C4d deposition is associated with chronic allograft nephropathy in rats and could be influenced by immunosuppressants

Aims

Deposition of C4d in peritubular capillaries (PTC) has been considered to be a marker of humoral immunity in renal transplant. This study is to investigate C4d deposition in rat renal allografts undergoing CAN and the effects of immunosuppressants on it.

Methods

Fisher 344 rat renal grafts were orthotopically transplanted into Lewis rats following the procedure of Kamada with our modification. All the recipients were given CsA 10 mg/kg⁻¹ · d⁻¹ × 10 d and then divided into 5 groups (each n = 9); (1) Vehicle: vehicle orally, (2) CsA: 6 mg/kg⁻¹ · d⁻¹, (3) RAPA: 0.8 mg/kg⁻¹ · d⁻¹, (4) FK 506: 0.15 mg/kg⁻¹ · d⁻¹, (5) MMF: 20 mg/ kg⁻¹ · d⁻¹. At 4 weeks, 8 weeks, 12 weeks, the rats were sacrificed, renal allografts were harvested and sera were collected. The deposition of C4d was detected by immunofluorescence and analyzed by Integrated Optical Density (IOD). The pathological changes were accessed according to the Banff 97 criteria.

Results

C4d deposition in PTC was found in all the allografts at 4 weeks, while there was no obvious manifestations of CAN in all the groups; the differences of Banff Score between all groups were not significant (P > .05). The values of IOD in RAPA and MMF group were lower than those in other 3 groups (P = .002, .006). The differences between RAPA and MMF, and between other 3 groups were not significant (P > .05). The intensity of C4d increased along with the progression of CAN, the heaviest C4d deposits in PTC were found at 12 weeks, and meanwhile the severest CAN was found. Comparing with Vehicle group, CsA and FK 506 had no effect on C4d deposition (P > .05), however, MMF and RAPA obviously decreased the C4d deposition (P = .000). The intensity of C4d deposition had a significant correlation with the severity of CAN (r = 0.894, P = .000).

Conclusions

Our study suggests that the deposition of C4d in allografts appears earlier than pathological changes of CAN and has a correlation with the progression of CAN. MMF and RAPA can attenuate CAN by inhibiting humoral immunity. In contrast, CsA and FK 506 have no effect on humoral immunity.     

C4d deposition is correlated with the level of antivimentin antibody in rat kidneys undergoing chronic allograft nephropathy

Aims

Antivimentin antibody is often produced as an autoantibody after transplantation. C4d deposition, a marker of humoral immunity during transplantation, is believed to reflect alloantibodies. This study investigated the relationship between C4d deposition and humoral immunity to vimentin among rat kidneys undergoing chronic allograft nephropathy (CAN).

Methods

Fisher 344 rat renal grafts were orthotopically transplanted into Lewis rats following the procedure of Kamada with our modification. All recipients were administered cyclosporine (CsA) (10 mg/kg⁻¹ · d⁻¹ × 10 d) before being divided into 3 groups of oral treatments: (1) vehicle, (2) CsA (6 mg/kg⁻¹ · d⁻¹), and (3) mycophenolate mofetil (MMF; 20 mg/kg⁻¹ · d⁻¹). At 4, 8 and 12 weeks after transplantation, the rats were killed, the renal allografts harvested, and the sera collected. Serum creatinine (SCr) was measured and pathologic changes assessed according to the Banff 97 criteria. The antivimentin antibody was quantified by enzyme-linked immunosorbent assay. The deposition of C4d detected by immunofluorescence was analyzed by integrated optical density (IOD).

Results

Antivimentin antibody was observed in sera of all transplanted rats. The level of antivimentin antibody (IgGΔOD) increased gradually during the development of CAN from 4 weeks. Simultaneously, C4d deposition in peritubular capillaries also progressively strengthened. There was a strong positive correlation between the content of antivimentin antibody and C4d deposition (r = 0.892; P = .000). MMF simultaneously decreased antivimentin antibody formation and C4d deposition. In contrast, CsA had no significant effect.

Conclusions

We demonstrated the production of antivimentin antibodies and the deposition of C4d during the development of CAN. There was a positive correlation between them. Whether humoral immunity to vimentin contributes to C4d deposition is not clear and further studies are needed to elucidate this issue.     

