Male Sprague Dawley (SD), Zucker lean (ZL) and Zucker diabetic fatty (ZDF) rats were purchased from Charles River Laboratories, Inc. Prior to the experimental procedures, animals were acclimatized for at least 5 days and housed under a 12-h light/dark cycle with ad libitum access to water and standard chow, CRF-1 (Oriental Yeast Co., Ltd.). For the puromycin aminonucleoside (PAN)-induced glomerular nephritis model, 73 6-week-old male SD rats received a tail vein injection of 100 mg/kg/5 ml PAN (Sigma-Aldrich; Merck KGaA) in saline buffer. In addition, 24 saline buffer-injected rats were used as the control. Prednisolone was administered as a single oral dose of 10 mg/kg/10 ml prednisolone (Shionogi & Co., Ltd.) 1 h prior to PAN injection using distilled water as the vehicle control. These animals were kept for 1 week and then were euthanized. For the type 2 diabetes model, 12-week-old male ZL (n=6) and ZDF (n=12) rats were used at the beginning of the experiment. These animals were kept for 20 weeks and then were euthanized. All animals were observed at least once daily for monitoring of health. No unforeseen deaths of animals occurred in these studies.

Total urine samples were collected over 24 h from each animal in the metabolic cages at each time-point. Urine samples were used immediately or stored at -20˚C until required. Urinary protein, albumin and creatinine levels were measured using a Rat Urinary Protein assay kit (Chondrex, Inc.), a LBIS™ Rat Albumin ELISA kit (FUJIFILM Wako Pure Chemical Corporation) and a LabAssay™ Creatinine kit (FUJIFILM Wako Pure Chemical Corporation) respectively, according to the manufacturer's protocol.

Blood samples were collected from the tail vein at each time-point. Blood samples were diluted with distilled water (1:10) and blood glucose was measured using a Glucose Cii Test Wako kit (FUJIFILM Wako Pure Chemical Corporation) according to the manufacturer's protocol.

Urinary NVs were isolated from urine samples using differential centrifugation at 3,000 x g for 10 min at 4˚C. Then, supernatants were centrifuged at 100,000 x g for 1 h at 4˚C and NVs were retrieved from the pellets after gently discarding the supernatants. For nanoparticle tracking analysis (NTA), pellets were suspended in PBS. NTA was performed using the Nanosight LM20 instrument (Nanosight Ltd.) to analyze the distribution of vesicle size and the concentration of particles, as previously reported (10).

For the NVs isolated from the urine of PAN nephritis model rats, total RNA was purified using the RNeasy micro kit (QIAGEN, Inc.) according to the manufacturer's protocol. Briefly, isolated NVs were lysed using lysis buffer containing 1% β-mercaptoethanol, and total RNA was purified using MinElute spin columns with on-column DNase digestion (Qiagen, Inc.). RNA was quantified using a NanoDrop 1000 (Thermo Fisher Scientific, Inc.) and cDNA was synthesized using a SuperScript™ VILO™ cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). Reverse transcription-quantitative (RT-q)PCR was performed using the Biomark HD system (Fluidigm Corporation) with specific TaqMan Gene Expression assays for hypoxanthine phosphoribosyltransferase (Hprt1; Rn01527840_m1), desmin (Rn00574732_m1), aquaporin 1 (Aqp1; Rn00562834_m1), nephrin (Rn00674268_m1), podocin (Rn00709834_m1), and regulator of calcineurin 1 (Rcan1; Rn00596606_m1) (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. For analysis of NVs from ZDF and ZL rats, mRNA was extracted from the isolated NVs using oligo(dT)-immobilized microplates (Hitachi Chemical Diagnostics, Inc.) and quantified by RT-qPCR as previously described (11). All mRNA levels were normalized to the level of Hprt1.

Rats were anesthetized using 5% sevoflurane via an inhalation anesthetic system, and the kidneys were immediately excised following euthanasia by exsanguination. After removal of kidney capsules, renal cortexes were minced into very fine fragments. Glomeruli were collected using standard sieving methods as previously described (12). Total RNA was isolated from glomeruli was purified using a RNeasy micro kit, and RT-qPCR was performed using the Biomark HD system for specific TaqMan Gene Expression assays as described above.

Kidneys were collected from euthanized animals. These samples were immediately fixed in 10% neutralized buffered formalin for 1 week at room temperature. Fixed tissues were then embedded in paraffin for immunohistochemical analysis. Immunostaining of desmin was performed as described previously (13). Briefly, paraffin sections were deparaffinized and incubated overnight at 4˚C with an anti-desmin mouse monoclonal antibody (1:200; cat. no. M0760; Dako; Agilent Technologies, Inc.), and subsequently incubated for 30 min at room temperature with horseradish peroxidase-conjugated anti-mouse IgG goat polyclonal antibody (Nichirei Biosciences Inc.).

