Klotho coreceptors inhibit signaling by paracrine fibroblast growth factor 8 subfamily ligands.

It has been recently established that Klotho coreceptors associate with fibroblast growth factor (FGF) receptor tyrosine kinases (FGFRs) to enable signaling by endocrine-acting FGFs. However, the molecular interactions leading to FGF-FGFR-Klotho ternary complex formation remain incompletely understood. Here, we show that in contrast to αKlotho, ßKlotho binds its cognate endocrine FGF ligand (FGF19 or FGF21) and FGFR independently through two distinct binding sites. FGF19 and FGF21 use their respective C-terminal tails to bind to a common binding site on ßKlotho. Importantly, we also show that Klotho coreceptors engage a conserved hydrophobic groove in the immunoglobulin-like domain III (D3) of the "c" splice isoform of FGFR. Intriguingly, this hydrophobic groove is also used by ligands of the paracrine-acting FGF8 subfamily for receptor binding. Based on this binding site overlap, we conclude that while Klotho coreceptors enhance binding affinity of FGFR for endocrine FGFs, they actively suppress binding of FGF8 subfamily ligands to FGFR.

Dietary and genetic evidence for enhancing glucose metabolism and reducing obesity by inhibiting klotho functions.

Klotho is a multifunctional protein involved in numerous biological functions, ranging from mineral ion metabolism to signaling activities. Recent studies have identified klotho as a target gene for peroxisome proliferator-activated receptor-γ (PPAR-γ), a master regulator of adipocyte differentiation, and an adipogenesis-promoting factor. In a similar line of observation, eliminating klotho function from mice resulted in the generation of lean mice with almost no detectable fat tissue. In contrast to the klotho-knockout mice (11.7±0.3 g at 9 wk), leptin-deficient (ob/ob) mice are severely obese (49.3±0.6 g at 9 wk), due to excessive fat accumulation. To study the in vivo role of klotho in obesity, we have generated and characterized ob/ob mice lacking klotho activity [ob/ob-klotho double-knockout (DKO) mice]. The ob/ob mice started to get bigger from 3 wk onward and gained almost 2 times more weight than their wild-type (WT) counterparts (WT vs. ob/ob: 34.8±1.3 vs. 65.5±1.2 g at 21 wk). The generated ob/ob-klotho DKO mice were not only viable throughout their adulthood but also showed markedly reduced fat tissue accumulation compared to their ob/ob littermates. The ob/ob-klotho DKO mice had significantly (P<0.01) less retroperitoneal, mesenteric, and epididymal fat accumulation, compared to their ob/ob counterparts. Similarly, the fatty liver that was consistently observed in the ob/ob mice was eliminated in the ob/ob-klotho DKO mice. Such structural improvement in the liver was also evident from markedly reduced fasting blood glucose levels in ob/ob-klotho DKO mice, compared to their ob/ob counterparts (ob/ob vs. ob/ob-klotho DKO: 266 ± 36 vs. 65±2 mg/dl). Finally, to study whether the absence of klotho can induce resistance to high-fat-diet-induced obesity, we provided a high-fat (60%) diet to klotho-knockout mice and compared them with normal-fat (20%) diet-fed klotho-knockout mice. No significant difference in body weight was detected in klotho-knockout mice fed either the normal-fat diet or high-fat diet, while WT mice fed the high-fat diet gradually gained body weight, compared to the normal-fat-diet-fed counterparts. The results of our dietary and genetic manipulation studies provide in vivo evidence for a role of klotho in obesity and offer a novel target to manipulate obesity and associated complications.

Long-term outcomes and the quality of life of Hirschsprung disease in adolescents who have reached 18 years or older--a 47-year single-institute experience.

