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) 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.
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 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 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 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 (<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.
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
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.
3. Nanoparticle Tracking Analysis (NTA)
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 size and had a mean concentration of 2.22 x 109 particles/mL (Figure 3B). To determine if NTA could assess nanocrystals in human urine, healthy adults were asked to consume a controlled oxalate diet followed by a high oxalate load. Twenty-four hour urine samples before and after the load were collected to assess urinary nanocrystal size and concentration. Pre-oxalate urine samples contained some urinary nanocrystals (1.65 x 108 ± 3.29 x 107 particles/mL) between 110-300 nm (Figure 4). In contrast, there was a significant increase (p<0.0001) in urinary nanocrystals present in post-oxalate samples (7.05 x 108 ± 1.08 x 108 particles/mL; 100-320 nm) (Figure 4). To confirm the reproducibility of the method, samples were measured three times and there was no significant variation in technical replicates (Figure 5).
Figure 1: Protocol for isolating and staining human urinary nanocrystals. Please click here to view a larger version of this figure.
Figure 2: Description of Nanoparticle Tracking Analysis (NTA). (A) Computer and instrument set up used for these studies. (B) Samples are injected into an inlet tubing using a syringe pump at a continuous rate prior to filling the optical surface. Samples are then observed by the objective lens and captured by the camera as samples flow through the platform before exiting through the outlet tubing to be discarded. Please click here to view a larger version of this figure.
Figure 3: NTA detects Fluo-4 AM labeled calcium oxalate (CaOx) and calcium phosphate (CaP) crystals. Representative graphs of (A) CaOx and (B) CaP crystals showing size distribution and concentration. Please click here to view a larger version of this figure.
Figure 4: NTA detects Fluo-4 AM labeled 24-hour human urinary nanocrystals. Representative graph of Fluo-4 AM labeled urinary nanocrystals in 24-hour pre-oxalate and post-oxalate samples from a healthy adult on a controlled oxalate diet. Please click here to view a larger version of this figure.
Figure 5: Technical replicates of human nanocrystals in 24-hour urine collections using NTA. Technical replicates of Fluo-4 AM labeled urinary nanocrystals in 24-hour (A) pre-oxalate and (B) post-oxalate samples from a healthy adult on a controlled oxalate diet. Please click here to view a larger version of this figure.
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 nanocrystals in the urine of humans who participated in a dietary feeding study which consisted of ingesting a high oxalate load. The amount of oxalate consumed was equivalent to real world consumption of oxalate (e.g., ½ spinach salad).
NTA is a well characterized high resolution tool that uses Brownian motion to measure particles in solution30. It has been used to assess biological nanoparticles in a variety of biological samples31,32,33. In addition, NTA can accurately predict the size as well as concentration of particles in any type of biological sample. This method does not require any labeling; however, labeling may be used to detect specific particles. Fluo-4 AM was used in this study to efficiently and specifically detect calcium containing nanocrystals in urine samples. Calcium fluorescent probes were initially used to measure free cytosolic calcium34. Fluo-4 is an analogue of Fluo-3 whose fluorescence increases >100-fold upon binding to free calcium35. In addition, Fluo-4 has been shown to assess calcium particles in the synovial fluid of patients with arthritis using flow cytometry36. Thus, we used Fluo-4 AM for these studies.
All samples were continuously injected into the platform for accurate detection. Determining the concentration and particle size depends on the flow rate, as a high flow rate (i.e., 50 µL/min) can affect accurate assessment of the concentration, as well as the particle size compared to a static setting and a lower flow rate (i.e. 20 µL/min)37. Thus, a steady slow flow rate provides accurate measurement of the number of particles present in samples. Other important parameters that might affect the particle count and size include the camera level, detection threshold, and focus38,39,40. A consistent particle measurement in samples (CV approx. 20%) was observed in the current study, which was consistent with findings from another study39. Lastly, the presence of nanocrystals in human urine has been confirmed using electron microscopy29. This research demonstrates NTA can successfully measure urinary nanocrystals from humans.
One advantage of this protocol is the use of Fluo-4 AM to evaluate calcium containing particles in solution. Another advantage is the minimal variability observed in detecting nanocrystals within samples. One limitation of NTA in this setting, is the inability to distinguish the morphology of nanocrystals. However, this method could be beneficial to detect crystalluria for predicting stone risk in individuals with a history of calcium containing kidney stones. This protocol cannot replace current methodologies but may provide new insight about urinary nanocrystals. The use of NTA to assess urinary calcium containing crystals is a novel approach that should highlight the importance of nanocrystalluria beyond standard microscopy and methods mentioned above. Additional investigations are warranted to explore the reliability of this method in the kidney stone population.
The authors have nothing to disclose.
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).
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. |