Data are presented as mean ± standard error of the mean unless otherwise stated. A Student's t-test was used for pairwise comparisons. When comparing >2 groups, statistical differences were evaluated using a one-way ANOVA followed by a Dunnett's multiple comparison test. Repeated measure-based parameters were evaluated using a two-way ANOVA followed by Bonferroni's correction. P<0.05 was considered to indicate a statistically significant difference. For correlation analysis, Pearson correlation coefficients were calculated. All statistical analyses were performed using GraphPad Prism version 7.04 (GraphPad Software, Inc.).

Notable proteinuria developed 3 days after the induction of PAN nephritis in the rat model. Urinary protein excretion significantly increased at days 3 and 7 (P<0.05 and P<0.01, respectively) without a significant change in urine volume (Fig. 1A). The size distribution and concentration of NVs in urine were assessed using NTA to confirm the presence of NVs and compare the vesicle profiles between normal and diseased animals. As previously reported (14,15), most of the obtained vesicles were <200 nm in diameter. The size distribution of NVs was not altered (Fig. 1B), but the concentration of urinary NVs was slightly increased in the PAN nephritis model, even though the difference was not significant (Fig. 1C).

Whether urinary NVs could indicate site-specific damages in glomeruli were next determined. In fact, PAN is an antibiotic well known to cause glomerular specific damage (16). Therefore, the mRNA levels of desmin, a sensitive biomarker for glomerular injury in rodents (17,18), were evaluated in urinary NVs. The relative content of desmin mRNA in urinary NVs of the PAN nephritis model increased 5.8-fold (P=0.44) on day 3 and 17.3-fold (P<0.01) on day 7 (Fig. 2A). To confirm whether this change reflected the status of kidney tissue, mRNA and protein levels of desmin in the glomeruli were assessed by RT-qPCR and immunohistochemistry, respectively. The mRNA levels of desmin in glomeruli showed a similar increasing trend to that of urinary NVs, particularly on day 7 (P<0.01; Fig. 3A), whereas increased desmin immunoreactivity in glomeruli was observed in the PAN nephritis model (Fig. 3B), corroborating the observations from urinary NVs. Meanwhile, the mRNA levels of Aqp1, a proximal tubular marker, in urinary NVs was not altered in the PAN nephritis model (Fig. 2B).

To evaluate the potential of mRNAs in urinary NVs as pharmacological biomarkers, the effect of prednisolone on the PAN nephritis model was assessed. The induced urinary protein excretion of the PAN nephritis model was significantly mitigated by treatment with prednisolone (P<0.01; Fig. 4A), confirming previously reported observations (19). Fig. 4B shows the effect of prednisolone on the mRNA levels of urinary NVs. The expression of two podocyte injury markers, the mRNA levels of desmin and the podocin to nephrin ratio (PNR), the latter being a potential podocyte loss prediction marker reported by Fukuda et al (20), significantly increased in the PAN nephritis model (P<0.01), whereas a decreasing tendency and a significant decrease in the prednisolone-treated group was observed (P=0.10 and P<0.01, respectively). The mRNA levels of Rcan1, which is upregulated during active calcineurin signaling and is therefore considered a target of immunosuppressants for nephritis treatment (21), also increased in urinary NVs of the PAN nephritis model (P<0.01), and it was reversed by prednisolone treatment (P<0.01). Additionally, the correlations between mRNA levels and disease severity were assessed (Fig. 5). A positive correlation was observed between PNR and urinary protein excretion, as well as between Rcan1 levels and urinary protein excretion (r²=0.75 and 0.64, respectively; P<0.01 in both cases). A similar but weaker correlation was observed between desmin mRNA levels and urinary protein excretion (r²=0.12; P=0.07).

The relationship between kidney functionality and PNR in another type of kidney disease model was assessed. ZDF rats, a type 2 diabetes model, showed a significant increase in blood glucose on weeks 0 and 20 (P<0.01; Fig. 6A). A progressive increase in urinary albumin excretion was also observed in ZDF rats (P<0.01; Fig. 6B). In addition, PNR in ZDF rats on week 20 increased compared to that in the ZL rats (P<0.01; Fig. 6C). Furthermore, PNR was positively correlated with urinary albumin excretion, similar to the PAN nephritis model (r²=0.66; P<0.01; Fig. 7).