Use and isolation of urinary exosomes as biomarkers for diabetic nephropathy

Abstract

Diabetes represents a major threat to public health and the number of patients is increasing alarmingly in the global scale. Particularly, the diabetic kidney disease (nephropathy, DN) together with its cardiovascular complications cause immense human suffering, highly increased risk of premature deaths, and lead to huge societal costs. DN is first detected when protein appears in urine (microalbuminuria). As in other persisting proteinuric diseases (like vasculitis) it heralds irreversible damage of kidney functions up to non-functional (end-stage) kidney and ultimately calls for kidney replacement therapy (dialysis or kidney transplantation). While remarkable progress has been made in understanding the genetic and molecular factors associating with chronic kidney diseases, breakthroughs are still missing to provide comprehensive understanding of events and mechanisms associated. Non-invasive diagnostic tools for early diagnostics of kidney damage are badly needed. Exosomes - small vesicular structures present in urine are released by all cell types along kidney structures to present with distinct surface assembly. Furthermore, exosomes carry a load of special proteins and nucleic acids. This "cargo" faithfully reflects the physiological state of their respective cells of origin and appears to serve as a new pathway for downstream signaling to target cells. Accordingly, exosome vesicles are emerging as a valuable source for disease stage-specific information and as fingerprints of disease progression. Unfortunately, technical issues of exosome isolation are challenging and, thus, their full potential remains untapped. Here, we review the molecular basis of exosome secretion as well as their use to reveal events along the nephron. In addition to novel molecular information, the new methods provide the needed accurate, personalized, non-invasive, and inexpensive future diagnostics.

Keywords: diabetic nephropathy; exosomes; extracellular vesicles; podocyte; urine.

Figure 1

Exosomes biogenesis; (A) multivesicular body formation; intraluminal vesicles formation pathway based on: (B) ESCRT complex involvement, (C) transformation of sphingomyelin into ceramide, and (D) triggered by phospholipid lysobisphosphatidic acid (LBPA) in acidic pH.

Figure 2

Schematic picture of the urogenital route with one nephron and bladder. Bowman’s capsule (BC), proximal convoluted tubule (PCT), loop of Henle (LH), distal convoluted tubule (DCT), collecting duct (CD), and bladder (BL). List of specific biomarkers for each nephron segment are presented in frames. WT-1, Wilm’s tumor 1; CR1, complement receptor 1; AQP1, aquaporin 1; SGLT2, sodium-glucose linked transporter 2; CA-IV, carbonic anhydrase IV; THP, Tamm-Horsfall protein; KSP-cadherin, kidney specific cadherin; AQP2, aquaporin 2.

Figure 3

Hydrostatic dialysis systems set up for large (0.5–1 l) medium (200–600 ml) and small volume (10–100 ml) of urine. The dialysis membrane tube (MWCO 1,000 kDa) is connected to the separating funnel and/or a normal funnel. The bottom end is sealed with a universal dialysis tube closure. The separating funnel is filled in with supernatant from 2,000 g centrifugation (A). The hydrostatic pressure of urine in funnel pushes the solvent (water) trough the meshwork of dialysis membrane and liquid below the MWCO, which fells to the bottom of the cylinder (B). When urine is concentrated up to 7–8 ml (C) the funnel is re-filled with milliQ water (~200 ml equivalent to 25 volumes of concentrated urine), which flushes away all analytes left inside the tube (D). This dialysis step is kept going until the solution is completely clear from yellow pigments (as internal natural control) and concentrated to a desired final volume (E). The whole process has been named hydrostatic dialysis. Finally, it is possible to observe the diffusion of urine from the tube and precipitation at the bottom of the cylinder (E).