Water Loading and Uromodulin Secretion in Healthy Individuals and Idiopathic Calcium Stone Formers.

BACKGROUND: Uromodulin is a protein made only by the kidney and released in urine, circulating in polymerizing and nonpolymerizing forms. This protein's multiple functions include inhibition of stone formation in the urine. The physiological determinants of uromodulin production are incompletely understood. METHODS: We investigated changes in uromodulin levels and key factors governing its production and release in urine and serum. We performed an experiment to determine whether water loading, a common intervention to prevent stone formation, will alter the rate of uromodulin production. During a 2-day period, 17 stone forming participants and 14 control participants were subjected to water loading (day 1) and normal fluid intake (day 2). Uromodulin levels were measured on timed hourly collections in urine and plasma during the period of the study. RESULTS: Water loading increased urinary uromodulin secretion (33±4 versus 10±4 µ g/min at baseline, P < 0.0001) in stone formers and control participants. Despite high urine volumes, most participants maintained relatively stable urinary uromodulin concentrations. Native Western blots for polymerizing and nonpolymerizing uromodulin suggest that polymerizing uromodulin was the predominant form at higher urinary flow volumes. Urine flow rates and sodium excretion were significant correlates of urinary uromodulin production. Water loading did not affect serum uromodulin levels, which were also not associated with urinary uromodulin. CONCLUSIONS: Water loading increases the secretion of polymerizing urinary uromodulin. This increased secretion reduces the variability of urinary uromodulin concentrations despite high urine volumes. Serum uromodulin levels were not affected by this treatment.

Evolving Concepts in Uromodulin Biology, Physiology, and Its Role in Disease: a Tale of Two Forms.

Uromodulin (or Tamm-Horsfall protein) is a glycoprotein uniquely produced in the kidney by tubular cells of the thick ascending limb of the loop of Henle and early distal tubules. This protein exhibits bidirectional secretion in the urine and in the renal interstitium and circulation. The role of this protein in maintaining renal and systemic homeostasis is becoming increasingly appreciated. Furthermore, perturbations of its functions may play a role in various diseases affecting the kidney and distant organs. In this review, we will discuss important advances in understanding its biology, highlighting the recent discoveries of its secretion and differential precursor processing that generates 2 forms: (1) a highly polymerizing form that is apically excreted in the urine and generates filaments and (2) a nonpolymerizing form that retains a polymerization inhibitory pro-peptide and is released basolaterally in the kidney interstitium and circulation, but can also be found in the urine. We will also discuss factors regulating its production and release, taking into account its intricate physiology, and propose best practices to report its levels. We also discuss breaking advances in its role in hypertension, acute kidney injury and progression to chronic disease, immunomodulation and regulating renal and systemic oxidative stress. We anticipate that this work will be a great resource for researchers and clinicians. This review will highlight the importance of defining what regulates the 2 forms of uromodulin, so that modulation of uromodulin levels and function could become a novel tool in our therapeutic armamentarium against kidney disease.

The kidney protects against sepsis by producing systemic uromodulin.

Sepsis is a significant cause of mortality in hospitalized patients. Concomitant development of acute kidney injury (AKI) increases sepsis mortality through unclear mechanisms. Although electrolyte disturbances and toxic metabolite buildup during AKI could be important, it is possible that the kidney produces a protective molecule lost during sepsis with AKI. We have previously demonstrated that systemic Tamm-Horsfall protein (THP; uromodulin), a kidney-derived protein with immunomodulatory properties, falls in AKI. Using a mouse sepsis model without severe kidney injury, we showed that the kidney increases circulating THP by enhancing the basolateral release of THP from medullary thick ascending limb cells. In patients with sepsis, changes in circulating THP were positively associated with a critical illness. THP was also found de novo in injured lungs. Genetic ablation of THP in mice led to increased mortality and bacterial burden during sepsis. Consistent with the increased bacterial burden, the presence of THP in vitro and in vivo led macrophages and monocytes to upregulate a transcriptional program promoting cell migration, phagocytosis, and chemotaxis, and treatment of macrophages with purified THP increases phagocytosis. Rescue of septic THP-/- mice with exogenous systemic THP improved survival. Together, these findings suggest that through releasing THP, the kidney modulates the immune response in sepsis by enhancing mononuclear phagocyte function, and systemic THP has therapeutic potential in sepsis.NEW & NOTEWORTHY Specific therapies to improve outcomes in sepsis with kidney injury have been limited by an unclear understanding of how kidney injury increases sepsis mortality. Here, we identified Tamm-Horsfall protein, known to protect in ischemic acute kidney injury, as protective in preclinical sepsis models. Tamm-Horsfall protein also increased in clinical sepsis without severe kidney injury and concentrated in injured organs. Further study could lead to novel sepsis therapeutics.

