Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis

Summary

The objective of this study was to determine whether nanoparticle tracking analysis (NTA) could detect and quantify urinary calcium containing nanocrystals from healthy adults. The findings from the current study suggest NTA could be a potential tool to estimate urinary nanocrystals during kidney stone disease.

Abstract

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) crystals. Crystalluria occurs when urine becomes supersaturated with minerals (e.g., calcium, oxalate, phosphate) and precedes kidney stone formation. Standard methods to assess crystalluria in stone formers include microscopy, filtration, and centrifugation. However, these methods primarily detect microcrystals and not nanocrystals. Nanocrystals have been suggested to be more harmful to kidney epithelial cells than microcrystals in vitro. Here, we describe the ability of Nanoparticle Tracking analysis (NTA) to detect human urinary nanocrystals. Healthy adults were fed a controlled oxalate diet prior to drinking an oxalate load to stimulate urinary nanocrystals. Urine was collected for 24 hours before and after the oxalate load. Samples were processed and washed with ethanol to purify samples. Urinary nanocrystals were stained with the calcium binding fluorophore, Fluo-4 AM. After staining, the size and count of nanocrystals were determined using NTA. The findings from this study show NTA can efficiently detect nanocrystalluria in healthy adults. These findings suggest NTA could be a valuable early detection method of nanocrystalluria in patients with kidney stone disease.

Introduction

Urinary crystals form when urine becomes supersaturated with minerals. This can occur in healthy individuals but is more common in individuals with kidney stones¹. The presence and accumulation of urinary crystals can increase one's risk of developing a kidney stone. Specifically, this occurs when crystals bind to Randall's plaque, nucleate, accumulate, and grow over time^2,3,4. Crystalluria precedes kidney stone formation and assessment of crystalluria may have predictive value in kidney stone formers^3,5. Specifically, crystalluria has been suggested to be useful to predict the risk of stone recurrence in patients with a history of calcium oxalate containing stones^6,7.

Crystals have been reported to negatively impact renal epithelial and circulating immune cell function^{8,9,10,11,12,13}. It has been previously reported that circulating monocytes from calcium oxalate (CaOx) kidney stone formers have suppressed cellular bioenergetics compared to healthy individuals¹⁴. In addition, CaOx crystals reduce cellular bioenergetics and disrupt redox homeostasis in monocytes⁸. Consumption of meals rich in oxalate may cause crystalluria which could lead to renal tubule damage and alter the production and function of urinary macromolecules that are protective against kidney stone formation^15,16. Several studies have demonstrated that urinary crystals can vary in shape and size depending on the pH and temperature of the urine^17,18,19. Further, urinary proteins have been shown to modulate crystal behavior²⁰. Daudon et al.¹⁹, proposed that crystalluria analysis could be helpful in the management of patients with kidney stone disease and in assessing their response to therapies. A few conventional methods currently available to evaluate the presence of crystals include polarized microscopy^21,22, electron microscopy²³, particle counters³, urine filtration²⁴, evaporation^3,5 or centrifugation²¹. These studies have provided valuable insight to the kidney stone field regarding crystalluria. However, a limitation of these methods has been the inability to visualize and quantify crystals less than 1 µm in size. Crystals of this size may influence the growth of CaOx stones by attaching to Randall's plaque.

Nanocrystals have been shown to cause extensive injury to renal cells compared to larger microcrystals²⁵. The presence of nanocrystals has been reported in urine using a nanoparticle analyzer^26,27. Recent studies have used fluorescently labeled bisphosphate probes (alendronate-fluorescein/alendronate-Cy5) to examine nanocrystals using nanoscale flow cytometry²⁸. The limitation of this dye is that it is not specific and will bind to almost all types of stones except cysteine. Thus, accurately assessing the presence of nanocrystals in individuals may be an effective tool to diagnose crystalluria and/or predict stone risk. The purpose of this study was to detect and quantify calcium containing nanocrystals (<1 µm in size) using nanoparticle tracking analysis (NTA). To achieve this, NTA technology was used in combination with a calcium binding fluorophore, Fluo-4 AM to detect and quantify calcium containing nanocrystals in the urine of healthy adults.

