Intestinal Npt2b Plays a Major Role in Phosphate Absorption and Homeostasis

Intestinal phosphate absorption occurs through both a paracellular mechanism involving tight junctions and an active transcellular mechanism involving the type II sodium-dependent phosphate cotransporter NPT2b (SLC34a2). To define the contribution of NPT2b to total intestinal phosphate absorption, we generated an inducible conditional knockout mouse, Npt2b^−/− (Npt2b^fl/fl:Cre^+/−). Npt2b^−/− animals had increased fecal phosphate excretion and hypophosphaturia, but serum phosphate remained unchanged. Decreased urinary phosphate excretion correlated with reduced serum levels of the phosphaturic hormone FGF23 and increased protein expression of the renal phosphate transporter Npt2a. These results demonstrate that the absence of Npt2b triggers compensatory renal mechanisms to maintain phosphate homeostasis. In animals fed a low phosphate diet followed by acute administration of a phosphate bolus, Npt2b^−/− animals absorbed approximately 50% less phosphate than wild-type animals, confirming a major role of this transporter in phosphate regulation. In vitro analysis of active phosphate transport in ileum segments isolated from wild-type or Npt2b^−/− mice demonstrated that Npt2b contributes to >90% of total active phosphate absorption. In summary, Npt2b is largely responsible for intestinal phosphate absorption and contributes to the maintenance of systemic phosphate homeostasis.Inorganic phosphate is an essential mineral critical for cellular processes and bone mineralization. Severe disruptions in serum phosphate have pathologic consequences.^1,2 Hypophosphatemic disorders are associated with rickets, osteomalacia, and a host of secondary dysfunctions.³ In contrast, hyperphosphatemia associated with chronic kidney disease (CKD) is linked tightly to increased risk of cardiovascular morbidity and mortality.^4–6 Recent studies show that elevated phosphate concentrations within the high normal range in individuals with functional kidneys also are correlated with increased cardiovascular risk and mortality.^7,8 Thus, an elevated serum phosphate level is an emerging health risk.Despite the importance of maintaining a relatively narrow serum phosphate range, nearly 70% of dietary phosphate is absorbed, resulting in transient postprandial increases in serum phosphate concentrations.⁹ Normalization of serum phosphate appears to be managed primarily within the renal proximal tubule by the type II sodium-dependent phosphate cotransporters NPT2a (SLC34a1) and NPT2c (SLC34a3). Genetic knockout mouse models demonstrate that 80% and 20% of total urinary phosphorus are managed by the Npt2a and Npt2c transporters, respectively.^10,11 Chronic and acute regulation of these renal transporters is modulated by changes in dietary and serum phosphate levels and by three major hormones: parathyroid hormone (PTH), 1,25-dihydroxy vitamin D₃ (1,25(OH)₂D₃), and fibroblast growth factor 23 (FGF23).¹The emergence of data demonstrating that regulation of the renal phosphate transporters can adequately maintain systemic phosphate levels has reduced contemporary interest in intestinal phosphate regulation. Furthermore, early studies of intestinal transport suggest that paracellular transport driven by a passive diffusional process predominates under standard dietary conditions.^12,13 An alternative transcellular mechanism in the small intestine is dependent on active transport through the sodium-dependent phosphate cotransporter NPT2b (SLC34a2).¹⁴ Npt2b has a relatively low K_m for phosphate, suggesting that transport would be readily saturated under standard conditions and therefore may be important only under conditions of hypophosphatemia or low dietary phosphate intake.¹⁵ Furthermore, individuals with inactivating NPT2b mutations have pulmonary alveolar microlithiasis (PAM) but do not have serum or urinary phosphate abnormalities.^16,17 Despite this cumulative evidence downplaying the relative importance of NPT2b, its expression is increased by either 1,25(OH)₂D₃ or low dietary phosphate and decreased by nicotinamide. Interestingly, nicotinamide treatment in late stage hyperphosphatemic CKD patients has been shown to lower serum phosphate concentrations,¹⁸ raising the possibility that phosphate transport in the intestine by Npt2b may be important under the pathologic conditions associated with loss of renal function.To assess Npt2b''s relative importance under defined conditions, we have generated a tamoxifen-inducible ubiquitous Npt2b deletion. Surprisingly, deletion of the intestinal transporter leads to altered compensatory hormonal responses that maintain serum phosphate levels within normal limits. These data demonstrate that Npt2b plays an active role in systemic phosphate regulation.     

Adenylate Cyclase 6 Determines cAMP Formation and Aquaporin-2 Phosphorylation and Trafficking in Inner Medulla

Arginine vasopressin (AVP) enhances water reabsorption in the renal collecting duct by vasopressin V₂ receptor (V₂R)-mediated activation of adenylyl cyclase (AC), cAMP-promoted phosphorylation of aquaporin-2 (AQP2), and increased abundance of AQP2 on the apical membrane. Multiple isoforms of adenylate cyclase exist, and the roles of individual AC isoforms in water homeostasis are not well understood. Here, we found that levels of AC6 mRNA, the most highly expressed AC isoform in the inner medulla, inversely correlate with fluid intake. Moreover, mice lacking AC6 had lower levels of inner medullary cAMP, reduced abundance of phosphorylated AQP2 (at both serine-256 and serine-269), and lower urine osmolality than wild-type mice. Water deprivation or administration of the V₂R agonist dDAVP did not increase urine osmolality of AC6-deficient mice to the levels of wild-type mice. Furthermore, AC6-deficient mice lacked dDAVP-promoted inner medullary cAMP formation and phosphorylation of serine-269 and had attenuated increases in both phosphorylation of serine-256 and apical membrane AQP2 trafficking. In summary, AC6 expression determines inner medullary cAMP formation and AQP2 phosphorylation and trafficking, the absence of which causes nephrogenic diabetes insipidus.The anti-diuretic hormone arginine-vasopressin (AVP) is the primary regulator of water reabsorption in the renal collecting duct (CD) and is critically involved in the regulation of water balance and maintenance of plasma osmolality.¹ AVP acts on the CD through the G_s protein–coupled vasopressin V₂ receptor (V₂R) to stimulate adenylyl cyclase (AC) and thus the synthesis of cAMP.² cAMP activates protein kinase A (PKA), which phosphorylates the water channel aquaporin-2 (AQP2) in its COOH-terminal tail on serine residue 256 (S256), thereby resulting in apical plasma membrane accumulation of AQP2.^3–6 In addition, cAMP-independent activation of AQP2 has been reported, which may involve V₂R action of phosphoinositide-specific phospholipase C.⁷ Other non–PKA-targeted AQP2 phosphorylation sites include S261, S264, and S269.⁸ Phosphorylation of AQP2 at S264 and S269 requires prior phosphorylation of S256.⁹ Immunohistochemistry showed that pS269-AQP2 localizes exclusively in the apical plasma membrane of the connecting tubule and CD.^8,10 AVP-stimulated AQP2 phosphorylation thus induces plasma membrane accumulation and retention of the channel.^8,10,11 Genetic defects in the V₂R or AQP2 impair AVP-induced increases in tubular water permeability and cause nephrogenic diabetes insipidus (NDI).^12–14 In comparison, less is known about the role of genetic variation of the proteins involved in signaling from V₂R to AQP2.Generation of cAMP involves the activation of ACs, of which nine different membrane-bound isoforms have been identified (AC1 to 9).¹⁵ Studies on AC isoform expression in the kidney have been almost exclusively confined to the rat, in which mRNA analyses have shown that all membrane-bound isoforms, except for AC1 and 8, are expressed.¹⁶ The same pattern was found for mRNA expression of ACs in inner medullary CD (IMCD) suspensions of rats.¹⁷Based on in situ hybridization studies and the relative expression of AC6 mRNA in CD principal cells of rats, as well as studies using small interfering RNA designed to knock down AC6 in primary cultured mouse IMCD, it has been proposed that this AC isoform may contribute to AVP-stimulated cAMP formation,^18,19 although studies in rat IMCD have indicated that AVP-promoted Ca²⁺/calmodulin-dependent cAMP accumulation involves AC3 activity.¹⁷ In these experiments, we examined mice that lack AC6 (AC6^−/−) to gain insights regarding the role of AC6 in the formation of cAMP, phosphorylation of AQP2 at S256 and S269, and urinary concentration in vivo. The results indicate an important contribution of AC6 to AVP action and urinary concentration in vivo and that AC6^−/− mice have NDI.     

