The present work demonstrates the successful application of laser capture microdissection followed by mass spectrometry in the characterization of glomerular proteomes from clinical kidney biopsies. The high degree of clustering within the normal group and the fibronectin glomerulopathy group is an indication of the reproducibility of the method. There was more variability in the diabetic nephropathy and lupus nephritis patient groups, however this is not unexpected. Kidney biopsies in diabetics are generally done in patients with atypical clinical courses, and in lupus nephritis there is a considerable spectrum of pathologic changes within individual lupus nephritis classes. Glomerular immunohistochemistry was used to validate differential glomerular expression of selected proteins identified by the proteomic approach. Additional validation was provided by the finding that several of the differentially expressed proteins revealed by proteomic analysis are consistent with proteins known to be involved in glomerular injury, for example complement activation products and immunoglobulins in lupus nephritis, or fibronectin in fibronectin glomerulopathy.
The most interesting findings from this study are the newly discovered changes in protein expression. This demonstrates that our approach is a robust platform that may inform disease pathogenesis. For example, despite the diversity of glomerular diseases examined, we found a number of glomerular protein changes that were common in the disease glomeruli but not the normal glomeruli, as follows:
1) Several podocyte proteins, particularly those associated with the slit diaphragm, were significantly diminished in fibronectin glomerulopathy, diabetic nephropathy and lupus nephritis. As all of these diseases are proteinuric, and because the attenuated podocyte proteins are important in the maintenance of the glomerular filtration barrier, decreased expression likely contributes to proteinuria. Interestingly, laser capture microdissection followed by microarray analysis of glomeruli from patients with focal segmental glomerulosclerosis showed a down-regulation of mature podocyte genes 5
. It is not presently known if the loss of these podocyte proteins and genes is a non-specific response to podocyte injury and/or proteinuria, or is a primary event in the development of proteinuria. The latter scenario would imply a common pathogenic mechanism for different diseases.
2) Complement components were found in fibronectin glomerulopathy, diabetic nephropathy and lupus nephritis. The presence of complement proteins in the glomeruli of lupus nephritis patients is expected, though the patterns of Class IV and Class V are slightly different. Class IV lesions uniquely contained C1q, no C4 protein, and abundant C3 protein. The presence of C3 without C4 suggests significant alternative pathway activation, and is in agreement with our recent report of C3 consumption, but not C4 consumption, being independently associated with renal flare 34
. In contrast, Class V lesions contained no C1q but detectable C4 protein, suggesting the involvement of the lectin pathway. Other complement components were also present in Class V lesions, including abundant C9, likely reflecting the presence of the complement membrane attack complex as previously reported 35
In diabetic nephropathy there are some data that complement may be involved in disease progression, possibly through the lectin binding pathway, and C3 and the membrane attack complex (C5b-9) have been found in glomeruli (reviewed in 36
The presence of complement proteins in fibronectin glomerulopathy glomeruli is somewhat surprising, because immunohistochemical analysis of fibronectin glomerulopathy reveals inconsistent involvement of immunoglobulins or complement 13
. Fibronectin binds C1q via its collagenous tail 37, 38
, which could explain the presence of C1q, but this binding does not result in complement activation 39
. Complement activation could occur through the alternative pathway, especially if the expression or function of complement regulators is compromised. Interestingly, a genome-wide linkage study identified a fibronectin glomerulopathy genetic susceptibility locus within the regulators of complement activation gene cluster 40
. However, no studies to date have identified abnormal expression or function of regulators of complement activation gene cluster proteins in patients with fibronectin glomerulopathy 41
. Another possible explanation for the presence of complement proteins in fibronectin glomerulopathy is through glomerular complement expression. This has been demonstrated for C3 by in situ hybridization in glomeruli from lupus nephritis patients, but not from healthy controls 42
. Finally, this could simply represent non-specific trapping of complement proteins, but if so this is significantly more than in normal glomeruli (see raw spectral counts, ). The presence of complement proteins in the glomeruli of fibronectin glomerulopathy raises the possibility that complement-mediated damage may be involved in this non-immune glomerular lesion. A recent proteomic evaluation of chronic allograft nephropathy also unexpectedly found complement pathway activation and reached a similar conclusion 43
. Limiting complement activation may therefore improve clinical outcomes in diseases not previously thought to involve clinically relevant complement activation.
