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,

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 Scien....

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

List of materials used in this article

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.