Cdc42 Deficiency Causes Ciliary Abnormalities and Cystic Kidneys

Ciliogenesis and cystogenesis require the exocyst, a conserved eight-protein trafficking complex that traffics ciliary proteins. In culture, the small GTPase Cdc42 co-localizes with the exocyst at primary cilia and interacts with the exocyst component Sec10. The role of Cdc42 in vivo, however, is not well understood. Here, knockdown of cdc42 in zebrafish produced a phenotype similar to sec10 knockdown, including tail curvature, glomerular expansion, and mitogen-activated protein kinase (MAPK) activation, suggesting that cdc42 and sec10 cooperate in ciliogenesis. In addition, cdc42 knockdown led to hydrocephalus and loss of photoreceptor cilia. Furthermore, there was a synergistic genetic interaction between zebrafish cdc42 and sec10, suggesting that cdc42 and sec10 function in the same pathway. Mice lacking Cdc42 specifically in kidney tubular epithelial cells died of renal failure within weeks of birth. Histology revealed cystogenesis in distal tubules and collecting ducts, decreased ciliogenesis in cyst cells, increased tubular cell proliferation, increased apoptosis, increased fibrosis, and led to MAPK activation, all of which are features of polycystic kidney disease, especially nephronophthisis. Taken together, these results suggest that Cdc42 localizes the exocyst to primary cilia, whereupon the exocyst targets and docks vesicles carrying ciliary proteins. Abnormalities in this pathway result in deranged ciliogenesis and polycystic kidney disease.Cilia are thin rod-like organelles, found on the surface of many eukaryotic cells, with complex functions in signaling, cell differentiation, and growth control. Cilia extend outward from the basal body, a cellular organelle related to the centriole. In kidney cells, a single primary cilium projects from the basal body, is nonmotile, and exhibits an axoneme microtubule pattern of 9+0. In the mammalian kidney, primary cilia have been observed on renal tubule cells in the parietal layer of the Bowman capsule, the proximal tubule, the distal tubule, and in the principal, but not intercalated, cells of the collecting duct.¹Multiple proteins that, when mutated, result in the development of polycystic kidney disease (PKD) have been localized to renal primary cilia. These include polycystin-1 and -2, the causal proteins in autosomal dominant PKD (ADPKD) (reviewed by Smyth et al.²). Research into pkd2 function in zebrafish has further strengthened the idea that polycystin-2 functions in cilia. Knockdown of pkd2 by morpholino (MO)^3–5 or in mutants^5,6 produces phenotypes that are consistent with a role in cilia function, such as curved tails, pronephric cysts, and edema.Although we are beginning to identify the roles ciliary proteins play in diverse biologic processes, relatively little is known about how these proteins are transported to the cilium.⁷ The exocyst, originally identified in Saccharomyces cerevisiae,⁸ is a highly conserved 750-kD eight-protein complex known for the targeting and docking of vesicles carrying membrane proteins.⁹ It is composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 (also known as EXOC1–8).¹⁰ Notably, in addition to being found near the tight junction, exocyst proteins were localized to the primary cilium in kidney cells.^11,12 Sec10 and Sec15 are the most vesicle-proximal of the exocyst components. Sec10 directly binds to Sec15, which, in turn, directly binds Sec4/Rab8, a Rab GTPase found on the surface of transport vesicles. Sec10 then acts as a “linker” by binding the other exocyst components through Sec5.