Unveiling spontaneous renal tubule-like structures from human adult renal progenitor cell spheroids derived from urine

Abstract

The rapidly developing field of renal spheroids and organoids has emerged as a valuable tool for modeling nephrotoxicity, kidney disorders, and kidney development. However, existing studies have relied on intricate and sophisticated differentiation protocols to generate organoids and tubuloids, necessitating the external administration of multiple growth factors within precise timeframes. In our study, we demonstrated that human adult renal progenitor cells (ARPCs) isolated from the urine of both healthy subjects and patients can form spheroids that naturally generated very long tubule-like structures. Importantly, the generation of these tubule-like structures is driven solely by ARPCs, without the need for the external use of chemokines or growth factors to artificially induce this process. These tubule-like structures exhibit the expression of structural and functional renal tubule markers and bear, in some cases, striking structural similarities to various nephron regions, including the distal convoluted tubule, the loop of Henle, and proximal convoluted tubules. Furthermore, ARPC spheroids express markers typical of pluripotent cells, such as stage-specific embryonic antigen 4 (SSEA4), secrete elevated levels of renin, and exhibit angiogenic properties. Notably, ARPCs isolated from the urine of patients with IgA nephropathy form spheroids capable of recapitulating the characteristic IgA1 deposition observed in this disease. These findings represent significant advancements in the field, opening up new avenues for regenerative medicine in the study of kidney development, mechanisms underlying renal disorders, and the development of regenerative therapies for kidney-related ailments.

Keywords: IgA nephropathy; regenerative medicine; renal progenitors; spheroids; tubule-like structures.

Graphical Abstract

Figure 1.

Generation of renal spheroids and tubule-like structures from ARPCs. (A) ARPCs formed spheroids after 2 days in 3D culture conditions. Each circle in the image represents the microcavity of the 3D plates containing a spheroid. (B) Primary and (C) immortalized RPTECs cannot form spheroids in 3D plates, and they appear to break up and disperse in the microcavities. Both tARPC (D) and gARPC (E) were able to form spheroids. (F) Spheroids derived from the mixed-cell population of uARPCs/CD133^- cells cultured in normal plates for 30-45 days without changing their morphology. (G) Spheroids from gARPCs formed circular protrusions similar to those of podocyte cell bodies connected by primary processes. (H-I) Spheroids derived from the mixed-cell population of uARPCs/CD133^- cells spontaneously generated tubular-like structures starting from one or both poles. (J-M) Tubule-like structures spontaneously generated by the uARPCs/CD133^- mixed cell population showing many similarities with the nephron unit of the kidney. (J) Image of a tubule-like structure acquired with a stereomicroscope. (M) Images acquired by a phase contrast microscope of the same tubule-like structure observed in (J) via a stereomicroscope. The scale bars represent 100 μm.

Figure 2.

Expression of stem cell markers in ARPC spheroids. (A-C) Whole-mount double-label immunofluorescence shows that uARPC spheroids expressed high levels of the functional and constitutional marker ARPC CD133. The spheroids were also stained with beta-actin to determine their structure and integrity. Spheroids expressed low levels of the transcription factors NanoG (B) and Oct3/4 (C) and elevated levels of the SOX2 (D), GATA-3 transcription factors (E), and of the embryonic stem cell marker SSEA4 (F). Spheroids coexpressing CD133/SSEA4 can spontaneously form the typical groove of organoids (F, arrows). The scale bars represent 25 μm.

Figure 3.

Cytofluorometric analysis showing the expression of stem cell markers in the uARPCs/CD133⁻ mixed cell population and in cells derived from spheroids. (A-C) Compared with monolayer ARPCs, spheroids expressed higher levels of SOX2, OCT3/4, and GATA3. (D) NanoG was not expressed or was expressed at low levels in the uARPCs/CD133^- mixed cell population or in spheroids. (E) Both the uARPC/CD133^- mixed cell population in the monolayer and cells from spheroids expressed high levels of the SSEA-4 marker. For each analyzed marker, the percentage data from triplicate samples are shown in histograms as mean ± SEM. *P <.05; ***P <.0005, ****P <.0001.

Figure 4.

