Urine samples were collected from 25 male DD1 patients with confirmed genetic diagnosis and from 10 healthy donors of the same age and sex from different hospitals in Spain for processing at the Hospital Universitari Vall d'Hebron (Barcelona, Spain) (Fig. 1A). The study was approved by the Vall d'Hebron Hospital Ethics Committee (PR(AG)149/2020/ 314/C/2020). The estimated glomerular filtration rate (eGFR) was calculated using the CKD-EPI formula.13

Figure 1.

Sample collection and uEV isolation. (A) Map of Spain with the geographical distribution of the centers that participated in this study. (B) Graphic representation of the protocol for extracting uEV from urine of patients or controls.

uEV: urinary exosome-like vesicles.

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Isolation of uEV was performed according to the protocol previously described by our group.14 This process is shown in the scheme shown in Fig. 1B.

The quantification and distribution of the uEV measurement was analyzed using the nanoparticle tracking analysis (NTA) technique (NanoSight NS300, Malvern Instruments, UK). This equipment uses light scattering and Brownian motion to obtain the dimensions and concentration of suspended particles.15,16 These parameters were recorded with a high sensitivity camera and the images obtained were analyzed with NTA v3.1 software (NanoSight Ltd., Malvern Instruments, UK) at the Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Universitat Autònoma de Barcelona (Spain).

Cryogenic transmission electron microscopy (Cryo-TEM) was performed at the Microscopy Center of the Universitat Autònoma de Barcelona. 10 μL of uEV dsiluted in 1x PBS were applied on Formvar-Carbon EM grids and frozen in liquid ethanol cooled to −179 °C. Samples were analyzed on a Jeol JEM 2011 transmission electron microscope at an acceleration voltage of 200 kV.

Suspended uEVs were incubated with 1:1 lysis buffer (Tris−HCl 100 mM pH 7.5, EDTA 2 mM, pH 8, NaCl 300 mM, SDS 0.2%, NP-40.2%, sodium deoxycholate 0.5%, PIC 1:200, NaF 1 mM, Na 3VO 1 mM) at 4 °C under constant rotation for 1 h. Then, samples were sonicated at maximum amplitude for 5 cycles of 5 s, and then centrifuged at 13,000×g for 15 min at 4 °C. Supernatants were collected and stored at −20 °C. The uEV lysates were processed and analyzed by western blot.

For RNA extraction from previously characterized uEV lysates we used the miRNeasy Mini kit (Qiagen). Quantification of RNA levels was performed using the BioAnalyzer 2100 in combination with the RNA 6000 Pico LabChip kit.

Results are presented as mean ± standard error of the mean (SEM). Student's t-test (2-tailed) was used for statistical analysis. A p value below 0.05 was considered to indicate statistically significant differences. Statistical analyses were performed with commercial software (GraphPad Prism, version 9 for MacOS, GraphPad Software, La Jolla California, USA).

DD1 is an ultra-rare disease with a low number of diagnosed patients. To obtain sufficient patients for this study, a national call was made through the Spanish Society of Nephrology (SEN) and the Spanish Society of Pediatric Nephrology (AENP). A total of 25 patients with confirmed mutations in the CLCN5 gene from 14 hospitals in Spain participated in this study, from whom samples and clinical data were collected (Fig. 1A). Ten age- and race-matched healthy controls were also included (all individuals studied were male). Patient demographic and clinical data confirmed that there were no significant differences in age between the two groups (p = 0.67) but showed that 44% of them (p = 0.012) had reduced renal function (GFRe <90 mL/min/1.73 m2) and 52% had nephrocalcinosis (p = 0.0052) (Table 1). There were no significant differences in the rest of the parameters between patients and controls.

Urine samples from patients and controls were processed to obtain uEVs as previously described (Fig. 1B).14 Exosome-enriched uEVs obtained from patients and controls were morphologically characterized using the cryo-TEM technique, which allows direct visualization of vesicles without the addition of heavy metals or fixatives, that could cause artifacts. Our results revealed single-, double- or multilayered vesicles as well as uEVs of different sizes without obvious impurities (Fig. 2A).

Figure 2.

Morphological characterization of uEVs in patients and controls. (A) Details of representative samples with different scales (500 nm, 200 nm and 100 nm from left to right) showing single (white arrow), double (continuous black arrow) or multilayered (dashed black arrow) vesicles. (B) Quantification of uEV concentration per mL of urine in controls (black circles) or patients (gray circles). Each point represents one subject. The mean ± SEM is also shown in the graph. (C) Quantification of uEV diameter of controls (black circles) and patients (gray circles). Each point represents the mean uEV diameter for a single subject. The graph also shows the mean ± SEM.

CD81: differentiation group 81; GRP78: glucose regulatory protein 78; HepG2: hepatocellular carcinoma cell line G2; uEV: urinary exosome-like vesicles; SN: supernatant.

** p < 0.01.

***p < 0.001.

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The size distribution and relative uEV concentration were evaluated by nanoparticle tracking analysis (NTA) in randomly selected samples (n = 12 patients, n = 5 controls).15,16 It was very interesting to note that the relative concentration of uEV/mL was significantly lower in patients than in controls (0.26 ± 0.04 × 1016 vs. 1.19 ± 0.27 × 1016 uEV/mL; p = 0.0004) (Fig. 2B). As seen in Fig. 2C, most of the detected vesicles were less than 200 nm in diameter, as expected for exosomes. Curiously, exosomes from patients were significantly larger than those from control subjects (mean diameter 187.8 ± 4.053 vs. 143.6 ± 2.994 nm, respectively; p < 0.0001) (Fig. 2C).