The C3a Anaphylatoxin Receptor Is a Key Mediator of Insulin Resistance and Functions by Modulating Adipose Tissue Macrophage Infiltration and Activation

OBJECTIVE

Significant new data suggest that metabolic disorders such as diabetes, obesity, and atherosclerosis all posses an important inflammatory component. Infiltrating macrophages contribute to both tissue-specific and systemic inflammation, which promotes insulin resistance. The complement cascade is involved in the inflammatory cascade initiated by the innate and adaptive immune response. A mouse genomic F2 cross biology was performed and identified several causal genes linked to type 2 diabetes, including the complement pathway.

RESEARCH DESIGN AND METHODS

We therefore sought to investigate the effect of a C3a receptor (C3aR) deletion on insulin resistance, obesity, and macrophage function utilizing both the normal-diet (ND) and a diet-induced obesity mouse model.

RESULTS

We demonstrate that high C3aR expression is found in white adipose tissue and increases upon high-fat diet (HFD) feeding. Both adipocytes and macrophages within the white adipose tissue express significant amounts of C3aR. C3aR^−/− mice on HFD are transiently resistant to diet-induced obesity during an 8-week period. Metabolic profiling suggests that they are also protected from HFD-induced insulin resistance and liver steatosis. C3aR^−/− mice had improved insulin sensitivity on both ND and HFD as seen by an insulin tolerance test and an oral glucose tolerance test. Adipose tissue analysis revealed a striking decrease in macrophage infiltration with a concomitant reduction in both tissue and plasma proinflammatory cytokine production. Furthermore, C3aR^−/− macrophages polarized to the M1 phenotype showed a considerable decrease in proinflammatory mediators.

CONCLUSIONS

Overall, our results suggest that the C3aR in macrophages, and potentially adipocytes, plays an important role in adipose tissue homeostasis and insulin resistance.The complement system is an integral part of both the innate and adaptive immune response involved in the defense against invading pathogens (1). Complement activation culminates in a massive amplification of the immune response leading to increased cell lysis, phagocytosis, and inflammation (1). C3 is the most abundant component of the complement cascade and the convergent point of all three major complement activation pathways. C3 is cleaved into C3a and C3b by the classical and lectin pathways, and iC3b is generated by the alternative pathway (2,3). C3a has potent anaphylatoxin activity, directly triggering degranulation of mast cells, inflammation, chemotaxis, activation of leukocytes, as well as increasing vascular permeability and smooth muscle contraction (3). C3a mediates its downstream signaling effects by binding to the C3a receptor (C3aR), a Gi-coupled G protein–coupled receptor. Several studies have demonstrated a role for C3a and C3aR in asthma, sepsis, liver regeneration, and autoimmune encephalomyelitis (1,3). Therefore, targeting C3aR may be an attractive therapeutic option for the treatment of several inflammatory diseases.Increasing literature suggests that metabolic disorders such as diabetes, obesity, and atherosclerosis also possess an important inflammatory component (4–7). Several seminal reports have demonstrated that resident macrophages can constitute as much as 40% of the cell population of adipose tissue (7–9) and can significantly affect insulin resistance (10–18). Several proinflammatory cytokines, growth factors, acute-phase proteins, and hormones are produced by the adipose tissue and implicated in insulin resistance and vascular homeostasis (4–7,19). An integrated genomics approach was performed with several mouse strains to infer causal relationships between gene expression and complex genetic diseases such as obesity/diabetes. This approach identified the C3aR gene as being causal for omental fat pad mass (20). The C3aR^−/− mice were shown to have decreased adiposity as compared with wild-type mice on a regular diet (20). Monocytes and macrophages express the C3aR (21–28). Increased C3a levels also correlate with obesity, diabetes, cholesterol, and lipid levels (29–34). We therefore sought to investigate the specific role of the C3aR in insulin resistance, obesity, and macrophage function utilizing both normal diet and the diet-induced obesity model.     

Atrap Deficiency Increases Arterial Blood Pressure and Plasma Volume

The angiotensin receptor-associated protein (Atrap) interacts with angiotensin II (AngII) type 1 (AT1) receptors and facilitates their internalization in vitro, but little is known about the function of Atrap in vivo. Here, we detected Atrap expression in several organs of wild-type mice; the highest expression was in the kidney where it localized to the proximal tubule, particularly the brush border. There was no Atrap expression in the renal vasculature or juxtaglomerular cells. We generated Atrap-deficient (Atrap−/−) mice, which were viable and seemed grossly normal. Mean systolic BP was significantly higher in Atrap−/− mice compared with wild-type mice. Dose-response relationships of arterial BP after acute AngII infusion were similar in both genotypes. Plasma volume was significantly higher and plasma renin concentration was markedly lower in Atrap−/− mice compared with wild-type mice. ¹²⁵I-AngII binding showed enhanced surface expression of AT1 receptors in the renal cortex of Atrap−/− mice, accompanied by increased carboanhydrase-sensitive proximal tubular function. In summary, Atrap−/− mice have increased arterial pressure and plasma volume. Atrap seems to modulate volume status by acting as a negative regulator of AT1 receptors in the renal tubules.Angiotensin II (AngII) is the primary end point of the renin-angiotensin (RAS) cascade. AngII exerts multiple functions, including mediation of vasoconstriction, stimulation of aldosterone release, and promotion of renal salt/water reabsorption. These classical effects of AngII, which eventually all result in a rise of arterial blood pressure (BP), are thought to be primarily mediated by AngII type 1 (AT1) receptors. Because changes in the activity of the systemic RAS cascade similarly affect all different accessible target tissues, it is reasonable to assume that strategies that allow for local and temporal modification of the sensitivity of AT1 receptors have evolved. In this context, modulation of AT1 receptor expression, receptor desensitization, and internalization all have been described to modify locally the AT1-related effects of AngII.^1–8 In addition, a growing number of proteins seem to bind to the AT1 receptor and either enhance or suppress AT1 receptor function.^9–13Of the known proteins that can interact with AT1 receptors, the function of the angiotensin receptor–associated protein (Atrap) is the best characterized.^10,14 Atrap is a 19-kD protein with three potential transmembrane domains, and it binds to the C-terminal intracellular portion of the AT1 receptor.¹⁵ Atrap catalyzes the internalization of the AT1 receptor in cultured vascular smooth muscle cells.^14,16 Also, Atrap inhibits AngII-mediated intracellular signaling in vitro.¹⁷ Overall, the data suggest an inhibitory effect of Atrap on AT1 receptor function in vitro.Regarding the in vivo function of Atrap, a recently generated transgenic mouse line with overexpression of Atrap in the heart, aorta, and femoral artery showed reduced inflammatory vascular remodeling and reduced heart hypertrophy as compared with transgene-negative controls.¹⁸ The authors concluded that Atrap attenuates AT1-mediated signaling under pathophysiologic conditions.¹⁸To assess the in vivo function of Atrap, we generated Atrap-deficient (Atrap−/−) mice. Vascular responsiveness to AngII was virtually unaltered in Atrap−/− mice compared with wild-type mice; however, loss of Atrap resulted in increased plasma volume and elevated arterial BP. Renal cortical AngII binding and acetazolamide-sensitive tubular function were enhanced in Atrap−/− mice compared with wild-type controls. We propose that Atrap is a negative modulator of renal AngII signaling and that loss of Atrap results in enhanced renal reabsorptive function, leading to volume expansion and hypertension.     

A Mouse Model of Human Hyperinsulinism Produced by the E1506K Mutation in the Sulphonylurea Receptor SUR1