¹³ Our previous studies suggested that the exocyst would no longer be able to bind Sec15, and thereby target/dock transport vesicles, without Sec10, and would, instead, disintegrate and be degraded. Importantly, we showed that knockdown of Sec10 in Madin-Darby canine kidney (MDCK) cells abrogated ciliogenesis, while Sec10 overexpression enhanced ciliogenesis. Furthermore, Sec10 knockdown caused abnormal cystogenesis when the cells were grown in a collagen matrix and decreased the levels of other exocyst components and the intraflagellar transport protein 88. This was in contrast to knockdown of exocyst components Sec8 and Exo70, which had no effect on ciliogenesis, cystogenesis, or levels of other exocyst components.¹² On the basis of these data, and its known role in trafficking proteins to the plasma membrane,^14–17 we proposed that Sec10 and the exocyst are required to build primary cilia by targeting and docking vesicles carrying ciliary proteins.A possible mechanism to target the exocyst to nascent primary cilia, so it can participate in ciliogenesis, is through the Par complex. We previously showed that the exocyst co-localizes with Par3¹² and directly interacts with Par6,¹⁸ both components of the Par complex, which also includes atypical PKC. Cdc42 is associated with the Par complex.^19,20 In addition to their well studied function at cell-cell contacts, the Par complex has been immunolocalized to primary cilia and is necessary for ciliogenesis.^21,22 The exocyst is regulated by multiple Rho and Rab family GTPases (reviewed by Lipschutz and Mostov⁹), including Cdc42, which regulates polarized exocytosis via interactions with the exocyst in yeast.²³ Using inducible MDCK cell lines that express constitutively active or dominant negative forms of Cdc42,^24,25 we established that Cdc42 is centrally involved in three-dimensional collagen gel cystogenesis and tubulogenesis.²⁶ Whether and how Cdc42 might participate in ciliogenesis and cooperate with the exocyst in ciliary membrane trafficking are open questions.Toward this end, we showed, in cell culture, that Cdc42 co-immunoprecipitated and co-localized with Sec10 and that Cdc42 was necessary for ciliogenesis in renal tubule cells, in that Cdc42-dominant negative expression, small hairpin RNA knockdown of Cdc42, and small hairpin RNA knockdown of Tuba, a guanine nucleotide exchange factor (GEF) for Cdc42, all inhibited ciliogenesis. Exocyst Sec8 and polycystin-2 also no longer localized to the primary cilium, or the ciliary region, after Cdc42 and Tuba knockdown.¹⁸ As noted, we showed that Sec10 directly binds to Par6, and others have shown that Cdc42 also directly binds to Par6.^27,28 Knockdown of both Sec10²⁹ and Cdc42 increased mitogen-activated protein kinase (MAPK) activation.¹⁸Here, using two different living organisms, we confirm and extend our in vitro findings. We show that cdc42 knockdown in zebrafish phenocopies many aspects of sec10 and pkd2 knockdown—including curved tail, glomerular expansion, and MAPK activation—suggesting, in conjunction with our previous data,^12,18,29 that cdc42 may be required for sec10 (and possibly pkd2) function in vivo. Other ciliary phenotypes include hydrocephalus and loss of photoreceptor cilia. We also demonstrate a synergistic genetic interaction between zebrafish cdc42 and sec10 for these cilia-related phenotypes, indicating that cdc42 and sec10 function in the same pathway. Demonstrating that the phenotypes were not due to off-target effects from the cdc42 MOs, we rescued the phenotypes with mouse Cdc42 mRNA. Cdc42 kidney-specific knockout mice died of kidney failure within weeks of birth; histologic examination revealed cystogenesis in distal tubules and collecting ducts and decreased ciliogenesis in cyst cells. Cdc42 conditional knockout kidneys showed increased tubular epithelial cell proliferation, increased apoptosis, increased interstitial fibrosis, and MAPK pathway activation, all features of the nephronophthisis form of PKD. These data, along with our previously published results, support a model in which Cdc42 localizes the exocyst to the primary cilium, whereupon the exocyst then targets and docks vesicles carrying proteins necessary for ciliogenesis; if this does not occur, the result is abnormal ciliogenesis and PKD.     

