Modifiable factors related to 7-year renal outcomes in subjects with type 2 diabetes and chronic kidney disease stage 3.

BACKGROUND AND AIMS: Subjects with diabetes are prone to a rapid decline in renal function and major adverse cardiovascular events when they reach chronic kidney disease (CKD) stage 3. This study aimed to identify modifiable risk factors associated with the progression of CKD in this population. SETTINGS AND DESIGN: An observational cohort study. METHODS AND MATERIALS: A total of 320 type 2 diabetic patients with CKD stage 3 registered in the shared-care-system in our hospital in 2010 were regularly followed up for 7 years. Demographic, laboratory, medication, and fundus examination data of these subjects were collected and analyzed. STATISTICAL ANALYSIS USED: Cox regression was used to identify factors associated with changes in CKD stage. RESULTS: During the 7-year follow-up period, 204 cases (63.7%) remained at CKD stage 3 while 79 cases (24.7%) progressed to stage 4 or 5 and 37 cases (11.6%) improved to stage 1 or 2. The change in estimated glomerular filtration rate (eGFR) in the first 2 years and variations in glycated hemoglobin (HbA1c) over 7 years were independent factors of both progression (hazard ratio (HR) 1.098 and 1.710, respectively) and improvement (HR 0.919 and 0.231, respectively) of CKD stage. Variations in systolic blood pressure (SBP) was also found as an independent factor for progression of renal function (HR 1.052). CONCLUSIONS: Our results demonstrated that fluctuations in HbA1c and SBP, and changes in eGFR during the first 2 years of treatment were associated with the long-term renal outcomes in type 2 diabetic patients with CKD stage 3.

AMT, an inducible nitric oxide synthase inhibitor, enhances islet engraftment.

We co-transplanted silica gel-entrapping 4H-1,3-Thiazin-2-amine,5,6-dihydro-6-methyl monohydrochloride (AMT) with islets to evaluate the effects of AMT on early graft dysfunction in a syngeneic mouse model. The mean diameter of AMT-embedding silica gel particles was 595 +/- 275 nm. The cumulative release of AMT was 29% at 1 hour and 45% at 72 hours. Sixteen streptozotocin-induced diabetic mice were separated into 3 groups. Group A received 50 islets (n = 4). Group B received 50 islets and blank silica gel (n = 6). Group C received 50 islets plus silica-gel containing 6.4 microg AMT (n = 6). Mice in group C required significantly less time for temporary posttransplantation hyperglycemia than those in groups A and B (A, 39 +/- 7 vs B, 40 +/- 5 vs C, 24 +/- 2 days; P < .05). The insulin contents of grafts retrieved at 13 weeks were 1.17 +/- 0.11 (n = 4), 1.01 +/- 0.16 (n = 6), and 1.68 +/- 0.30 microg (n = 6) for mice in groups A, B, and C, respectively. Pancreatic remnant insulin did not differ significantly between the 3 groups (A, 0.32 +/- 0.04 [n = 4] vs B, 0.29 +/- 0.06 [n = 6] vs C, 0.40 +/- 0.05 microg [n = 6]; P > .05). In vitro study revealed that 4 and 20 nmol/L of sol-gel-embedded AMT protected 87% and 96% RIN-m5F cells from 1 ng/mL interleukin-1beta-mediated destruction, respectively. Silica-gel-entrapped AMT protects islet graft from a nonspecific inflammatory destruction, which is partly mediated via interleukin-1beta.

Hyperglycemia in vitro up-regulates growth-related cell cycle proteins of adult mouse pancreatic islets.

