Alginate@TiO2 hybrid microcapsules with high in vivo biocompatibility and stability for cell therapy.

Designing new materials to encapsulate living therapeutic cells for the treatment of the diseases caused by protein or hormone deficiencies is a great challenge. The desired materials need to be biocompatible towards both entrapped cells and host organisms, have long-term in vivo stability after implantation, allow the diffusion of nutrients and metabolites, and ensure perfect immune-isolation. The current work investigates the in vivo biocompatibility and stability of alginate@TiO2 hybrid microcapsules and the immune-isolation of entrapped HepG2 cells, to assess their potential for cell therapy. A comparison was made with alginate-silica hybrid microcapsules (ASA). These two hybrid microcapsules are implanted subcutaneously in female Wistar rats. The inflammatory responses of the rats are monitored by the histological examination of the implants and the surrounding tissues, to indicate their in vivo biocompatibility towards the hosts. The in vivo stability of the microcapsules is evaluated by the recovery rate of the intact microcapsules after implantation. The immune-isolation of the entrapped cells is assessed by their morphology, membrane integrity and intracellular enzymatic activity. The results show high viability of the entrapped cells and insignificant inflammation of the hosts, suggesting the excellent biocompatibility of alginate@TiO2 and ASA microcapsules towards both host organisms and entrapped cells. Compared to the ASA microcapsules, more intact alginate@TiO2 hybrid microcapsules are recovered 2-day and 2-month post-implantation and more cells remain alive, proving their better in vivo biocompability, stability, and immune-isolation. The present study demonstrates that the alginate@TiO2 hybrid microcapsule is a highly promising implantation material for cell therapy.

Protective effect of nitric oxide in aristolochic acid-induced toxic acute kidney injury: an old friend with new assets.

Aristolochic acid (AA) nephropathy (AAN), a progressive tubulointerstitial injury of toxic origin, is characterized by early and transient acute tubular necrosis. This process has been demonstrated to be associated with reduced nitric oxide (NO) production, which can disrupt the regulation of renal function. In this study, we tested the hypothesis that L-arginine (L-Arg) supplementation could restore renal function and reduce renal injury after AA intoxication. C57BL/6 J male mice were randomly subjected to daily i.p. injection of either sterile saline solution or AA (2.5 mg kg(-1)) for 4 days. To determine whether AA-induced renal injuries were linked to reduced NO production, L-Arg, a substrate for NO synthase, was supplemented (5%) in drinking water. Mice intoxicated with AA exhibited features of rapid-onset acute kidney injury, including polyuria, significantly increased plasma creatinine concentrations, proteinuria and fractional excretion of sodium (P < 0.05), along with severe proximal tubular cell injury and increased NADPH oxidase 2 (Nox2)-derived oxidative stress (P < 0.05). This was associated with a significant reduction in NO bioavailability. L-Arg supplementation in AA-treated mice significantly increased NO bioavailability, which in turn improved renal function (creatininaemia, polyuria, proteinuria, fractional excreted sodium and N-acetyl-ß-D-glucosaminidase enzymuria) and renal structure (tubular necrosis and tubular cell apoptosis). These changes were associated with significant reductions in Nox2 expression and in production of reactive oxygen species and with an increase in antioxidant concentrations. Our results demonstrate that preservation of NO bioavailability leads to renal protection in AA-induced acute kidney injury by reducing oxidative stress and maintaining renal function.

Lack of hyaluronidases exacerbates renal post-ischemic injury, inflammation, and fibrosis.