Loss-of-function mutations in the K_ATP channel genes KCNJ11 and ABCC8 cause neonatal hyperinsulinism in humans. Dominantly inherited mutations cause less severe disease, which may progress to glucose intolerance and diabetes in later life (e.g., SUR1-E1506K). We generated a mouse expressing SUR1-E1506K in place of SUR1. K_ATP channel inhibition by MgATP was enhanced in both homozygous (homE1506K) and heterozygous (hetE1506K) mutant mice, due to impaired channel activation by MgADP. As a consequence, mutant β-cells showed less on-cell K_ATP channel activity and fired action potentials in glucose-free solution. HomE1506K mice exhibited enhanced insulin secretion and lower fasting blood glucose within 8 weeks of birth, but reduced insulin secretion and impaired glucose tolerance at 6 months of age. These changes correlated with a lower insulin content; unlike wild-type or hetE1506K mice, insulin content did not increase with age in homE1506K mice. There was no difference in the number and size of islets or β-cells in the three types of mice, or evidence of β-cell proliferation. We conclude that the gradual development of glucose intolerance in patients with the SUR1-E1506K mutation might, as in the mouse model, result from impaired insulin secretion due a failure of insulin content to increase with age.Congenital hyperinsulinism of infancy (HI) is a rare genetic disorder characterized by enhanced insulin secretion that leads to persistent hypoglycemia soon after birth (1,2). It occurs in ∼1 in 50,000 live births within the general population and at higher levels in communities that practice consanguineous marriage. The severity of the disease varies from a mild form, which responds to treatment with drugs (such as diazoxide or octreotide), to a severe drug-resistant form, which may require removal of most of the pancreas. Early diagnosis is important to avoid irreversible brain damage due to the hypoglycemia. Loss-of-function mutations in either the pore-forming (Kir6.2, encoded by KCNJ11) or regulatory (SUR1, ABCC8) subunit of the β-cell plasma membrane ATP-sensitive potassium (K_ATP) channel are the most common causes of HI (3–6).The K_ATP channel plays a crucial role in insulin secretion by coupling the energy state of the β-cell to the plasma membrane potential (5). It achieves this by sensing changes in intracellular adenine nucleotides, being inhibited by ATP binding to Kir6.2 and activated by MgATP (and MgADP) binding and hydrolysis at the nucleotide-binding domains of SUR1 (7,8). As a consequence, the K_ATP channel is open when metabolism is low, keeping the β-cell membrane hyperpolarized and preventing insulin secretion. An increase in β-cell metabolism, consequent on elevation of the plasma glucose concentration, leads to an increase in intracellular ATP and K_ATP channel closure. This produces a membrane depolarization that opens voltage-gated Ca²⁺ channels, stimulating Ca²⁺ influx and exocytosis of insulin granules. Loss-of-function mutations in either Kir6.2 or SUR1 result in permanent membrane depolarization, persistent Ca²⁺ influx, and thus continuous, unregulated insulin release (2–6).Most reported mutations in KCNJ11 and ABCC8 that cause HI are inherited recessively; these mutations cause the most severe form of the disease. However, a few dominantly expressed mutations have also been described (9–15). Children with these mutations generally have a milder phenotype than those with recessive mutations, and their hypoglycemia is well controlled by the K_ATP channel opener diazoxide. Members of one family, who are heterozygous carriers of the SUR1-E1506K mutation, have mild neonatal HI but are at increased risk of diabetes in middle age (9,14); 4 out of 11 had overt diabetes, and 5 of those without diabetes showed impaired glucose tolerance. Similarly, a child with a heterozygous SUR1-R370S mutation causing neonatal hyperinsulinism developed diabetes at 10 years of age (15). Studies of other dominantly inherited mutations have not shown a link between HI mutations and late-onset diabetes, although the disease severity may diminish later in life as many patients no longer require diazoxide therapy and become normoglycemic (12).Despite their impaired glucose tolerance, blood glucose levels were normal in heterozygous carriers of the SUR1-E1506K mutation without diabetes, and only slightly increased in those with diabetes (14). Electrophysiological studies indicate that the E1506K mutation does not impair membrane trafficking but results in channels that are no longer activated by MgATP (9,16). As a consequence, homozygous whole-cell K_ATP currents are absent, and heterozygous K_ATP currents are 30–50% smaller than wild type (WT). The smaller K_ATP currents would be expected to result in membrane depolarization, thus accounting for the increased insulin secretion in human neonates. Why this translates into reduced insulin secretion later in life is unclear, as is why impaired insulin secretion was observed in all carriers of the E1506K mutation but diabetes in only some of them.Unexpectedly, genetic deletion of SUR1 in mice did not mimic human hyperinsulinism (6,17–20). SUR1^−/− mice exhibited hypoglycemia on the first day of life but normal blood glucose levels subsequently (17), and by 12 weeks of age, they showed impaired glucose tolerance (17,18). The β-cell resting membrane potential was depolarized (17), and the basal intracellular calcium concentration was elevated (18), as expected if K_ATP channels were blocked. Reported data on insulin secretion from SUR1^−/− islets are controversial. Early studies suggested that basal insulin secretion was not elevated (17) and that glucose-induced insulin secretion from isolated islets (17) and perfused pancreas (18) was severely impaired. However, subsequent studies showed that basal insulin secretion is elevated (as expected from the raised [Ca²⁺]_i), that after overnight culture in 10 mmol/L glucose, glucose-stimulated insulin release is greater than from WT islets (19,20), and that the amplifying effect of glucose on insulin secretion is intact. What remains unclear, however, is why SUR1^−/− mice have a different phenotype from the human disease, and why some human mutations cause diabetes later in life.To address these questions, we generated a mouse carrying a human HI mutation, SUR1-E1506K, which causes neonatal hypoglycemia and predisposes to diabetes late in life (9,14). We show here that both homozygous and heterozygous mice secrete more insulin than their WT littermates early in life, but with age, homozygous mice secrete less insulin and become increasingly glucose intolerant.     

Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice

OBJECTIVE

Oxyntomodulin (OXM) is a glucagon-like peptide 1 (GLP-1) receptor (GLP1R)/glucagon receptor (GCGR) dual agonist peptide that reduces body weight in obese subjects through increased energy expenditure and decreased energy intake. The metabolic effects of OXM have been attributed primarily to GLP1R agonism. We examined whether a long acting GLP1R/GCGR dual agonist peptide exerts metabolic effects in diet-induced obese mice that are distinct from those obtained with a GLP1R-selective agonist.

RESEARCH DESIGN AND METHODS

We developed a protease-resistant dual GLP1R/GCGR agonist, DualAG, and a corresponding GLP1R-selective agonist, GLPAG, matched for GLP1R agonist potency and pharmacokinetics. The metabolic effects of these two peptides with respect to weight loss, caloric reduction, glucose control, and lipid lowering, were compared upon chronic dosing in diet-induced obese (DIO) mice. Acute studies in DIO mice revealed metabolic pathways that were modulated independent of weight loss. Studies in Glp1r^−/− and Gcgr^−/− mice enabled delineation of the contribution of GLP1R versus GCGR activation to the pharmacology of DualAG.

RESULTS

Peptide DualAG exhibits superior weight loss, lipid-lowering activity, and antihyperglycemic efficacy comparable to GLPAG. Improvements in plasma metabolic parameters including insulin, leptin, and adiponectin were more pronounced upon chronic treatment with DualAG than with GLPAG. Dual receptor agonism also increased fatty acid oxidation and reduced hepatic steatosis in DIO mice. The antiobesity effects of DualAG require activation of both GLP1R and GCGR.

CONCLUSIONS

Sustained GLP1R/GCGR dual agonism reverses obesity in DIO mice and is a novel therapeutic approach to the treatment of obesity.Obesity is an important risk factor for type 2 diabetes, and ∼90% of patients with type 2 diabetes are overweight or obese (1). Among new therapies for type 2 diabetes, peptidyl mimetics of the gut-derived incretin hormone glucagon-like peptide 1 (GLP-1) stimulate insulin biosynthesis and secretion in a glucose-dependent manner (2,3) and cause modest weight loss in type 2 diabetic patients. The glucose-lowering and antiobesity effects of incretin-based therapies for type 2 diabetes have prompted evaluation of the therapeutic potential of other glucagon-family peptides, in particular oxyntomodulin (OXM). The OXM peptide is generated by post-translational processing of preproglucagon in the gut and is secreted postprandially from l-cells of the jejuno-ileum together with other preproglucagon-derived peptides including GLP-1 (4,5). In rodents, OXM reduces food intake and body weight, increases energy expenditure, and improves glucose metabolism (6–8). A 4-week clinical study in obese subjects demonstrated that repeated subcutaneous administration of OXM was well tolerated and caused significant weight loss with a concomitant reduction in food intake (9). An increase in activity-related energy expenditure was also noted in a separate study involving short-term treatment with the peptide (10).OXM activates both, the GLP-1 receptor (GLP1R) and glucagon receptor (GCGR) in vitro, albeit with 10- to 100-fold reduced potency compared with the cognate ligands GLP-1 and glucagon, respectively (11–13). It has been proposed that OXM modulates glucose and energy homeostasis solely by GLP1R agonism, because its acute metabolic effects in rodents are abolished by coadministration of the GLP1R antagonist exendin(9–39) and are not observed in Glp1r^−/− mice (7,8,14,15). Other aspects of OXM pharmacology, however, such as protective effects on murine islets and inhibition of gastric acid secretion appear to be independent of GLP1R signaling (14). In addition, pharmacological activation of GCGR by glucagon, a master regulator of fasting metabolism (16), decreases food intake in rodents and humans (17–19), suggesting a potential role for GCGR signaling in the pharmacology of OXM. Because both OXM and GLP-1 are labile in vivo (T_1/2 ∼12 min and 2–3 min, respectively) (20,21) and are substrates for the cell surface protease dipeptidyl peptidase 4 (DPP-4) (22), we developed two long-acting DPP-4–resistant OXM analogs as pharmacological agents to better investigate the differential pharmacology and therapeutic potential of dual GLP1R/GCGR agonism versus GLP1R-selective agonism. Peptide DualAG exhibits in vitro GLP1R and GCGR agonist potency comparable to that of native OXM and is conjugated to cholesterol via a Cys sidechain at the C-terminus for improved pharmacokinetics. Peptide GLPAG differs from DualAG by only one residue (Gln³→Glu) and is an equipotent GLP1R agonist, but has no significant GCGR agonist or antagonist activity in vitro. The objective of this study was to leverage the matched GLP1R agonist potencies and pharmacokinetics of peptides DualAG and GLPAG in comparing the metabolic effects and therapeutic potential of a dual GLP1R/GCGR agonist with a GLP1R-selective agonist in a mouse model of obesity.     

