Podocyte-specific GLUT4-deficient mice have fewer and larger podocytes and are protected from diabetic nephropathy.

Podocytes are a major component of the glomerular filtration barrier, and their ability to sense insulin is essential to prevent proteinuria. Here we identify the insulin downstream effector GLUT4 as a key modulator of podocyte function in diabetic nephropathy (DN). Mice with a podocyte-specific deletion of GLUT4 (G4 KO) did not develop albuminuria despite having larger and fewer podocytes than wild-type (WT) mice. Glomeruli from G4 KO mice were protected from diabetes-induced hypertrophy, mesangial expansion, and albuminuria and failed to activate the mammalian target of rapamycin (mTOR) pathway. In order to investigate whether the protection observed in G4 KO mice was due to the failure to activate mTOR, we used three independent in vivo experiments. G4 KO mice did not develop lipopolysaccharide-induced albuminuria, which requires mTOR activation. On the contrary, G4 KO mice as well as WT mice treated with the mTOR inhibitor rapamycin developed worse adriamycin-induced nephropathy than WT mice, consistent with the fact that adriamycin toxicity is augmented by mTOR inhibition. In summary, GLUT4 deficiency in podocytes affects podocyte nutrient sensing, results in fewer and larger cells, and protects mice from the development of DN. This is the first evidence that podocyte hypertrophy concomitant with podocytopenia may be associated with protection from proteinuria.

Regional differences in islet distribution in the human pancreas--preferential beta-cell loss in the head region in patients with type 2 diabetes.

While regional heterogeneity in islet distribution has been well studied in rodents, less is known about human pancreatic histology. To fill gaps in our understanding, regional differences in the adult human pancreas were quantitatively analyzed including the pathogenesis of type 2 diabetes (T2D). Cadaveric pancreas specimens were collected from the head, body and tail regions of each donor, including subjects with no history of diabetes or pancreatic diseases (n = 23) as well as patients with T2D (n = 12). The study further included individuals from whom islets were isolated (n = 7) to study islet yield and function in a clinical setting of islet transplantation. The whole pancreatic sections were examined using an innovative large-scale image capture and unbiased detailed quantitative analyses of the characteristics of islets from each individual (architecture, size, shape and distribution). Islet distribution/density is similar between the head and body regions, but is >2-fold higher in the tail region. In contrast to rodents, islet cellular composition and architecture were similar throughout the pancreas and there was no difference in glucose-stimulated insulin secretion in islets isolated from different regions of the pancreas. Further studies revealed preferential loss of large islets in the head region in patients with T2D. The present study has demonstrated distinct characteristics of the human pancreas, which should provide a baseline for the future studies integrating existing research in the field and helping to advance bi-directional research between humans and preclinical models.

Remote processing of pancreas can restore normal glucose homeostasis in autologous islet transplantation after traumatic whipple pancreatectomy: technical considerations.

An emergency autologous islet transplant after a traumatic Whipple operation and subsequent total pancreatectomy was performed for a 21-year-old patient who was wounded with multiple abdominal gunshot wounds. After Whipple pancreatectomy, the remnant pancreas (63.5 g), along with other damaged organs, was removed by the surgeons at Walter Reed Army Medical Center (WRAMC) and shipped to Diabetes Research Institute (DRI) for islet isolation. The pancreas was preserved in UW solution for 9.25 h prior to islet isolation. Upon arrival, the organ was visually inspected; the pancreatic head was missing, the rest of the pancreas was damaged and full of blood; the tail looked normal. A 16-gauge catheter was inserted into the main duct and directed towards tail of the pancreas after the dissection of main duct in the midbody of the pancreas. The pancreas was distended with collagenase solution (Roche MTF) through the catheter. During 10 min of intraductal delivery of enzyme, the gland was distended uniformly. No leakage of the solution was observed. The pancreas was transferred to a Ricordi chamber for automated mechanical and enzymatic digestion. Islets were purified using a COBE 2991 cell processor. Islet equivalents (IEQ; 221,250) of 40% purity and 90% viability were recovered during the isolation, which were shipped back to WRAMC and infused by intraportal injection into the patient. Immediate islet function was demonstrated by the rapid elevation of serum C peptide followed by insulin independence with near normal oral glucose tolerance test (OGTT) 1 and 2 months later. It is possible to restore near normal glucose tolerance with autologous islet transplantation after total pancreatectomy even with suboptimal number of islets while confirming that islets processed at a remote site are suitable for transplantation.

The use of multiparametric monitoring during islet cell isolation and culture: a potential tool for in-process corrections of critical physiological factors.

Variables such as pH, pCO2, and PO2 have been established in the literature as critical factors that could affect the outcome of the islet cell processing and, therefore, the quality of the cells that could be transplanted. This report describes a highly accurate continuous multiparametric monitoring system and its evaluation for continuous monitoring of physiological variables during critical steps of the islet isolation procedure as well as during in vitro culture of the insulin-producing cells. Close monitoring of these variables could be of assistance to improve the outcome of islet cell processing, allowing to identify as soon as possible problems that could be corrected during the procedure, as well as during in vitro preservation, or shipment to remote sites.

Improved human islet isolation outcome from marginal donors following addition of oxygenated perfluorocarbon to the cold-storage solution.

Last year, from the approximately 6,000 organ donors, only approximately 1,500 pancreata were used for clinical transplantation. Factors that contribute to this poor pancreas use include strict donor selection criteria and the requirement for short cold-ischemia time (CIT). Numerous pancreata have not been used because of long ischemia times postprocurement. Given the oxygen-rich environment of the islets in the native pancreas, it is conceivable that islets are highly susceptible to irreversible damage following prolonged ischemia. The use of continuously oxygenated perfluorohydrocarbons (PFCs), known for their high oxygen-solubility coefficients, in a two-layer culture with standard University of Wisconsin preservation media, has extended the acceptable range CIT, and, furthermore, there has been no evidence of adverse effects from PFCs on the outcome of transplanted cells, whereas they often enhance islet cell function. The purpose of this study was to use the two-layer culture method to improve donor-organ use from marginal donors. Fifteen organs were procured using the two-layer method, and 18 without using it, from donors greater than 50 years of age. Despite nonsignificant differences in age, weight of the donors, weight of the organ and CIT, the PFC group yielded an average of twofold more islet equivalents than those harvested from the control group. As a result, from the control group, only 2 of 18 organs were used for clinical islet transplantation, whereas 8 of 15 were used from the PFC group. To this end, the two-layer method may help clinicians overcome the problem of organ underuse.