Renal ischemia-reperfusion injury (IRI) is a pathological process that may lead to acute renal failure and chronic dysfunction in renal allografts. During IRI, hyaluronan (HA) accumulates in the kidney, but suppression of HA accumulation during IRI protects the kidney from ischemic insults. Here we tested whether Hyal1-/- and Hyal2-/- mice display exacerbated renal damage following unilateral IRI due to a higher HA accumulation in the post-ischemic kidney compared with that in the kidney of wild-type mice. Two days after IRI in male mice there was accumulation of HA and CD44 in the kidney, marked tubular damage, infiltration, and increase creatininemia in wild-type mice. Knockout mice exhibited higher amounts of HA and higher creatininemia. Seven days after injury, wild-type mice had a significant decrease in renal damage, but knockout mice still displayed exacerbated inflammation. HA and CD44 together with α-smooth muscle actin and collagen types I and III expression were increased in knockout compared with wild-type mice 30 days after IRI. Thus, both HA-degrading enzymes seem to be protective against IRI most likely by reducing HA accumulation in the post-ischemic kidney and decreasing the inflammatory processes. Deficiency in either HYAL1 or HYAL2 leads to enhanced HA accumulation in the post-ischemic kidney and consequently worsened inflammatory response, increased tubular damage, and fibrosis.

Hyaluronidase 1 and hyaluronidase 2 are required for renal hyaluronan turnover.

Hyaluronidase 1 (HYAL1) and hyaluronidase 2 (HYAL2) are the major hyaluronidases acting synergistically to degrade hyaluronan (HA). In the kidney, HA is distributed heterogeneously. Our goal was to determine the consequences of a lack of either HYAL1 or HYAL2 (using specific knockout mice) on renal function and on renal HA accumulation. Experiments were performed in Hyal1(-/-) and Hyal2(-/-) mice and in their wild-type controls. HA concentration was measured in the plasma and kidney tissue and its distribution through the different kidney zones was examined by immunohistochemistry. Relative mRNA expressions of HYAL1, HYAL2 and the 3 main HA synthases were evaluated by quantitative RT-PCR. Results: Kidney function was not impaired in the knockout mice but they displayed elevated HA concentrations in the plasma and in the kidney. Hyal1(-/-) mice presented an accumulation of HA inside the proximal tubular cells whereas Hyal2(-/-) mice showed HA accumulation in the interstitial space. In the cortex and in the outer medulla, HYAL1 mRNA expression was up-regulated in Hyal2(-/-) mice. From our study we conclude that somatic hyaluronidases are not required for renal function. However, HYAL1 is necessary for the breakdown of intracellular HA in the cortex, whereas HYAL2 is essential for the degradation of extracellular HA in all kidney regions.

Inhibition of hyaluronan is protective against renal ischaemia-reperfusion injury.

BACKGROUND: Ischaemia-reperfusion injury (IRI) to the kidney is a complex pathophysiological process that leads to acute renal failure and chronic dysfunction in renal allografts. It was previously demonstrated that during IRI, hyaluronan (HA) accumulates in the cortical and external medullary interstitium along with an increased expression of its main receptor, CD44, on inflammatory and tubular cells. The HA-CD44 pair may be involved in persistent post-ischaemic inflammation. Thus, we sought to determine the role of HA in the pathophysiology of ischaemia-reperfusion (IR) by preventing its accumulation in post-ischaemic kidney. METHODS: C57BL/6 mice received a diet containing 4-methylumbelliferone (4-MU), a potent HA synthesis inhibitor. At the end of the treatment, unilateral renal IR was induced and mice were euthanized 48 h or 30 days post-IR. RESULTS: 4-MU treatment for 14 weeks reduced the plasma HA level and intra-renal HA content at 48 h post-IR, as well as CD44 expression, creatininemia and histopathological lesions. Moreover, inflammation was significantly attenuated and proliferation was reduced in animals treated with 4-MU. In addition, 4-MU-treated mice had a significantly reduced expression of α-SMA and collagen types I and III, i.e. less renal fibrosis, 30 days after IR compared with untreated mice. CONCLUSION: Our results demonstrate that HA plays a significant role in the pathogenesis of IRI, perhaps in part through reduced expression of CD44. The suppression of HA accumulation during IR may protect renal function against ischaemic insults.

A hybrid assembly by encapsulation of human cells within mineralised beads for cell therapy.

