/fl mice. The arrow in the panel of 4 weeks aged podocin-Cre ILK-fl/fl mice showed segmental lesions (×630). Star in same panel shows normal glomerulus. The representative results of podocin-Cre ILK-fl/fl mice and control from 3 mice in each group are shown.

Fig. 6. Podocyte slit diaphragm proteins and basement membrane components expression in the podocin-Cre ILK-fl/fl mice

Immunofluorescence analysis for (A-B) Nephrin, (C-D) Podocin, (E-J) Collagen type IV α3-5, (K-N) Laminin β1-2, (O-P) Entactin, (Q-R) CD31. (S-T) Fibronectin (red) co-stained with WT1 (green). In panel (K-L), it is clear that laminin β1 is localized in GBM in podocin-Cre ILK-fl/fl mice but not in the control mice. The representative results of podocin-Cre ILK-fl/fl mice (podCRE(+) ILKfl/fl) and control (podCRE(-) ILKfl/fl) from 3 mice in each group are shown. The kidney samples from 2 weeks old mice were used here (A-T). (U-V) TEM analysis in P1 mice. podocin-Cre ILK-fl/fl mice shows occasional foot process effacement. (W-X) TEM analysis for mice 2 weeks of age. Nodular thickening (arrow heads in panel (W)) in the GBM of podocin-Cre ILK-fl/fl mice are apparent. (Y-Z) SEM analysis for mice 2 weeks of age. Microvillous formations in podocin-CRE ILK-fl/fl mice are present. The representative results of podocin-Cre ILK-fl/fl mice (podCRE(+) ILKfl/fl) and control (podCRE(-) ILKfl/fl) from 2 mice in each group are shown.

Fig. 7. Activation of integrin β1-FAK signaling in podocin-Cre ILK-fl/fl mice

Phospho- FAK^Tyr397 is induced in podocin-Cre ILK-fl/fl mice (A and D) compared to control mice (B and E). In panel (C), negative control was shown (secondary antibody only without primary antibody in podocin-Cre ILK-fl/fl mice). The primary antibodies from Sigma (#1) or from Invitrogen (#2) are used in panel (A-B) or (D-E), respectively. (F-G) Total FAK expression is also increased in the podocin-Cre ILK-fl/fl mice. Enlarged image in panel (F) clearly indicates increased expression of FAK in podocytes. Merged image with entactin are also shown for indicating the glomerlar structure (D-G). (H) Western blot anaysis for phosphorylated FAK^Tyr397 and total-FAK. Podocin is shown as the control for glomerular proteins. Actin and Coomassie Brilliant Blue staining are shown as the loading control. (I-L) Integrin β1 and α3 expressions in the glomerulus in the podocin-Cre ILK-fl/fl mice. (I-J) Integrin β1 is localized in mesangial area, endothelial cell and capillary walls. There is no apparent difference in the expression of integrin β1 between podocin-Cre ILK-fl/fl and control mice. (K-L) Compared to control mice, integrin α3 is occasionally upregulated in capillary walls of glomerulus in podocin-Cre ILK-fl/fl mice. The representative results of podocin-Cre ILK-fl/fl mice (podCRE(+) ILKfl/fl) and control (podCRE(-) ILKfl/fl) from 3 mice in each group are shown. The samples from 2 weeks old mice are used (A-L).