Different Effect of Cyclosporine and Tacrolimus on Renal Expression of P-Glycoprotein in Human Kidney Transplantation

Objective

To investigate the differential effects of cyclosporine (CsA) and tacrolimus (TAC) on renal expression of P-glycoprotein (P-gp) in a cohort of kidney transplant recipients.

Methods

We enrolled 78 cadaveric kidney transplant recipients recuring basal immunosuppressive protocol with prednisone + mycophenolate mofetil + calcineurin inhibitor (CsA or TAC).

Results

We performed a 3-year analysis of 60 patients. There was no difference in age, gender, or cold ischemic time between two groups, Serum creatinine, urine protein, and blood fat levels of the CsA group were significantly higher than the TAC group (P < .05), while the creatinine clearance was remarkably lower than the TAC group (P < .05). The incidence of tubular atrophy, arteriohyalinosis, and interstitial fibrosis and nephrotoxic lesions among the CsA group were higher than the TAC group, as well as the chronic allograft nephropathy (CAN) Banff score (P < .05).^® P-gp was predominantly present in the a tubular apical membrane, basal membrane, and cytoplasm. The intensity and extent of tubular staining score in the CsA group were lower compared with the TAC group (P < .01 and P < .05, respectively).

Conclusion

Less P-gp expression in the CsA group than the TAC group may be the molecular action pathway of the high incidence of CsA nephrotoxicity and CsA-induced CAN. This study perhaps unraveled a novel interpretation that the differences of CsA and TAC on long-term allograft survival were due to increases dynamic effects of CsA at the exposures employed in this study.     

Evolution of glomerular filtration rate, renal injury markers, anemia, and angiotensin blockers use after change from calcineurin inhibitors to sirolimus in transplant patients with neoplasia versus chronic allograft nephropathy

Background

The change from calcineurin inhibitors (CNI) to sirolimus (SRL) is a safe alternative in transplant patients with neoplasia (NEO) whereas the results of conversion for chronic allograft nephropathy (CAN) are controversial, depending on the histologic score, degree of proteinuria, and glomerular filtration rate (GFR). Our aim in this study was to compare GFR, proteinuria, albuminuria, blood pressure (BP) effects, and anemia after switching to sirolimus (SRL) among renal transplant recipients with CAN versus NEO.

Methods

Fifty-five kidney transplant recipients with conversion from CNI to SRL owing to CAN or NEO were analyzed for the variables at 6 months before, at the time of, and at 6 months and 1, 2, and 3 years after the switch to SRL.

Results

There were no differences between CAN and NEO in the slope of estimated GFR (mL/min/1.73 m² by Cockcroft-Gault formula) at 1 year (−5.5 vs 3.7; P = .007) and at 2 years (−3.86 vs −10.3; P = .01). The values of proteinuria (mg/24 h/1.73 m²) before (665 ± 136 vs 329 ± 69; P = .036) as well as at 1 (1,122 ± 306 vs 863 ± 190; P = .478) and at 2 years after conversion (1,360 ± 430 vs 457 ± 154; P = .045) showed some significant differences, as did the use of both antiangiotensin agents, angiotensin-converting enzyme inhibitor and angiotensin receptor blocker at the moment of switch (35% vs 0%; P = .005) at 1 year (69% vs. 6% P = .02) and at 2 years (67% vs 28%; P = .047). There were no differences in graft survival (log rank: P = .515). By logistic regression analysis, the best covariate associated with GFR >45 mL/min at 2 years was GFR >60 mL/min at the moment of switch to SRL (odds ratio, 1.33; 95% confidence interval, 1.002-1.74).

Conclusions

The evolution of renal damage was more important in the CAN group requiring greater use of 2 angiotensin antagonists for control of proteinuria. We probably need histologic and serologic biomarkers to show which patients with CAN will show a bad evolution after the change to SRL.     

A potent anti-angiogenic factor, vasohibin-1, ameliorates experimental bronchiolitis obliterans

Background

Bronchiolitis obliterans (BO) is a major cause of morbidity and mortality after lung transplantation. BO is pathologically characterized by neovascularized fibro-obliteration of the allograft airway. A recent study has shown that aberrant angiogenesis during fibro-obliteration contributes to the pathogenesis of BO. Vasohibin-1 (VASH1) has been isolated as a vascular endothelial growth factor-inducible gene in endothelial cells (ECs) that inhibits migration and proliferation of ECs and exhibits anti-angiogenic activity in vivo.