Klotho Prevents Renal Calcium Loss

Disturbed calcium (Ca²⁺) homeostasis, which is implicit to the aging phenotype of klotho-deficient mice, has been attributed to altered vitamin D metabolism, but alternative possibilities exist. We hypothesized that failed tubular Ca²⁺ absorption is primary, which causes increased urinary Ca²⁺ excretion, leading to elevated 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and its sequelae. Here, we assessed intestinal Ca²⁺ absorption, bone densitometry, renal Ca²⁺ excretion, and renal morphology via energy-dispersive x-ray microanalysis in wild-type and klotho^−/− mice. We observed elevated serum Ca²⁺ and fractional excretion of Ca²⁺ (FE_Ca) in klotho^−/− mice. Klotho^−/− mice also showed intestinal Ca²⁺ hyperabsorption, osteopenia, and renal precipitation of calcium-phosphate. Duodenal mRNA levels of transient receptor potential vanilloid 6 (TRPV6) and calbindin-D_9K increased. In the kidney, klotho^−/− mice exhibited increased expression of TRPV5 and decreased expression of the sodium/calcium exchanger (NCX1) and calbindin-D_28K, implying a failure to absorb Ca²⁺ through the distal convoluted tubule/connecting tubule (DCT/CNT) via TRPV5. Gene and protein expression of the vitamin D receptor (VDR), 25-hydroxyvitamin D-1-α-hydroxylase (1αOHase), and calbindin-D_9K excluded renal vitamin D resistance. By modulating the diet, we showed that the renal Ca²⁺ wasting was not secondary to hypercalcemia and/or hypervitaminosis D. In summary, these findings illustrate a primary defect in tubular Ca²⁺ handling that contributes to the precipitation of calcium-phosphate in DCT/CNT. This highlights the importance of klotho to the prevention of renal Ca²⁺ loss, secondary hypervitaminosis D, osteopenia, and nephrocalcinosis.Characterization of a mouse that showed a phenotype comparable to human aging led to the identification of the hormone klotho.¹ Klotho^−/− mice have atherosclerosis, osteopenia, soft tissue calcifications, pulmonary emphysema, and altered glucose metabolism.¹ It has been suggested that the etiology of many of these findings is a primary defect in phosphorous [P(i)] and calcium (Ca²⁺) homeostasis.^2,3 Klotho^−/− mice have elevated serum levels of Ca²⁺.^1,4,5 The mechanism mediating hypercalcemia is poorly understood. A possible explanation invokes the role of klotho in vitamin D homeostasis. Klotho has been proposed to participate in a negative feedback circuit to inhibit 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] synthesis.^6,7 Specifically, klotho is necessary to transduce the signal of fibroblast growth factor 23 (FGF23) through the FGF receptor, thereby suppressing CYP1b expression, the enzyme that mediates the conversion of 25-hydroxyvitamin D into 1,25(OH)₂D₃. Thus, the absence of klotho results in increased serum levels of 1,25(OH)₂D₃ and reduced serum concentrations of the calciotropic hormone parathyroid hormone.^4,7,8 This would drive increased resorption of Ca²⁺ from bone, hyperabsorption from the intestine, increased serum levels of Ca²⁺, and consequently increase renal Ca²⁺ excretion. Definitive proof of this is lacking because the molecular control of Ca²⁺ homeostasis in klotho^−/− mice has yet to be delineated.Consistent with the above hypothesis is the observation that klotho^−/− mice display hypercalciuria^4,5,9 and that normalization of serum 1,25(OH)₂D₃ levels reverts many, but not all, of their abnormalities.⁶ The published literature supports an alternative, complementary hypothesis.^9–11 A primary defect in tubular Ca²⁺ handling might cause hypervitaminosis D and renal Ca²⁺ wasting observed in klotho^−/− mice. Consistent with this idea, in vitro, klotho mediates an increase in cell surface expression of transient receptor potential vanilloid 5 (TRPV5)^10,11 the distal convoluted tubule/connecting tubule (DCT/CNT) channel responsible for the transcellular absorption of Ca²⁺.¹² This process is itself implicit to Ca²⁺ homeostasis as TRPV5 is the predominant regulator of urinary Ca²⁺ excretion.¹³ Therefore, we set out to test the hypothesis that klotho^−/− mice have a primary renal Ca²⁺ leak that contributes to a secondary increase in 1,25(OH)₂D₃ synthesis and its consequences.     

Pathogenesis of A?β+ Ketosis-Prone Diabetes

A⁻β⁺ ketosis-prone diabetes (KPD) is an emerging syndrome of obesity, unprovoked ketoacidosis, reversible β-cell dysfunction, and near-normoglycemic remission. We combined metabolomics with targeted kinetic measurements to investigate its pathophysiology. Fasting plasma fatty acids, acylcarnitines, and amino acids were quantified in 20 KPD patients compared with 19 nondiabetic control subjects. Unique signatures in KPD—higher glutamate but lower glutamine and citrulline concentrations, increased β-hydroxybutyryl-carnitine, decreased isovaleryl-carnitine (a leucine catabolite), and decreased tricarboxylic acid (TCA) cycle intermediates—generated hypotheses that were tested through stable isotope/mass spectrometry protocols in nine new-onset, stable KPD patients compared with seven nondiabetic control subjects. Free fatty acid flux and acetyl CoA flux and oxidation were similar, but KPD had slower acetyl CoA conversion to β-hydroxybutyrate; higher fasting β-hydroxybutyrate concentration; slower β-hydroxybutyrate oxidation; faster leucine oxidative decarboxylation; accelerated glutamine conversion to glutamate without increase in glutamate carbon oxidation; and slower citrulline flux, with diminished glutamine amide–nitrogen transfer to citrulline. The confluence of metabolomic and kinetic data indicate a distinctive pathogenic sequence: impaired ketone oxidation and fatty acid utilization for energy, leading to accelerated leucine catabolism and transamination of α-ketoglutarate to glutamate, with impaired TCA anaplerosis of glutamate carbon. They highlight a novel process of defective energy production and ketosis in A⁻β⁺ KPD.Ketosis-prone diabetes (KPD) is characterized by presentation with diabetic ketoacidosis (DKA) in persons who do not fit traditional categories of types 1 or 2 diabetes (1–5). We have defined four subgroups of KPD based on presence or absence of β-cell autoantibodies (A⁺ or A⁻), and recovery or lack of recovery of β-cell functional reserve following the index episode of DKA (β⁺ or β⁻) (1,6,7).The A⁻β⁺ KPD subgroup represents a novel syndrome of severe but reversible β-cell dysfunction (1,3,5,8,9). Approximately 50% of these patients develop DKA without a precipitating factor at diagnosis of diabetes. These new-onset, unprovoked A⁻β⁺ KPD patients display male predominance (10) and low frequencies of human leukocyte antigen class II susceptibility alleles for autoimmune diabetes (11). β-Cell function increases markedly within 1–3 months after the index DKA, with sustained glycemic improvement and insulin independence (1,9,11,12). The cause of the unprovoked ketoacidosis is unknown. Over 5–10 years, patients may relapse to unprovoked ketosis (3,5).This syndrome provides a model to identify novel mechanisms of obesity, ketosis, and reversible β-cell dysfunction. We used a metabolomics approach to identify unique alterations in A⁻β⁺ KPD patients, with a kinetics approach to specify the pathophysiology.     

