ICAM-1 and VCAM-1
Endothelial cells are important players in promoting the microvascular disease that defines some of the pathologic lesions of diabetic nephropathy. Endothelial cell expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) is augmented when cells are exposed to serum from diabetic patients, and the enhanced levels correlate well with worsening albuminuria.
Soluble forms of ICAM-1 (sICAM-1) and VCAM-1 (sVCAM-1) are produced either de novo or as cleavage products from established cellular lesions, and their plasma levels correlate with the severity of diabetic nephropathy. Notably, ACE inhibitor and ARB therapies can significantly reduce circulating sVCAM-1 levels while statins can reduce sICAM levels in patients with type 2 diabetes. Thus, ICAM-1 and VCAM-1 have the potential for use as biomarkers in diabetic nephropathy.
Integrins. Integrins are cell surface receptors, consisting of approximately 25 a- and b-heterodimeric complexes that recognize and adhere to specific ECM proteins (Nephron Exp Nephrol. 2003;94: e77-e84). The b1-integrin family functions as cellular receptors that transmit the external environment via adhesion to fibronectin (a1b1, a5b1, avb1, a8b1, a3b1), collagen (a1b1, a2b1, a3b1), vitronectin (a5b1, a8b1), and some laminin isoforms (a1b1, a2b1, a3b1) through recognition of a common arginine-glycine-aspartate (RGD) motif on ECM proteins. Together, the a- and b-subunits determine the specificity of ligand binding and maintain the structural integrity of the glomerulus.
However, the cytoplasmic tail of the b1-subunit promotes intracellular “inside-out” and “outside-in” signaling processes that modulate cell functions by regulating cellular proliferation, migration, apoptosis, and differentiation, as well as ECM gene transcription and ECM assembly upon ligand-binding and integrin-clustering at the cell membrane. Thus, integrins sense and transmit the external cellular environment.
Their role in renal development has been highlighted by in vitro and in vivo gene mutational studies and by the use of function-blocking antibodies that demonstrate abnormal or absent renal morphogenesis and induce significant renal diseases. In the diabetic kidney, expression of specific integrin subunits and their respective ligand ECM proteins correlates with disease severity (J Am Soc Nephrol. 1996;7:2636-2645).
A more striking observation is that changes in the expression of glomerular b1 integrin and its intracellular signaling actually precede alterations in ECM morphology in short-term and long-term streptozotocin-induced diabetic rats.
However, only specific integrins can support ECM assembly, and this process can be blocked by inhibition of phosphoinositol 3-kinase activity. Studies in our laboratory suggest that blockade of certain integrin functions may alter the course of diabetic nephropathy. Thus, the monitoring of integrin function and expression may be an early and predictable biomarker of diabetic nephropathy, and these cell adhesion molecules may be appropriate targets in matrix remodeling.
The a3b1 is a promiscuous integrin that is primarily expressed by podocytes for attachment to the glomerular basement membrane. Because diabetic nephropathy is also associated with injury or loss of podocytes, expression of a3b1 is attenuated with short-term and long-term diabetes in animals and humans. We and others (Dev Biol 2008;313:584-593) have observed significant proteinuria and anomalous postnatal glomeruli in podocyte-specific b1-knock-out mice.
Interestingly, integrins have been more thoroughly explored as biomarkers in cancer biology. A number of studies have demonstrated elegant, noninvasive, sensitive imaging of integrin distribution and function in various tissues, but these have not been investigated in the kidney. These studies highlight the enormous potential for using integrins as biomarkers in diabetic nephropathy and suggest that the groundwork is set for further studies in this area.
Osteopontin. Osteopontin (OPN) is a large phosphoglycoprotein adhesion molecule that is expressed in almost every tissue. Originally isolated from bone, OPN also contains an RGD recognition sequence. This sequence becomes exposed upon cleavage of a conserved thrombin cleavage site that modulates both RGD-dependent and RGD-independent OPN-receptor interactions.
OPN serves as a ligand for CD44 and avb3, avb1, avb5, a9b1 and a4b1 integrins, and its roles in wound healing and development of renal fibrosis have been demonstrated in animal models of kidney damage. Although OPN is primarily expressed in the tubules of normal kidneys, renal damage enhances its tubular expression and further promotes its glomerular expression.