BACKGROUND: The design of new technologies for treatment of human disorders such as protein deficiencies is a complex and difficult task. Particularly, the construction of artificial organs, based on the immunoisolation of protein-secreting cells, requires the use of suitable materials which have to be biocompatible with the immunoisolated cells and avoid any inappropriate host response. METHODOLOGY/PRINCIPAL FINDINGS: This work investigates the in vivo behavior of mechanically resistant hybrid beads which can be considered as a model for artificial organ for cell therapy. This hybrid system was designed and fabricated via the encapsulation of living cells (HepG2) within alginate-silica composites. Two types of beads (alginate-silica hybrid (AS) or alginate/silica hybrid subsequently covered by an external layer of pure alginate (ASA)), with or without HepG2 cells, were implanted into several female Wistar rats. After four weeks, the potential inflammatory local response that might be due to the presence of materials was studied by histochemistry. The results showed that the performance of ASA beads was quite promising compared to AS beads, where less abnormal rat behaviour and less inflammatory cells in histological sections were observed in the case of ASA beads. CONCLUSIONS/SIGNIFICANCE: The current study highlights that alginate-silica composite materials coated with an extra-alginate shell offer much promise in the development of robust implantation devices and artificial organs.

Do segmental reflexes play a role in the distribution of external intercostal EMG activity in the rabbit?

External intercostal muscle is considered as an inspiratory muscle. Its electromyographic (EMG) activity is exquisitely sensitive to proprioceptive afferents and is predominant in zones with the greatest respiratory effect. In the current series of studies, we assessed the contribution of segmental reflexes to this particular distribution of activity in anesthetized rabbits. We first established a parallel between gradients of activity and mechanical advantage. The sensitivity of external intercostal EMG activity to rib displacement was then assessed in the 3rd and 5th interspaces and in the dorsal, middle and ventral muscle bundles of the 3rd interspace. Finally, persistence of the dorso-ventral gradient was tested after a dorsal rhizotomy (interspaces 3-5). There were no differences in the sensitivity to rib motion in the studied positions. After rhizotomy, the dorso-ventral gradient of activity persisted at rest and with inspiratory resistive loading. We, therefore, conclude that the neuromechanical matching of respiratory drive in the external intercostal muscles is not the result of segmental reflexes.

Synthesis and fragmentation of hyaluronan in renal ischaemia.

BACKGROUND: The turnover of hyaluronan (HA), especially the production of low-molecular-weight fragments of HA, was examined in a model of unilateral renal ischaemia-reperfusion (IR) in rats. METHODS: HA was extracted from the outer and inner stripe of the outer medulla (OSOM and ISOM) at different times following IR. Its fragmentation was measured using membrane filtration and size-exclusion chromatography. Quantitative reverse transcription-polymerase chain reaction, zymography and immunohistochemistry were used to assess the expression and localization of various forms of HA synthase (HAS) and hyaluronidase (HYAL). Macrophage infiltration was evaluated using immunohistochemistry. RESULTS: HA accumulated at Day 1 mostly as high-molecular-weight (HMW) species with an elution profile similar to a reference 2500 kDa HA and at Day 14 mostly as medium- to low-size fragments. Within 1 day, HAS1 messenger RNA was up-regulated > 50- and 35-fold in OSOM and ISOM, respectively. Thereafter, HAS1 tended to normalize, while HAS2 increased steadily. Both synthetic enzymes were localized around tubules and in the interstitium. Conversely, HYAL1, HYAL2 and global hyaluronidase activity were repressed during the first 24 h. The patterns were identical in the OSOM and ISOM despite markedly different amounts of HA at baseline. There was no obvious correlation between HA deposits and macrophage infiltration. CONCLUSIONS: In the post-ischaemic kidney, HA starts to accumulate at Day 1 mostly as HMW species. Later on, a large proportion becomes degraded into smaller fragments. This pattern is explained by coordinated changes in the expression of HA synthases and hyaluronidases, especially an early induction of HAS1. The current data open the door to timed pharmacological interventions blocking the production of HA fragments.