Hydrogel-Mediated Local Delivery of Induced Nephron Progenitor Cell-Sourced Molecules as a Cell-Free Approach for Acute Kidney Injury

Abstract

Acute kidney injury (AKI) constitutes a severe condition characterized by a sudden decrease in kidney function. Utilizing lineage-restricted stem/progenitor cells, directly reprogrammed from somatic cells, is a promising therapeutic option in personalized medicine for serious and incurable diseases such as AKI. The present study describes the therapeutic potential of induced nephron progenitor cell-sourced molecules (iNPC-SMs) as a cell-free strategy against cisplatin (CP)-induced nephrotoxicity, employing hyaluronic acid (HA) hydrogel-mediated local delivery to minimize systemic leakage and degradation. iNPC-SMs exhibited anti-apoptotic effects on HK-2 cells by inhibiting CP-induced ROS generation. Additionally, the localized biodistribution facilitated by hydrogel-mediated iNPC-SM delivery contributed to enhanced renal function, anti-inflammatory response, and renal regeneration in AKI mice. This study could serve as a 'proof of concept' for injectable hydrogel-mediated iNPC-SM delivery in AKI and as a model for further exploration of the development of cell-free regenerative medicine strategies.

Keywords: cell-free regenerative medicine; induced nephron progenitor cells; injectable hydrogel.

Figure 1

Preparation and profiling of iNPC-SMs. (A) Schematic representing the beneficial characteristics of iNPCs generated from urine-derived cells and the preparation process of iNPC-SMs as a therapeutic agent for renal regeneration. (B) ELISA analysis of paracrine factors (HGF, IGF1, FGF2, and VEGF) involved majorly in renal diseases and repair process, secreted from iNPCs in a cell density- and time-dependent manner. * p < 0.05, ** p < 0.01, *** p < 0.001. (C,D) Antibody-based protein array of iNPC-SMs including growth factors and cytokines. The array assessed proteins associated with immunomodulation, angiogenesis, proliferation, migration, and renal development, with potential implications for renal regeneration and recovery. Blot density was analyzed using ImageJ. Data are presented as mean ± SD from three independent experiments.

Figure 2

In vitro protective effects of iNPC-SMs against CP-induced cytotoxicity. (A) Time-dependent changes in viability of HK2 cells with various concentrations of CP. (B) Cell viability of HK2 cells exposed to different culture conditions supplemented with CP and iNPC-SM for 24 or 72 h. Cells were seeded at a density of 5 × 10³ cells per well in a 96-well plate. Viability was analyzed using a CCK -8 assay. (C) Real-time PCR analysis of cell arrest- and apoptosis-associated genes (p53, p21, Caspase 3/7, Bax, and Bcl2) in HK2 cells exposed to CP and CP/iNPC-SM. (D) Western blot analysis of apoptotic markers (p53, Caspase 3, Cleaved Caspase 3, and Bax) in HK2 cells exposed to basal medium, CP, and CP/iNPC-SM. Blot density was analyzed using ImageJ. Data are represented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. (E) Schematic illustration depicting the anti-apoptotic effect of iNPC-SMs via inhibiting ROS generation on CP-induced cytotoxicity.

Figure 3

Preparation and characterization of hyaluronic acid-based injectable hydrogel (HAgel). (A) Schematic representation for the synthesis of HA-Tyr conjugate and the preparation process of HAgel. (B) ¹H NMR spectrum of tyramine (a), hyaluronic acid (b), and HA-Tyr conjugate (c) dissolved in D₂O. (C) Gelation times of HAgel at various concentrations of HRP and H₂O₂. (D) Digital image and scanning electron microscopic (SEM) image of HAgel. Scale bars = 100 μm. (E) Mechanical properties (storage modulus: G’, loss modulus: G”) of HAgel in the presence of HRP and H₂O₂. (F) Cumulative release profile of iNPC-SMs from HAgel in PBS at various concentrations of HA-Tyr conjugate. Data are represented as mean ± SD.

Figure 4

Hydrogel-mediated delivery of iNPC-SMs to AKI mice. (A) In vivo biodistribution of iNPC-SMs in the kidney, testis, spleen, lung, liver, and heart of C57B/6NTac mice injected with Naked SM and SM-HAgel. The SM-HAgel group exhibits a greater and sustained retention of iNPC-SM in the kidney from 6 to 24 h post-injection compared to the naked SM group. (B) Representative images of the kidneys on day 10 in normal mice and CP-treated mice injected with PBS, HAgel, Naked SM, and SM-HAgel. (C–E) Body weights (C), survival rates (D), and kidney weight (E) until day 10 of normal mice and CP-treated mice injected with PBS, HAgel, Naked SM, and SM-HAgel (n = 9). Significant differences in body weight changes compared to PBS are indicated. ** denotes significant differences for SM-HAgel, and * indicates significance for Naked SM. (F–G) Renal function analysis of BUN and creatinine in normal mice and CP-treated mice injected with PBS, HAgel, Naked SM, and SM-HAgel. Data are represented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

The therapeutic actions of iNPC-SMs in AKI mice. (A) Schematic representing the therapeutic actions of HAgel-mediated delivery of iNPC-SMs in acute kidney injury. (B,C) Quantitative analysis of pro-oxidant markers (malondialdehyde and MDA) and antioxidant enzymes (catalase and CAT) in normal and CP-treated mice injected with PBS, HAgel, Naked SM, and SM-HAgel. (D–I) Real-time PCR analysis of pro-inflammatory markers (TNF-α and IL6), anti-inflammatory marker (Bcl2), and the markers associated with renal regeneration (FGF2, VEGF, and Bmp7) in normal and CP-treated mice injected with PBS, HAgel, Naked SM, and SM-HAgel. Data are represented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.