BACKGROUND AND AIMS: Hirschsprung disease is considered to be curable when performing definitive surgery. However, the postoperative bowel function is not always satisfactory. The long-term postoperative function of bowel evacuation and the quality of life of such patients are considered to be important. In this study, we evaluated the general condition, bowel function, and social performance in adults who were older than 17 years who had suffered from Hirschsprung disease in childhood. METHODS: From 1963 to 2009, 184 patients with Hirschsprung disease underwent definitive surgery, mostly Z-shaped anastomosis at Kyushu University Hospital. As a result, 146 (95.4%) of 153 of those patients survived and reached 17 years of age. Their present status and symptoms, anorectal functions, genitourinary functions, and social performance were evaluated during the clinical follow-up based on a questionnaire survey. RESULTS: In our series, the evacuation score was rated as "excellent" (score of 7-8/8) in 66.7%, "good" (5-6/8) in 19.0%, "fair" (3-4/8) in 11.9%, and "poor" (0-2/8) in 2.4%. Therefore, 85.7% were considered to have a satisfactory bowel function. However, only 21.4% had a completely normal score of 8/8. Incontinence occurred in 16.7%, and soiling was present in 19.0% of the questionnaire respondents. The genitourinary function was considered to be within the reference range, and urinary problems were minimal. Among the respondents, 45.2% were married, and 68.4% of those married had children. The educational and professional careers of the respondents were successful. CONCLUSIONS: In general, the bowel function was satisfactory. However, the ratio of patients with completely normal bowel function was low. Because incontinence and soiling impair the quality of life, pediatric surgeons should therefore continue trying to achieve a complete bowel function after definitive surgery for the treatment of Hirschsprung disease.

Effectiveness of basic endoscopic surgical skill training for pediatric surgeons.

PURPOSE: Pediatric surgeons require highly advanced skills when performing endoscopic surgery, but their experience with such cases tend to be limited in comparison to general surgeons. The aim of this study was to evaluate the effectiveness of basic endoscopic surgery training for less-experienced young pediatric surgeons and then compare their skills with those of general surgeons. METHODS: The surgeons (n = 477) subjected to this study underwent a 2-day endoscopic skill training program, consisting of lectures, box training, VR simulator training, tissue training, and live tissue training. The trainees were divided into two groups: P (pediatric surgeons, n = 33) and G (general surgeons, n = 444). The trainees were required to make a continuous suture along a circle measuring 2.5 cm in diameter and the findings were evaluated both before and after training. A statistical analysis was conducted using the unpaired t test. RESULTS: The number of experienced cases totaled 20.8 ± 23.9 in P and 60.6 ± 80.5 in G (p < 0.001). The number of completed sutures before training was 1.4 ± 1.1 in P and 1.9 ± 1.5 in G (p < 0.05). The number of completed sutures after training was 4.1 ± 1.3 in P and 3.9 ± 1.9 in G (p > 0.05). The economy and speed of the forceps improved, however, the number of errors increased. CONCLUSION: Less-experienced pediatric surgeons improved their surgical skill and ability until reaching almost the same level as that observed in more experienced general surgeons during training, however, the number of errors after training increased in comparison to before training. As a result, this program needs to be modified to reduce the number of errors while enabling pediatric surgeons to master the safe and precise surgical techniques needed in this field.

Direct vasodilative effect of FK506 on porcine mesenteric artery in small bowel transplantation.

BACKGROUND: Tacrolimus (FK506) is widely used as an immunosuppressive drug in small bowel transplantation. However, its precise effects on the vascular tone of the transplanted organ have not been studied. This study aimed to clarify the effects of FK506 on the porcine mesenteric artery. METHODS: The effects of FK506 on the changes in cytosolic Ca(2+) concentration ([Ca(2+)]i) and force using fura-2 fluorometry were investigated in mesenteric arterial strips of the porcine small intestine. The effects of FK506 on the activity of voltage-dependent Ca(2+) channels and receptor-operated Ca(2+) channels using high K(+) (118 mmol/L K(+)) depolarization and thromboxane A(2) analog (U46619) stimulation were also examined. RESULTS: FK506 inhibited the force development induced by 118 mmol/L K(+) depolarization and 1 micromol/L U46619 stimulation in a concentration-dependent manner. The extent of inhibition of this contraction was greater than that of the K(+)-induced contraction, and its inhibitory potency was about 10-fold. FK506 (10 micromol/L) inhibited the increases in [Ca(2+)]i (24.9% +/- 7.4%) and the force development (52.0% +/- 5.6%) induced by 1 micromol/L U46619, respectively. CONCLUSIONS: FK506 induces arterial relaxation by decreasing [Ca(2+)]i. Pretreatment of a graft with FK506 may reduce the risk of vasospasm, ischemia-reperfusion injury, and thrombosis in small bowel transplantation.

Clinical features of Hirschsprung's disease associated with Down syndrome: a 30-year retrospective nationwide survey in Japan.