The kidney releases a nonpolymerizing form of uromodulin in the urine and circulation that retains the external hydrophobic patch domain.

Uromodulin [Tamm-Horsfall protein (THP)] is a glycoprotein uniquely produced in the kidney. It is released by cells of the thick ascending limbs apically in the urine and basolaterally in the renal interstitium and systemic circulation. Processing of mature urinary THP, which polymerizes into supramolecular filaments, requires cleavage of an external hydrophobic patch (EHP) at the COOH-terminus. However, THP in the circulation is not polymerized, and it remains unclear if nonaggregated forms of THP exist natively in the urine. We propose that an alternative processing path, which retains the EHP domain, can lead to a nonpolymerizing form of THP. We generated an antibody that specifically recognizes THP with retained EHP (THP + EHP) and established its presence in the urine in a nonpolymerized native state. Proteomic characterization of urinary THP + EHP revealed its COOH-terminus ending at F617. In the human kidney, THP + EHP was detected in thick ascending limb cells and less strongly in the renal parenchyma. Using immunoprecipitation followed by proteomic sequencing and immunoblot analysis, we then demonstrated that serum THP has also retained EHP. In a small cohort of patients at risk for acute kidney injury, admission urinary THP + EHP was significantly lower in patients who subsequently developed acute kidney injury during hospitalization. Our findings uncover novel insights into uromodulin biology by establishing the presence of an alternative path for cellular processing, which could explain the release of nonpolymerizing THP in the circulation. Larger studies are needed to establish the utility of urinary THP + EHP as a sensitive biomarker of kidney health and susceptibility to injury.NEW & NOTEWORTHY In this work, we discovered and characterized a novel form of uromodulin that does not polymerize because it retains an external hydrophobic patch at the COOH-terminus. These findings establish an alternative form of cellular processing of this protein and elucidate new aspects of its biology. We also provide evidence suggesting that measuring urinary nonpolymerizing uromodulin could be a promising assay to assess the risk of acute kidney injury.

Uromodulin (Tamm-Horsfall protein): guardian of urinary and systemic homeostasis.

Biology has taught us that a protein as abundantly made and conserved among species as Tamm-Horsfall protein (THP or uromodulin) cannot just be a waste product serving no particular purpose. However, for many researchers, THP is merely a nuisance during urine proteome profiling or exosome purification and for clinicians an enigmatic entity without clear disease implications. Thanks to recent human genetic and correlative studies and animal modeling, we now have a renewed appreciation of this highly prevalent protein in not only guarding urinary homeostasis, but also serving as a critical mediator in systemic inter-organ signaling. Beyond a mere barrier that lines the tubules, or a surrogate for nephron mass, mounting evidence suggests that THP is a multifunctional protein critical for modulating renal ion channel activity, salt/water balance, renal and systemic inflammatory response, intertubular communication, mineral crystallization and bacterial adhesion. Indeed, mutations in THP cause a group of inherited kidney diseases, and altered THP expression is associated with increased risks of urinary tract infection, kidney stone, hypertension, hyperuricemia and acute and chronic kidney diseases. Despite the recent surge of information surrounding THP's physiological functions and disease involvement, our knowledge remains incomplete regarding how THP is normally regulated by external and intrinsic factors, how precisely THP deficiency leads to urinary and systemic pathophysiology and in what clinical settings THP can be used as a theranostic biomarker and a target for modulation to improve patient outcomes.

Circulating uromodulin inhibits systemic oxidative stress by inactivating the TRPM2 channel.

