SINGLE CENTER EXPERIENCE WITH ROBOTIC KIDNEY TRANSPLANTATION FOR RECIPIENTS WITH BMI 40 kg/m2: A COMPARISON WITH THE UNOS REGISTRY.

BACKGROUND: Obesity represents a barrier to kidney transplantation, but the increasing prevalence among renal failure patients has forced some centers to carefully consider such candidates. Morbidly obese patients may be at increased risk of delayed graft function, higher postoperative complications, and inferior graft outcomes. Nevertheless, mortality on the waiting list remains significantly higher than after transplant. We have applied minimally invasive surgery to perform kidney transplant in individuals with body mass index (BMI) of 40 kg/m or greater. We compared our results to the national United Network of Organ Sharing database. METHODS: The United Network of Organ Sharing registry was reviewed for adult living donor kidney transplant recipients with BMI of 40 kg/m or greater performed from September 2009 to December 2014. We compared transplants performed with robotic technique (RKT) versus patients performed with open surgery at all US centers including our own (open kidney transplant). Subgroup analysis in patients with BMI of 45 kg/m or greater was conducted. We compared outcomes including patient and graft survival, renal function, and technical complications. RESULTS: Robotic kidney transplantation group had a significantly higher mean BMI overall. The 1-year patient and graft survival rates were similar between groups. Renal function was also similar at 6, 12, and 36 months. Thrombosis caused 1.3% of the graft losses in open kidney transplant and 0% in the RKT group. Interestingly, 52.8% of the overall experience in patients with BMI of 45 kg/m or greater was performed with the robotic technique. CONCLUSIONS: Robotic surgery offers similar patient and graft survivals with comparable renal function to open technique. Robotic kidney transplantation permits transplantation in extreme BMI categories without additional technical complications. Further studies are required to establish the role of RKT for obese candidates but preliminary data are encouraging

Ang/Tie expression in isolated islets correlates with changes in vessel area.

<p>Isolated WT mouse and human islets were cultured for 3 days in control condition (11.1 mM glucose for mouse or 5.5 mM for human) or treated with diabetic conditions of 22.2 mM glucose + 0.5 mM palmitic acid or mixture of cytokines: 2 ng/mL IL-1β, 1000 U/ml IFN-ɣ and TNF-α (cyto). <b>(A,D)</b> GSIS is shown by the stimulatory index assessed by 16.7/2.8 mM glucose stimulation. <b>(B,C,E,F)</b> Graph shows ratio of vessel area to islet area for mouse <b>(B,C)</b> and human <b>(E,F)</b> islets, fixed and immune-labelled for vessel (CD-31,red) and islet (insulin, green). <b>(G-L)</b> qPCR analysis of treated mouse and human islets for mouse CD-31 <b>(G,J)</b>, Ang-1,-2 <b>(H,K)</b>, Tie-1,-2 <b>(I,L)</b>. All genes have been normalized to PPIA or 18s as housekeeping control. *p<0.05, treated vs. control 11.1 mM (mouse) or 5.5 mM (human). <b>(M)</b> Representative western blot from treated human islet lysates (left panel) and densitometric analyses of Ang-2 (right panel). Data are means +/-SE from 3–5 independent experiments from 3–5 different organ donors (human islets) or 3–5 independent mouse islet isolations.</p

Islet vessel area increases in T2DM.

<p><b>(A)</b> Representative images of pancreatic sections from non-diabetic controls and patients with T2D, immune-labelled for CD31 (red) and insulin (green). <b>(B)</b> Graphs show ratio of vessel area to islet area (control: n = 6; T2D: n = 10). <b>(C)</b> Plot shows no correlation of vessel density with BMI. <b>(D-H)</b> qPCR analysis of Ang-2, Tie-1, Tie-2, CD-31 from isolated mouse islets from C57BL/6 WT mice kept on normal diet (ND) or high-fat high-sucrose diet (HFD) for <b>(D)</b> 8 weeks (n = 4/group), <b>(E)</b> 16 weeks (n = 9/group) and <b>(F)</b> 24 weeks (n = 7/group), <b>(G) of</b> eNOS and <b>(H) of</b> ICAM-1. <b>(I)</b> qPCR analysis of Ang-1, Ang-2, Tie-1 and Tie-2 of isolated islets from non-diabetic (control, n = 8) and from patients with T2D (n = 7). *p<0.05, HFD vs ND or T2D vs. control</p

Islet hypervascularization in T2D and effects of Ang-2 overexpression.