Expression of renal tubular markers in tubular-like structures. (A) Whole-mount immunofluorescence showing the expression of CD249 in tubular-like structures. (B) Whole-mount immunofluorescence showing that the tubule-like structures generated from spheroids were positive for CD13 (aminopontinidase N). (C) Whole-mount immunofluorescence showing the expression of ZO-1 in tubular-like structures. (D) Whole-mount immunofluorescence showing the expression of uromodulin in tubule-like structures generated from spheroids. (E) Whole-mount immunofluorescence showing the expression of lotus tetragonolobus lectin in some tubular-like segments. The scale bars represent 15 μm in A, and 25 μm in B-E. Cell nuclei were visualized using DAPI.

Figure 5.

Expression of renal tubule markers in tubular-like structures generated by ARPC spheroids. (A) Whole-mount double-label immunofluorescence showing the coexpression of CD13 with ZO-1. (B) Whole-mount double-label immunofluorescence showing the coexpression of uromodulin with lotus tetragonolobus lectin. (C) Optical microscope image of spheroid sections. (D) Hematoxylin–eosin staining of spheroid sections. (E) Immunofluorescence image showing uromodulin in spheroids and sections with a tubule-like structure. (F) Immunofluorescence showing the expression of CD249 in spheroids and tubule-like structure sections. Nuclei were counterstained with DAPI. The scale bars represent 30 μm in A, 20 μm in B and E-F, and 100 μm in C and D. (G) ELISA showing that after 10 days of culture, ARPC-derived spheroids had elevated levels of renin compared with those in RPTECs and compared with those in ARPC monolayers. *P <.05; **P <.005.

Figure 6.

Angiogenic properties of ARPC and ARPC-derived spheroids. Microscopic (A-C) and macroscopic (D-F) images of CAMs transplanted with a silicon ring alone (A, D) or with a silicon ring containing inside RPTECs (B, E) and ARPCs (C, F). The A-C sections are H&E stained; the arrowheads indicate some vessels and the arrow in C a group of ARPCs cells near the vessel. In G, histograms showing the postimage analysis of vessel total area, vessel total length, and number of vessel branching points, expressed as percent variation in RPTECs and ARPCs compared with the control (silicon ring alone). (H) Histograms showing the postimage analysis of vessel total area, vessel total length, and number of vessel branching points, expressed as percent variation in ARPCs and ARPC-derived spheroids compared with controls. (I-K) Microscopy images of CAMs transplanted with a silicon ring alone (control, I) or a silicon ring containing inside ARPCs (J) or ARPC-derived spheroids (K). The arrowheads indicate some CAM vessels and the arrows in J and K indicate a group of ARPCs and ARPC-derived spheroids, respectively, near the vessels. (L-N) Fluorescence microscopy images of a CAM vessel (arrowhead) in a control (L), in PKH26-labeled ARPC-transplanted CAM vessels (arrowhead, M) and in CAM vessels (arrowhead) transplanted with spheroids generated from PKH26-labeled ARPCs. The arrows in (M) indicate the ARPCs around the vessels. The arrows in (N) indicate the spheroids near the vessel. Scale bar: A-K 100 μm; L-N 25 μm.

Figure 7.

Immunofluorescence staining of IgA1 deposits in ARPC-derived spheroids from IgAN patients and healthy subjects. ARPC-derived IgAN spheroids exhibited IgA1 deposits 4 days (A), 8 days (B), and 15 days (C) after the addition of IgAN patient serum. (D) IgA1 deposition in the glomerulus of a patient diagnosed with IgAN. (E) IgA1 immunostaining of IgAN spheroids cultured with FBS for 15 days. (F) IgA1 immunostaining of IgAN spheroids cultured without FBS for 15 days. (G) IgA1 immunostaining of ARPC-derived spheroids from healthy controls cultured with IgAN patient serum for 15 days. (H) IgA1 immunostaining of ARPC-derived spheroids from healthy controls cultured with inactivated IgAN patient serum for 15 days. (I) Fluorescence levels of IgA1 deposits in IgAN spheroids cultured with IgAN patient serum for 4 days (1), 8 days (2), or 15 days (3); in IgAN patient glomeruli (4); in IgAN spheroids cultured with FBS (5) or without FBS (6); in healthy subjects with inactivated IgAN patient serum (7); or in those cultured with IgAN patient serum (8). The scale bars represent 50 μm in A-C and 25 μm in E-H. Results are representative of 3 independent experiments (3 different IgAN patients). The immunofluorescent quantification from triplicate samples is shown in the histogram as mean ± SEM. **P <.01; ***P <.001.