Expression of agrin, dystroglycan, and utrophin in normal renal tissue and in experimental glomerulopathies

Abstract

The dystrophin-glycoprotein complex, which comprises alpha- and beta-dystroglycan, sarcoglycans, and utrophin/dystrophin, links the cytoskeleton to agrin and laminin in the basal lamina in muscle and epithelial cells. Recently, agrin was identified as a major heparan sulfate proteoglycan in the glomerular basement membrane. In the present study, we found mRNA expression for agrin, dystroglycan, and utrophin in kidney cortex, isolated glomeruli, and cultured podocytes and mesangial cells. In immunofluorescence, agrin was found in the glomerular basement membrane. The antibodies against alpha- and beta-dystroglycan and utrophin revealed a granular podocyte-like staining pattern along the glomerular capillary wall. With immunoelectron microscopy, agrin was found in the glomerular basement membrane, dystroglycan was diffusely found over the entire cell surface of the podocytes, and utrophin was localized in the cytoplasm of the podocyte foot processes. In adriamycin nephropathy, a decrease in the glomerular capillary wall staining for dystroglycan was observed probably secondary to the extensive fusion of foot processes. Immunoelectron microscopy showed a different distribution pattern as compared to the normal kidney, with segmentally enhanced expression of dystroglycan at the basal side of the extensively fused podocyte foot processes. In passive Heymann nephritis we observed no changes in the staining intensity and distribution of the dystrophin-glycoprotein complex by immunofluorescence and immunoelectron microscopy. From these data, we conclude that agrin, dystroglycan, and utrophin are present in the glomerular capillary wall and their ultrastructural localization supports the concept that these molecules are involved in linking the podocyte cytoskeleton to the glomerular basement membrane.

Figure 1.

Reverse transcriptase-polymerase chain reaction for C-terminal agrin (A), N-terminal agrin (B), utrophin (C), and dystroglycan (D) on cDNA from cortex, glomeruli, and cultured podocytes and mesangial cells. M50 bp marker. Lanes 1 and 2, kidney cortex; lanes 3 and 4, glomeruli; lanes 5 and 6, podocytes; lanes 7 and 8, mesangial cells; lane 9, buffers only. Lanes 1, 3, 5, and 7: cDNA (reverse-transcribed RNA); lanes 2, 4, 6, and 8: nonreverse-transcribed RNA.

Figure 2.

Indirect IF on normal rat kidney tissue with anti-N-terminal agrin mAb MI-90 (A), anti-C-terminal agrin mAb Agr-131 (B), anti-α-dystroglycan mAb IIH6 (C), and anti-utrophin mAb DRP-2 (D). Magnification, ×375.

Figure 3.

Indirect IF on normal human kidney tissue with anti-N-terminal agrin mAb MI-90 (A), anti-β-dystroglycan mAb 43-DAG (B), and anti-utrophin mAb DRP-2 (C). Magnification, ×475.

Figure 4.

Immunoelectron microscopy on normal rat kidney tissue with anti-N-terminal agrin mAb MI-90 (A, B, and C), anti-C-terminal agrin mAb Agr-131 (D, E, and F), anti-α-dystroglycan mAb IIH6 (G, H, and I), and anti-utrophin mAb DRP-2 (J, K, and L). Magnifications: A, ×4500; B, ×12000; C, ×5500; D, ×6000; E, ×12,000; F, ×7000; G, ×3500; H, ×7000; I, ×6000; J, ×3500; K, ×12,000; L, ×12,000.

Figure 4.

Immunoelectron microscopy on normal rat kidney tissue with anti-N-terminal agrin mAb MI-90 (A, B, and C), anti-C-terminal agrin mAb Agr-131 (D, E, and F), anti-α-dystroglycan mAb IIH6 (G, H, and I), and anti-utrophin mAb DRP-2 (J, K, and L). Magnifications: A, ×4500; B, ×12000; C, ×5500; D, ×6000; E, ×12,000; F, ×7000; G, ×3500; H, ×7000; I, ×6000; J, ×3500; K, ×12,000; L, ×12,000.

Figure 5.

Immunoelectron microscopy on normal rat kidney tissue with anti-C-terminal agrin antibody 45. Note that the perimesangial GBM is negative. Magnification, ×8000.

Figure 6.

Staining of anti-α-dystroglycan mAb IIH6 in ADN. Indirect IF (A) and IEM (B, C, and D). Magnifications: A, ×375; B, ×3500; C, ×7500; D, ×3500.

Figure 7.

Staining of anti-α-dystroglycan mAb IIH6 in PHN. Glomerular localization of immune complexes is demonstrated by IF staining of sheep IgG (A). Distribution of α-dystroglycan is shown by IF (B) and IEM (C and D). Magnification: A and B, ×530; C, ×4200; D ×13,500.

Figure 8.

Schematic representation of the possible interaction between agrin, DG, and utrophin in the glomerular capillary wall. Reproduced with permission from Groffen et al.