Intra‐operative cardiac arrests during liver transplantation – a retrospective review of the first 15 yr in Asian population

Although many report intra‐operative cardiac arrests (ICAs) in liver transplantation (LT), the incidence, major causes, and outcome remain unclear. We aimed to investigate retrospectively, the incidence, nature, and outcome of ICA in Asian population and to identify risk factors for ICA. Consecutive 1071 LTs in an institution during 1996–2011 (adult 920, pediatric 151/living donor liver transplantation, LDLT 841, deceased donor liver transplantation, DDLT 230) were reviewed. ICA occurred in 14 adult LTs (1.5%), but none in pediatrics. ICA occurred 1.0% and 3.3% in LDLT and DDLT, respectively. Stages of ICA incidence were three at pre‐anhepatic, one at anhepatic, and 10 at neohepatic stage. Post‐reperfusion syndrome (PRS) with hyperkalemia and bleeding were the major causes of ICA. While LDLT showed miscellaneous causes for ICA at various stages, DDLT incurred ICAs at neohepatic stage only. Interestingly, we did not find pulmonary thromboembolism (PTE) to incur ICA. Risk factor analysis showed no association of pre‐operative patient condition, donor types, and intra‐operative parameters. In this review, the incidence of ICA was low in Asian population with LDLT predominance, and while PTE was not the cause of ICA, the neohepatic stage with PRS and bleeding was the most vulnerable period to anticipate ICA.     

Role of Cholecystectomy and Lymph Node Dissection in Patients with T2 Gallbladder Cancer

Background

Incidental findings of gallbladder cancer (GBCA) have dramatically increased as an initial presentation of the disease because of the expansion of laparoscopic cholecystectomy. However, the optimal management of T2 GBCA remains at issue.

Methods

We compared our 10-year experience with the consensus surgical strategy for T2 GBCA. Between January 2000 and December 2009, 70 patients at Severance Hospital, Yonsei University Health System, Seoul, Korea, underwent surgical treatment for GBCA stage T2. The medical records of 70 patients with T2 GBCA were retrospectively reviewed.

Results

Radical cholecystectomy was performed on only 32 (45.8 %) patients. In patients with T2 GBCA and positive lymph nodes (LN), the overall survival rate between cholecystectomy with LN dissection and radical cholecystectomy did not show a significant difference. Twenty patients experienced recurrence during the follow-up period. Among the 11 patients who underwent cholecystectomy with liver resection, only 2 (18.2 %) patients had an intrahepatic recurrence. Of the 9 patients who underwent cholecystectomy without liver resection, 3 (33.3 %) patients had an intrahepatic recurrence. However, recurrences at the gallbladder bed occurred only in one and two patients, respectively, and were not significantly different between the two groups.

Conclusions

There was a large gap between clinical practice and treatment guidelines. Though relatively few patients enrolled in this study experienced recurrence, cholecystectomy and LN dissection without liver resection showed similar survival and recurrence patterns compared with those of radical cholecystectomy. To improve consistency between clinical practice and consensus guidelines, the role of limited resection for T2 lesions needs further evaluation.     

Efficacy of endoscopic mucosal resection using a dual-channel endoscope compared with endoscopic submucosal dissection in the treatment of rectal neuroendocrine tumors

Background

Conventional endoscopic mucosal resection (EMR) for removing rectal neuroendocrine tumors (NETs) has a high risk of incomplete removal because of submucosal tumor involvement. EMR using a dual-channel endoscope (EMR-D) may be a safe and effective method for resection of polyps in the gastrointestinal tract. The efficacy of EMR-D in the treatment of rectal NET has not been evaluated thoroughly.

Methods

From January 2005 to September 2011, a total of 70 consecutive patients who received EMR-D or endoscopic submucosal dissection (ESD) to treat a rectal NET <16 mm in diameter were included to compare EMR-D with ESD for the treatment of rectal NETs.

Results

The EMR-D group contained 44 patients and the ESD group contained 26 patients. The endoscopic complete resection rate did not differ significantly between the EMR-D and ESD groups (100 % for each). The histological complete resection rate also did not differ significantly between groups (86.3 vs. 88.4 %). The procedure time was shorter for the EMR-D group than for the ESD group (9.75 ± 7.11 vs. 22.38 ± 7.56 min, P < 0.001). Minor bleeding occurred in 1 EMR-D patient and in 3 ESD patients (2.3 vs. 7.6 %). There was no perforation after EMR-D or ESD.

Conclusions

Compared with ESD, EMR-D is technically simple, minimally invasive, and safe for treating small rectal NETs contained within the submucosa. EMR-D can be considered an effective and safe resection method for rectal NETs <16 mm in diameter without metastasis.     