To study effects of glucose on growth-related proteins of adult islets, we cultured mice islets in medium containing either 5.5 mmol/L (LG) or 20 mmol/L (HG) glucose. Total islet proteins were processed for sodium dodecyl sulfate polyacrylamide gel and Western blotting using antibodies against beta-actin (housekeeping), p27kip1 (G1/G0 checkpoint), cyclin D1 (G1/S), cyclin B1 (G2/M), and FoxM1. At day 1, protein levels p27, B1, D1, and FoxM1 of islets on LG and HG were 0.48- and 0.63-fold; 7.09- and 11.58-fold; 1.25- and 1.38-fold; and 1.75- and 1.75-folds, the value of day 0, determinations respectively. At day 3, the proteins of p27, B1, D1, and FoxM1 of islets in LG and HG were 0.84- and 0.84-fold; 3.08- and 17.17-fold; 1.41- and 1.54-fold; and 0.83- and 1.17-fold of those on day 0, respectively. On day 7 the values were 1.19- and 1.09-fold; 3.15- and 14.81-fold; 0.86- and 1.44-fold; and 2.75- and 3.42-fold that of day 0, respectively. At day 1, the ratios of protein in islets after HG verse LG were 1.25, 2.38, 0.94, and 1.00 for p27, B1, D1, and FoxM1, respectively. At days 3 and 7, the protein ratios of HG/LG were 0.81 and 0.82, 5.47 and 2.64, 0.81 and 1.51, and 1.11 and 1.24 for p27, B1, D1, and FoxM1, respectively. In conclusion, adult mouse islets rapidly respond to cultivation by reducing p27 and increasing B1; HG attenuates p27 elevation but enhances B1 and D1 elevations, which favor islet entry into the cell cycle.

Enhancing engraftment of neonatal porcine xenoislet with CTLA4Ig and nordihydroguaiaretic acid.

This study examined the combinatory effect on graft survival of neonatal pig pancreatic cell clusters (NPCC) with nordihydroguaiaretic acid (NDGA), a 5-lipoxygenase inhibitor, with systemic CTLA4Ig expression, with local CTLA4Ig and with interleukin-1 (IL-1) receptor antagonist (IL-1ra) expression using a pig to mouse model. About 2000 NPCCs, which were infected with both adenoviruses carrying CTLA4Ig and IL1-1ra genes (each 500 pfu/NPCC), were transplanted beneath the kidney capsule of diabetic BALB/c mice. Two days before transplantation, the recipient mice were either injected with (group C, n = 4; group D, n = 6) or without (group A, n = 7; group B, n = 9) 1 x 10(13) pfu/kg body weight of adenovirus carrying the CTLA4Ig gene. Mice in groups B and D received daily injections of NDGA (20 mg/kg body weight) subcutaneously for 4 weeks. Blood glucose levels less than 200 mg/dL were defined to be normoglycemic and the transplant termed as a functioning graft for the purpose of calculating mean graft function time (MFT). Four weeks posttransplantation, an intraperitoneal glucose tolerance test (IPGTT) was performed to calculate the area under the curve (AUC). Blood glucose levels in groups C and D were significantly lower than groups A and B at 1, 2, and 3 weeks after transplantation. Graft MFT and AUC of IPGTT in group D were significantly different from those in groups A and B. Our data suggested that a high dosage of systemic expression of CTLA4Ig was effective to enhance xenograft survival and that in it was reinforced by a combination with the macrophage inhibitor NDGA.

Effects of insulin sensitizers on islet transplantation.

To solve the problems of islet engraftment, we investigated the effects of insulin sensitizers, metformin and rosiglitazone, on the in vitro and in vivo function of mouse islets. The in vitro study was done by culturing 30 isolated C57BL/6 mouse islets with glucose (100 or 300 mg/dL) or rosiglitazone (4.5 mumol/L) for 2, 4, 8, or 12 hours. The in vivo study was performed by syngeneically transplanting 150 C57BL/6 mouse islets under the kidney capsule of streptozotocin-diabetic mice. The metformin group was treated with 200 mg/kg/d in water and the control group was pair-fed the same volume of liquid diet. In the in vitro study, insulin release stimulated by 300 mg/dL glucose (n = 6) was the highest at all time points. That stimulated by rosiglitazone (n = 6) was greater than by 100 mg/dL glucose (n = 6) only at 8 hours. In the recipients treated with metformin (n = 17) and controls (n = 13), the blood glucose decreased and body weight increased gradually after transplantation. However, there was no significant difference between the two groups. Their tolerance to intraperitoneal glucose challenge at 2 and 4 weeks was also comparable. At 4 weeks, 12/17 (71%) in the metformin group and 8/13 (62%) in the control group achieved normoglycemia (P = .60). At 4 weeks, the insulin content of the graft was 8.35 +/- 3.42 mg in the metformin group and 5.28 +/- 4.28 mg in the control group (P = .59). Our data indicate that (1) rosiglitazone stimulated isolated islets to release insulin but was less effective than high levels of glucose; and (2) metformin treatment had no beneficial effect on islet recipients.

Islet transplantation at subcutaneous and intramuscular sites.

