The authors thank all study participants and the UAB CCTS Bionutrition Core and UAB High Resolution Imaging Service Center for their contributions. This work was supported by NIH grants DK106284 and DK123542 (TM), and UL1TR003096 (National Center for Advancing Translational Sciences).

All experiments outlined in this work were approved by the University of Alabama at Birmingham (UAB) Institutional Review Board. Healthy adults (33.6 &#177; 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 pr...

<ol>
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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 samples27,28. This study shows a specific and sensitive method for detecting calcium containing nanocrysta...

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&#39;s risk of developing a kidney stone. Specifically, this occurs when crystals bind to Randall&#39;s plaque, nucleate, accumulate, and grow over time2,3,4. Crystalluria precedes kidney stone formation and assessment of crystalluria may have predictive value in kidney stone formers3,5. Specifically, crystalluria has been suggested to be useful to predict the risk of stone recurrence in patients with a history of calcium oxalate containing stones6,7.
Crystals have been reported to negatively impact renal epithelial and circulating immune cell function8,9,10,11,12,13. 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 formation15,16. Several studies have demonstrated that urinary crystals can vary in shape and size depending on the pH and temperature of the urine17,18,19. 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 microscopy21,22, electron microscopy23, particle counters3, urine filtration24, evaporation3,5 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 &#181;m in size. Crystals of this size may influence the growth of CaOx stones by attaching to Randall&#39;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 analyzer26,27. 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 (&#60;1 &#181;m 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Benchtop Centrifuge</td>
			<td>Jouan Centrifuge</td>
			<td>CR3-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Oxalate monohydrate</td>
			<td>Synthesized in the lab as previously described29.</td>
			<td> </td>
			<td>Store at RT; Stock 10 mM</td>
		</tr>
		<tr>
			<td>Calcium Phosphate crystals (hydroxyapatite nanopowder)</td>
			<td>Sigma</td>
			<td>677418</td>
			<td>Store at RT; Stock 10 mM</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fischer Scientific</td>
			<td>AC615095000</td>
			<td>Store at RT; Stock 100%</td>
		</tr>
		<tr>
			<td>Fluo-4 AM*</td>
			<td>AAT Bioquest, Inc.</td>
			<td>20550</td>
			<td>Store at Freezer (-20&#176;C); Stock 5 mM</td>
		</tr>
		<tr>
			<td>Gold Nanoparticles</td>
			<td>Sigma</td>
			<td>742031</td>
			<td>Store at 2-8&#176;C</td>
		</tr>
		<tr>
			<td>NanoSight Instrument</td>
			<td>Malvern Instruments, UK</td>
			<td>NS300</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>98-4730</td>
			<td></td>
		</tr>
		<tr>
			<td>Virkon Disinfectant</td>
			<td>LanXESS Energizing Company, Germany</td>
			<td>LSP</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">*Fluorescence dyes are light sensitive; stock and aliquots should be stored in the dark at -20&#176;C.</td>
		</tr>
	</tbody>
</table>

estimation of urinary nanocrystals in humans using calcium fluorophore labeling and nanoparticle tracking analysis

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.

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...

Watch this Scientific Journal Video about Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis at JoVE.com

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

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.

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) ...