Purpose

This study examines whether VASH1 inhibits fibro-obliteration of the allograft in a murine intrapulmonary tracheal transplantation model.

Method

Tracheal allografts of BALB/c mouse were transplanted into the left lung of recipient C57BL/6J mouse. We performed gene transfer to the recipient lungs using an adenovirus vector encoding human VASH1 (Ad-VASH1) or beta- garactosidase (Ad-LacZ) as the control. Tracheal allografts were harvested and pathological on days 21 and 28.

Result

Ad-VASH1 treatment reduced the vascular area on day 21 (4.6% versus 13.0%, P = .037) and day 28 (5.4% versus 13.4%, P = .022) compared with the control group. This was accompanied by significantly inhibited luminal obliteration of the tracheal allografts in the animals transferred with Ad-VASH1 compared with the control (69% versus 93%, P = .028) on day 21. We were not able to observe this effect on day 28 (92% versus 97%, P = .48).

Conclusion

Transgene expression of VASH1 in the recipient lung significantly attenuated luminal obliteration of the tracheal allograft; this was associated with significantly reduced aberrant angiogenesis in the fibro-obliterative tissue in a murine model intrapulmonary tracheal transplantation.     

Adrenal cortical cell transplantation

Purpose

The adrenal cortex is a critical component of the hypothalamic-pituitary-adrenal/gonadal axis that coordinates the stress response and maintains homeostasis. The authors hypothesize that adrenal cortical cells can be transplanted in adrenal insufficiency states to regenerate the adrenal cortex.

Methods

Murine adrenal glands were dissociated into adrenal cortical cells. Cells cultured in a collagen matrix were transplanted under the renal capsule. The implants were retrieved 1, 2, 4, and 8 weeks later. Total RNA was extracted from the retrieved specimens and was analyzed by real-time polymerase chain reaction.

Results

All animals survived the surgical procedure. At implant retrieval, a distinct organoid could be visualized under the renal capsule. The expressions of adrenal-specific markers including Sf1, Dax1, Star, Cyp11a, Cyp11b1, and Cyp21 were detectable in the retrieved specimens up to 8 weeks posttransplantation.

Conclusion

Primary adrenal cortical cells retained their gene expressions after heterotopic transplantation. Ex vivo gene transfer followed by adrenal cortical cell transplantation may lead to curative therapy for patients with adrenal insufficiency.     

Differential expression of angiopoietin-2 and vascular endothelial growth factor in androgen-independent prostate cancer models

OBJECTIVE

To investigate the relationship between microvessel density (MVD), blood vessel morphology and the expression of angiopoietin (Ang)‐1, Ang‐2, tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (Tie)‐2, and vascular endothelial growth factor (VEGF) in androgen‐dependent (AD) and androgen‐independent (AI) prostate cancer models, to gain insight into the regulation of angiogenesis at different stages of prostate cancer.

MATERIALS AND METHODS

MVD and blood vessel morphology were evaluated by CD34 immunohistochemical staining. The mRNA and protein secretion of the Angs, Tie‐2 and VEGF were measured by real‐time polymerase chain reaction and enzyme‐linked immunosorbent assays, respectively, in LNCaP (AD) and LNCaP‐19, C4‐2, C4–2B₄ and PC‐3 (AI) prostate cancer xenografts in mice.

RESULTS

LNCaP, C4‐2 and C4–2B₄ xenografts had high expression of Ang‐2 and VEGF, similar MVD and blood vessel morphology_. However, the most angiogenic cell line LNCaP‐19 expressed low levels of both factors and had different vessel morphology. PC‐3 xenografts had a similar MVD to LNCaP, C4‐2 and C4–2B₄, but the Ang‐2 and VEGF expression as well as the vessel morphology were similar to LNCaP‐19.

CONCLUSION

The differences in MVD, blood vessel morphology and the expression of Ang‐2 and VEGF show that prostate cancer cells display angiogenic heterogeneity, which indicates different roles of these factors in the regulation of angiogenesis in different stages of prostate cancer.     