The subcutaneous site is ideal for clinical islet transplantation because it has the advantage of being accessible and can be biopsied when needed. Sadly, the results at subcutaneous sites were disappointing. The reason for this failure is not known, but poor vascularization may play a role. We tested the hypothesis that islet grafts would do better if more vasculature or oxygen could be supplied. Six hundred isolated C57BL/6 mouse islets were syngeneically transplanted into inbred streptozotocin-diabetic recipients at a subcutaneous site on the back with (Group A, n = 6) or without (Group B, n = 8) postoperative hyperbaric oxygen (2.4 ATA, 100% O(2)) therapy, or at a calf muscle (Group C, n = 9). During 13-week posttransplantation follow-up, recipients' blood glucose decreased and body weight increased significantly in all 3 groups (P < .05). However, there was no significant difference among the 3 groups. At 13 weeks, the insulin contents of the graft was also comparable among the 3 groups. Our data indicate the following: (1) postoperative hyperbaric oxygen therapy did not improve the outcome of islet transplantation at a subcutaneous site; and (2) a muscular site was not superior to a subcutaneous site for islet transplantation.

Effects of intrahepatic canine islet autotransplantation on the portal vein pressure and liver function.

Currently, the most common method used for human islet transplantation is intrahepatic implantation via the portal vein, which may affect portal vein pressure and liver function. The aim of this study was to investigate the effects of intrahepatic canine islet autotransplantation on portal vein pressure and liver function. After total pancreatectomy was performed in 30 mongrel dogs, islets were isolated and transplanted back into the portal vein of the same dog. In our series, 12 dogs achieved normoglycemia (fasting glucose <200 mg/dL) without exogenous insulin after transplantation. The portal vein pressure increased from 4.6 +/- 1.5 to 7.7 +/- 2.9 cm H(2)O after islet infusion (P < .05). Alanine transferase amino transferase (ALT) levels gradually increased after pancreatectomy with the peak at 4 weeks after islet infusion. But the changes of portal vein pressure and ALT were not significantly different between successful and failed islet transplantation. In summary, elevation of portal vein pressure and liver enzymes were noted after intrahepatic canine islet autotransplantation. However, they did not influence the transplant outcome.

A single-dose of cobalt-protoporphyrin protects islet beta cells from glucocorticoid suppression.

This study examined whether treating donor mice with a single-dose of cobalt protoporphyrin (CoPP) could induce heme oxygenase-1 (HO-1) and thus protect islet cells from suppression by high-dose glucocorticoid. Islets were isolated from mice receiving either a single dose of CoPP (20 mg/kg body weight) (CoPP-islets) or isotonic sodium chloride solution (control islets) at 24 hours before isolation. Following incubation in the absence or presence of methylprednisolone (100 and 1000 ng/mL) for 24 hours, glucose-stimulated insulin secretion and insulin content of cultured islets were determined. Data were expressed as the mean +/- standard error. HO-1 protein level of CoPP-islets was significantly higher than that of normal islets at 12 hours (P < .005) and 30 hours (P < .05) but not at 56 hours after CoPP administration (P = NS). The expression of CPP-32, an apoptosis inducer, was significantly inhibited in CoPP-islets at 24 hours after CoPP administration. Compared to the control islets, CoPP-islets secreted significantly more insulin in response to glucose stimulation following 24-hour incubation with 100 and 1000 ng/mL of methylprednisolone (P < .05 and P < .05). The insulin content of both control and CoPP-islets did not differ significantly after 24-hour incubation with methylprednisolone. In conclusion, a single-dose treatment with cobalt-protoporphyrin for the induction of heme oxygenase-1 protects islets against the suppressive effect of methylprednisolone.

Attenuation of primary nonfunction for syngeneic islet graft using sodium 4-phenylbutyrate.

