Nanoparticle Tracking Analysis for the Enumeration and Characterization of Mineralo-Organic Nanoparticles in Feline Urine

Abstract

Urinary stone disease, particularly calcium oxalate, is common in both humans and cats. Calcifying nanoparticles (CNP) are spherical nanocrystallite material, and are composed of proteins (fetuin, albumin) and inorganic minerals. CNP are suggested to play a role in a wide array of pathologic mineralization syndromes including urolithiasis. We documented the development of a clinically relevant protocol to assess urinary CNP in 9 healthy cats consuming the same diet in a controlled environment using Nanoparticle Tracking Analysis (NTA®). NTA® is a novel method that allows for characterization of the CNP in an efficient, accurate method that can differentiate these particles from other urinary submicron particulates. The predominant nanoscale particles in feline urine are characteristic of CNP in terms of their size, their ability to spontaneously form under suitable conditions, and the presence of an outer layer that is rich in calcium and capable of binding to hydroxyapatite binders such as alendronate and osteopontin. The expansion of this particle population can be suppressed by the addition of citrate to urine samples. Further, compounds targeting exosomal surfaces do not label these particulates. As CNP have been associated with a number of significant urologic maladies, the method described herein may prove to be a useful adjunct in evaluating lithogenesis risk in mammals.

Fig 1. Representative histogram showing size distribution and cumulative percentage (left) of submicron particulate matter in urine from healthy cats.

A representative frame from the captured video analyzed by NTA is shown as well (right).

Fig 2. Fluo-4-AM positive particulate matter from a healthy cat.

A representative histogram showing averaged sizing and relative abundance data (with SE in red) and individual analyses from the triplicate assessment (right) cumulative percentage (left) of submicron particulate matter in healthy feline urine.

Fig 3. Representative histogram showing size distribution and cumulative percentage (left) of submicron particulate matter from urine obtained from a healthy cat.

After a 4 hr incubation at 37 deg C. A representative frame from the captured video analyzed by NTA is shown as well (right).

Fig 4

A: Representative histogram showing size distribution and cumulative percentage (left) of submicron particulate matter in healthy feline urine after labeling with DyLight 488 conjugated alendronate. A representative frame from the captured video analyzed by NTA is shown as well (right). Note the strongly preferential binding to primary CNP that lack an outer layer of protein or mucus. B: Representative histogram showing averaged sizing and relative abundance data (left; SE in red) and individual analyses from the assessment (right) of this representative sample of healthy feline urine after labeling with DyLight 488 conjugated osteopontin. Note the strongly preferential binding to primary naturally-occurring CNP that lack an outer layer of protein or mucus.

Fig 5. DyLight488-conjugated alendronate binding of CNP generated in vitro.

A representative histogram showing averaged sizing and relative abundance data (left; SE in red) and individual analyses from the assessment (right) of this representative sample in triplicate. Note that CNP generated in vitro do not acquire the protein/mucus coating known to occur in vivo and the larger forms retain the ability to bind to alendronate.

Fig 6. Transmission electron microscopy (TEM) images of calcifying nanoparticles (CNP).

(A) CNP synthesized de novo in the laboratory using supersatured solutions of calcium and phosphorus and a solution of bovine fetuin A (see detailed description in the methodology section). (B) CNP identified in the urine of healthy, colony cats. In each case 5 microliters of samples were applied to a coated copper mesh and excess fluid wicked away. After complete air-drying, each prepared mesh was evaluated with electron microscopy at 80KV.