Adenovirus-mediated anti-sense ERK2 gene therapy inhibits tubular epithelial-mesenchymal transition and ameliorates renal allograft fibrosis

Purpose

Epithelial-mesenchymal transition (EMT) plays an important role in progress of renal allograft fibrosis. The adenovirus-mediated anti-sense extracellular signal-regulated kinase 2 (Adanti-ERK2) gene therapy was used to block ERK signaling pathway, and its effect on EMT and renal allograft fibrosis both in vivo and in vitro was explored.

Methods

We first generated an in vitro EMT model by connective tissue growth factor (CTGF) stimulation in a HK-2 cell culture system, and then applied Adanti-ERK2 gene therapy on it. The transition of epithelial marker (E-cadherin) to mesenchymal markers (??-SMA, Vimentin) and the cell mobility function alteration were monitored for the observation of EMT progress. In vivo, a rat renal transplant model with Fisher-Lewis combination was employed and the Adanti-ERK2 gene therapy was given. The tubular EMT changes and pathology of allograft fibrosis were examined.

Results

In vitro, Adanti-ERK2 gene therapy inhibited CTGF-induced tubular EMT and attenuated the cell motility function induced by CTGF. In vivo, Adanti-ERK2 gene therapy attenuated tubular EMT, modulated the infiltration of macrophages and CD8⁺, CD4⁺T lymphocytes, and ameliorated fibrosis effectively in the renal allografts 24 weeks after transplantation.

Conclusions

Adanti-ERK2 gene therapy inhibits tubular EMT and attenuates renal allograft fibrosis. It is possible to develop promising molecular drug(s) in the future based on ERK signaling pathway.     

An association of multiple endocrine neoplasia 2B, a RET mutation; constipation; and low substance P-nerve fiber density in colonic circular muscle

Background

Multiple endocrine neoplasia (MEN) 2B is a rare hereditary syndrome that results from an activating mutation of the RET proto-oncogene. The RET gene is involved in the development of the enteric nervous system. Patients with MEN 2B have enlarged enteric ganglia and may be affected by gastrointestinal dysmotility. A deficiency of the neurotransmitter substance P (SP) has been identified in both pediatric and adult patients with chronic constipation.

Methods

Three patients, in whom constipation was the presenting symptom and MEN 2B had been provisionally diagnosed, underwent genetic analysis. Seromuscular colonic biopsies were taken for immunofluorescence imaging in all 3 patients. A retrospective review of the patient notes was undertaken.

Results

All 3 patients had constipation refractory to conservative treatment. Genetic analyses in the 3 patients confirmed an identical RET mutation (Met918Thr). Immunofluorescence imaging in all 3 patients identified grossly enlarged myenteric plexus ganglia but surprisingly a low density of SP-labeled nerve fibers in the colonic circular muscle. Nitric oxide synthase and vasoactive intestinal peptide labeling were not reduced.

Conclusion

The results show an association between MEN 2B and its most common RET mutation, colonic dysmotility, and low density of SP in the colonic circular muscle. Larger numbers of patients need to be studied to investigate whether low SP is primarily associated with the constipation or RET mutation and if it is a common feature of MEN 2B.     

Posttransplant inflammation associated with onset of chronic kidney disease

Background

Chronic allograft nephropathy (CAN), a major complication in renal transplant patients, is an important cause of graft loss. Inflammation as measured in the pretransplant and posttransplant phases, using various markers, has been associated with worse renal function and a greater risk of cardiovascular disease and of long-term graft loss.

Objective

The objective of our study was to evaluate whether worsening inflammation in the first 3 months postoperatively was a risk factor for developing CAN.

Patients and methods

We performed a cross-sectional study in 207 patients. The following markers of inflammation (MIF) were determined pretransplant and at 3 months after grafting: C-reactive protein (CRP) (mg/L), interleukin (IL)-6 (pg/mL), IL-10 (pg/mL), tumor necrosis factor (TNF)-α (pg/mL), and its soluble receptor (ng/mL), soluble-IL2R (UI/mL), pregnancy-associated plasma protein A (PAPP-A; mUI/L), and IL-4 (pg/mL). We also calculated the ratio at 3 months versus the pre value of MIF.