Finally, the expression of two exosome-associated markers (ALIX and CD81)17 was evaluated by western blotting to confirm the presence of urinary exosomes (Fig. 3A). Both markers were detected in all exosome samples (both patients and controls) and were absent in the supernatant of these same samples, which were used as a negative control to confirm the absence of contamination by cellular debris. In addition, we used an endoplasmic reticulum marker (GRP78) to demonstrate the absence of intracellular structures in the exosome suspensions.

Figure 3.

RNA and protein obtained from uEVs. (A) Alix and CD81 (exosome markers) levels were analyzed by western blot in patient and control samples. HepG2 cell line lysate was used as a positive control for the detection of Grp78, a reticulum marker. (B) RNA concentration (ng/μL) in samples from controls (black circles) and patients (gray circles). Each point represents a single subject. The graph also shows the mean ± SEM. (C) Representative electropherogram showing good integrity of RNA obtained from uEVs. Peaks corresponding to 18S or 28S ribosomal RNA are marked on the graph.

FU: arbitrary fluorescence units; nt: nucleotides.

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RNA extracted from the uEVs was quantified using the BioAnalyzer 2100. Total RNA quantification showed no differences between controls and patients (Fig. 3B) and similar electropherograms were obtained in all samples, as well as good RNA integrity in all cases. As exhibited in the example in Fig. 3C, a high peak corresponding to 250 nt RNA and two lower but larger peaks corresponding to 18S or 28S ribosomal RNA were observed (Fig. 3C).

The aim of this study was to identify and select Spanish patients with a confirmed genetic diagnosis of DD1, as well as sex and age matching healthy controls for the isolation and characterization of exosome-enriched uEVs. In addition, RNA has been extracted from the uEVs to further study the differential expression profile between patients and controls foridentification of diagnostic and prognostic biomarkers, and possible therapeutic targets against DD1. Indeed, urine sample analysis is irreplaceable as a non-invasive method for the diagnosis and monitoring of some diseases, particularly renal diseases. In contrast to tissue biopsy, an invasive and complicated procedure that allows only a partial sample of the organ to be obtained, exosome-enriched uEVs provide a complete representation of the entire urinary system and their study is attractive in the field of biomarker discovery. The potential use of uEVs as diagnostic and prognostic markers in clinical studies has been evaluated, as well as their contribution in pathophysiological processes using experimental models.18–20

Urine samples from patients from all over Spain were frozen, after an initial centrifugation, until processing. Although some authors have pointed out that direct freezing at −80 °C could better preserve exosomes,21 others have shown that exosomes and their charges are stable in urine under different storage conditions, by obtaining comparable results with freshly processed samples or after repeated freeze-thaw cycles.22 For uEV isolation, the differential centrifugation method was preferred to other techniques.23 No previous data have been published on uEV in DD1 patients and, therefore, the efficacy of exosome isolation per urine sample in these polyuric patients, who, in addition, have impaired endocytosis and endolysosomal pathways, both of which are involved in exosome formation, was unknown.

This technique has been widely used in our laboratory and has already proven effective in the isolation of exosomes to study another rare renal tubulopathy with polyuria.14

Quantification of uEVs by NTA revealed a substantial concentration, although highly variable concentrations between individual samples. This variation was independent of the glomerular filtrate or the degree of proteinuria, but other factors could play a role, such as the amount of Tamm Horsfall protein. Interestingly, patients were observed to have almost five times lower uEV/mL urine concentration than controls. This fact may be related to the polyuria observed in people with DD1.

However, we also observed significant differences in the size of the exosomes of the patients compared to those of the controls, as mentioned above. One possible hypothesis would be that some defects in the endolysosomal pathway, resulting from ClC-5 dysfunction, produced by an unknown mechanism the release of larger vesicles in DD1 patients than in controls. In fact, this change in size could also explain the reduction in the number of particles between patients and controls: fewer exosomes are released, but they are larger.

The tubular dysfunction that occurs in Dent's disease could have an impact on the production and release of exosomes, since their biogenesis begins in the endosomal system, where early endosomes mature into late endosomes or multivesicular bodies.24 The electrogenic antiporter (2Cl−/H+) ClC-5 is localized in early endosomes and has a very relevant role in vesicle trafficking and intracellular transport,4,5 which is altered when mutations occur in the CLCN5 gene, as occurs in DD1. Likewise, it has been pointed out that the fate of multivesicular bodies, either their degradation or exosome secretion, depends on cellular homeostasis, which is probably altered in patients with Dent's disease. Multivesicular bodies may be directed to lysosomes, where their contents are degraded, or transported to the plasma membrane for the release of exosomes.25 Little is known about the molecular and cellular mechanisms that regulate this balance and the role of ClC-5 in these processes remains to be determined.

In other diseases, such as neurodegenerative dementias, a reduction in the concentration of extracellular vesicles in plasma has also been seen, with an increase in their size.26 However, no mechanism explaining this change has yet been reported in the literature. On the other hand, Paulaitis et al. have characterized exosome size distributions based on the dynamic scale of multivesicular body growth in the exosome biogenesis pathway.27 These authors find statistically significant differences in the scaling exponents characterizing exosome size distributions in serum samples from cancer patients compared to those from healthy donors. The authors consider that such changes could reflect alterations in the membrane composition of exosomes and propose that the scaling exponent could be considered as a biophysical biomarker comparable to and complementary to biochemical biomarkers of exosomes, such as specific membrane proteins such as tetraspanins.