Treatment of Renal Transplant Complications with a Mesh Hood Fascial Closure Technique

Early renal allograft dysfunction may be caused by a number of technical factors including thrombosis, kinking of vessels, and a Page kidney situation in which the allograft is compressed within a shallow false pelvis and limited retroperitoneal space. Without early recognition, compromised graft function, obstruction, or graft loss may ensue. We describe a technique using a polypropylene-assisted mesh hood fascial closure (MHFC) to prevent and treat this potential complication. MHFC was performed both primarily to prevent this phenomenon, and secondarily to treat this complication. Between April 2001 and October 2002, 16 patients undergoing 17 renal transplants underwent MHFC. The mean recipient body weight was 17% less than the mean donor weight. The mean follow-up period was 9 months. The mean serum creatinine level after primary MHFC was 148.4 micromol/L. Three of 4 patients with early allograft dysfunction regained function after secondary MHFC and had a mean serum creatinine level of 155.3 micromol/L. Wound complications were seen in 5 (31%) patients with no wound or mesh infections and 1 patient was diagnosed with a lymphocele. We conclude that the use of mesh in the primary closure of the incision after renal transplantation is safe and has minimal complications

Relationship of clusterin with renal inflammation and fibrosis after the recovery phase of ischemia-reperfusion injury

Background: Long-term outcomes after acute kidney injury (AKI) include incremental loss of function and progression towards chronic kidney disease (CKD); however, the pathogenesis of AKI to CKD remains largely unknown. Clusterin (CLU) is a chaperone-like protein that reduces ischemia-reperfusion injury (IRI) and enhances tissue repair after IRI in the kidney. This study investigated the role of CLU in the transition of IRI to renal fibrosis. Methods: IRI was induced in the left kidneys of wild type (WT) C57BL/6J (B6) versus CLU knockout (KO) B6 mice by clamping the renal pedicles for 28 min at the body temperature of 32 °C. Tissue damage was examined by histology, infiltrate phenotypes by flow cytometry analysis, and fibrosis-related gene expression by PCR array. Results: Reduction of kidney weight was induced by IRI, but was not affected by CLU KO. Both WT and KO kidneys had similar function with minimal cellular infiltration and fibrosis at day 14 of reperfusion. After 30 days, KO kidneys had greater loss in function than WT, indicated by the higher levels of both serum creatinine and BUN in KO mice, and exhibited more cellular infiltration (CD8 cells and macrophages), more tubular damage and more severe tissue fibrosis (glomerulopathy, interstitial fibrosis and vascular fibrosis). PCR array showed the association of CLU deficiency with up-regulation of CCL12, Col3a1, MMP9 and TIMP1 and down-regulation of EGF in these kidneys. Conclusion: Our data suggest that CLU deficiency worsens renal inflammation and tissue fibrosis after IRI in the kidney, which may be mediated through multiple pathways.Medicine, Faculty ofOther UBCNon UBCUrologic Sciences, Department ofReviewedFacult

RAPA potentiates HF-induced cell death in FACS analysis.

<p>Cell death in cultured splenocytes was determined by FACS analysis with annexin-V-PE and 7-AAD staining. (A) Anti-CD3 antibody-stimulated splenocytes were treated with 1 nM of RAPA alone or in combination with various concentrations of HF for 48 hrs. Data are presented as a typical dot plot showing the percentage of annexin-V-PE and/or 7-AAD positive staining of cell populations. (B) Anti-CD3 antibody-stimulated splenocytes were treated with HF alone or in combination with 1 nM of RAPA for 48 hrs. (C) Anti-CD3 antibody-stimulated splenocytes were treated with RAPA alone or in combination with 2.5 nM of HF for 48 hrs. Apoptosis was represented by the sum of annexin-V stained cell populations (single annexin-V-PE positive cells in lower right quadrant, and double-annexin-V-PE/7-AAD positive cells in upper right quadrant). Data are presented as mean ± SD of 3–4 separate experiments, and were statistically analyzed by ANOVA. P ˂ 0.0001 (HF <i>vs</i>. HF + RAPA, n = 3); P ˂ 0.0001 (RAPA <i>vs</i>. RAPA + HF, n = 4).</p

HF antagonistically interacts with CsA in the suppression of TCR stimulated T cell proliferation.

<p>T cell proliferation in response to the stimulation of anti-CD3 antibody binding to TCR complex was measured by MTT assay. (A) Anti-CD3 antibody-stimulated splenocytes were treated with HF alone or in combination with 5 nM of CsA for 48 hrs. (B) Anti-CD3 antibody-stimulated splenocytes were treated with CsA alone or in combination with 2.5 nM of HF for 48 hrs. Data are presented as mean ± SD of five separate experiments, and were statistically analyzed by ANOVA. P = 0.0014 (HF <i>vs</i>. HF + CsA); P ˂ 0.0001 (CsA <i>vs</i>. CsA + HF). γ = 1.8375 based on IC₅₀ values in these two experiments.</p

RAPA potentiates HF-induced cell death in Western blot.

<p>The cleaved form of PARP as a biomarker for cell apoptosis in protein extracts of splenocytes was analyzed by Western blot. Anti-CD3 antibody-stimulated splenocytes were treated with RAPA (1 nM) alone, HF (2.5 nM) alone or a mixture of RAPA (1 nM) and HF (2.5 nM) for 48 hrs. Equal amount of protein (approximately 250 μg) extracted from whole cell pellets was fractioned by 7% of SDS-PAGE, and the cleaved PARP protein bands was identified based on specifically binding of anti-cleaved PARP antibody, and their molecular size (cleaved PARP: 89 kDa). The protein content in each sample was confirmed by re-probing the blot with anti-β-actin IgG antibody and was measured by a densitometry. Imaging data are a representative of three separate experiments. The ratio unit (RU) of PARP band to actin band from the same sample on the same blot was presented as mean ± SD of three determinants. P ˂ 0.0001 (HF vs. HF + RAPA), P ˂ 0.0001 (RAPA vs. HF + RAPA), P = 0.0009 (HF vs. RAPA).</p

HF reduces CsA-induced cell death in cultured HK-2 cells.