Argon plasma coagulation is safe and effective for treating smaller gastric lesions with low-grade dysplasia: a comparison with endoscopic submucosal dissection

Background and aims

The best therapeutic modality has not been established for gastric low-grade adenomas or dysplasia (LGD), which can progress to invasive carcinoma despite a low risk. This study aims to investigate the clinical efficacy, safety, and local recurrence after argon plasma coagulation (APC) treatment of gastric LGD compared with endoscopic submucosal dissection (ESD).

Patients and methods

A total of 320 patients with gastric LGD ≤2.0 cm treated with APC or ESD between 2004 and 2011 were retrospectively analyzed. We compared local recurrence rate, complication rate, procedure time, and admission to hospital between APC and ESD groups.

Results

Of the 320 patients, 116 patients were treated with APC and 204 with ESD. During follow-up, local recurrence was more common in the APC group (3.8 %, 4/106) than the ESD group (0.5 %, 1/188; log-rank test P = 0.036). However, all patients with local recurrence (n = 5) were treated by additional APC, and followed up without further recurrences. ESD was complicated by two perforations (1.0 %, 2/204) compared with no perforations in the APC group (0 %, 0/116). Bleeding complications were not different between the APC (1.7 %, 2/116) and ESD (2.0 %, 4/204) groups. Procedure time was shorter in the APC (7.8 ± 5.1 min) than the ESD (53.1 ± 38.1 min) group (P < 0.001). The proportion of hospitalization was less in the APC group (31.0 %, 36/116) than the ESD group (100.0 %, 204/204) (P < 0.001).

Conclusions

APC can be a good treatment option for patients with LGD ≤2.0 cm.     

Comparing the short-term outcomes of totally intracorporeal gastroduodenostomy with extracorporeal gastroduodenostomy after laparoscopic distal gastrectomy for gastric cancer: a single surgeon’s experience and a rapid systematic review with meta-analysis

Background

Since delta-shaped gastroduodenostomy was introduced, many surgeons have utilized laparoscopic distal gastrectomy (LDG) with totally intracorporeal Billroth I (ICBI) for gastric cancer, because it is expected to have several advantages over laparoscopic-assisted distal gastrectomy with extracorporeal Billroth I (ECBI). In this study, we compared these two reconstruction options to evaluate their outcomes.

Methods

The data of 166 gastric cancer patients who underwent LDG performed by a single surgeon between April 2009 and February 2012 were analyzed retrospectively. The subjects were divided into ECBI (n = 106) and ICBI (n = 60) groups, and then the clinical characteristics, surgical outcomes, symptoms, and change in BMI at 3 months after surgery were compared. Furthermore, a rapid systematic review and meta-analysis were conducted.

Results

The operative time was significantly shorter in the ICBI group (197.4 ± 45.5 vs. 157.1 ± 43.9 min), but blood loss was similar between the groups. Regarding surgical outcomes, there were no significant differences in the length of hospital stay, soft diet initiation, visual analogue scale, frequency of analgesics injection, and postoperative white blood cell counts and C-reactive protein levels between the groups. The surgical complication rates were 5.7 and 13.3 % in the ECBI and ICBI groups, respectively, and one case of anastomosis leakage was observed in each group. At 3 months after surgery, reflux symptoms were more frequent in the ICBI group, but other gastrointestinal symptoms and the change of BMI were similar between the groups. The meta-analysis revealed no significant differences in the operative time, time to first flatus, length of hospital stay, frequency of analgesic usages, and rates of anastomosis complications between the groups.

Conclusions

We could not demonstrate the clinical superiority of ICBI over ECBI based on our data and a rapid systematic review and meta-analysis. The anastomosis method may be selected according to patient conditions and the surgeon’s preference.     

Endoscopic clip closure versus surgery for the treatment of iatrogenic colon perforations developed during diagnostic colonoscopy: a review of 115,285 patients

Background

Although the incidence of perforation after endoscopic procedures of the colon is low, the rising number of diagnostic colonoscopies could pose relevant health problems. Optimizing treatment may reduce the probability of severe complications. This study aimed to determine perforation frequency and the management of perforations that occurred during diagnostic colonoscopy.

Methods

A retrospective review of patient records was performed for all patients with iatrogenic colonic perforations after sigmoidoscopy/colonoscopy from 2000 to 2011 in three institutions of The Catholic University of Korea. The patients’ demographic data, endoscopic procedure information, perforation location, therapy, and outcomes along with different therapeutic strategies were recorded.