Tetrahydrobiopterin Has a Glucose-Lowering Effect by Suppressing Hepatic Gluconeogenesis in an Endothelial Nitric Oxide Synthase–Dependent Manner in Diabetic Mice

Endothelial nitric oxide synthase (eNOS) dysfunction induces insulin resistance and glucose intolerance. Tetrahydrobiopterin (BH₄) is an essential cofactor of eNOS that regulates eNOS activity. In the diabetic state, BH₄ is oxidized to 7,8-dihydrobiopterin, which leads to eNOS dysfunction owing to eNOS uncoupling. The current study investigates the effects of BH₄ on glucose metabolism and insulin sensitivity in diabetic mice. Single administration of BH₄ lowered fasting blood glucose levels in wild-type mice with streptozotocin (STZ)-induced diabetes and alleviated eNOS dysfunction by increasing eNOS dimerization in the liver of these mice. Liver has a critical role in glucose-lowering effects of BH₄ through suppression of hepatic gluconeogenesis. BH₄ activated AMP kinase (AMPK), and the suppressing effect of BH₄ on gluconeogenesis was AMPK-dependent. In addition, the glucose-lowering effect and activation of AMPK by BH₄ did not appear in mice with STZ-induced diabetes lacking eNOS. Consecutive administration of BH₄ in ob/ob mice ameliorated glucose intolerance and insulin resistance. Taken together, BH₄ suppresses hepatic gluconeogenesis in an eNOS-dependent manner, and BH₄ has a glucose-lowering effect as well as an insulin-sensitizing effect in diabetic mice. BH₄ has potential in the treatment of type 2 diabetes.Nitric oxide (NO) is a biological messenger produced by NO synthase (NOS), which includes endothelial (eNOS), inducible (iNOS), and neuronal (nNOS) isoforms. eNOS-derived NO is well-known to have a pivotal role in physiological regulation of endothelial function (1,2). eNOS dysfunction occurs in conditions of diabetes and is known to induce insulin resistance and glucose intolerance (3–5). Insulin resistance caused by eNOS dysfunction is thought to be induced by endothelial dysfunction, leading to decreased skeletal muscle blood flow and glucose uptake (4). On the other hand, glucose transport in isolated skeletal muscle is lower in eNOS-deficient (eNOS^−/−) mice, indicating that eNOS expressed in skeletal muscle also regulates its glucose uptake (4). Moreover, eNOS^−/− mice are insulin resistant at the level of liver (5). These studies suggest that eNOS plays a central role in the regulation of glucose metabolism and insulin sensitivity and represents several therapeutic targets for type 2 diabetes.The function of eNOS is regulated by multiple factors such as mRNA expression of eNOS, l-arginine, influx of Ca²⁺, and tetrahydrobiopterin (BH₄) (2,6,7). BH₄ is an essential cofactor for eNOS catalysis and functions as an allosteric modulator of arginine binding (7,8). Binding of BH₄ to eNOS elicits a conformational change that increases the affinity for binding of arginine-based ligands. BH₄ binding also plays a role in dimer formation of the active and stabilized form of eNOS (8). BH₄ is converted to 7,8-dihydrobiopterin (BH₂) by exposure to oxidative stress such as diabetes (8,9). Increase in BH₂ induces dysfunction of eNOS, as BH₂ is inactive for NOS cofactor function and competes with BH₄ for BH₄ binding (8,9). Furthermore, in states of diabetes and high glucose, de novo synthesis of BH₄, which is rate limited by GTP cyclohydrolase I (GTPCH I), is impaired (10–13). Thus, the availability of BH₄ is reduced and the function of eNOS is altered so that the enzyme produces superoxide anion (O₂⁻) rather than NO, a phenomenon called “eNOS uncoupling” (7,8,14). Supplementation of BH₄ can improve endothelial dysfunction by elevating the BH₄-to-BH₂ ratio, leading to recoupling of eNOS, and has been used in clinical trials with patients with atherosclerotic diseases for the expected vasodilatation effects of BH₄ through NO production (15). However, it is unclear whether BH₄ improves glucose metabolism and insulin sensitivity in diabetic conditions.In the current study, we investigated the effects of BH₄ on blood glucose levels and insulin sensitivity in diabetic mice. Fasting blood glucose levels are regulated by the level of hepatic gluconeogenesis, elevation of which is the major cause of fasting hyperglycemia in diabetes (16,17). We demonstrate here that BH₄ lowers fasting blood glucose levels and suppresses gluconeogenesis in liver in an eNOS-dependent manner. In addition, BH₄ has an ameliorating effect on glucose intolerance as well as insulin resistance in diabetic mice. Using primary hepatocytes isolated from mouse liver, we have clarified the mechanism by which BH₄ suppresses hepatic gluconeogenesis. These data suggest that BH₄ has potential as a novel therapeutic approach to diabetes.     

Six-Month Prophylaxis Is Cost Effective in Transplant Patients at High Risk for Cytomegalovirus Infection

The risk of late-onset cytomegalovirus (CMV) infection remains a concern in seronegative kidney and/or pancreas transplant recipients of seropositive organs despite the use of antiviral prophylaxis. The optimal duration of prophylaxis is unknown. We studied the cost effectiveness of 6- versus 3-mo prophylaxis with valganciclovir. A total of 222 seronegative recipients of seropositive kidney and/or pancreas transplants received valganciclovir prophylaxis for either 3 or 6 mo during two consecutive time periods. We assessed the incidence of CMV infection and disease 12 mo after completion of prophylaxis and performed cost-effectiveness analyses. The overall incidence of CMV infection and disease was 26.7% and 24.4% in the 3-mo group and 20.9% and 12.1% in the 6-mo group, respectively. Six-month prophylaxis was associated with a statistically significant reduction in risk for CMV disease (HR, 0.35; 95% CI, 0.17 to 0.72), but not infection (HR, 0.65; 95% CI, 0.37 to 1.14). Cost-effectiveness analyses showed that 6-mo prophylaxis combined with a one-time viremia determination at the end of the prophylaxis period incurred an incremental cost of $34,362 and $16,215 per case of infection and disease avoided, respectively, and $8,304 per one quality adjusted life-year gained. Sensitivity analyses supported the cost effectiveness of 6-mo prophylaxis over a wide range of valganciclovir and hospital costs, as well as variation in the incidence of CMV disease. In summary, 6-mo prophylaxis with valganciclovir combined with a one-time determination of viremia is cost effective in reducing CMV infection and disease in seronegative recipients of seropositive kidney and/or pancreas transplants.Cytomegalovirus (CMV) infection remains one of most common opportunistic infections in solid organ transplant patients despite availability of specific and efficacious anti-viral drugs.^1,2 Solid organ transplant patients who have a negative CMV serology and receive an organ from a positive CMV serologic donor (D+/R−) have the highest incidence of CMV disease with and without prophylaxis.^2–5 Although the risk for CMV disease persists for life, the majority of cases occur shortly after completion of prophylaxis, often within the first year after transplant.⁶ CMV disease causes significant morbidity, increases mortality, and is associated with inferior transplant outcomes, particularly in the case of kidney transplantation.^7–10 Furthermore, the presence of CMV disease is one of the most frequent infectious causes of hospitalization early after transplantation, increasing the total cost of kidney transplantation and reducing its overall effectiveness.^7,11–13Valganciclovir (VGCV) is an effective anti-CMV agent for prophylaxis and treatment of CMV disease that is widely used in transplantation.^2,14–16 Although the recommended dose for CMV prophylaxis is 900 mg daily adjusted for renal function, a recent study showed that VGCV at 450 mg daily provides similar drug exposure compared with oral ganciclovir (GCV) at 1000 mg three times daily in kidney transplant patients, a dose similarly effective for CMV prophylaxis.^2,17 In most studies, VGCV prophylaxis consisted of 100 d after transplant, after which time the risk of CMV infection and disease increased.^2,18,19 Extending the duration of VGCV prophylaxis beyond the early post-transplant period may abrogate this transient increase in the risk of infection and disease.^20,21 In this regard, the optimal duration of prophylaxis for CMV D+/R− patients has not been determined and is the subject of ongoing study.²² Cost, efficacy, and safety are important factors in determining the optimal duration of VGCV prophylaxis. Over the past two decades, various strategies have been used including pre-emptive versus universal prophylaxis and shorter versus longer period of prophylaxis.^20,21,23,24 Although several clinical studies comparing universal prophylaxis versus pre-emptive anti-viral therapy have found similar efficacy and cost in managing CMV infection across various combinations of donor and recipient CMV serologic status, two meta-analyses did find that the use of universal prophylaxis was associated with reduced risk for CMV disease and death.^23–26This study is based on a single center experience comparing two CMV prophylaxis strategies. We report here the clinical outcome and cost-effectiveness analyses of 6- versus 3-mo VGCV prophylaxis in CMV D+/R− de novo kidney and/or pancreas transplant patients.     

GAD Antibody Positivity Predicts Type 2 Diabetes in an Adult Population

OBJECTIVE

To evaluate the significance of GAD antibodies (GADAs) and family history for type 1 diabetes (FH_T1) or type 2 diabetes (FH_T2) in nondiabetic subjects.

RESEARCH DESIGN AND METHODS

GADAs were analyzed in 4,976 nondiabetic relatives of type 2 diabetic patients or control subjects from Finland. Altogether, 289 (5.9%) were GADA⁺—a total of 253 GADA⁺ and 2,511 GADA⁻ subjects participated in repeated oral glucose tolerance tests during a median time of 8.1 years. The risk of progression to diabetes was assessed using Cox regression analysis.