High serum concentrations of kidney-derived protein uromodulin [Tamm-Horsfall protein (THP)] have recently been shown to be independently associated with low mortality in both older adults and cardiac patients, but the underlying mechanism remains unclear. Here, we show that THP inhibits the generation of reactive oxygen species (ROS) both in the kidney and systemically. Consistent with this experimental data, the concentration of circulating THP in patients with surgery-induced acute kidney injury (AKI) correlated with systemic oxidative damage. THP in the serum dropped after AKI and was associated with an increase in systemic ROS. The increase in oxidant injury correlated with postsurgical mortality and need for dialysis. Mechanistically, THP inhibited the activation of the transient receptor potential cation channel, subfamily M, member 2 (TRPM2) channel. Furthermore, inhibition of TRPM2 in vivo in a mouse model mitigated the systemic increase in ROS during AKI and THP deficiency. Our results suggest that THP is a key regulator of systemic oxidative stress by suppressing TRPM2 activity, and our findings might help explain how circulating THP deficiency is linked with poor outcomes and increased mortality.

Tamm-Horsfall Protein Regulates Mononuclear Phagocytes in the Kidney.

Tamm-Horsfall protein (THP), also known as uromodulin, is a kidney-specific protein produced by cells of the thick ascending limb of the loop of Henle. Although predominantly secreted apically into the urine, where it becomes highly polymerized, THP is also released basolaterally, toward the interstitium and circulation, to inhibit tubular inflammatory signaling. Whether, through this latter route, THP can also regulate the function of renal interstitial mononuclear phagocytes (MPCs) remains unclear, however. Here, we show that THP is primarily in a monomeric form in human serum. Compared with wild-type mice, THP-/- mice had markedly fewer MPCs in the kidney. A nonpolymerizing, truncated form of THP stimulated the proliferation of human macrophage cells in culture and partially restored the number of kidney MPCs when administered to THP-/- mice. Furthermore, resident renal MPCs had impaired phagocytic activity in the absence of THP. After ischemia-reperfusion injury, THP-/- mice, compared with wild-type mice, exhibited aggravated injury and an impaired transition of renal macrophages toward an M2 healing phenotype. However, treatment of THP-/- mice with truncated THP after ischemia-reperfusion injury mitigated the worsening of AKI. Taken together, our data suggest that interstitial THP positively regulates mononuclear phagocyte number, plasticity, and phagocytic activity. In addition to the effect of THP on the epithelium and granulopoiesis, this new immunomodulatory role could explain the protection conferred by THP during AKI.

Quantitative Three-Dimensional Tissue Cytometry to Study Kidney Tissue and Resident Immune Cells.

Analysis of the immune system in the kidney relies predominantly on flow cytometry. Although powerful, the process of tissue homogenization necessary for flow cytometry analysis introduces bias and results in the loss of morphologic landmarks needed to determine the spatial distribution of immune cells. An ideal approach would support three-dimensional (3D) tissue cytometry: an automated quantitation of immune cells and associated spatial parameters in 3D image volumes collected from intact kidney tissue. However, widespread application of this approach is limited by the lack of accessible software tools for digital analysis of large 3D microscopy data. Here, we describe Volumetric Tissue Exploration and Analysis (VTEA) image analysis software designed for efficient exploration and quantitative analysis of large, complex 3D microscopy datasets. In analyses of images collected from fixed kidney tissue, VTEA replicated the results of flow cytometry while providing detailed analysis of the spatial distribution of immune cells in different regions of the kidney and in relation to specific renal structures. Unbiased exploration with VTEA enabled us to discover a population of tubular epithelial cells that expresses CD11C, a marker typically expressed on dendritic cells. Finally, we show the use of VTEA for large-scale quantitation of immune cells in entire human kidney biopsies. In summary, we show that VTEA is a simple and effective tool that supports unique digital interrogation and analysis of kidney tissue from animal models or biobanked human kidney biopsies. We have made VTEA freely available to interested investigators via electronic download.

Immunofluorescence laser micro-dissection of specific nephron segments in the mouse kidney allows targeted downstream proteomic analysis.