Results

In the 12-year period, a total of 115,285 diagnostic sigmoidoscopic/colonoscopic procedures were performed. A total of 27 perforations occurred. Sixteen patients underwent endoscopic clipping, of which three patients failed and were referred for surgery. Fourteen patients in total underwent surgery for perforation. Endoscopic clip closure was successful in 81 % of the patients. No perforation-related major morbidity or mortality occurred.

Conclusion

Endoscopic repair using clips can be effective for the treatment of colon perforations that occur during diagnostic colonoscopy.     

Which should go first during same-day upper and lower gastrointestinal endoscopy? A randomized prospective study focusing on colonoscopy performance

Background

Same-day bidirectional endoscopy is commonly performed in clinical practice. However, the optimal sequence of procedures for same-day bidirectional endoscopy has not been established. The purpose of this study was to compare colonoscopy performance and quality between patients who underwent colonoscopy before or after esophagogastroduodenoscopy (EGD).

Methods

A total of 1,103 patients were prospectively randomized into either the EGD–colonoscopy or colonoscopy–EGD sequence groups. Three patients who had incomplete cecal intubation due to structural obstruction were excluded from the analysis. During colonoscopy, colonoscopic parameters including difficult cecal intubation (cecal intubation failure and prolonged insertion), insertion time, and adenoma detection rate were measured. Out of 1,100 patients, 524 patients without sedation completed a questionnaire designed to assess subjective discomfort experienced.

Results

The colonoscopy completion rate was 99.5 %, and the rate of difficult cecal intubation was 14.5 %. The time from insertion to reaching the cecum (minutes:seconds, 06:32 ± 04:26 vs. 06:40 ± 04:09, p = 0.649), difficult cecal intubation ratio (76 of 550 vs. 83 of 550, p = 0.593), and colonoscopic adenoma detection rate (29.8 vs. 25.5 %, p = 0.106) did not differ between the groups. On multivariate analysis, difficulty with cecal intubation increased specifically in women, in patients aged 55 years and over, in patients with poor bowel preparation, and in patients who had undergone previous abdominal surgery. Subjective discomfort after EGD was higher in the colonoscopy–EGD sequence group.

Conclusions

The procedural sequence did not affect colonoscopy performance and quality in same-day bidirectional endoscopy, and factors such as old age, female gender, poor bowel preparation, and previous abdominal surgery were confirmed to adversely affect colonoscopy. In addition, the EGD–colonoscopy sequence induced less subjective discomfort during EGD.     

Endoscopic resection of granular cell tumors in the gastrointestinal tract: a single center experience

Background

The frequency of granular cell tumors (GCTs) identified in the gastrointestinal tract has recently increased with the increased use of routine endoscopy. Endoscopic treatment is increasingly used as an alternative to traditional surgical resection, but there are few reports on the efficacy, safety, and long-term prognosis of endoscopic treatment for GCTs. The aim of this study was to assess the efficacy, safety, and long-term prognosis of endoscopic resection for the gastrointestinal GCTs.

Methods

We examined a total of 27 GCTs in 25 patients who were treated by endoscopic resection from January 2007 to February 2011. For endoscopic resection, endoscopic mucosal resection (EMR) or endoscopic submucosal dissection (ESD) was used.

Results

Twenty GCTs were located in the esophagus, 5 in the stomach, and 2 in the colon. The median size of the GCTs was 10 mm; the largest size, located in the ascending colon, measured 18 mm. EMR with a ligation device was performed in 20 cases, conventional EMR in 5 cases, and ESD in 2 cases. En bloc resection was performed in 25 cases (92.6 %), and endoscopic complete resection piecemeal resection was achieved in 25 cases (92.6 %). Pathologic complete resection was achieved in 22 lesions (81.5 %). Intraprocedural bleeding was noted in three patients, with no occurrence of perforation or postprocedure stricture. No recurrence was observed during the mean follow-up period of 15 months (range 9–31 months).

Conclusions

Endoscopic resection appears to be a safe and effective treatment for GCTs in the gastrointestinal tract.