RESULTS

Subjects within the highest quartile of GADA⁺ (GADA⁺_high) had more often first-degree FH_T1 (29.2 vs. 7.9%, P < 0.00001) and GADA⁺ type 2 diabetic (21.3 vs. 13.7%, P = 0.002) or nondiabetic (26.4 vs. 13.3%, P = 0.010) relatives than GADA⁻ subjects. During the follow-up, the GADA⁺ subjects developed diabetes significantly more often than the GADA⁻ subjects (36/253 [14.2%] vs. 134/2,511 [5.3%], P < 0.00001). GADA⁺_high conferred a 4.9-fold increased risk of diabetes (95% CI 2.8–8.5) compared with GADA⁻—seroconversion to positive during the follow-up was associated with 6.5-fold (2.8–15.2) and first-degree FH_T1 with 2.2-fold (1.2–4.1) risk of diabetes. Only three subjects developed type 1 diabetes, and others had a non–insulin-dependent phenotype 1 year after diagnosis. GADA⁺ and GADA⁻ subjects did not clinically differ at baseline, but they were leaner and less insulin resistant after the diagnosis of diabetes.

CONCLUSIONS

GADA positivity clusters in families with type 1 diabetes or latent autoimmune diabetes in adults. GADA positivity predicts diabetes independently of family history of diabetes, and this risk was further increased with high GADA concentrations.Latent autoimmune diabetes in adults (LADA) was introduced nearly 2 decades ago to separate a GAD antibody (GADA)-positive subgroup of adult patients initially diagnosed with type 2 diabetes (1,2). Using this definition with the add-on criteria of no exogenous insulin during the first 6–12 months, the prevalence of LADA among unselected “type 2 diabetic patients” is ∼25% in subjects younger than 35 years and between 4 and 13% in subjects older than 35 years at diagnosis in populations of European origin (3–9). In follow-up studies, a progressive defect in insulin secretion was observed in ∼50–60% of LADA patients within 6–10 years (3,10), which led to the inclusion of these patients as a slowly progressing form of type 1 diabetes in the last World Health Organization (WHO) classification of diabetes (11). However, both the existence of LADA as a distinct subgroup of diabetes and the criteria that should be used to diagnose it have been challenged (e.g., (12,13). The LADA group is heterogeneous, and most studies have been cross-sectional, whereas prospective studies including patients at or before diagnosis and population-based studies are few (3,4,14–16). Genetic background, especially for type 1 diabetes, may be a confounding factor, and we have shown that LADA was more frequent in families with both type 1 and type 2 diabetes than in families with type 2 diabetes only (17). Moreover, some data support that type 1 and type 2 diabetes cluster in same families (17–20), although this has been contradicted in a large U.K. study on parents of type 1 diabetic patients (21).In children, progression to diabetes has been associated with high antibody levels and early development of multiple autoantibodies, whereas subjects with a later appearance of antibodies had a slower progression (22–25). We have previously hypothesized that GADAs would be a marker of a subclinical autoimmune process and showed that GADA positivity was associated with a decrease in maximal insulin secretory capacity in nondiabetic subjects (26). If that is the case, GADAs should also be a predictor of future diabetes in adults. This was not supported by two studies on the general population (16,27), but a Swedish study reported a sixfold increased risk for diabetes in GADA⁺ subjects (15).In a prospective follow-up study of a large cohort of relatives of type 2 diabetic patients and population control subjects from Finland, we have now evaluated the predictive value of GADAs and family history for type 1 or type 2 diabetes in conjunction with the traditional risk factors for diabetes.     

Renal Dendritic Cells Ameliorate Nephrotoxic Acute Kidney Injury

Inflammation contributes to the pathogenesis of acute kidney injury. Dendritic cells (DCs) are immune sentinels with the ability to induce immunity or tolerance, but whether they mediate acute kidney injury is unknown. Here, we studied the distribution of DCs within the kidney and the role of DCs in cisplatin-induced acute kidney injury using a mouse model in which DCs express both green fluorescence protein and the diphtheria toxin receptor. DCs were present throughout the tubulointerstitium but not in glomeruli. We used diphtheria toxin to deplete DCs to study their functional significance in cisplatin nephrotoxicity. Mice depleted of DCs before or coincident with cisplatin treatment but not at later stages experienced more severe renal dysfunction, tubular injury, neutrophil infiltration and greater mortality than nondepleted mice. We used bone marrow chimeric mice to confirm that the depletion of CD11c-expressing hematopoietic cells was responsible for the enhanced renal injury. Finally, mixed bone marrow chimeras demonstrated that the worsening of cisplatin nephrotoxicity in DC-depleted mice was not a result of the dying or dead DCs themselves. After cisplatin treatment, expression of MHC class II decreased and expression of inducible co-stimulator ligand increased on renal DCs. These data demonstrate that resident DCs reduce cisplatin nephrotoxicity and its associated inflammation.Innate immune responses are pathogenic in both ischemic and toxic acute renal failure. In response to renal injury, inflammatory chemokines and cytokines are produced both by renal parenchymal cells, such as proximal tubule epithelial cells, and resident or infiltrating leukocytes.^1–4 The elaborated chemokines and cytokines, including TNF-α, IL-18, keratinocyte-derived chemokine, and monocyte chemoattractant protein 1, subsequently recruit additional immune cells to the kidney, such as neutrophils, T cells, monocytes, and inflammatory dendritic cells (DCs), which may cause further injury through pathways that are not fully defined.^2,5–12 DCs are sentinels of the immune system and under steady-state conditions induce tolerance by various mechanisms, including production of TGF-β, IL-10, or indoleamine 2,3-dioxygenase^13–16; expression of PDL-1, PDL-2, or FcγR2B^17,18; clonal deletion of autoreactive T cells¹⁹; and induction of T regulatory cells via the inducible co-stimulator (ICOS) pathway.^20–23 In response to pathogens or products of tissue injury, DCs mature and initiate immunity or inflammatory diseases.^24,25 Monocytes recruited to inflamed tissue can also differentiate into inflammatory DCs and mediate defense against pathogens or contribute to inflammatory tissue responses.^12,26–28Although DCs represent a major population of immune cells in the kidney,²⁹ their role in renal disease is poorly defined. Liposomal clodronate has been used to study the pathophysiologic role of phagocytic cells, which include DCs and macrophages.^3,30–32 An alternative DC-specific approach uses expression of the simian diphtheria toxin receptor (DTR) driven by the CD11c promoter to target DCs for DT-mediated cell death.²⁴ This model has been used extensively to study the role of DCs in various physiologic and pathophysiologic contexts^32,33; however, its application in kidney disease has been limited to recent studies of immune complex–mediated glomerulonephritis.^12,23We have reported that inflammation plays an important role in the pathogenesis of cisplatin-induced acute kidney injury (AKI).^1,4,5,34 Given the dearth of information regarding the role of renal DCs in AKI, this study examined the renal DC population and the impact of its depletion on cisplatin nephrotoxicity. We show that DCs are the most abundant population of renal resident leukocytes and form a dense network throughout the tubulointerstitium. Renal DCs displayed surface markers that distinguished them from splenic DCs. Using a conditional DC depletion model, we determined that DC ablation markedly exacerbates cisplatin-induced renal dysfunction, structural injury, and infiltration of neutrophils.     

Phosphatidylinositol-4-phosphate-5-kinase alpha deficiency alters dynamics of glucose-stimulated insulin release to improve glucohomeostasis and decrease obesity in mice

OBJECTIVE

Phosphatidylinositol-4-phosphate-5-kinase (PI4P5K) has been proposed to facilitate regulated exocytosis and specifically insulin secretion by generating phosphatidylinositol-4,5-bisphosphate (PIP₂). We sought to examine the role of the α isoform of PI4P5K in glucohomeostasis and insulin secretion.

RESEARCH DESIGN AND METHODS

The response of PI4P5Kα^−/− mice to glucose challenge and a type 2-like diabetes-inducing high-fat diet was examined in vivo. Glucose-stimulated responses and PI4P5Kα^−/− pancreatic islets and β-cells were characterized in culture.

RESULTS

We show that PI4P5Kα^−/− mice exhibit increased first-phase insulin release and improved glucose clearance, and resist high-fat diet-induced development of type 2-like diabetes and obesity. PI4P5Kα^−/− pancreatic islets cultured in vitro exhibited decreased numbers of insulin granules docked at the plasma membrane and released less insulin under quiescent conditions, but then secreted similar amounts of insulin on glucose stimulation. Stimulation-dependent PIP₂ depletion occurred on the plasma membrane of the PI4P5Kα^−/− pancreatic β-cells, accompanied by a near-total loss of cortical F-actin, which was already decreased in the PI4P5Kα^−/− β-cells under resting conditions.