Sodium 4-phenylbutyrate (4-SPB), an aromatic derivative of butyric acid, was examined to elucidate its effect on islet engraftment in a syngeneic transplantation model using C57BL/6 mice. Diabetic mice that received subrenal implantation of 150 islets on day 0 and oral administration of twice daily 4-SPB (500 mg/kg body weight) on days -2 through 28 displayed a significantly shorter duration of posttransplantation temporary hyperglycemia than diabetic mice that received islets in isotonic sodium chloride solution (NaCl), namely 16 +/- 2 (n = 12) vs 23 +/- 2 days (n = 7; P < .05). Four weeks after transplantation, the insulin content (IC) of grafts from mice treated with islets and 4-SPB was substantially higher than that of grafts from mice treated with islets and NaCl, namely 2.59 +/- 0.37 (n = 8) vs 1.36 +/- 0.36 mug (n = 13; P < .01). The IC of pancreatic remnants showed no significant difference between groups after 2 and 4 weeks of incubation. In vitro studies demonstrated that the net glucose-stimulated insulin secretion (GSIS) and the ratio of net GSIS to the IC of islets cultured with 4-SPB (1 mM) did not differ significantly from those cultured with NaCl. The lipopolysaccharide-stimulated secretions of IL-1beta, IL-10, and IFNgamma from peritoneal exudate monocytes were significantly reduced by co-incubation with 4-SPB (1 mM). In conclusion, our data suggest that daily administration of 4-SPB reduces primary nonfunction and enhances islet engraftment in a syngeneic mouse transplantation model.

Survival prolongation of microencapsulated allogeneic islet by nanosized nordihydroguaiaretic acid.

Immunoisolation such as alginate-poly-L-lysine-alginate (APA) microencapsulation may protect entrapped islet graft cells from destruction by cellular and humoral immunities, but cannot avoid aggregation of macrophages and fibroblasts around microcapsules, which has been known to cause late dysfunction. Nordihydroguaiaretic acid (NDGA) is a lipoxygenase inhibitor that prevents the activation and chemotaxis of macrophages. In this study, we used the dialysis method without surfactant to prepare poly (DL-lactide-co-glycolide) (PLGA) nanoparticles to entrap NDGA. We determined the formulation conditions suitable for sustained release when coencapsulated with the islets. Nanoparticle sizes of 0.2-0.3 microm were suitable for sustained release in electromagnetic driven APA microcapsules. In the toxicity study, we coincubated islets with PLGA-NDGA nanoparticles in vitro for 2 and 4 weeks. The glucose stimulated insulin secretion and insulin contents of islets were not influenced significantly. To test whether nanosized NDGA provides extra protection for APA islets, about 160-200 allogeneic islets of C57BL/6 mice were either encapsulated alone using APA or coencapsulated with PLGA-NDGA. At 2 and 4 weeks after implantation into the peritoneal cavities of healthy BALB/c mice, the intraperitoneal islet grafts were recovered using lavage. Mice that received islets of APA-PLGA-NDGA preparations showed a higher recovery rate of functioning grafts than those that received islets prepared using APA alone (10.1%, n = 4 vs 5.2%, n = 3). In conclusion, nanosized NDGA prolonged the graft survival of APA microencapsulated allogeneic islets.

Enhancing islet engraftment with rosiglitazone.

To study the role of a peroxisome proliferator-activated receptor agonist, rosiglitazone, on islet engraftment, streptozotocin-induced diabetic C57BL/6 mice were fed daily rosiglitazone (2.4 mg/kg) for 9 and 31 days starting 2 days before transplantation with 75 and 150 syngeneic islets, respectively. After receiving 75 islets and 9 days of rosiglitazone, half of the treated diabetic mice became normoglycemic at 4 weeks, while none were normoglycemic among those mice that did not receive treatment. After transplanting 150 islets and receiving 31 days of rosiglitazone, 80% of the treated diabetic mice became normoglycemic while the incidence was only 25% for the controls. The insulin content of the islet grafts in the rosiglitazone groups was 0.8 times (75-islet group) and 1.3 times (150-islet group) higher than that of control mice. The insulin content of pancreatic remnants did not differ significantly among all groups. An in vitro study revealed that the glucose-stimulated insulin secretion and insulin content of cultured islets was not different in the presence versus absence of 4.5 or 22.5 micromol/L rosiglitazone. In vitro study revealed that rosiglitazone inhibited the lipopolysaccharide-induced secretion of interleukin-1 beta and interferon-gamma from peritoneal exudate cells. In conclusion, our data suggest that short-term administration of rosiglitazone enhances islet engraftment.

Cobalt-protoporphyrin treatment enhances murine isoislets engraftment.

