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Comparing Approaches to Normalize, Quantify, and Characterize Urinary Extracellular Vesicles.

Author(s): Blijdorp CJ, Tutakhel OAZ, Hartjes TA, van den Bosch TPP, van Heugten MH, Rigalli JP, Willemsen R, Musterd-Bhaggoe UM, Barros ER, Carles-Fontana R, Carvajal CA, Arntz OJ, van de Loo FAJ, Jenster G, Clahsen-van Groningen MC, Cuevas CA, Severs D, Fenton RA, van Royen ME, Hoenderop JGJ, Bindels RJM, Hoorn EJ

Publication: J Am Soc Nephrol, 2021, Vol. 32, Page 1210-1226

PubMed ID: 33782168 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

    This study investigated the relationship between creatinine concentration and particle counts in urine from healthy volunteers and patients with polycystic kidney disease and compared urine flow rate, particle counts, particle size, osmolality, osmole excretion rate, expression of exosomal markers, and the concentration and excretion rate of creatinine, urea and electrolytes in urine collected from healthy volunteers during water deprivation and in the hours following water loading (drinking 20 mL/kg in 30 min). The authors compared particle counts using three different methods [nanoparticle tracking analysis (NTA), EVQuant, and CD9 time-resolved fluorescence-immunoassay (CD9-TR-FIA)] and investigated the effects of adding 1% or 0.01% sodium dodecyl sulfate (SDS) to urine on particle and epitope detection. The authors also investigated the source of urinary particles by comparing expression levels of tubular segment markers in CD9⁺ and CD63⁺ particles. Urine was collected from 11 healthy men who were water deprived starting at 10 PM, voided urine at 7 AM (discarded), 10 am (water deprivation timepoint 1), and noon (water deprivation timepoint 2), after which the men received a water load of 20 mL/kg in 30 min and a standardized meal and urine was collected at 2, 3, 5 and 7 PM (water-loading timepoints 1-4). To control for collection timing, urine was collected from 3 healthy men who drank to thirst at the same times of day as the water deprivation/loading study. Additionally, random spot urine was collected from 8 healthy men, 7 healthy women, 10 men with polycystic kidney disease and 16 women with polycystic kidney disease. Within 2 h of collection, urine was centrifuged at 2000 g for 10 min at 4°C and the supernatant was aliquoted and stored with protease inhibitor cocktail at -80°C until analysis. Urine creatinine, electrolytes, and urea were quantified using a Cobas 8000 autoanalyzer and osmolality was measured using an osmometer. Particle size and quantity were analyzed using nanoparticle tracking analysis. Total and CD9, CD63 and Aquaporin-expressing EVs were also counted using the fluorescence-based EVQuant assay.  EVs were pelleted from urine by centrifugation at 17,000 g (duration not specified), dissolved in 200 mg/ml dithiothreitol in isolation buffer (10 mM triethanolamine, 250 mM sucrose, pH 7.6), centrifuged at 17,000 g (duration not specified), followed by 200,000 g for 2 hours. The EVs in the ultracentrifugation pellet were analyzed by NTA and EVQuant. The ultracentrifugation pellet was dissolved in phosphate buffered saline (PBS) and CD9 and CD63 expressing EVs were immunoprecipitated. Levels of ALIX, TSG101, CD63, CD81, and CD9 were quantified in the ultracentrifugation pellet by Western blot. The morphology of the ultracentrifugation pellet particles was assessed by transmission electron microscopy (TEM). The relative expression of NHE3 (proximal tubule marker), NKCC2 (thick ascending limb marker), parvalbumin (distal convoluted tubule marker), AQP2 (collecting duct marker), or WT1 (Glomerular marker) in CD9 and CD63 captured particles was evaluated by Western blot.

