Healthy Women Have Higher Systemic Uromodulin Levels: Identification of Uromodulin as an Estrogen Responsive Gene.

Serum uromodulin levels are higher in healthy female participants than healthy male participants. Serum uromodulin levels in participants with normal kidney function do not correlate with eGFR but do correlate with body mass index. Estrogen increases uromodulin production, likely because of noncanonical and half estrogen response elements in the gene.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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)].

Decoy receptor 3 (DcR3) is proteolytically processed to a metabolic fragment having differential activities against Fas ligand and LIGHT.

Fas ligand (FasL) and Fas receptor are members of the tumor necrosis factor (TNF) receptor and ligand family that play an important role in regulating apoptosis in normal physiology. Decoy receptor 3 (DcR3) is a novel member of the TNF receptor superfamily, which binds to and blocks the activities of the ligands FasL and LIGHT. We have demonstrated that DcR3 was degraded rapidly to a major circulating metabolic fragment after subcutaneous administration in primates and mice.

Glycosylation of erythropoietin affects receptor binding kinetics: role of electrostatic interactions.

Erythropoietin (EPO) is a cytokine produced by the kidney whose function is to stimulate red blood cell production in the bone marrow. Previously, it was shown that the affinity of EPO for its receptor, EPOR, is inversely related to the sialylation of EPO carbohydrate. To better understand the properties of EPO that modulate its receptor affinity, various glycoforms were analyzed using surface plasmon resonance.

Competitive, reversible inhibition of cytosolic phospholipase A2 at the lipid-water interface by choline derivatives that partially partition into the phospholipid bilayer.

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA2 using membranes isolated from [3H]arachidonate-labeled U937 cells as substrate, 2-(2'-benzyl-4-chlorophenoxy)ethyl-dimethyl-n-octadecyl-ammonium chloride (compound 1) was found to inhibit the enzyme in a dose-dependent manner (IC50 = 5 microM). It was over 70 times more selective for the cPLA2 as compared with the human nonpancreatic secreted phospholipase A2, and it did not inhibit other phospholipases.

Presence of glycerol masks the effects of phosphorylation on the catalytic efficiency of cytosolic phospholipase A2.

Cytosolic phospholipase A2 catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. The enzymatic activity of cPLA2 is affected by several mechanisms, including substrate presentation and the phosphorylation state of the enzyme. Using covesicles of 1-palmitoy1-2-arachidonoyl-[arachidonoyl-1-14C]-8n-glycero-3 -phosphocholine and 1,2-dimyristoyl-phosphatidylmethanol as substrate, the effects of phosphorylation on the interfacial binding and catalytic constants were investigated.

Cooperativity and binding in the mechanism of cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2 ester of phospholipids and is believed to be responsible for the receptor-regulated release of arachidonic acid from phospholipid pools. The enzyme was assayed using vesicles containing arachidonate-containing phospholipid substrate, such as 1-palmitoyl-2-arachidonoylphosphatidylcholine (PAPC) or 1-stearoyl-2-arachidonoylphosphatidylinositol (SAPI), dispersed within vesicles of 1,2-dimyristoylphosphatidylmethanol (DMPM). We report here that the enzyme shows an apparent cooperative effect with respect to the mole fraction of arachidonate-containing phospholipids within these covesicles.