Results

CAN was diagnosed after the first year in 23 patients (11.3%) always by renal biopsy performed for clinical indications. Patients with CAN showed worse inflammation, eg, MIF ratios over one, with statistically significant differences for the ratios of TNF-α and PAPP-A (P = .032 and P = .051 respectively). Upon multivariate logistic regression analysis, using CAN as the dependent variable and age, sex, donor age, months on dialysis, acute tubular necrosis, acute rejection, and MIF ratios as covariates, we observed that an acute rejection episode (OR = 13.03; CI = 2.8-60.9; P = .001), CRP ratio (OR = 1.36; CI = 1.07-1.73; P = .013), and PAPP-A ratio (OR = 1.80; CI = 0.92-3.53; P = .005) were independent markers of CAN.

Conclusions

Among other factors, inflammation may determine the onset of CAN as diagnosed by renal biopsy.     

Impact of Donor Age on Renal Allograft Function and Survival

Background

The lack of cadaveric donors coupled with a rapidly growing number of potential recipients have stimulated the implementation of several strategies, including the acceptance of older donors, to increase the organ pool and reduce the waiting list for kidney transplantation. However several studies have demonstrated higher incidences of delayed graft function and poor graft outcomes among kidneys harvested from older donors.

Objective

The objective of this study was to evaluate the influence of donor age on the function and long-term survival of renal allografts.

Patients

We performed a retrospective review of the clinical data from 441 adult kidney transplantation from cadaveric heart-beating donors performed in our unit from May 1989 to May 2007.

Results

Recipients of kidney allografts from older donors were significantly older (49.2 vs 43.7 years; P < .0001) and had a higher incidence of delayed graft function (15.1% vs 5.4%; P = .005). Renal function was superior following kidney transplantation using younger donors not only at 3 months (P < .0001) and 12 months (P < .0001) posttransplantation, but also upon long-term follow-up at 60 months (P < .0001) and 96 months (P = .030). Allograft survival censored for death with a functioning graft and patient survival were not different when comparing older versus younger donors. Multivariate analysis confirmed the lack of correlation between donor age and allograft failure.

Conclusion

Donor age showed no influence on allograft survival. However, kidney allografts from older donors displayed lower first year and long-term renal function.     

Expression of Toll-like receptors 2 and 4 is downregulated after operation

Background

Toll-like receptors (TLR) that recognize microbial pathogens play a critical role in innate immunity; however, their expression and function after surgery remain unknown. The aim of this study was to examine TLR2 and TLR4 expression on monocytes and their responses to each agonist after surgical insults.

Methods

Blood samples were obtained from 83 patients who underwent gastrointestinal surgery. TLR2, TLR4, and inducible nitric oxide synthase expressions on peripheral blood mononuclear cells (PBMCs) were analyzed by flow cytometry. Macrophage-activating lipopeptide-2 or lipopolysaccharide-induced tumor necrosis factor-α and interleukin-6 production was measured by enzyme-linked immunosorbent assay.

Results

TLR2 and TLR4 decreased and showed the lowest values on the postoperative days 3 and 1, respectively. Macrophage-activating lipopeptide-2-stimulated tumor necrosis factor-α and interleukin-6 production was decreased immediately after the operation (P<.05), increased to a maximum value on postoperative day 1, and then decreased gradually. Lipopolysaccharide-stimulated tumor necrosis factor-α production was also suppressed immediately (P<.05) after operation then showed a gradual increase to maximum values on postoperative day 3. Inducible nitric oxide synthase in cultured PBMC that was obtained immediately after operation was upregulated (P<.05).

Conclusion

Expressions of TLR2 and TLR4 were downregulated by operation, and agonist-induced cytokine production was suppressed transiently and soon increased through the activation of PBMC. The present study may offer new insights for postoperative modulation of innate immunity under surgical stress.     

Ang1、Ang2及其受体Tie2在血管瘤组织中的表达及意义

目的探讨血管瘤的血管生成与促血管生成素家族（Ang/Tie2）之间的关系。方法通过免疫组化SP法、RT-PCR检测增生期血管瘤17例、消退期血管瘤13例和小儿正常皮肤10例的组织中促血管生成素1（Ang1）、促血管生成素2（Ang2）及其受体Tie2的表达情况。结果增生期血管瘤组织Ang2、Tie2表达高于消退期血管瘤（P〈0.01）;Ang2、Tie2在小儿正常皮肤表达较弱或阴性;Ang1在血管瘤和小儿正常皮肤表达均较弱或阴性,二者比较差异无统计学意义（P〉0.05）。结论Ang/Tie2体系在血管瘤的增生和消退过程中可能起重要的作用。     

Lateral semicircular canal fenestration for congenital conductive hearing loss: Solution for a dilemma

Objective

To determine functional results after lateral semicircular canal fenestration on congenital conductive hearing loss.