   Summary of Findings:

   Average urine creatinine concentration was correlated with the average particle count in the water loading experiment when analyzed by NTA or EVQuant and when data for each timepoint was averaged (R²=0.99, P<0.001, both) and when each individual was analyzed (R²=0.96, both).  Creatinine concentration and particle counts were also correlated in random spot urine specimens from healthy individuals and those with polycystic kidney disease by NTA (R²=0.91 and R²=0.87 for healthy women and men; R²=0.81 and R²=0.63 for women and men with polycystic kidney disease) and EVQuant (R²=0.95 and R²=0.47 for healthy women and men; R²=0.52 and R²=0.41 for women and men with polycystic kidney disease).

   Not surprisingly, urine flow rate increased and urine osmolality, creatinine concentration, and particle count decreased in the hours following water loading (drinking 20 mL/kg in 30 min) relative to during water deprivation. Levels of the exosomal markers ALIX, TSG101, CD63, CD81, and CD9 decreased and the amount of THP increased in the ultracentrifugation pellet following water loading relative to during water deprivation. While creatinine excretion rate (µmol/min) was unaffected by water loading, the osmole excretion rate increased following water loading and was due to an increased excretion rate of potassium (P=0.0005), phosphate (P<0.0001) and urea (P<0.0001). While water loading did not affect the particle excretion rate when analyzed by EVQuant or CD9–TR-FIA, NTA showed a 50% increase in the particle excretion rate (P<0.001) and a 11 nm increase in the median particle width (P<0.001) following water loading. TEM confirmed the increased median particle size that was observed following water loading (71 nm after versus 47 nm before, P<0.001).  When THP was added to urine, the number of particles detected by NTA increased and when urine osmolality was changed, the measured particle size and, consequently, the number of particles detected increased, indicating the effects of water loading on particle count and size are likely attributable to the increase in THP and osmole excretion rate.

   Particle counts were very strongly correlated between NTA and EVQuant (R²=0.95), but the particle number was 2.8-fold higher when quantified using EVQuant than NTA (P<0.001). Particle counts by CD9-TR-FIA were correlated with those by EVQuant (R²=0.79) and NTA (R²=0.79) and were lower when quantified by NTA than CD9-TR-FIA.  Overall, 32% of particles were CD9⁺ and 8% were CD63⁺, with 10% of CD9⁺ particles also CD63⁺ and 33% of CD63⁺ particles also CD9⁺. Particles immunoprecipitated with both CD9 and CD63 antibodies also expressed parvalbumin and NCC (distal convoluted tubule markers) and AQP2 (a marker for the collecting duct), but not the markers for the proximal tubule (NHE3) and thick ascending limb (NKCC2).

   When SDS was added to try and allow for intracellular epitope detection, 1% SDS decreased particle counts (P<0.01) and 0.01% SDS led to a non-significant decrease in particle counts and a slight shift toward larger particles relative to when no SDS was added. Further, the particles from 0.01% SDS-treated urine appeared similar to those from untreated urine. Treatment with 0.01% SDS increased detection using the intracellular AQP2 antibody by 3.5-fold and increased the percentage of AQP+ particles using EVQuant.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Frozen

   Diagnoses:

   • Other diagnoses
   • Normal

   Platform:

   Analyte	Technology Platform
   Morphology	Electron microscopy
   Morphology	Light scattering
   Electrolyte/Metal	Clinical chemistry/auto analyzer
   Protein	Western blot
   Cell count/volume	Light scattering
   Cell count/volume	Fluorescent microscopy
   Small molecule	Clinical chemistry/auto analyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Polycystic kidney disease Healthy
   Preaquisition	Patient gender	Female Male
   Storage	Short-term storage solution	No SDS 0.01% SDS 1% SDS
   Biospecimen Acquisition	Time of biospecimen collection	After 12 h water deprivation After 14 h Water deprivation 2 h after water loading 3 h after water loading 5 h after water loading 7 h after water loading
   Fluorescent microscopy Specific	Technology platform	TEM EVQuant CD9–TR-FIA

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