Laser micro-dissection (LMD) is a very useful tool that allows the isolation of finite areas from tissue specimens for downstream analysis of RNA and protein. Although LMD has been adapted for use in kidney tissue, the use of this powerful tool has been limited by the diminished ability to identify specific tubular segments in the kidney. In this study, we describe a major improvement in the methodology to isolate specific cells in the mouse kidney using immunofluorescence LMD (IF-LMD). Using IF-LMD, we can reproducibly isolate not only glomeruli, but also S1-S2 proximal segments, S3 tubules, and thick ascending limbs. We also demonstrate the utility of a novel rapid immunofluorescence staining technique, and provide downstream applications for IF-LMD such as real-time PCR and cutting-edge proteomic studies. This technical breakthrough may become an invaluable tool for understanding cellular and molecular events in the heterogeneous kidney milieu.

Tamm-Horsfall Protein Regulates Granulopoiesis and Systemic Neutrophil Homeostasis.

Tamm-Horsfall protein (THP) is a glycoprotein uniquely expressed in the kidney. We recently showed an important role for THP in mediating tubular cross-talk in the outer medulla and in suppressing neutrophil infiltration after kidney injury. However, it remains unclear whether THP has a broader role in neutrophil homeostasis. In this study, we show that THP deficiency in mice increases the number of neutrophils, not only in the kidney but also in the circulation and in the liver, through enhanced granulopoiesis in the bone marrow. Using multiplex ELISA, we identified IL-17 as a key granulopoietic cytokine specifically upregulated in the kidneys but not in the liver of THP(-/-) mice. Indeed, neutralization of IL-17 in THP(-/-) mice completely reversed the systemic neutrophilia. Furthermore, IL-23 was also elevated in THP(-/-) kidneys. We performed real-time PCR on laser microdissected tubular segments and FACS-sorted renal immune cells and identified the S3 proximal segments, but not renal macrophages, as a major source of increased IL-23 synthesis. In conclusion, we show that THP deficiency stimulates proximal epithelial activation of the IL-23/IL-17 axis and systemic neutrophilia. Our findings provide evidence that the kidney epithelium in the outer medulla can regulate granulopoiesis. When this novel function is added to its known role in erythropoiesis, the kidney emerges as an important regulator of the hematopoietic system.

The role of tumor necrosis factor alpha in regulating the expression of Tamm-Horsfall Protein (uromodulin) in thick ascending limbs during kidney injury.

BACKGROUND: Tamm-Horsfall Protein (THP) is a glycoprotein expressed exclusively by cells of the thick ascending loop (TAL) of Henle. THP has a protective role in acute kidney injury (AKI), and its expression is downregulated in the early stages of injury. Tumor necrosis factor alpha (TNFα) is a cytokine endogenously expressed by the TAL and is also induced by AKI. Therefore, we hypothesized that TNFα is a key regulator of THP expression. METHODS: We used a mouse model of AKI (ischemia-reperfusion injury, IRI) and a cell culture system of a TAL cell line (MKTAL). RESULTS: We show that TNFα is upregulated by TAL cells early after AKI in vivo. The expression of THP and its transcription factor Hepatocyte nuclear factor 1ß (HNF1ß) were concomitantly decreased at the peak of injury. Furthermore, recombinant TNFα inhibits significantly, and in a dose-dependent manner, the expression of THP, but not HNF1ß in MKTAL cells. Interestingly, neither TNFα neutralization nor genetic deletion of TNFα increased THP or HNF levels after injury in vivo. CONCLUSION: Our data suggest that TNFα can inhibit the expression of THP in TAL cells via an HNF1ß-independent mechanism, but the downregulation of THP expression in the early AKI does not depend on TNFα. We propose that TNFα regulates THP expression in a homeostatic setting, but the impact of TNFα on THP during kidney injury is superseded by other factors that could inhibit HNF1ß-mediated expression of THP.

An intricate network of conserved DNA upstream motifs and associated transcription factors regulate the expression of uromodulin gene.