PURPOSE: To identify the clinical features in diagnosis and treatment of Hirschsprung's disease (HD) associated with Down syndrome (DS), the authors retrospectively analyzed data for patients with DS from the past 3 nationwide surveys in Japan. This survey was already approved by the Japanese Society of Pediatric Surgeons. METHODS: Patient data were collected in 3 phases-phase I (1978-1982), n = 47; phase II (1988-1992), n = 79; and phase III (1998-2002), n = 90. In total, data on 216 patients (5.6%) of 3852 were collected and analyzed. RESULTS: The incidence of DS in patients with HD was 2.9%, 7.1%, and 8.2% in phases I, II, and III, respectively, with a corresponding male/female ratio of 5:1, 2.4:1, and 5:1. The ratio of the extent of aganglionosis was nearly consistent across all phases. In phases I, II, and III, the incidence of total colonic aganglionosis was 2.1%, 0%, and 2.2%; and that of cardiovascular anomalies, 36.1%, 45.6%, and 55.6%; and that of preoperative enterocolitis, 31.0%, 26.6%, and 24.4%. The 2 most common surgical procedures were the Soave procedure, including transanal endorectal pull-through, and Duhamel procedure including Z-shaped anastomosis. The mortality rate decreased over time, from 26.1% in phase I to 11.4% in phase II and 7.8% in phase III. Almost all mortality cases were associated with cardiovascular anomalies: 54.5%, 62.5%, and 85.7% in phases I, II, and III, respectively. CONCLUSIONS: The incidence of HD with DS has increased over time. The number of male patients and cardiac anomalies has also increased in the last 10 years. Total colonic aganglionosis was rare. A marked decrease in the overall mortality rate was observed.

Mechanism of Rho-kinase-mediated Ca2+-independent contraction in aganglionic smooth muscle in a rat model of Hirschsprung's disease.

PURPOSE: Lack of ganglion cells is the main cause of bowel movement disorder in Hirschsprung's disease. Because smooth muscle is the primary organ, the properties of intestinal smooth muscle need to be investigated. We therefore investigated the reactivity of the contractile system and the mechanism of contraction in aganglionic intestinal smooth muscle. METHODS: Colonic smooth muscle strips from endothelin-B receptor gene-deficient [EDNRB(-/-)] rats were loaded with the Ca(2+) indicator dye fura-PE3/AM and changes in fluorescence intensity were monitored. The intracellular calcium concentration ([Ca(2+)]i) and force development in the strips were measured simultaneously. RESULTS: The force induced by 10 microM substance P (SP) was higher than that induced by 60 mM K(+) depolarization (control), whereas [Ca(2+)]i elevation induced by 10 microM SP was less than that induced by 60 mM K(+) in all segments. Pretreatment with the Rho-kinase inhibitor Y-27632 inhibited force development more strongly in EDNRB(-/-) aganglionic segments than in EDNRB(+/+) ganglionic segments. However, [Ca(2+)]i was higher in EDNRB(-/-) aganglionic segments than in EDNRB(+/+) ganglionic segments. CONCLUSIONS: The Ca(2+)-independent pathway involving Rho-kinase was hyperactivated in EDNRB(-/-) aganglionic segments. This phenomenon is assumed to compensate for Ca(2+) channel downregulation and Ca(2+)-dependent contraction. From a clinical point of view, the motility of aganglionic intestine would be controllable with the control of Ca(2+)-independent contraction before definitive operations in Hirschsprung's disease.

Total colonic aganglionosis with or without small bowel involvement: a 30-year retrospective nationwide survey in Japan.

PURPOSE: We conducted a nationwide survey in Japan, to identify recent trends in the diagnosis and treatment of patients who had total colonic aganglionosis with or without small bowel involvement (TCSA). These data were compared with 2 similar studies conducted within the last 30 years. METHODS: Patient data were collected in 3 phases; phase I included 135 patients between 1978 and 1982; phase II, 107 patients between 1988 and 1992; and phase III, 101 patients between 1998 and 2002. RESULTS: The incidence of TCSA was 1:59,059, 1:58,084, and 1:58,375, and the male-female ratios were 1.5:1, 1.5:1, and 2.2:1 in each phase, respectively. Patients with associated anomalies increased from 15.2% (phase I) to 22.8% (phase III). The incidence of preoperative enterocolitis decreased over time. For treatment, Duhamel's procedure and ascending colon patch methods have increased over time, whereas Martin's procedure has decreased. The mortality rate dropped from 40.9% to 15.8%; however, a high mortality rate persists in those cases with small bowel involvement (35.5%). CONCLUSIONS: A marked decrease in the overall mortality rate was observed during the study period. However, further efforts are still required especially in cases involving aganglionosis extending orally to 75 cm from Treitz's band.

Intracellular calcium mobilization of the aganglionic intestine in the endothelin B receptor gene-deficient rat.

