Immunohistochemical Characterization of Alport Syndrome using alpha 2 (IV) and alpha 5 (IV) Specific Monoclonal Antibodies

In the kidney, the glomerular basement membrane (GBM) contains type IV collagen. This collagen is composed of two specific protein networks: heterotrimers of α3, α4, and α5 chains and heterotrimers of α1 and α2 chains (1). Genetic mutations in the α5(IV) chain, which is encoded by the COL4A5 gene located on the X chromosome, disrupt the formation of the α3-4-5 network and lead to abnormal GBM development. This structural defect causes a type of nephritis known as X-linked Alport Syndrome (XLAS). Alport Syndrome can be diagnosed through pathogenesis tests that detect these mutations via immunostaining.  Furthermore, kidney function tests are used to evaluate the disease by measuring urinary protein levels (2). Similarly, the skin's epidermal basement membrane (EBM) contains type IV collagen made of α1/α2 and α5/α6 heterotrimers. Because the α5(IV) chain is present in both the kidney and the skin, immunohistochemistry tests of skin biopsy sections is a highly effective diagnostic method. This approach is often preferred because a skin biopsy is much less invasive than a kidney biopsy (3).

To support this research, Chondrex, Inc. provides two epitope-defined biotinylated rat monoclonal antibodies (mAbs) as well as an assay protocol. One mAb detects the α2(IV) chain (Cat # 70751, H25) and the other detects the α5(IV) chain (Cat # 70781, H53). In samples from patients with Alport Syndrome, immunohistochemistry tests using the anti-α5(IV) mAb reveals abnormal distributions and abnormal structural patterns of collagen due to the genetic mutation. Conversely, immunostaining with the anti-α2(IV) mAb shows normal structural patterns, providing a clear comparison for diagnosis, as shown in Figure 1 and Table 1 (1, 4-6).

Figure 1. Typical Type IV Collagen Staining Patterns in Clinical Specimens

NOTE 1: XLAS: X-linked Alport Syndrome, ARAS: Autosomal Recessive Alport Syndrome
NOTE 2:  the staining results may vary due to sample preparation. Chondrex, Inc. recommends establishing your own control references.
 

Table 1. Interpretation of Immunostaining Results

REFERENCES

1.      I. Naito, S. Nomura, S. Inoue, M. Kagawa, S. Kawai, et al., Normal Distribution of Collagen IV in Renal Basement Membranes in Epstein's Syndrome. J Clin Pathol 50, 919-22 (1997).

2.      C. Kashtan, A. F. Michael, Alport syndrome. Kidney International. 50, 1445–1463 (1996).

3.      E. Lagona, L. Tsartsali, S. Kostaridou, A. Skiathitou, E. Georgaki, F. Sotsiou, et al., Skin Biopsy for the Diagnosis of Alport Syndrome. Hippokratia 12, 116-8 (2008).

4.      M. Kagawa, Y. Kishiro, I. Naito, T. Nemoto, H. Nakanishi, et al., Epitope-defined Monoclonal Antibodies Against type-IV Collagen for Diagnosis of Alport's Syndrome. Nephrol Dial Transplant 12, 1238-41 (1997).

5.      D. Borza, O. Bondar, Y. Ninomiya, Y. Sado, I. Naito, et al., The NC1 Domain of Collagen IV Encodes a Novel Network Composed of the Alpha 1, Alpha 2, Alpha 5, and Alpha 6 Chains in Smooth Muscle Basement Membranes. J Biol Chem 276, 28532-40 (2001).

6.      K. Yoshioka, S. Hino, T. Takemura, S. Maki, J. Wieslander, et al., Type IV Collagen Alpha 5 Chain. Normal Distribution and Abnormalities in X-linked Alport Syndrome Revealed by Monoclonal Antibody. Am J Pathol 144, 986-96 (1994).

7.      Kashtan, Alport Syndromes: Phenotypic Heterogeneity of Progressive Hereditary Nephritis. Pediatr Nephrol 14, 502-12 (2000).