We have performed in vivo and ex vivo renal imaging using a RGD-labeled NIR dye (800CW) using two different optical imaging systems. Our data indicate that the strong interaction between integrin αvβ3 and its peptide ligand, RGD, can be used to non-invasively monitor renal disease progression in an anti-GBM nephritis model. In addition, our study implicates a potential role for imaging-guided disease monitoring for renal diseases with high integrin αvβ3 expression.
Integrin αvβ3, also known as the vitronectin receptor, consists of a 125 kD αv subunit and a 105 kD β3 subunit. It is expressed at low levels in most normal tissues, but, of particular interest, highly expressed in inflamed sites and on activated macrophages. In various forms of glomerulonephritis, including IgA nephropathy, lupus nephritis, membranoproliferative glomerulonephritis, as well as diabetic nephropathy, high Integrin αvβ3 expression has been reported 
. In patients with anti-GBM nephritis, an abnormal pattern of integrin αvβ3 expression was also consistently found on crescentic cells 
. Besides its role in the trafficking of inflammatory cells, this integrin may also function through other mechanisms. Several studies focus on the interaction of integrin αvβ3 with a variety of extracellular matrix components. Integrin αvβ3 has been identified as a key player in tubulo-interstitial nephritis, where it impacts tubule cell survival and proper tubular homeostasis 
. The adhesive crosstalk between glomerular mesangial cells and fibrinogen is also integrin αvβ3 dependent 
. Recently, Hayashida T et al
also reported that Integrin αvβ3 can promote renal fibrogenesis through Rac-1 mediated ERK activity 
. Thus, integrin αvβ3 may contribute to the tissue damage seen in various renal diseases through its crosstalk with a variety of extracellular matrix components. In rheumatoid arthritis, a disease characterized by inflammatory arthritis, blocking the function of integrin αvβ3 can significantly reduce inflammatory cell infiltration and restore joint function 
The recognition motif, RGD (Arg-Gly-Asp), is a tripeptide sequence that binds integrin αvβ3 receptors. Interest in using a labeled RGD peptide ligand for the study and/or monitoring of diseases related to the αvβ3 receptor over-expression is increasing. Several groups have labeled RGD successfully with fluorescent dyes for in vitro
and in vivo
imaging, mostly in tumor-related diseases 
. These studies unanimously support the notion that the interaction between integrin αvβ3 and RGD can be used to study and/or monitor the progression of diseases with enhanced integrin αvβ3 expression. In agreement with previous studies, our IHC and qPCR findings demonstrated the overexpression of integrin αvβ3 in the renal tissue following anti-GBM nephritis induction, rendering it a facile tool for imaging.
To the best of our knowledge, no in vivo
optical renal imaging based on integrin αvβ3 has been reported to date. One possible reason is that in the visible electromagnetic spectrum, tissue absorption and light scattering result in small penetration depths of a few millimeters. This makes the use of fluorescent agents in the visible spectrum suboptimal for deep organ imaging, including the imaging of the kidney. However, NIR fluorophores (700–900 nm), which have high molar extinction coefficients, good quantum yields, and low non-specific tissue binding can penetrate organs to a depth of several centimeters. Hence, these agents make deep organ in vivo
imaging possible 
. Recently, Nakamura et al
. demonstrated that Cy7-labeled recombinant-gelatin can be used to monitor renal inflammation 
. However, this study was not performed in a true “in vivo
” setting because the images were captured after the skin was removed.
The RGD 800CW probe created by LI-COR Biosciences is a NIR fluorescent agent that can bind specifically to integrin, with predominant renal excretion. We were able to successfully set up in vivo optical imaging based on this probe using the Pearl® Impulse small-animal imaging system. Our study shows that there is significantly higher accumulation of the RGD probe within nephritic kidneys, and that the accumulation can persist as long as 14 days following injection, whereas there is only a transient, low dye signal in the nephritic mice receiving the 800CW dye alone or the BSA-conjugated 800CW dye. These findings were consistent in both the in vivo and ex vivo imaging studies using two independent imaging platforms. In the healthy control group with no disease, we believe that the RGD probe is rapidly washed out after administration because there is no evidence of a renal-enhanced fluorescent signal captured at any time point.
Importantly, the change in the disease course was paralleled by the change in dye accumulation in the diseased kidney. As shown in , the high disease peak in our mouse model is around D13–D19 after anti-GBM serum challenge, as evidenced by increased Scr levels. The longitudinal changes in the fluorescent signal captured by the Pearl® Impulse imaging system () suggest that the high peak of the fluorescent signal in the nephritis group is around D12–D19 (D1–D7 after injection of the RGD probe), although the signal decreases slowly following this.
In contrast to CT and MRI, molecular and cellular information offered by molecular imaging approaches such as optical imaging may permit the earlier diagnosis of diseases, and quantitative monitoring of disease progression 
. Optical fluorescent imaging has unique advantages including high picomolar molecular sensitivity, absence of ionizing radiation, relatively low cost and the possibility of using it in many modalities with different scales, when compared to other molecular imaging technologies 
. The clinical translation of optical NIR imaging has been successfully reported in oncology practice, in which intra-operative fluorescence imaging can be used to detect sentinel lymph node involvement in patients with cervical and vulval cancer 
or to guide surgical resection 
, as well as in differentiating between malignant and benign masses in the breast 
. Moreover, NIR fluorescent dyes can be conjugated with antibody, peptide, or other small molecule, which allows for specific binding to specific target organs or tissues for potential therapeutic or diagnostic monitoring. Hence, we believe that NIR imaging is likely to emerge as a powerful tool for monitoring renal disease in the near future.