BACKGROUND AND AIM: Up to now, numerous reports have analyzed the pathogenesis of Hirschsprung's disease (HD) by means of physiologic, pathologic, or molecular biologic methods. However, very little is still known about the smooth muscle cell itself. The endothelin B receptor gene-deficient (EDNRB(-/-)) rat, which is suitable for research of HD, has an aganglionic segment of the total colon. Our purpose is to investigate the myogenic mechanisms using simultaneous measurements of the intracellular Ca2+ concentration ([Ca2+]i) and tension and reverse transcriptase polymerase chain reaction for L-type Ca2+ channel (L-VOC) expression. METHODS: The muscle strips of the rat distal colon were loaded with a Ca2+ indicator dye, fura-PE3/AM, for 3 to 4 hours. The changes in the fluorescence intensity of Ca2+-fura-PE3 complex of the strips were monitored with a front surface fluorometer (CAM-230). The fluorescence intensities at 340- and 380-nm excitation and their ratio (F340/F380) were recorded as the level of [Ca2+]i. The comparison of L-VOC alpha1c subunit messenger RNA (mRNA) expression in both wild and homozygous rat was performed by reverse transcriptase polymerase chain reaction. RESULTS: The peak levels of force development induced by carbachol were 139.1% +/- 5.0% in EDNRB(-/-) rat, whereas the peak levels were 242.1% +/- 27.7% in EDNRB(+/+) rat. The changes in the [Ca2+]i elevation induced by carbachol were 101.7% +/- 12.2% in the homozygous rat, whereas these were 143.8% +/- 8.9% in the wild-type rat. Both results in the homozygous rat significantly decreased in comparison with those of the wild rat (P < .05). The expression of the L-VOC channel mRNA also decreased in the homozygous rat. CONCLUSIONS: This is the first report to show the [Ca2+]i mobilization in the smooth muscles of the rat model of HD. The decrease in both [Ca2+]i and force development was thus considered to be due to the decrease in the Ca2+ channel expression.

Morphological and physiological changes of interstitial cells of Cajal after small bowel transplantation in rats.

Intestinal dysmotility has been reported to be associated with a decreased number of interstitial cells of Cajal (ICCs). However, the chronological changes in ICCs after small bowel transplantation (SBT) have not yet been elucidated. In this study, we aimed to evaluate the chronological change of ICCs after SBT. Orthotopic syngeneic SBT was performed in rats. Graft specimens were obtained at postreperfusion, and on 1, 3, 7, 14, and 30 postoperative day (POD). Thereafter, immunohistochemical staining was performed and the spontaneous contractions measured. During the initial period after SBT, the temporal impairment of ICCs was found. In an immunohistochemical study, c-Kit-positive cells appeared to decrease on POD 0, 1, and 3. Thereafter, the number of cells increased gradually up to POD 7. In contrast, the recovery of the spontaneous contractile amplitude took more time. The frequency of the electrical signal was preserved at almost exactly the same levels throughout this experimental period. Although the network of ICCs was found to be temporarily impaired after SBT in an immunohistochemical examination, this change was reversible. Moreover, the recovery of the function of the intestinal motility associated with ICCs was delayed after the early postoperative period.

New classification of hypoganglionosis: congenital and acquired hypoganglionosis.

AIM: Hypoganglionosis has been associated with fewer intestinal ganglion cells. However, current reports questioned the validity of this clinical entity. The aim of this study is to demonstrate the existence of hypoganglionosis. MATERIALS AND METHODS: We have experienced 24 cases of functional intestinal obstruction with abnormalities of the intestinal ganglia. A precise histological examination was performed using quantitative morphometric studies. These results were compared with age-matched controls (n = 13). RESULTS: Based on histological examination, disorders in the ganglion cells could be classified into 3 categories: immaturity of ganglia (n = 13), congenital hypoganglionosis (n = 7), and acquired hypoganglionosis (n = 4). In congenital hypoganglionosis, the number as well as the size of ganglion cells are small at birth. The size of ganglion cells tends to increase over time, but their numbers do not increase; as a result, the symptoms of dysmotility do not improve. On the other hand, acquired hypoganglionosis is late onset and characterized as a degeneration of ganglion cells and gliosis histologically. After performing a resection of the affected bowel, the prognosis is usually good. CONCLUSION: Congenital and acquired hypoganglionosis are 2 distinct entities. The histological findings as well as the clinical characteristics of these 2 types of hypoganglionosis are different.