PURPOSE: Uromodulin is a kidney specific glycoprotein whose expression can modulate kidney homeostasis. However, the set of sequence specific transcription factors that regulate the uromodulin gene UMOD and their upstream binding locations are not well characterized. We built a high resolution map of its transcriptional regulation. MATERIALS AND METHODS: We applied in silico phylogenetic footprinting on the upstream regulatory regions of a diverse set of human UMOD orthologs to identify conserved binding motifs and corresponding position specific weight matrices. We further analyzed the predicted binding motifs by motif comparison, which identified transcription factors likely to bind these discovered motifs. Predicted transcription factors were then integrated with experimentally known protein-protein interactions available from public databases and tissue specific expression resources to delineate important regulators controlling UMOD expression. RESULTS: Analysis allowed the identification of a reliable set of binding motifs in the upstream regulatory regions of UMOD to build a high confidence compendium of transcription factors that could bind these motifs, such as GATA3, HNF1B, SP1, SMAD3, RUNX2 and KLF4. ENCODE deoxyribonuclease I hypersensitivity sites in the UMOD upstream region of the mouse kidney confirmed that some of these binding motifs were open to binding by predicted transcription factors. The transcription factor-transcription factor network revealed several highly connected transcription factors, such as SP1, SP3, TP53, POU2F1, RARB, RARA and RXRA, as well as the likely protein complexes formed between them. Expression levels of these transcription factors in the kidney suggest their central role in controlling UMOD expression. CONCLUSIONS: Our findings will form a map for understanding the regulation of uromodulin expression in health and disease.

Rational design of a fibroblast growth factor 21-based clinical candidate, LY2405319.

Fibroblast growth factor 21 is a novel hormonal regulator with the potential to treat a broad variety of metabolic abnormalities, such as type 2 diabetes, obesity, hepatic steatosis, and cardiovascular disease. Human recombinant wild type FGF21 (FGF21) has been shown to ameliorate metabolic disorders in rodents and non-human primates. However, development of FGF21 as a drug is challenging and requires re-engineering of its amino acid sequence to improve protein expression and formulation stability. Here we report the design and characterization of a novel FGF21 variant, LY2405319. To enable the development of a potential drug product with a once-daily dosing profile, in a preserved, multi-use formulation, an additional disulfide bond was introduced in FGF21 through Leu118Cys and Ala134Cys mutations. FGF21 was further optimized by deleting the four N-terminal amino acids, His-Pro-Ile-Pro (HPIP), which was subject to proteolytic cleavage. In addition, to eliminate an O-linked glycosylation site in yeast a Ser167Ala mutation was introduced, thus allowing large-scale, homogenous protein production in Pichia pastoris. Altogether re-engineering of FGF21 led to significant improvements in its biopharmaceutical properties. The impact of these changes was assessed in a panel of in vitro and in vivo assays, which confirmed that biological properties of LY2405319 were essentially identical to FGF21. Specifically, subcutaneous administration of LY2405319 in ob/ob and diet-induced obese (DIO) mice over 7-14 days resulted in a 25-50% lowering of plasma glucose coupled with a 10-30% reduction in body weight. Thus, LY2405319 exhibited all the biopharmaceutical and biological properties required for initiation of a clinical program designed to test the hypothesis that administration of exogenous FGF21 would result in effects on disease-related metabolic parameters in humans.

Fundamentals of FGF19 & FGF21 action in vitro and in vivo.