<p>A healthy islet is surrounded by intact capillaries, maintained by the Ang/Tie system and extracellular matrix supporting the function and survival of the islet. An increased insulin demand leads to more islet blood flow, β-cell mass and vascular expansion and consequent compensation. A transient Ang-2 upregulation promotes angiogenesis, leading islet endothelium to a non-quiescent state inhibiting Tie-2 signaling. Towards human T2D progression, β-cell failure and apoptosis occurs together with increased islet and endothelial inflammation and islet hypervascularization. Ang-2 overexpression on the other hand prevents β-cell mass and vascular expansion in response to HFD with persistent islet and endothelial inflammation.</p

Ang-2 over-expression impairs islet function but protects from cytokine treatment in isolated islets.

<p>Isolated islets from RIP-rtTA;tet-O-Ang-2 (Ang2-rtTA) and RIP-rtTA control (rtTA) mice were cultured for 3 days in presence of 10 μg/ml doxycycline to achieve Ang-2 overexpression. Mouse or human islets were cultured in 11.1 (mouse) or 5.5 mM glucose (human) or treated with diabetic conditions of 22.2 mM glucose + 0.5mM palmitic acid or mixture of cytokines: 2 ng/mL IL-1β, 1000 U/ml IFN-ɣ and TNF-α (cyto). <b>(A)</b> Western blot from treated mouse islets shows Ang-2 overexpression in islets by myc-Ang-2. <b>(B)</b> GSIS is shown by the stimulatory index assessed by 16.7/2.8 mM glucose stimulation and normalized to control. <b>(C,D)</b> Treated mouse islets fixed post-GSIS and apoptotic cells detected by double staining for TUNEL and insulin. Representative images from different treatments. <b>(E,F)</b> qPCR analysis for CD31 <b>(E)</b> and ICAM <b>(F)</b> from mouse islets overexpressing Ang-2. <b>(G,H)</b> Representative western blots (upper panel) and densitometric analyses of proteins (lower panels) showing myc-Ang-2, ICAM-1, cleaved caspase 3 and actin/tubulin as housekeeping control, in human islets overexpressing Ang-2 by Ad-Ang-2 or control Ad-GFP <b>(G;</b> MOI = 50) or treated with 100 nM Tie-2 inhibitor for 72h (<b>H)</b>. Data are means +/-SE from 3–5 independent experiments from 3–5 different organ donors (human islets) or 3–5 independent mouse islet isolations. *p<0.05, treated vs. 11.1 mM glucose control, #p<0.05, Ang2-rtTA vs. rtTA.</p

Coexpression of neurogenin 3 (NGN3) and CD133 in cultured human exocrine tissue.

<p><b>A-D</b>, Expression of NGN3 and CD133 in exocrine tissue. <b>A</b>, CD133 expression. <b>B</b>, NGN3 expression. <b>C</b>, Nuclei costained with 4',6-diamidino-2-phenylindole (DAPI). <b>D</b>, Overlay of 3 channels. 1-μm confocal sections. Scale bar is 50 μm. <b>E-H</b>, FACS analysis of exocrine cells after 4 days in culture. Gates indicated by red lines. % cells in each gate shown in red. <b>E</b>, CD133+ gate defined by isotype negative control. <b>F</b>, CD133+ cells following anti-CD133 stain. <b>G</b>, Cells within the CD133+ gate following staining with NGN3 isotype negative control. <b>H</b>, Cells within the CD133+ gate following staining with anti-NGN3. <b>I-P</b>, Parallel fluorescence microscopy imaging of cell populations in E-H. <b>I</b>, Cells stained with NGN3 isotype negative control. <b>J</b>, Cells stained with anti-CD133. <b>K</b>, Cells stained with Hoechst 33352. <b>L</b>, Overlay of images in I-K. <b>M</b>, Cells stained with anti-NGN3. <b>N</b>, Cells stained with anti-CD133. <b>O</b>, Cells stained with Hoechst 33352. <b>P</b>, Overlay of images in M-O. Scale bars are 20 μm. <b>Q</b>, Change in the percentage and total number of CD133+ cells over time in culture. Mean ± SEM percentage of CD133+ cells (black bar) and total number of CD133+ cells (white bar) indicated along Y-axis as a percentage of initial level on day 0 of culture. Significance determined by ANOVA with Bonferroni-Holm post hoc analysis, ***, P<0.001, **, P<0.01, (n = 6 exocrine cultures).</p