Protocol

All experiments outlined in this work were approved by the University of Alabama at Birmingham (UAB) Institutional Review Board. Healthy adults (33.6 ± 3.3 years old; n=10) were enrolled in the study if they had a normal blood comprehensive metabolic panel, non-tobacco users, non-pregnant, a BMI between 20-30 kg/m², and free of chronic medical conditions or acute illnesses. Healthy participants signed a written informed consent form prior to the start of the study.

1. Clinical protocol and urine collection

1. Have participants consume a low oxalate diet prepared by the UAB Center for Clinical and Translational Sciences Bionutrition Core for 3 days and fast overnight before collecting their urine (24-hour sample).
2. The following day, have participants return their 24-hour urine sample (pre-oxalate) before consuming an oxalate load (smoothie containing fruits and vegetables, ~8 mM oxalate). Have participants subsequently collect their urine for 24 hours (post-oxalate sample) and return their urine the following day.
3. Maintain all urine samples at room temperature (RT) prior to processing as described below and shown in Figure 1.

2. Urine Processing

NOTE: All materials and equipment used are listed in Table of Materials.
CAUTION: Wear personal protective equipment at all times while handling clinical samples and reagents. Specifically, gloves, face and eye shields, respiratory protection, and protective clothing.

1. Measure and record urine pH and volume. Mix thoroughly prior to adding 50 mL of urine into a labeled sterile 50 mL conical tube.
2. Centrifuge sample at 1200 x g for 10 min at RT using a benchtop centrifuge.
   NOTE: Keep the sample at RT to prevent further crystal formation as cooler temperatures can promote crystallization.
3. Discard the supernatant and wash and resuspend the pellet again with 5 mL of 100% ethanol. Centrifuge the sample at 1200 x g for 10 minutes at RT using a benchtop centrifuge.
4. Discard the supernatant and resuspend the pellet in 1 mL of 100% ethanol. Store the sample at -20 °C for later processing OR stain the sample as described below.
   ​NOTE: There is no significant difference in data points (i.e., particle size/concentration) between stored or freshly stained samples.