Fibroblast growth factors 19 (FGF19) and 21 (FGF21) have emerged as key regulators of energy metabolism. Several studies have been conducted to understand the mechanism of FGF19 and FGF21 action, however, the data presented has often been inconsistent and at times contradictory. Here in a single study we compare the mechanisms mediating FGF19/FGF21 actions, and how similarities/differences in actions at the cellular level between these two factors translate to common/divergent physiological outputs. Firstly, we show that in cell culture FGF19/FGF21 are very similar, however, key differences are still observed differentiating the two. In vitro we found that both FGF's activate FGFRs in the context of ßKlotho (KLB) expression. Furthermore, both factors alter ERK phosphorylation and glucose uptake with comparable potency. Combination treatment of cells with both factors did not have additive effects and treatment with a competitive inhibitor, the FGF21 delta N17 mutant, also blocked FGF19's effects, suggestive of a shared receptor activation mechanism. The key differences between FGF21/FGF19 were noted at the receptor interaction level, specifically the unique ability of FGF19 to bind/signal directly via FGFR4. To determine if differential effects on energy homeostasis and hepatic mitogenicity exist we treated DIO and ob/ob mice with FGF19/FGF21. We find comparable efficacy of the two proteins to correct body weight and serum glucose in both DIO and ob/ob mice. Nevertheless, FGF21 and FGF19 had distinctly different effects on proliferation in the liver. Interestingly, in vivo blockade of FGF21 signaling in mice using ΔN17 caused profound changes in glycemia indicative of the critical role KLB and FGF21 play in the regulation of glucose homeostasis. Overall, our data demonstrate that while subtle differences exist in vitro the metabolic effects in vivo of FGF19/FGF21 are indistinguishable, supporting a shared mechanism of action for these two hormones in the regulation of energy balance.

Different roles of N- and C- termini in the functional activity of FGF21.

Fibroblast growth factor 21 is a member of endocrine FGFs subfamily, along with FGF19 and FGF23. It is emerging as a novel regulator with beneficial effects on a variety of metabolic parameters, including glucose and lipid control. FGF21 activity depends on membrane protein betaKlotho that physically complexes with various FGF receptors, thus conferring them the ability to bind FGF21 and activate downstream signaling pathways. FGF21, like other FGFs, folds to a beta-trefoil-like core region, with disordered N- and C-termini. In order to investigate their role in the activity of FGF21, we have constructed a series of deletion mutants and tested them for their ability to (1) bind betaKlotho, analyzed by surface plasmon resonance spectroscopy (2) signal through MAPK phosphorylation and inhibit apoptosis in 3T3-L1/betaKlotho fibroblasts (3) stimulate GLUT1 mRNA upregulation and glucose uptake in 3T3-L1 adipocytes. Binding studies with betaKlotho revealed that the interaction with the co-receptor involves the C-terminus, as progressive removal of amino acids from the carboxy end decreased affinity for betaKlotho. By contrast, removal of up to 17 amino acids from the N-terminus had no effect on the interaction with betaKlotho. Terminal deletions had greater effect on function, as deletions of six amino acids from the amino-terminus and only four from the carboxy-terminus each significantly impacted activity (10-fold). Of the extreme terminal truncations, with no detectable activity, DeltaN17 acted as competitive antagonist while DeltaC20 did not. Our structure/function studies show that the C-terminus is important for betaKlotho interaction whereas the N-terminus likely interacts directly with FGF receptors.

FGF-21/FGF-21 receptor interaction and activation is determined by betaKlotho.

Fibroblast growth factor-21 (FGF-21) is a metabolic regulator that can influence glucose and lipid control in diabetic rodents and primates. We demonstrate that betaKlotho is an integral part of an activated FGF-21-betaKlotho-FGF receptor (FGFR) complex thus a critical subunit of the FGF-21 receptor. Cells lacking betaKlotho did not respond to FGF-21; the introduction of betaKlotho to these cells conferred FGF-21-responsiveness and recapitulated the entire scope of FGF-21 signaling observed in naturally responsive cells. Interestingly, FGF-21-mediated effects are heparin independent suggesting that betaKlotho plays a role in FGF-21 activity similar to the one played by heparin in the signaling of conventional FGFs. Moreover, in addition to conferring specificity for FGF-21, betaKlotho appears to support FGF-19 activity and mediates the receptor selectivity profile of FGF-19. All together, these results indicate that betaKlotho and FGFRs form the cognate FGF-21 receptor complex, mediating FGF-21 cellular specificity and physiological effects.

pI-shifted insulin analogs with extended in vivo time action and favorable receptor selectivity.