CONCLUSIONS

Our findings suggest that PI4P5Kα plays a complex role in restricting insulin release from pancreatic β-cells through helping to maintain plasma membrane PIP₂ levels and integrity of the actin cytoskeleton under both basal and stimulatory conditions. The increased first-phase glucose-stimulated release of insulin observed on the normal diet may underlie the partial protection against the elevated serum glucose and obesity seen in type 2 diabetes-like model systems.Failure of pancreatic β-cells to release adequate amounts of insulin contributes to the onset of type 2 diabetes and obesity (1). Elevated serum glucose transported into pancreatic β-cells is metabolized to increase cytosolic ATP levels, which then promote closure of ATP-sensitive K⁺ (KATP) channels, causing membrane depolarization. Membrane depolarization triggers opening of L-type Ca²⁺ channels, influx of Ca²⁺, and exocytosis. The first phase of exocytosis entails fusion of primed insulin granules predocked at the plasma membrane (2). A second phase involving mobilization of distal insulin vesicles occurs after approximately 10 min (3), preceded by actin cytoskeletal reorganization (4–6) and generation of lipid second messengers (7,8). The majority of type 2 diabetes is only weakly associated with specific genetic defects and is characterized by inadequate release of insulin, in addition to insulin resistance exhibited by fat and muscle target cells. Prolonged stimulation of β-cells by elevated levels of glucose, such as is encountered in the typical western-style high-fat diet, eventually suffices to trigger changes in insulin secretion in many individuals with otherwise seemingly normal physiology and genetics.Lipid kinases and their phosphoinositide products play important roles in secretory vesicle trafficking (9). Type I phosphatidylinositol-4-phosphate-5-kinases (PI4P5Ks) α, β, and γ generate the signaling lipid phosphatidylinositol-4,5-bisphosphate (PIP₂). Elegant studies in neurons and neuroendocrine cells on the role of PI4P5Kγ (10,11) and PIP₂ (12–14) have revealed that PIP₂ generation at the plasma membrane is critical during regulated exocytosis. PIP₂ directly facilitates some types of Ca⁺⁺ signaling (15,16), recruits proteins that facilitate the fusion process (13,14), and is required for docked secretory vesicles to undergo priming to become part of the ready-releasable pool and then fuse into the plasma membrane (10,11). Although less well understood mechanistically, decreased levels of PIP₂ have also been shown to inhibit insulin secretion in pancreatic β-cell model systems (7,17–19).However, PIP₂ also carries out functions that potentially oppose regulated exocytosis (20). First, PIP₂ supports the open state of the KATP channel (21,22); thus, because it is ATP-driven closure of the KATP channel that triggers secretion, PIP₂ deficiency might be anticipated to decrease K⁺ efflux, triggering membrane depolarization and thus increasing Ca⁺⁺ currents, resulting in increased secretion (23–25). In support of this model, expression of a dominant-negative isoform of PI4P5K to lower levels of PIP₂ changes the responsiveness of mutant KATP channels with decreased ATP sensitivity. However, expression of the dominant-negative PI4P5K does not alter function of wild-type (WT) KATP channels, suggesting that under normal physiologic conditions, the level of PIP₂ on the plasma membrane is not high enough to strongly affect KATP channel activity (26). PIP₂ has also been suggested to restrain fusion of docked vesicles by inhibiting SNARE complex function (14,27), with the restraint being alleviated through sequestration of the PIP₂ by Syntaxin-1 (14) or Ca²⁺-triggered PIP₂ destruction (28).Taken together, the action of PIP₂ is complex, and both its synthesis and its turnover are required at different steps in the fusion process.Another function undertaken by PIP₂ is to promote assembly of actin filaments (F-actin) (29,30). F-actin at the periphery of the cell (cortical F-actin) has also been proposed to have both positive and negative regulatory functions in exocytosis. Cortical F-actin has been proposed to act as a barrier to block access of undocked secretory vesicles to the plasma membrane; consistent with this model, cortical F-actin is disassembled during regulatory exocytosis events (31), and pharmacologic agents that disassemble F-actin enhance movement of insulin granules to the plasma membrane and insulin release, whereas agents that stabilize the actin cytoskeleton decrease insulin release (4,18,32–34). F-actin may also affect SNARE complex function by binding to and inhibiting Syntaxin-4 function in a glucose stimulation-relieved manner (32,33). To further complicate matters, F-actin can also undertake a positive role in regulated exocytosis by mediating translocation of more internal secretory vesicles to the periphery, particularly in poorly granulated or recently degranulated cells (4,35). Thus, dynamic regulation of the actin cytoskeleton is also important in the progression of regulated exocytosis and can play positive or negative roles depending on the setting.Changes in PIP₂ and cortical F-actin can have positive and negative effects on the secretory process, making it difficult a priori to predict the outcomes of their physiologic and experimental manipulation. Critically, individual PI4P5K isoforms may generate subpools of PIP₂ that regulate distinct components of the fusion process. Deletion of PI4P5Kγ markedly decreases levels of PIP₂ at the plasma membrane in resting neurons and neuroendocrine cells, and inhibits secretion at the stage of vesicle priming and fusion without notable effect on the actin cytoskeleton (10,11). In contrast, deletion of PI4P5Kα increases mast cell degranulation triggered by cross-linking of the IgE receptor, accompanied by minor decreases in cellular PIP₂, but significantly increased levels of Ca²⁺ signaling and decreased levels of total F-actin (36). PI4P5Kα knockdown using RNAi has also been reported to alter Ca²⁺ signaling, disrupt F-actin, and affect insulin release in a pancreatic β-cell line (37).This report examines insulin secretion in PI4P5Kα^−/− mice and finds that first-phase insulin release is augmented, and on a high-fat diet, fasting and stimulated serum insulin levels are even more elevated, conferring faster glucose clearance and resistance to the development of obesity. In this setting, K⁺ and Ca⁺⁺ signaling is seemingly normal; in contrast, the determinative factor seems to be a dramatic stimulation-dependent loss of PIP₂ at the plasma membrane that leads to reorganization of the actin cytoskeleton resulting in a near-total loss of cortical F-actin and decreased numbers of insulin granules docked at the plasma membrane.     

Podocyte-Specific VEGF-A Gain of Function Induces Nodular Glomerulosclerosis in eNOS Null Mice