Relative mRNA expression of pancreas development transcription factors by CD133+ cells isolated after 4 days of exocrine tissue culture.

<p>Mean ± SEM relative expression level of genes in CD133+ cells compared to the CD133-depleted (CD133D) population shown on Y-axis as fold difference. Significance determined by Student’s t-test from 2^-Δct values. ***, p<0.001, **, p<0.01, *, p<0.05 (n = 4 exocrine cultures). Genes ranked in order of overexpression in the CD133+ population. Inset shows gene expression using an enlarged scale. Genes are: Neurogenin 3 (NGN3), One cut homeobox 2 (ONECUT2), NK6 homeobox 1 (NKX6.1), GLIS family zinc finger 3 (GLIS3), motor neuron and pancreas homeobox 1 (MNX1), HNF1 homeobox B (HNF1B), pancreatic and duodenal homeobox 1 (PDX1), SRY (sex determining region Y)-box 9 (SOX9), Hairy enhancer of split 1 (HES1), One cut homeobox 1 (ONECUT1), Forkhead box A2 (FOXA2), Forkhead box O1 (FOXO1), v-maf avian musculoaponeurotic fibrosarcoma oncogene family, protein B (MAFB), GATA binding protein 4 (GATA4), Pancreas specific transcription factor 1A (PTF1A), Neuronal differentiation 1 (NEUROD1), ISL LIM homeobox 1 (ISL1), paired box 6 (PAX6). No expression of NK2 homeobox 2 or paired box 4 was detected.</p

Relative mRNA expression level of endocrine development transcription factors by differentiating pancospheres (PS) over time.

<p>Mean ± SEM (n = 3 exocrine cultures) relative expression level reported as fold difference from CD133+ starting population shown on Y-axis. Days after initiating PS formation shown along X-axis. Proliferative (Phase I) and maturation (Phase II) phases shown at bottom. <b>A</b>, Upregulated genes are: marker of proliferation Ki-67 (KI67), Neuronal differentiation 1 (NEUROD1), Neurogenin 3 (NGN3), v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA), v-maf avian musculoaponeurotic fibrosarcoma oncogene family, protein B (MAFB), paired box 4 (PAX4), paired box 6 (PAX6). <b>B</b>, Down regulated genes are: SRY (sex determining region Y)-box 9 (SOX9), pancreatic and duodenal homeobox 1 (PDX1), ISL LIM homeobox 1 (ISL1), NK6 homeobox 1 (NKX6.1), GLIS family zinc finger 3 (GLIS3), motor neuron and pancreas homeobox 1 (MNX1), Hairy enhancer of split 1 (HES1). <b>C</b>, Isolation and endocrine differentiation of NGN3+ cells. After death, pancreas is removed and transported to an islet isolation facility where a biopsy is taken for histology. The pancreas is then digested and separated into islets and exocrine tissue. Exocrine culture is initiated on day 2 post mortem. On day 6 post mortem, tissue is digested to single cells, which are labeled and sorted for expression of CD133. CD133+ cells are plated on pancosphere day 0. Samples are collected on pancosphere days 4, 6, 9, 13, 15, 19 and 21. Cell proliferation marker KI67 expression peaks at pancosphere day 9. On pancosphere day 19 IGF II is withdrawn and cells begin final maturation. Days between each step are indicated below arrows. Exocrine tissue culture and phases of pancosphere differentiation are shown above the timeline.</p

Expression of Notch pathways genes.