A long-acting (basal) insulin capable of delivering flat, sustained, reproducible glycemic control with once daily administration represents an improvement in the treatment paradigm for both type 1 and type 2 diabetes. Optimization of insulin pharmacodynamics is achievable through structural modification, but often at the expense of alterations in receptor affinity and selectivity. A series of isoelectric point (pI)-shifted insulin analogs based on the human insulin sequence or the GlyA21 acid stable variant were prepared by semi-synthetic methods. The pI shift was achieved through systematic addition of one or more arginine (Arg) or lysine (Lys) residues at the N terminus of the A chain, the N terminus of the B chain, the C terminus of the B chain, or through a combination of additions at two of the three sites. The analogs were evaluated for their affinity for the insulin and IGF-1 receptors, and aqueous solubility under physiological pH conditions. Notably, the presence of positively charged amino acid residues at the N terminus of the A chain was consistently associated with an enhanced insulin to IGF-1 receptor selectivity profile. Increased IGF-1 receptor affinity that results from Arg addition to the C terminus of the B chain was attenuated by cationic extension at the N terminus of the A chain. Analogs 10, 17, and 18 displayed in vitro receptor selectivity similar to that of native insulin and solubility at physiological pH that suggested the potential for extended time action. Accordingly, the in vivo pharmacokinetic and pharmacodynamic profiles of these analogs were established in a somatostatin-induced diabetic dog model. Analog 18 (A0:Arg, A21:Gly, B31:Arg, B32:Arg human insulin) exhibited a pharmacological profile comparable to that of analog 15 (insulin glargine) but with a 4.5-fold more favorable insulin:IGF-1 receptor selectivity. These results demonstrate that the selective combination of positive charge to the N terminus of the A chain and the C terminus of the B chain generates an insulin with sustained pharmacology and a near-native receptor selectivity profile.

FGF-21 as a novel metabolic regulator.

Diabetes mellitus is a major health concern, affecting more than 5% of the population. Here we describe a potential novel therapeutic agent for this disease, FGF-21, which was discovered to be a potent regulator of glucose uptake in mouse 3T3-L1 and primary human adipocytes. FGF-21-transgenic mice were viable and resistant to diet-induced obesity. Therapeutic administration of FGF-21 reduced plasma glucose and triglycerides to near normal levels in both ob/ob and db/db mice. These effects persisted for at least 24 hours following the cessation of FGF-21 administration. Importantly, FGF-21 did not induce mitogenicity, hypoglycemia, or weight gain at any dose tested in diabetic or healthy animals or when overexpressed in transgenic mice. Thus, we conclude that FGF-21, which we have identified as a novel metabolic factor, exhibits the therapeutic characteristics necessary for an effective treatment of diabetes.

Pharmacokinetics, metabolic stability, and subcutaneous bioavailability of a genetically engineered analog of DcR3, FLINT [DcR3(R218Q)], in cynomolgus monkeys and mice.

Decoy receptor 3 (DcR3) is a novel member of the tumor necrosis factor receptor superfamily, which binds to and blocks the activities of the ligands, FasL and LIGHT (a cellular ligand for herpes virus entry mediator and lymphotoxin receptor), that play an important role in regulating apoptosis in normal physiology. DcR3 was rapidly degraded to a major circulating metabolic fragment, DcR3(1-218), after subcutaneous administration in primates and mice. DcR3 was molecularly engineered by changing the arginine residue at position 218 to glutamine to generate a potentially stable analog, DcR3(R218Q), which we termed FasLigand inhibitor protein [FLINT (LY498919)]. The influence of this modification on the kinetics and bioavailability of DcR3 was evaluated in primates and mice. After i.v. administration of FLINT and DcR3, both compounds were cleared from the plasma in a bi-phasic manner, with the terminal phase half-life being somewhat longer for FLINT than for DcR3. After s.c. administration, the exposure to the full-length form of FLINT was 5.7- to 6-fold greater than for DcR3. In both primates and mice, greater than 90% of circulating immunoreactivity after s.c. administration of FLINT was associated with intact molecule, whereas only 17 to 37% was associated with intact molecule after administration of DcR3. The absolute s.c. bioavailability of intact FLINT was approximately 4- to 6-fold higher than for DcR3. The improved s.c. bioavailability of FLINT is related to the increased metabolic stability afforded to the molecule as a result of the amino acid mutation at position 218 of the primary sequence of DcR3 and may translate to the need for lower therapeutic doses in a number of disease indications.