This protocol can specifically detect calcium-containing nanocrystals in human urine. This technique could potentially be useful to predict early stages of kidney stone formation as well as other diseases associated with crystal urea. Demonstrating the procedure will be Dr.Parveen Kumar, a researcher IV in my laboratory.Have participants consume a low-oxalate diet for three days and fast overnight. Then collect a 24-hour urine sample from the participants before having them consume an oxalate load. Feed the participants with a smoothie containing fruits and vegetables having an oxalate load of approximately eight millimoles.Have participants collect their urine for 24 hours post-oxalate consumption and return it on the following day. Maintain all urine samples at room temperature prior to processing. Measure and record urine pH and volume of the urine samples.Mix thoroughly and add 50 milliliters of urine into a labeled sterile 50 milliliter conical tube. Centrifuge the sample at 1, 200 times G for 10 minutes at room temperature using a bench-top centrifuge. Discard the supernatant, then wash and resuspend the pellet again with five milliliters of 100%ethanol.Centrifuge the sample at 1, 200 times G for 10 minutes at room temperature using a bench-top centrifuge. Discard the supernatant and resuspend the pellet in one milliliter of 100%ethanol. Store the sample at minus 20 degrees Celsius for later processing or immediately proceed with staining the samples.Dilute 100 nanometer-sized gold nanoparticles in ultrapure water at a ratio of 1:1, 000 and use them for optimizing the settings on the instrument. Dilute the urine samples 20 times in water prior to staining with five millimolar Fluo-4 AM for 30 minutes in the dark and analyze the samples using NTA. Prepare calcium oxalate and calcium phosphate crystals as described in the text manuscript prior to NTA analysis.Turn on the computer and the instrument, then open the software and turn on the camera. Click the capture icon in the top left corner of the window to start the capture mode. Clean the platform by first pumping air into it using a one milliliter syringe until the platform appears clean.Gently add water to the apparatus two to three times and use another one milliliter syringe to remove any air bubbles. Once the platform is clean, add water and check for any contamination on the surface by viewing the camera, then add gold nanoparticles as a control to the sample loading pump injector to set up the instrument. 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, then reduce the camera level.Next, adjust the screen to optimize the image. Left click the mouse button on the video image. Hold the left mouse button and drag the image up and down to get the entire view.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 for initial setup to ensure the gold nanoparticles are detected. Once detected, reduce the speed to 50 microliters per minute to visualize gold nanoparticles.Adjust the camera level to visualize the particles. For unstained samples, adjust the screen gain at level five to achieve the camera focus and set the camera level at eight. Once the focus is set, record the sample.After optimization, clean the apparatus again with water before assessing other samples to ensure that the tubing is clean and particles are not present. To analyze stained samples, adjust the camera to the filter position containing the suitable fluorescent filter and load diluted and stained samples onto the sample loading pump injector, reducing the speed to 20 microliters per minute for analysis. Adjust the screen gain to five and camera level to 13.Save the data after each measurement. Calculate the average number of nanoparticles for all five readings for each individual sample and analyze the data using standard deviation or standard error of the mean. Use T-tests for paired analysis.Fluo-4 AM was able to bind to both calcium oxalate crystals, which were between 50 and 270 nanometers in size and had a mean concentration of 1, 260 million particles per milliliter, and calcium phosphate crystals, which were between 30 and 225 nanometers and had a mean concentration of 2, 220 million particles per milliliter. 24-hour urine samples collected before the oxalate load contained some urinary nanocrystals between 110 to 300 nanometers in size. In contrast, there was a significant increase in urinary nanocrystals present in post-oxalate samples.To confirm the reproducibility of the method, samples were measured three times and there was no significant variation in technical replicates. When attempting this protocol, there may be some difficulty with adding samples without bubbles to the machine. To prevent this, add air and water to the machine, and then slowly add samples.Also, make sure to continually monitor your samples and that there are no bubbles present. This new method will be tested in individuals with calcium oxalate kidney stone disease to monitor disease progression and/or to predict kidney stone risk.

estimation-urinary-nanocrystals-humans-using-calcium-fluorophore

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JoVE Journal

Medicine

3.5K vistas.  University of Alabama at Birmingham. Este protocolo puede detectar específicamente nanocristales que contienen calcio en la orina humana. Esta técnica podría potencialmente ser útil para predecir los primeros tiempos de la formación de la piedra de riñón así como otras enfermedades asociadas a urea cristalina. Demostrando el procedimiento estará el Dr.Parveen Kumar, un investigador IV en mi laboratorio.Haga que los participantes consuman una dieta baja en oxalato durante tres días y ayunen durante la noche. Lueg...