**2. Current GFR markers**

In order to assess the status and to follow the progress of renal disease, there is a need to develop a simple, accurate, and continuous method for the determination of renal function by non-invasive procedures. At present, endogenous serum creatinine (**1**) (Fig. 1) concentration measured at frequent intervals over a 24-hour period has been the most common method of assessing renal function despite the well known serious limitations.16-18 The results from this analysis are frequently misleading since the value is affected by age, state of hydration, renal perfusion, muscle mass, dietary intake, and many other anthropometric and clinical variables. Theoretical methods for estimating GFR (eGFR)19-21 from body cell mass and plasma creatinine concentration have also been developed, but these methods also rely on the above anthropomorphic variables. Moreover, creatinine is

Exogenous Fluorescent Agents for the Determination of Glomerular Filtration Rate 253

Accordingly, there has been some effort on developing exogenous GFR tracer agents that absorb and emit in the visible or near infrared (NIR) region, which includes small molecules as well as macromolecular bioconjugates such FITC (fluorescien isothicyanate)-inulin and FITC- and Texas Red-dextrans.31-37 The key requirements for an ideal fluorescent tracer agent are: (a) must be excited at and emit in the visible region ( ≥ ~425 nm); (b) must be highly hydrophilic; (c) must be either neutral or anionic; (c) must have very low or no plasma protein binding; (d) must not be metabolized in vivo, and (e) must clear exclusively via glomerular filtration as demonstrated by equality of plasma clearance with and without a tubular secretion inhibitor such as probenecid.38 The selection of the lead clinical candidate(s) may be based on secondary considerations such as the ease of synthesis, lack of toxicity, and stability. The secondary screening criteria should further take into account the tissue optics properties and the degree of extracellular distribution of the fluorescent tracers. Volume of distribution is an important parameter in the assessment of hydration state of the patient, whereas the absorption/emission properties provide essential information for the

This chapter focuses on the most recent development on luminescent tracers for GFR measurement. There are basically two principal pathways for the design of fluorescent tracers for GFR determination. The first method involves enhancing the fluorescence of known renal agents that are intrinsically poor emitters such as lanthanide metal complexes; and the second involves transforming highly fluorescent dyes (which are intrinsically lipophilic) into hydrophilic, anionic species to force them to clear via the kidneys.32 In the first approach, several europium-DTPA complexes endowed with various molecular 'antenna' to induce ligand-to-metal fluorescence resonance energy transfer (FRET) were prepared and tested.32 Some of metal complexes (e.g. compound **7** exhibited high (c.a. 2000 fold) enhancement of europium fluorescence and underwent clearance exclusively through the kidneys, but whether they cleared exclusively via glomerular filtration remains uncertain. Moreover, the excitation maxima of these complexes remained in the violet or

**3. Development of fluorescent tracer agents** 

design of the probe.

UV-A region.

Fig. 2. Eu-DTPA-Quinoline Complex.

Fig. 1. Structures of Currently Known Exogenous GFR Markers.

partially cleared by tubular secretion along with glomerular filtration, and, as Diskin17 recently remarked, "Creatinine clearance is not and has never been synonymous with GFR, and all of the regression analysis will not make it so because the serum creatinine depends upon many factors other than filtration." More recently, endogenous cystatin-C has been suggested as an improvement over creatinine,15,20 but this marker also suffers from the same limitations as creatinine, and thus it remains questionable whether it is really an improvement.

In the past several decades, exogenous tracers such as inulin (**2**), iothalamate (**3**), iohexol (**4**), 99mTc-DTPA (diethylenetriaminepentaacetate) (**5**), and 51Cr-EDTA (ethylenediaminetetraacetate) (**6**) (Fig.1), have been developed to determine GFR, but all of them require either radiometric, HPLC (high performance liquid chromatography), or X-ray fluorescence methods for detection and quantification.22-29 Unfortunately, all of these markers suffer from various undesirable properties including the use of radioactivity, ionizing radiation, and the laborious ex-vivo handling of blood and urine samples, and the use of HPLC method that render them unsuitable for continuous monitoring of renal function in the clinical setting. Furthermore, inulin as well as other polysaccharides are polydisperse polymers, and availability of these substances in a reliable, uniform batches is a serious limiting factor for their use as GFR markers. Currently, iothalamate and iohexol are the accepted standard for the assessment of GFR. However, iothalmate requires the collection of blood samples and requires HPLC method, which is not well suited for continuous monitoring. Continuous monitoring of GFR has been accomplished via radiometric12 and magnetic resonance imaging30 techniques, but these are not suitable at the bedside. Hence, the availability of an exogenous marker for the measurement of GFR under specific yet changing circumstances would represent a substantial improvement over any currently available or widely practiced method. Moreover, a method that depends solely on the renal elimination of an exogenous chemical entity would provide an absolute and continuous pharmacokinetic measurement requiring less subjective interpretation based upon age, muscle mass, blood pressure, etc.