Study Design

Case series with chart review.

Setting

Amir-Alam otolaryngology tertiary referral center.

Subjects and Methods

Twenty patients with congenital oval window malformations who were not candidates for ossicular reconstruction underwent lateral semicircular canal fenestration. A skin graft was placed over the perforated fascial graft on the fenestrated area.

Results

The median preoperative mean air conduction (MAC) was 56.9 dB (50.0 dB median air-bone). Postoperative median MAC gain of 34.3 dB (P < 0.001) and the median air-bone gap of 18.8 dB were observed. The mean bone conduction (MBC) did not show any significant changes postoperatively (P = 0.12). No sensorineural hearing loss, tinnitus, or imbalance was observed.

Conclusion

We demonstrated hearing improvement after lateral semicircular canal fenestration. This technique can be considered as an alternative for patients with middle ear anomalies who are not candidates for ossicular reconstruction.     

Intra-abdominal administration of bevacizumab diminishes intra-peritoneal adhesions

Aim

To determine the effect of a single dose of bevacizumab on adhesion formation in the rat cecum abrasion model.

Methods

The cecum and parietal peritoneum of 38 male Wistar rats were abraded to promote adhesion formation. The rats were randomized into 2 groups: group 1 received bevacizumab (2.5 mg/kg) intraperitoneally, and group 2 received saline. On day 30 animals were killed, adhesions scored, and histopathological samples taken.

Results

There was no wound dehiscence; there were 2 incision hernias (5.3%), 1 per group. Thirty-seven animals developed adhesions (97.4%). Adhesion grade and severity scores were significantly different between groups 1 and 2 at 2.7:1.6 (P = .018) and 3.8:2.7 (P = .007), respectively. There was no difference in adhesion square area (27.7:25.0%; P = .16), location (P = 1.00), or number (2.1:1.3; P = .06). Histopathology confirmed the statistical difference between groups (P = .049), and a highly significant correlation between results was shown (r = .758; P = .0001).

Conclusion

A single dose of intraperitoneal bevacizumab significantly reduces grade and severity of abdominal adhesions in the cecum abrasion rat model.     

Quantification of intraepithelial lymphocytes in normal pediatric small intestinal allograft and native ilea

Background

The small bowel acts as one of the first lines of defense against intraluminal infections and antigenic stimuli. Pediatric small bowel transplant patients are at particular risk from such agents, especially viral enteridities. Quantification of intraepithelial lymphocytes (IEL) in architecturally normal small intestinal mucosal biopsies plays an important role in the diagnosis of conditions such as celiac disease and some viral infections. No studies to date have been done to quantify IEL numbers in pediatric small bowel allografts and in native pediatric ilea. Our study investigated the IEL:enterocyte (EC) ratio in pediatric allograft and native ilea.

Methods

Hematoxylin and eosin slides from 50 surveillance endoscopic biopsies of small bowel allografts taken from patients <8 years of age and 50 terminal ileal biopsies from aged-matched control populations were reviewed. IEL:EC ratios were averaged from five well-oriented villi in each case. IEC numbers were compared between biopsies from proximal (afferent) and distal (efferent) limbs of double-barrel allograft stomas, as well as native terminal ilea.

Results

Within small bowel allografts, the average number of villus tip IELs was 1.3/20 ECs (standard deviation [SD] 0.6) in the proximal limb and 1.0/20 ECs in the distal (SD 0.6 P < .01). This value was significantly lower than in the control ilea (2.1/20 EC [SD 0.6]; P < .01, each). The overall distribution of lymphocytes was in a similar pattern throughout the villus with IEL:EC ratios on villus sides from proximal allografts, distal allografts, and native ilea being 1:20, 0.8:20, and 2:20, respectively.

Conclusions

The results suggest that approximately one to two IEL per 20 ECs at villus tips may represent a “normal” intraepithelial inflammatory cell population in small bowel allografts. The value is lower than that seen in age-matched native ileal biopsies. IEL numbers are significantly higher in the proximal limb compared to the distal limb of double-barrel stomas.