3) Anti-oxidant proteins were conspicuously under-represented in fibronectin glomerulopathy, diabetic nephropathy and lupus nephritis glomeruli, but the proteomic approach cannot distinguish between a primary defect in anti-oxidant production, or a secondary loss of these proteins. Nonetheless, a reduction in glomerular anti-oxidants leaves the tissue open for oxidant injury.
4) Pathway analysis based on the differentially expressed proteins suggested that compared to normal, metabolic pathways were suppressed in all three types of glomerular disease. This suggests that any type of glomerular injury may attenuate metabolism of glomerular cells, but more importantly, indicates that metabolomic evaluation of glomerular disease may uncover novel mechanisms of injury and repair.
To further demonstrate the utility of laser capture microdissection followed by glomerular proteomics, we studied fibronectin glomerulopathy in more detail. This uncommon glomerular disease results from the deposition of excessive matrix material in glomeruli. The hypothesis that fibronectin glomerulopathy is mechanistically related to abnormal glomerular accumulation of fibronectin is based mainly on evidence from immunohistochemical staining, which has limitations. Immunohistochemical staining can only test constituents for which specific antibodies are available, and can be falsely positive due to nonspecific trapping of antibodies, or falsely negative if antigens are blocked. By isolating glomeruli from kidney biopsies of affected patients, and doing unbiased proteomic analysis of the recovered glomerular proteins, we confirmed that fibronectin is the predominant protein in the lesions of fibronectin glomerulopathy glomeruli. In addition to several common pathways of glomerular injury activated in fibronectin glomerulopathy described above, we found considerable deposition of fibulin in these glomeruli. Among the diseases examined, fibulin accumulation appears to be unique to fibronectin glomerulopathy because it is not present in normal glomeruli, diabetic nephropathy, or lupus nephritis. Interestingly, fibronectin binds to fibulin-1 but not fibulin-5 31, 41, 44, 45
. The fibulins appear to regulate several of the molecular activities of fibronectin. For example, fibronectin is important in cell-matrix interactions, and facilitates cell attachment, cell spreading, and cell migration 46
. Fibronectin also regulates the cytoskeleton, cellular morphology, and differentiation 30
. Fibulin-1 down-regulates fibronectin-facilitated cell spreading, adhesion and motility 47
. These effects may occur because fibulin-1 inhibits fibronectin-mediated extracellular signal-regulated kinase activation 47
. Although fibulin-5 does not directly bind to fibronectin, it binds to the same β1 integrins as fibronectin (α5β1, α4β1), and like fibulin-1, blocks fibronectin-mediated cell spreading, motility, and proliferation 48
The role of fibulins plus fibronectin in the pathogenesis of glomerular injury in fibronectin glomerulopathy is not yet known, however fibulins have been associated with other types of kidney disease. Specifically, fibulin-1 was identified as a candidate permeability factor in focal segmental glomerulosclerosis 49
, perhaps because it interferes with cell adhesion, and could therefore disrupt the podocyte-basement membrane relationship. Gene profiling of autosomal dominant polycystic kidney disease tissue showed markedly increased fibulin mRNA expression, and the protein was demonstrated in epithelial cells lining cysts 50
. Fibulin-1 has been shown to co-localize with ADAMTS-1 and increase its catalytic activity 51
. It is conceivable that in fibronectin glomerulopathy the accumulated fibronectin helps localize fibulins to glomeruli, and the fibulins inhibit cell spreading, adhesion, and motility, disrupting mesangial and/or podocyte function and facilitating the development of proteinuria.
In summary, the present study shows that the application of laser capture microdissection followed by liquid chromatography-tandem mass spectrometry analysis of kidney biopsies can provide large-scale quantitative proteomic profiles that may be important for characterizing glomerular diseases as well as determining mechanisms of disease pathogenesis. The ability to more clearly understand underlying molecular events critical to disease expression may open avenues for novel therapeutic interventions.