<p>Cell viability or apoptosis was measured by FACS analysis with annexin-V-PE and 7-AAD staining. A monolayer of HK-2 cells was treated with CsA (0–10 μM) alone or in combination with 1 μM HF for 48 hrs. Cell viability represented the percentage of viable cells (double-annexin-V-PE/7-AAD negative cells in lower left quadrant), and apoptosis was the sum of Annexin-V stained cell populations (single annexin-V-PE positive cells in lower right quadrant, and double-annexin-V-PE/7-AAD positive cells in upper right quadrant). Data are presented as mean ± SD of three separate experiments, and were statistically compared between CsA alone and CsA with HF in each group by two-tailed <i>t</i>-test.</p

Excess proline does not interfere synergistic interaction of RAPA with HF in the suppression of T cell proliferation.

<p>Anti-CD3 antibody-stimulated naïve splenocytes were treated with HF (2.5 nM) alone, RAPA (1 nM) alone or a mixture of RAPA (1 nM) and HF (2.5 nM) in the presence of 1 mM proline or vehicle for 48 hrs. T cell proliferation was measured by using MTT assay. Data are presented as the means ± SD of three separate experiments, and were statistically compared between vehicle and proline-treated samples in each group by two-tailed <i>t</i>-test.</p

HF synergistically interacts with RAPA in the suppression of TCR stimulated T cell proliferation.

<p>T cell proliferation in response to the stimulation of anti-CD3 antibody binding to TCR complex was measured by using MTT assay or trypan blue exclusion. (A) Anti-CD3 antibody-stimulated splenocytes were treated with HF alone or in combination with 1 nM of RAPA for 48 hrs. (B) Anti-CD3 antibody-stimulated splenocytes were treated with RAPA alone or in combination with 2.5 nM of HF for 48 hrs. Data are presented as mean ± SD of five separate experiments, and were statistically analyzed using two-way ANOVA. P < 0.0001 (HF <i>vs</i>. HF + RAPA); P < 0.0001 (RAPA <i>vs</i>. RAPA + HF). γ = 0.1905 based on IC₇₀ values in these two experiments. (C) Anti-CD3 antibody-stimulated splenocytes were treated with HF (1 or 2 nM) or RAPA (0.5 nM) alone or in combination with both drugs for 48 hrs, followed by counting viable cells using trypan blue exclusion assay. Data are presented as mean ± SD of three experiments. The Bliss independence: τ < α. (D) The splenocyte cultures were treated with HF alone, RAPA alone or both HF and RAPA for 18 hrs, and the cell growth was determined by using MTT assay. The difference of inhibition between groups was analyzed using two-tailed <i>t</i>-test. Data are presented as mean ± SD of five experiments. The Bliss independence: τ < α. (E) The splenocyte cultures were treated with HF alone or in combination with 1 nM RAPA for 36 hrs. (F) The splenocyte cultures were treated with RAPA alone or in combination with 2.5 nM HF for 36 hrs. The cell growth was determined using MTT assay. Data are presented as mean ± SD of five experiments. The difference of drug inhibitions between groups was analyzed using two-way ANOVA. P < 0.0001 (HF <i>vs</i>. HF + RAPA); P < 0.0001 (RAPA <i>vs</i>. RAPA + HF). γ = 0.201 based on IC₇₀ values in these two experiments.</p

Hyperbranched polyglycerol as a colloid in cold organ preservation solutions.

Hydroxyethyl starch (HES) is a common colloid in organ preservation solutions, such as in University of Wisconsin (UW) solution, for preventing graft interstitial edema and cell swelling during cold preservation of donor organs. However, HES has undesirable characteristics, such as high viscosity, causing kidney injury and aggregation of erythrocytes. Hyperbranched polyglycerol (HPG) is a branched compact polymer that has low intrinsic viscosity. This study investigated HPG (MW-0.5 to 119 kDa) as a potential alternative to HES for cold organ preservation. HPG was synthesized by ring-opening multibranching polymerization of glycidol. Both rat myocardiocytes and human endothelial cells were used as an in vitro model, and heart transplantation in mice as an in vivo model. Tissue damage or cell death was determined by both biochemical and histological analysis. HPG polymers were more compact with relatively low polydispersity index than HES in UW solution. Cold preservation of mouse hearts ex vivo in HPG solutions reduced organ damage in comparison to those in HES-based UW solution. Both size and concentration of HPGs contributed to the protection of the donor organs; 1 kDa HPG at 3 wt% solution was superior to HES-based UW solution and other HPGs. Heart transplants preserved with HPG solution (1 kDa, 3%) as compared with those with UW solution had a better functional recovery, less tissue injury and neutrophil infiltration in syngeneic recipients, and survived longer in allogeneic recipients. In cultured myocardiocytes or endothelial cells, significantly more cells survived after cold preservation with the HPG solution than those with the UW solution, which was positively correlated with the maintenance of intracellular adenosine triphosphate and cell membrane fluidity. In conclusion, HPG solution significantly enhanced the protection of hearts or cells during cold storage, suggesting that HPG is a promising colloid for the cold storage of donor organs and cells in transplantation