Molecular Pathways Underlying Adaptive Repair of the Injured Kidney: Novel Donation After Cardiac Death and Acute Kidney Injury Platforms.

OBJECTIVE: To test the hypothesis that gene expression profiling in peripheral blood from patients who have undergone kidney transplantation (KT) will provide mechanistic insights regarding graft repair and regeneration. BACKGROUND: Renal grafts obtained from living donors (LD) typically function immediately, whereas organs from donation after cardiac death (DCD) or acute kidney injury (AKI) donors may experience delayed function with eventual recovery. Thus, recipients of LD, DCD, and AKI kidneys were studied to provide a more complete understanding of the molecular basis for renal recovery. METHODS: Peripheral blood was collected from LD and DCD/AKI recipients before transplant and throughout the first 30 days thereafter. Total RNA was isolated and assayed on whole genome microarrays. RESULTS: Comparison of longitudinal gene expression between LD and AKI/DCD revealed 2 clusters, representing 141 differentially expressed transcripts. A subset of 11 transcripts was found to be differentially expressed in AKI/DCD versus LD. In all recipients, the most robust gene expression changes were observed in the first day after transplantation. After day 1, gene expression profiles differed depending upon the source of the graft. In patients receiving LD grafts, the expression of most genes did not remain markedly elevated beyond the first day post-KT. In the AKI/DCD groups, elevations in gene expression were maintained for at least 5 days post-KT. In all recipients, the pattern of coordinate gene overexpression subsided by 28 to 30 days. CONCLUSIONS: Gene expression in peripheral blood of AKI/DCD recipients offers a novel platform to understand the potential mechanisms and timing of kidney repair and regeneration after transplantation.

Age-related alterations in regeneration of the urinary bladder after subtotal cystectomy.

Prior work documented that surgical removal of approximately 70% of the bladder (subtotal cystectomy) in 12-week-old female rats induced complete functional regeneration of the bladder within 8 weeks. To determine whether animal age affects bladder regeneration, female F344 rats aged 12 weeks (young) and 12 months (old) underwent subtotal cystectomy, and then were evaluated from 1 to 26 weeks after subtotal cystectomy. At 26 weeks after subtotal cystectomy, bladder capacity in young animals was indistinguishable from that in age-matched controls, but bladder capacity in old animals was only approximately 56% of that in age-matched controls. There was no detectable difference in residual volume among treatment groups, but the diminished regeneration in old animals was associated with a corresponding increase in the ratio of residual volume to micturition volume. The majority of old animals exhibited evidence of chronic kidney damage after subtotal cystectomy. Maximal contraction of bladder strips to electrical field stimulation, as well as activation with carbachol, phenylephrine, and KCl, were lower in old than in young animals at 26 weeks after subtotal cystectomy. Immunostaining with proliferating cell nuclear antigen and Von Willebrand factor revealed delayed and/or diminished proliferative and angiogenic responses, respectively, in old animals. These results confirm prior work and suggest that multiple mechanisms may contribute to an age-related decline in the regenerative capacity of the bladder.

Cell and Growth Factor-Loaded Keratin Hydrogels for Treatment of Volumetric Muscle Loss in a Mouse Model.

Wounds to the head, neck, and extremities have been estimated to account for ∼84% of reported combat injuries to military personnel. Volumetric muscle loss (VML), defined as skeletal muscle injuries in which tissue loss results in permanent functional impairment, is common among these injuries. The present standard of care entails the use of muscle flap transfers, which suffer from the need for additional surgery when using autografts or the risk of rejection when cadaveric grafts are used. Tissue engineering (TE) strategies for skeletal muscle repair have been investigated as a means to overcome current therapeutic limitations. In that regard, human hair-derived keratin (KN) biomaterials have been found to possess several favorable properties for use in TE applications and, as such, are a viable candidate for use in skeletal muscle repair. Herein, KN hydrogels with and without the addition of skeletal muscle progenitor cells (MPCs) and/or insulin-like growth factor 1 (IGF-1) and/or basic fibroblast growth factor (bFGF) were implanted in an established murine model of surgically induced VML injury to the latissimus dorsi (LD) muscle. Control treatments included surgery with no repair (NR) as well as implantation of bladder acellular matrix (BAM). In vitro muscle contraction force was evaluated at two months postsurgery through electrical stimulation of the explanted LD in an organ bath. Functional data indicated that implantation of KN+bFGF+IGF-1 (n = 8) enabled a greater recovery of contractile force than KN+bFGF (n = 8)***, KN+MPC (n = 8)**, KN+MPC+bFGF+IGF-1 (n = 8)**, BAM (n = 8)*, KN+IGF-1 (n = 8)*, KN+MPCs+bFGF (n = 9)*, or NR (n = 9)**, (*p < 0.05, **p < 0.01, ***p < 0.001). Consistent with the physiological findings, histological evaluation of retrieved tissue revealed much more extensive new muscle tissue formation in groups with greater functional recovery (e.g., KN+IGF-1+bFGF) when compared with observations in tissue from groups with lower functional recovery (i.e., BAM and NR). Taken together, these findings further indicate the general utility of KN biomaterials in TE and, moreover, specifically highlight their potential application in the treatment of VML injuries.

Three-dimensional culture of hepatocytes on porcine liver tissue-derived extracellular matrix.

There is currently no optimal system to expand and maintain the function of human adult hepatocytes in culture. Recent studies have demonstrated that specific tissue-derived extracellular matrix (ECM) can serve as a culture substrate and that cells tend to proliferate and differentiate best on ECM derived from their tissue of origin. The goal of this study was to investigate whether three-dimensional (3D) ECM derived from porcine liver can facilitate the growth and maintenance of physiological functions of liver cells. Optimized decellularization/oxidation procedures removed up to 93% of the cellular components from porcine liver tissue and preserved key molecular components in the ECM, including collagen-I, -III, and -IV, proteoglycans, glycosaminoglycans, fibronectin, elastin, and laminin. When HepG2 cells or human hepatocytes were seeded onto ECM discs, uniform multi-layer constructs of both cell types were formed. Dynamic culture conditions yielded better cellular infiltration into the ECM discs. Human hepatocytes cultured on ECM discs expressed significantly higher levels of albumin over a 21-day culture period compared to cells cultured in traditional polystyrene cultureware or in a collagen gel "sandwich". The culture of hepatocytes on 3D liver-specific ECM resulted in considerably improved cell growth and maintained cell function; therefore, this system could potentially be used in liver tissue regeneration, drug discovery or toxicology studies.