Diabetic nephropathy (DN), a significant consequence of diabetes, is associated with adverse cardiovascular and renal disease as well as mortality. Although microalbuminuria is considered the best non-invasive marker for DN, better predictive markers are needed of sufficient sensitivity and specificity to detect disease in general and in early disease specifically. Even prior to appearance of microalbuminuria, urinary biomarkers increase in diabetics and can serve as accurate nephropathy biomarkers even in normoalbuminuria. In this review, a number of novel urine biomarkers including those reflecting kidney damage caused by glomerular/podocyte damage, tubular damage, oxidative stress, inflammation, and intrarenal renin-angiotensin system activation are discussed. Our review also includes emerging biomarkers such as urinary microRNAs. These short noncoding miRNAs regulate gene expression and could be utilized to identify potential novel biomarkers in DN development and progression. .

Diabetes mellitus is one of the most challenging health concerns of the 21st century. With at least 30% of the diabetic population remaining undiagnosed, effective and early diagnosis is of critical concern. Development of a diagnostic test, more convenient and reliable than those currently used, would therefore be highly beneficial. Urine as a diagnostic medium allows for non-invasive detection of biomarkers, including some associated with type 2 diabetes and its complications. This review provides a synopsis of those urinary biomarkers that potentially may provide a basis for the development of improved diagnostic tests. Three main pathways for the sourcing of potential makers are identified: kidney damage, oxidative stress and low-grade inflammation including atherosclerosis/vascular damage. This review briefly presents each pathway and some of the most relevant urinary biomarkers that may be used to monitor the development or progression of diabetes and its complications. In particular, biomarkers of renal dysfunction such as transferrin, type IV collagen and N-acetyl-beta-D-glucosaminidase might prove to be more sensitive than urinary albumin, the current gold standard, in the detection of incipient nephropathy and risk assessment of cardiovascular disease. Inflammatory markers including orosomucoid, tumour necrosis factor-alpha, transforming growth factor-beta, vascular endothelial growth factor and monocyte chemoattractant protein-1, as well as oxidative stress markers such as 8-hydroxy-2'deoxyguanosine may also be useful biomarkers for diagnosis or monitoring of diabetic complications, particularly kidney disease. However, the sensitivity of these markers compared with albumin requires further investigation.

Human free immunoglobulin light chain (FLC) kappa and lambda are useful clinical markers for light chain myeloma and AL amyloidosis. With the recent development of specific and reliable FLC immunoassays, the quantitative measurement of FLCs will be widely used in clinical practice. However, researchers have used various calibrators, mainly monoclonal FLCs; thus, no standardization has been performed among the assay methods. This prompted us to purify intact FLCs from the pooled urine specimens of healthy volunteers as the first reference materials for FLC assays. After precipitation with ammonium sulfate, FLCs were purified by the following steps of chromatography; cation exchange, gel filtration, and antibody-assisted immunoaffinity. SDS-PAGE and Western blotting analyses showed that the purity of FLC kappa and FLC lambda was more than 98%. These purified FLCs did not contain the other immunological types of light chains. These intact and purified FLCs are suitable as the first reference materials to standardize FLC assays.

Free forms of light chains (FLCs) in serum are clinically useful markers, but their practical use in laboratories has been limited due to the cross-reaction with light chains associated with heavy chains (intact immunoglobulins) on FLC assays. In this study, we attempted to use anti-FLC antiserum and anti-FLC monoclonal antibody (mAb) in a sandwich enzyme-linked immunosorbent assay (ELISA) for serum FLCs. This combination resulted in a synergistic effect, showing 10(5)-fold specificity for FLC kappa and 10(4)-fold for FLC lambda compared to IgG. The specific ELISAs had good sensitivity, detecting 0.78 microg/l of FLC kappa and 31.3 microg/l of FLC lambda. We then measured serum samples before and after the absorption of IgG, IgA, and IgM and found that there was no appreciable difference in the FLC values between them. These results indicate that the ELISAs quantify serum FLCs more precisely even in the presence of about a thousand-fold amount of intact immunoglobulin. Meanwhile, the use of two anti-FLC mAbs in an ELISA showed no response for FLCs probably because of the conflict of the two mAbs at the specific epitope in FLC. Thus, ELISAs with anti-FLC antiserum and anti-FLC mAb can provide a reliable method for the clinical test of serum FLCs.

For the measurement of human immunoglobulin free light chains (LCs) in clinical samples, highly specific assays for free LCs are required to discriminate them from LC portions of intact immunoglobulins (bound LCs). To develop specific enzyme-linked immunosorbent assays (ELISAs) for free LCs, two anti-free LC kappa and lambda monoclonal antibodies (MAbs) were raised by a mouse/mouse hybridoma technique. We compared the specificities of these two MAbs with those of six commercially available anti-free LC antisera, which are widely used in free LC immunoassays. Comparative titrations against free LCs and intact IgGs showed the MAbs had less cross-reactivity to intact IgGs, while the four of six antisera had high reactivity to intact IgGs. Using these MAbs, we developed LC kappa and LC lambda ELISAs with ranges from 7.8 to 500 micro g/l of free LCs and less cross-reactivity to intact IgGs (less than 0.12%). On the other hand, ELISAs with anti-free LC antisera showed low specificity and/or sensitivity. Thus, the use of these MAbs may provide reliable methods for specific measurements of free LCs in clinical samples.

The potential of immunoglobulin-free light chain detection and measurement in biological fluids, and particularly in urine, has not yet been fully explored in clinical medicine, because of differences in sensitivity and lack of standardization of both quantitative assays and qualitative analysis. The ability to identify monoclonal free light chain, i.e., Bence-Jones protein, reliably in urine is of great clinical interest even when it occurs only in such small amounts that they frequently remain undetected by the routine methods used in many laboratories. The standardized adoption of both qualitative and quantitative procedures with comparable sensitivity is suggested to allow precise characterization of the clonal nature, and therefore the clinical role, of any free light chain excess.