Indeed, OPN has been deemed a potential biomarker of diabetic nephropathy. Susztak et al showed that OPN expression correlated with increased expression of mesangial matrix expansion in mouse models of type 1 and type 2 diabetic nephropathy compared with nondiabetic mice (Diabetes. 2004;53:784-794). We have evidence that OPN is differentially regulated in diabetes (beyond targeting glucose control) and that deletion of OPN may protect against albuminuria in animal models of diabetic nephropathy.
We have also demonstrated that OPN modulates oxidative stress, inflammation, and fibrosis induced by the action of angiotensin II in OPN-null mice (Kidney Int. 2009;76:32-43). Moreover, long-term treatment with an ACE inhibitor attenuates OPN expression in diabetic rats. Importantly, OPN can be measured in serum and urine, highlighting its utility as a measurable biomarker in diabetic nephropathy.
Laminins. Laminins are glycoprotein CAMs expressed primarily in basement membrane, which is important in maintaining the integrity of the glomerular filtration barrier and normal renal function. Laminins are also critical to renal morphogenesis. Laminins are heterotrimeric structures consisting of combinations of five a, three b, and three g chains that share a common domain and several globular and rod-like domains.
The tissue-specific distribution of laminins is mainly determined by expression of the a chains; in particular, a number of a-chain mutations are associated with neonatal lethality and defective glomerulogenesis. Glomerular and glomerular epithelial cell laminin expression has been shown to increase in response to hyperglycemia, TGF-b, and high vs. normal glucose levels coincident with increased thickening of the glomerular basement membrane.
In addition, treatment with an ACE inhibitor has been shown to normalize expression of laminin in glomerular epithelial cells. In a recently described transgenic model of diabetic nephropathy expressing the dominant negative human glucose-dependent insulinotropic polypeptide receptor (GIPRdn) in pancreatic beta cells, mice bypassed the stage of insulin resistance and developed early-onset diabetes mellitus.
Kidney damage was characterized by podocyte hypertrophy, reduced volume density of podocytes, diffuse glomerular basement membrane thickening, and subsequent development of mesangial expansion and ECM accumulation, as well as proteinuria.
Early glomerular basement membrane thickening was associated with increased laminin expression (Am J Physiol Renal Physiol. 2009;296:F819-829). But laminin expression may also be a harbinger of diabetic microvascular disease, as others have revealed increased urinary and serum concentrations of laminin in patients with proteinuria as well as in diabetic patients with evidence of microangiopathy and absence of reduced renal function. In this regard, laminin may potentially be a feasible biomarker of diabetic nephropathy, and accessibility to circulating laminin levels can facilitate additional research in this area.
Emerging technologies
A number of current and relatively new technologies support investigation and confirmation of novel biomarkers in renal diseases and particularly in diabetic nephropathy. For example, biomarker arrays allow us to identify known genes in tissues of normal and diseased kidneys of humans, rats, and mice.
On the other hand, proteomics has emerged as a useful tool to identify novel biomarkers in tissues as well as in blood and urine, which are more easily accessible.
However, much of the work currently being performed in the area of renal-related proteomics tends to focus on acute renal injury. Newer technologies, such as capillary electrophoresis coupled with mass spectrometry (CE-MS), and bioinformatics, combined with clinical acumen may provide more relevance to CKD, particularly with regard to diagnosis, prognosis, and therapeutic applications in diabetic nephropathy.
The discovery and confirmation of feasible candidate biomarkers, especially small peptide molecules, of CKD could potentially advance research related to Nanomedicine, which has permeated and advanced therapies in several other human diseases but remains relatively uncharted in diabetic nephropathy.
It will become important as we establish more consistent and reliable biomarkers for clinical diabetic nephropathy, such as those described herein or studied by others, that efforts are directed toward the identification of biomarkers that are specifically expressed in patient populations that are at disproportionately high risk for diabetic nephropathy.
Acknowledgement
The author thanks Dr. Chi Pham for editorial input.
Dr. Nicholas is Associate Professor of Medicine in the Division of Nephrology and Division of Endocrinology, Diabetes, and Hypertension, David Geffen School of Medicine at the University of California at Los Angeles.