To study the effect of treatment with cobalt-protoporphyrin (CoPP) for the induction of the heme oxygenase-1 (HO-1) enzyme on islet engraftment donor mice received either a single intraperitoneal injection of CoPP (20 mg/kg body weight) 1 day prior to islet isolation or this injection plus a 9 day posttransplantation course of Copp. After a single injection of CoPP, the CoPP-induced islets contained higher HO-1 proteins than did the normal islets both at 12 (5.3 +/- 1.5 vs 0.1 +/- 0.1 ng/mg protein, P < .01) and at 30 hours (6.8 +/- 2.1 vs 0.4 +/- 0.3 ng/mg protein, P < .05), but not at 56 hours (1.9 +/- 0.8 vs 1.6 +/- 0.8 ng/mg protein, P > .05). In contrast, diabetic mice that received 75 CoPP-induced islets and a 9-day CoPP injection course posttransplantation showed better improvement in blood glucose levels and body weights than did the mice that only received CoPP-induced islets. Mice of both CoPP-treated groups displayed better improvement in glycemic control than mice that received control islets. At 8 weeks after transplantation, the insulin content of grafts from both CoPP groups was significantly higher than that in the control group. In conclusion, CoPP treatment for the induction of HO-1 enhances engraftment of islets in a syngeneic murine transplantation model.

Cobalt-Protoporphyrin treatment renders islets tolerant to interleukin-1 beta suppression.

This study examined whether treating donor mice with a single dose of cobalt protoporphyrin (CoPP) induced heme oxygenase-1 (HO-1) and protected islet cells from interleukin-1 beta (IL-1 beta) suppression. Islets were isolated from mice receiving a single dose of either CoPP (20 mg/kg of body weight, CoPP islets) or isotonic NaCl solution vehicle (control islets), 24 hours before isolation. Glucose-stimulated insulin secretion (GSIS) and insulin content (IC) of the islets were determined following incubation in the presence versus absence of murine IL-1 beta for 21 or 65 hours. The HO-1 protein level of CoPP-induced islets, as determined by an enzyme immunoassay, was significantly higher than that of control islets at 12 hours (P <.01) and 30 hours (P <.05), and returned to basal levels at 56 hours (P = NS). Following a 21-hour incubation with IL-1 beta, CoPP islets secreted significantly more insulin upon glucose stimulation and preserved significantly more IC than control islets. After 65-hour incubation with IL-1 beta, CoPP islets secreted significantly less insulin upon glucose stimulation than control islets and preserved significantly less IC compared to islets incubated without IL-1 beta. In conclusion, treatment with cobalt-protoporphyrin to induce heme oxygenase-1 protects islets against the suppressive effects of IL-1 beta.

Characteristics and transplantation of the porcine neonatal pancreatic cell clusters isolated from 1- to 3-day-old versus 1-month-old pigs.

Porcine neonatal pancreatic cell clusters (NPCCs) isolated from 1- to 3-day-old pigs (I-A) cured diabetic nude mice within 8 weeks after transplantation. To shorten the latent period between transplantation and reversal of hyperglycemia, we studied NPCCs isolated from 1-month-old pigs (I-B). One- to 3-day-old or 1-month-old pig pancreata were cut into fragments, digested by collagenase, and then studied for islet characteristics. In addition, 300 cultured NPCCs were transplanted under kidney capsule of nondiabetic nude mice. At 1 and 3 months after transplantation, the grafts were removed to measure the insulin content and beta-cell mass. Immediately after isolation, I-B was larger than I-A (0.211 +/- 0.006 vs 0.189 +/- 0.003 mm(2), P =.0003) and after a 6-day culture period, I-B contained more insulin than I-A (6.8 +/- 1.4 vs 2.3 +/- 0.2 microg/150 NPCCs, P =.02). However, the stimulation indices of I-A and I-B during static incubation with 500 mg/dL glucose (26.5 +/- 3.2 vs 23.9 +/- 1.7) or 500 mg/dL glucose plus 50 mol/L IBMX (41.9 +/- 4.4 vs 62.2 +/- 14.0) were not significantly different. Furthermore, neither I-A nor I-B showed first or second phase insulin secretion during sequential perifusion with 100 or 300 mg/dL glucose. In nondiabetic recipients, the insulin content of the graft at 1 month after transplantation was 0.3 +/- 0.0 and 0.3 +/- 0.1 microg, and the beta-cell mass of the graft at 3 months was 0.069 +/- 0.022 and 0.067 +/- 0.023 mg in mice receiving I-A or I-B, respectively (P >.05). Our data indicate NPCCs isolated from 1- to 3-day-old and 1-month-old pigs have different characteristics but similar transplantation effects.