<p><b>A</b>, Western blot analyses of Notch intercellular domain (NICD), hairy enhancer of split 1 (HES1) and endogenous control gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cells isolated from human exocrine tissue. Whole cell lysates from CD133+ (+) and CD133-depleted (D) cells. Nuclear (N) and cytoplasmic (C) extracts from CD133+ cells. <b>B</b>, Volcano plot of Notch pathway gene mean ± SEM mRNA level (n = 3 exocrine cultures) differences in expression level from CD133+ cells compared to CD133D shown on X-axis as Log2 of fold difference. Significance determined by Student’s t-test shown on Y-axis as p value. Magenta vertical lines mark a 2-fold difference in expression. Blue horizontal line marks the significance cutoff (p<0.05). Selected gene names shown. Genes are: receptor tyrosine-protein kinase erbB-2 (ERBB2), frizzled class receptor 7 (FZD7), E1A binding protein p300 (EP300), MFNG O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase (MFNG), H19, imprinted maternally expressed transcript (H19), LIM domain only 2 (rhombotin-like 1) (LMO2), inhibitor of DNA binding 1 (ID1), hairy enhancer of split 4 (HES4), cyclin D1 (CCND1), matrix metallopeptidase 7 (MMP7), mastermind-like 2 (MAML2), jagged 1 (JAG1), Notch 2 (NOTCH2), hes-related family bHLH transcription factor with YRPW motif-like (HEYL), snail family zinc finger 2 (SNAI2), recombination signal binding protein for immunoglobulin kappa J region-like (RBPJL). <b>C</b>, Normalized mRNA expression level of neurogenin 3 (NGN3), HES1 and pancreas transcription factor 1 subunit alpha (PTF1A) in exocrine tissue after 4 days of culture in the presence of 20 μM Notch inhibitor DAPT. Results reported as mean ± SEM percent of levels in DMSO carrier control. mRNA levels normalized to the level of cyclophillin A. Significance determined by Student’s t-test, ***, p<0.001 (n = 3 exocrine cultures). <b>D</b>, Expression of NGN3 protein following treatment with 20 ∞M DAPT and 47 ∞M Notch agonist JAG-1 peptide (JAG-1). Mean ± SEM percent of DMSO carrier only control or 47 ∞M scrambled JAG-1 peptide, respectively indicated on Y-Axis. Significance determined by Student’s t-test, ***, p<0.001 (n = 3 exocrine cultures). <b>E-H</b>, Orthogonal analysis of colocalized HES1 and NGN3 in nuclei of exocrine tissue after 4 days of culture. Nuclei counterstained with Hoechst 33342 (H). <b>E</b>, Overlay of 3 channels. 0.5 ∞m confocal section. Scale bar is 50 ∞m. <b>F-H</b>, Higher magnification of crosshair region in all three channels shown at right. Scale bars are 20 ∞m. <b>I</b>, Coprecipitation of ID proteins with HES1. Whole cell lysate from exocrine tissue after 4 days of culture immunoprecipitated with antibody to HES1. ID1, 2 and 4 detected following SDS PAGE and western blotting. Predicted molecular weights of ID proteins (ID) and immunoglobulin heavy chain used for precipitation (HC) shown at right. Molecular weight marker positions shown at left in kDa.</p

Expression of hormones, chromogranin A and PDX1 by CD133+ cells following <i>in vitro</i> differentiation.

<p><b>A</b>, Phase microscopic image of pancospheres on day 6 of formation. Scale bar is 100 μm. <b>B</b>, Pancreatic and duodenal homeobox 1 (PDX1) expression in a day 6 pancosphere. Scale bar is 50 μm. <b>C,D</b>, Orthogonal analyses of PDX1 / glucagon (GCG) and PDX1 / insulin C-peptide (CPEP) coexpression in day 21 pancospheres. 1 μm optical sections, scale bar is 50 μm. Inset box in D is magnified and rotated confocal reconstruction of cells indicated by lines. <b>E</b>, Coexpression of GCG and chromogranin A (CHGA) by cells within a day 21 pancosphere. Scale bar is 20 μm. <b>F</b>, Coexpression of CPEP, CHGA and PDX1 by cells within a day 21 pancosphere. Scale bar is 10 μm. B-F, Nuclei stained with Hoechst 33342 (H).</p