VEGF-A and nitric oxide are essential for glomerular filtration barrier homeostasis and are dysregulated in diabetic nephropathy. Here, we examined the effect of excess podocyte VEGF-A on the renal phenotype of endothelial nitric oxide synthase (eNOS) knockout mice. Podocyte-specific VEGF₁₆₄ gain of function in eNOS^−/− mice resulted in nodular glomerulosclerosis, mesangiolysis, microaneurysms, and arteriolar hyalinosis associated with massive proteinuria and renal failure in the absence of diabetic milieu or hypertension. In contrast, podocyte-specific VEGF₁₆₄ gain of function in wild-type mice resulted in less pronounced albuminuria and increased creatinine clearance. Transmission electron microscopy revealed glomerular basement membrane thickening and podocyte effacement in eNOS^−/− mice with podocyte-specific VEGF₁₆₄ gain of function. Furthermore, glomerular nodules overexpressed collagen IV and laminin extensively. Biotin-switch and proximity ligation assays demonstrated that podocyte-specific VEGF₁₆₄ gain of function decreased glomerular S-nitrosylation of laminin in eNOS^−/− mice. In addition, treatment with VEGF-A decreased S-nitrosylated laminin in cultured podocytes. Collectively, these data indicate that excess glomerular VEGF-A and eNOS deficiency is necessary and sufficient to induce Kimmelstiel-Wilson–like nodular glomerulosclerosis in mice through a process that involves deposition of laminin and collagen IV and de-nitrosylation of laminin.Vascular glomerular endothelial factor-A (VEGF-A) is essential for the development and maintenance of normal glomerular structure and function.¹ Podocytes are the most important source of glomerular VEGF-A.^1–4 Glomerular VEGF-A plays a critical role in the pathogenesis of diabetic nephropathy.^5–7 Transgenic mice with podocyte VEGF₁₆₄ gain of function develop a glomerular phenotype indistinguishable from early diabetic nephropathy, in the context of normal blood glucose and normal systemic VEGF-A.⁵ In the setting of type 1 diabetes, plasma VEGF-A increases but nodular glomerulosclerosis develops only in mice with podocyte VEGF₁₆₄ gain of function, demonstrating that local rather than systemic VEGF excess is critical for the progression of diabetic glomerulopathy to advanced disease.⁶Nitric oxide (NO) is a product of arginine oxidation: L arginine+O₂ → citrulline+NO, catalyzed by NO synthase (NOS). The major source of endogenous NO, NOS isoforms (neuronal NOS, inducible NOS, and endothelial NOS),^8–10 are expressed in the kidney.^11–14 VEGF-A activates endothelial NOS (eNOS), inducing NO generation, which stimulates soluble guanylate cyclase, thereby causing vasodilatation. VEGF-A activates eNOS via phosphatidylinositol-3-kinase/Akt.¹⁵ Signals downstream from VEGF-A and NO stimulate endothelial cell proliferation and migration in human endothelium, regulate endothelial integrity, and contribute to angiogenesis.^16–21 In diabetes, low NO bioavailability is associated with high VEGF-A levels.^7,8,22–25 Nakagawa et al. called this process “uncoupling of VEGF to NO,” connecting mechanistically the advanced nephropathy with the relationship between VEGF and NO in the kidney.²⁶ Experimental diabetes induced in eNOS knockout (KO) mice resulted in severe diabetic nephropathy: nodular glomerulosclerosis, decreased GFR, and hypertension, associated with increased VEGF mRNA renal expression.^26,27 Consistent with these findings, db/db mice treated with l-arginine and sepiapterin had improved albuminuria and glomerular basement membrane (GBM) thickness, associated with reversed eNOS dimerization and phosphorylation, suggesting that improving eNOS activity delays the progression of diabetic nephropathy.²⁸ However, the mechanisms whereby excess VEGF-A and eNOS insufficiency lead to advanced diabetic nephropathy remain unclear.At the cellular level, binding of NO to soluble guanylate cyclase leads to increased cyclic guanosine monophosphate (cGMP) production and activation of protein kinase G, phosphodiesterases, and cGMP-gated ion channels. However, extensive evidence demonstrates that NO exerts multiple biologic functions through cGMP–independent S-nitrosylation of proteins.^29–32 S-Nitrosylation is a reversible, covalent addition of NO to thiol groups on specific cysteine from proteins, forming nitroso-protein (SNO).^30–32 Nitrosylation induces redox-based conformational changes in target proteins that modulate signaling and function.³² Altered protein S-nitrosylation has been demonstrated in pulmonary, hematologic, neurologic, and cardiovascular diseases, as well as in cancer, preeclampsia, and diabetes.^31,33We hypothesized that deficient S-nitrosylation of specific proteins mediates the glomerular phenotype resulting from eNOS deletion and excess VEGF-A in vivo. Here we examined the effects of increased podocyte VEGF₁₆₄ in eNOS KO mice and evaluated whether S-nitrosylation is mechanistically involved in the ensuing glomerular phenotype. Our findings indicate that podocyte VEGF₁₆₄ gain of function in eNOS null mice is sufficient to induce nodular glomerulosclerosis, massive proteinuria, and renal failure in the absence of diabetic milieu. Podocyte VEGF₁₆₄ gain of function decreases glomerular laminin S-nitrosylation in eNOS null mice, linking this post-translational modification to nodular glomerulosclerosis.     

Reduced Hepatic Synthesis of Calcidiol in Uremia

Calcidiol insufficiency is highly prevalent in chronic kidney disease (CKD), but the reasons for this are incompletely understood. CKD associates with a decrease in liver cytochrome P450 (CYP450) enzymes, and specific CYP450 isoforms mediate vitamin D₃ C-25-hydroxylation, which forms calcidiol. Abnormal levels of parathyroid hormone (PTH), which also modulates liver CYP450, could also contribute to the decrease in liver CYP450 associated with CKD. Here, we evaluated the effects of PTH and uremia on liver CYP450 isoforms involved in calcidiol synthesis in rats. Uremic rats had 52% lower concentrations of serum calcidiol than control rats (P < 0.002). Compared with controls, uremic rats produced 71% less calcidiol and 48% less calcitriol after the administration of vitamin D₃ or 1α-hydroxyvitamin D₃, respectively, suggesting impaired C-25-hydroxylation of vitamin D₃. Furthermore, uremia associated with a reduction of liver CYP2C11, 2J3, 3A2, and 27A1. Parathyroidectomy prevented the uremia-associated decreases in calcidiol and liver CYP450 isoforms. In conclusion, these data suggest that uremia decreases calcidiol synthesis secondary to a PTH-mediated reduction in liver CYP450 isoforms.It has been known for decades that chronic renal failure (CRF) is associated with low serum 1,25-dihydroxyvitamin D₃ [calcitriol, or 1,25(OH)₂D₃], the active metabolite of vitamin D₃, because of a reduction in renal 1α-hydroxylase (CYP27B1). More recently, 25-hydroxyvitamin D₃ [calcidiol, or 25(OH)D₃] deficiency has also been demonstrated in patients with stages 3 and 4 chronic kidney disease (CKD) and in patients who are on dialysis.^1–8 In fact, low serum 25(OH)D₃ is so intimately associated with CRF that in one study, only 29 and 17% of patients with stages 3 and 4 CKD, respectively, had sufficient levels [defined as a serum 25(OH)D₃ concentrations >75 nmol/L or 30 ng/ml].² A more recent study showed a prevalence of calcidiol insufficiency and deficiency as high as 98% in predialysis patients with a mean GFR of 18.3 ml/min.⁴ Prevalence of low serum 25(OH)D₃ was 78 and 89% in two large cohorts of hemodialysis patients^9,10 and 87% in a large cohort of peritoneal dialysis patients.¹¹The metabolic consequences of calcidiol deficiency are important, because low levels of 25(OH)D₃ might contribute to low levels of 1,25(OH)₂D₃ and to secondary hyperparathyroidism.^1–8 Moreover, in addition to its role in bone metabolism, there is increasing evidence that vitamin D₃ is involved in the prevention of many chronic diseases, such as type 1 diabetes, hypertension, cardiovascular diseases, and cancer.^8,9,12–14 As a consequence, according to the 2003 Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines, calcidiol levels should be measured in patients with CKD, and deficiency should be treated with ergocalciferol (vitamin D₂) or cholecalciferol (vitamin D₃)⁵; however, a paucity of information exists concerning the effect of treatment of vitamin D₃ insufficiency in CRF on the frequency and severity of secondary hyperparathyroidism among patients with decreased 25(OH)D₃ concentrations. Furthermore, the efficacy of vitamin D₃ therapy on serum calcidiol levels of patients who experience kidney failure is variable and remains poor compared with patients without CKD.^{1–7,15–18}More important, the mechanisms underlying calcidiol deficiency remain poorly understood. Lower diet intake and reduced sun exposure have been proposed but never demonstrated.^1,2,4,6,7 Vitamin D₃ is normally synthesized in the skin under the influence of sunlight or taken orally as a vitamin supplement. It is hydroxylated in the liver to 25(OH)D₃, then hydroxylated in the kidney to form 1,25(OH)₂D₃, the most bioactive form of the vitamin (Figure 1). Both calcitriol and calcidiol are degraded in part by a C-24-hydroxylation achieved by a ubiquitous 24-hydroxylase.^19,20Open in a separate windowFigure 1.Vitamin D₃ biotransformation pathway.The enzymes responsible for the C-25-hydroxylation of vitamin D₃ in rats are liver cytochrome P450 (CYP450) isoforms, namely CYP2C11, 2J3, 2R1, 3A2, and 27A1.^21–26 Several studies have shown that in rats with CRF, total hepatic CYP450 content as well as the in vitro activity and expression of several liver CYP450 isoforms (mainly CYP2C11, 3A1, and 3A2) are decreased by >50%.^27–32 More recently, we showed that this decrease in hepatic CYP450 may be explained by the presence of serum uremic factors that accumulate in CRF serum^33,34 and that parathyroid hormone (PTH) is a major mediator implicated in the downregulation of liver CYP450 and other liver drug-metabolizing enzymes.^35,36Hence, an attractive hypothesis to explain the decreased synthesis of calcidiol in CRF is that uremic toxins and, more specific, elevated PTH could downregulate liver CYP450 isoforms implicated in the C-25-hydroxylation of vitamin D₃ (Figure 1). Indirect evidence supporting such a hypothesis is that low serum levels of 25(OH)D₃ have also been reported in primary hyperparathyroidism and found to be corrected by parathyroidectomy (PTX).³⁷ The objectives of this study were to determine (1) the effect of CRF on calcidiol levels in rats, (2) the ability of CRF rats to C-25-hydroxylate vitamin D₃ after administration of vitamin D₃ or 1α-hydroxyvitamin D₃, (3) the role of liver CYP450 downregulation in calcidiol deficiency in CRF, and (4) the potential role of secondary hyperparathyroidism in calcidiol synthesis in rats with CRF.