3. Nanoparticle Tracking Analysis (NTA)

1. Sample Preparation
   1. Gold nanoparticles: Use gold nanoparticles to optimize settings on the instrument. Dilute 100 nm sized gold nanoparticles 1:1000 in ultrapure water.
   2. Human Urine: Dilute urine samples 20 times in water prior to staining with 5 mM Fluo-4 AM (a calcium fluorescence dye) for 30 min in the dark. Analyze the samples using NTA.
   3. Prepare Calcium Oxalate (CaOx) crystals as previously described²⁹. Dilute 10 mM stock solution (14.6 mg in 10 mL of water) to 50 µM in water and stain the diluted samples using 5 mM Fluo-4 AM for 30 min in dark prior to analysis.
   4. Calcium Phosphate (CaP) crystals: Dilute 10 mM stock solution (50.4 mg in 10 mL of water) to 50 µM in water and stain the diluted samples using 5 mM Fluo-4 AM for 30 min in dark prior to NTA analysis.
2. Instrument Set-up, Camera Settings, and Data Collection
   NOTE: The computer and instrument setup used for this method are shown in Figure 2.
   1. Turn on the computer and then the instrument. Open the software and turn on the camera.
   2. Once the software window is open, click the capture icon in the top left corner on the window to start the capture mode. The camera initialization takes a few seconds.
   3. Clean the platform by first pumping air into it using a 1 mL syringe until the platform appears clean. Gently add water to the apparatus 2-3 times using another 1 mL syringe to remove any air bubbles.
      ​NOTE: Look for any air bubbles in the platform as well as in the tubing. It is important to not have bubbles throughout the apparatus prior to and while running samples. If bubbles are present, clean the platform again with air and water.
   4. Once the platform is clean, add water to check for any contamination on the surface by viewing the camera. Next, add gold nanoparticles as a control to the sample loading pump injector to set up the instrument.
   5. Adjust the camera level on the screen or on the knob to the right side of the instrument until the image starts to display colored pixels and then reduce the camera level.
   6. Then adjust the screen to optimize the image. Left-click the mouse button on the video image. Hold the left mouse button and drag image up and down to get the entire view.
      ​NOTE: The normal camera lens and filter is used to assess gold nanoparticles and unstained samples.
   7. Set up the infusion speed and focus the camera so that the gold nanoparticles are visible on the camera screen. Set the infusion speed to high (i.e., 500 µL/min) for initial set up to ensure the gold nanoparticles are detected. Once detected, reduce the speed to 50 µL/min.
   8. Adjust the camera level to visualize the particles. For unstained samples, adjust the screen gain at level 5 to achieve the camera focus, and set the camera level at 8. Once the focus is set, record the sample (i.e., 1 measurement for 60 seconds only).
      ​NOTE: The focus and continuous flow speed are important for obtaining clear and sharp images of the particles for counting.
   9. After optimization, clean the apparatus again with water before assessing samples. View the camera to ensure that the tubing is clean and particles are not present.
      ​NOTE: Wash the chamber between each sample until no particles are detected by the camera.
   10. To analyze stained samples, adjust the camera to the filter position containing the suitable fluorescent filter. Load diluted and stained samples onto the sample loading pump injector and reduce the speed to 20 µL/min for analysis of the sample.
   11. Next adjust the screen gain and camera level as these are important parameters. For stained (fluorescent) samples, set the screen gain to 5 and the camera level at level 13.
       ​NOTE: These parameters will vary based on the sample type and every sample will need to be optimized to gain focus.
   12. Use standard measurement to measure the samples for 5 captures per sample where one capture-duration is for 60 seconds.
   13. Save and store data after each measurement. The software will save image and video files for each measurement. The software provides output data (e.g., crystal size: 10 nM - 1000 nM and concentration) in both excel and pdf formats.
   14. Calculate the average number of nanoparticles for all 5 readings for each individual sample. Analyze the data using standard deviation or standard error of the mean and use t-tests for paired analysis.

Results

The findings from this study show NTA can efficiently detect the mean size and concentration of calcium containing urinary nanocrystals in human urine. This was achieved by using the fluorophore, Fluo-4 AM, and nanoparticle tracking analysis. Fluo-4 AM was able to bind to both CaOx and CaP crystals. As shown in Figure 3A, CaOx crystals were determined to be between 50-270 nm in size and have a mean concentration of 1.26 x 10⁹ particles/mL. CaP crystals were between 30-225 nm in si...

Discussion

NTA has been used in the present study to assess nanocrystals in human urine using a calcium binding probe, Fluo-4 AM. There is no standard method available to detect nanocrystals in the urine. Some research groups have detected nanocrystals in the urine and relied on the use of extensive protocols or methods that are limited in their ability to quantify the samples^27,28. This study shows a specific and sensitive method for detecting calcium containing nanocrysta...

Materials

Name	Company	Catalog Number	Comments
Benchtop Centrifuge	Jouan Centrifuge	CR3-12
Calcium Oxalate monohydrate	Synthesized in the lab as previously described29.		Store at RT; Stock 10 mM
Calcium Phosphate crystals (hydroxyapatite nanopowder)	Sigma	677418	Store at RT; Stock 10 mM
Ethanol	Fischer Scientific	AC615095000	Store at RT; Stock 100%
Fluo-4 AM*	AAT Bioquest, Inc.	20550	Store at Freezer (-20°C); Stock 5 mM
Gold Nanoparticles	Sigma	742031	Store at 2-8°C
NanoSight Instrument	Malvern Instruments, UK	NS300
Syringe pump	Harvard Apparatus	98-4730
Virkon Disinfectant	LanXESS Energizing Company, Germany	LSP
*Fluorescence dyes are light sensitive; stock and aliquots should be stored in the dark at -20°C.