The Epithelial Sodium Channel γ-Subunit Is Processed Proteolytically in Human Kidney

The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the γ-subunit and putative release of a 43-amino acid inhibitory tract from the γ-subunit ectodomain. We hypothesized that proteolytic processing of γENaC occurs in the human kidney under physiologic conditions and that proteinuria contributes to aberrant proteolytic activation. Here, we used monoclonal antibodies (mAbs) with specificity to the human 43-mer inhibitory tract (N and C termini, mAb_inhibit, and mAb4C11) and the neoepitope generated after proteolytic cleavage at the prostasin/kallikrein cleavage site (K181-V182 and mAb_prostasin) to examine human nephrectomy specimens. By immunoblotting, kidney cortex homogenate from patients treated with angiotensin II type 1 receptor antagonists (n=6) or angiotensin-converting enzyme inhibitors (n=6) exhibited no significant difference in the amount of full-length or furin-cleaved γENaC or the furin-cleaved–to–full-length ratio of γENaC compared with homogenate from patients on no medication (n=5). Patients treated with diuretics (n=4) displayed higher abundance of full-length and furin-cleaved γENaC, with no significant change in the furin-cleaved–to–full-length γENaC ratio. In patients with proteinuria (n=6), the inhibitory tract was detected only in full-length γENaC by mAb_inhibit. Prostasin/kallikrein-cleaved γENaC was detected consistently only in tissue from patients with proteinuria and observed in collecting ducts. In conclusion, human kidney γENaC is subject to proteolytic cleavage, yielding fragments compatible with furin cleavage, and proteinuria is associated with cleavage at the putative prostasin/kallikrein site and removal of the inhibitory tract within γENaC.     

A High-Fat Diet Attenuates AMPK α1 in Adipocytes to Induce Exosome Shedding and Nonalcoholic Fatty Liver Development In Vivo

Exosomes are important for intercellular communication, but the role of exosomes in the communication between adipose tissue (AT) and the liver remains unknown. The aim of this study is to determine the contribution of AT-derived exosomes in nonalcoholic fatty liver disease (NAFLD). Exosome components, liver fat content, and liver function were monitored in AT in mice fed a high-fat diet (HFD) or treated with metformin or GW4869 and with AMPKα1-floxed (Prkaα1^fl/fl/wild-type [WT]), Prkaα1^−/−, liver tissue-specific Prkaα1^−/−, or AT-specific Prkaα1^−/− modification. In cultured adipocytes and white AT, the absence of AMPKα1 increased exosome release and exosomal proteins by elevating tumor susceptibility gene 101 (TSG101)–mediated exosome biogenesis. In adipocytes treated with palmitic acid, TSG101 facilitated scavenger receptor class B (CD36) sorting into exosomes. CD36-containing exosomes were then endocytosed by hepatocytes to induce lipid accumulation and inflammation. Consistently, an HFD induced more severe lipid accumulation and cell death in Prkaα1^−/− and AT-specific Prkaα1^−/− mice than in WT and liver-specific Prkaα1^−/− mice. AMPK activation by metformin reduced adipocyte-mediated exosome release and mitigated fatty liver development in WT and liver-specific Prkaα1^−/− mice. Moreover, administration of the exosome inhibitor GW4869 blocked exosome secretion and alleviated HFD-induced fatty livers in Prkaα1^−/− and adipocyte-specific Prkaα1^−/− mice. We conclude that HFD-mediated AMPKα1 inhibition promotes NAFLD by increasing numbers of AT CD36-containing exosomes.     

Hypertrophy in the Distal Convoluted Tubule of an 11β-Hydroxysteroid Dehydrogenase Type 2 Knockout Model

Na⁺ transport in the renal distal convoluted tubule (DCT) by the thiazide-sensitive NaCl cotransporter (NCC) is a major determinant of total body Na⁺ and BP. NCC-mediated transport is stimulated by aldosterone, the dominant regulator of chronic Na⁺ homeostasis, but the mechanism is controversial. Transport may also be affected by epithelial remodeling, which occurs in the DCT in response to chronic perturbations in electrolyte homeostasis. Hsd11b2^−/− mice, which lack the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) and thus exhibit the syndrome of apparent mineralocorticoid excess, provided an ideal model in which to investigate the potential for DCT hypertrophy to contribute to Na⁺ retention in a hypertensive condition. The DCTs of Hsd11b2^−/− mice exhibited hypertrophy and hyperplasia and the kidneys expressed higher levels of total and phosphorylated NCC compared with those of wild-type mice. However, the striking structural and molecular phenotypes were not associated with an increase in the natriuretic effect of thiazide. In wild-type mice, Hsd11b2 mRNA was detected in some tubule segments expressing Slc12a3, but 11βHSD2 and NCC did not colocalize at the protein level. Thus, the phosphorylation status of NCC may not necessarily equate to its activity in vivo, and the structural remodeling of the DCT in the knockout mouse may not be a direct consequence of aberrant corticosteroid signaling in DCT cells. These observations suggest that the conventional concept of mineralocorticoid signaling in the DCT should be revised to recognize the complexity of NCC regulation by corticosteroids.     

Xanthophyll β-cryptoxanthin treatment inhibits hepatic steatosis without altering vitamin A status in β-carotene 9',10'-oxygenase knockout mice

Backgroundβ-cryptoxanthin (BCX), one of the major carotenoids detected in human circulation, can protect against the development of fatty liver disease. BCX can be metabolized through β-carotene-15,15''-oxygenase (BCO1) and β-carotene-9'',10''-oxygenase (BCO2) cleavage pathways to produce both vitamin A and apo-carotenoids, respectively, which are considered important signaling molecules in a variety of biological processes. Recently, we have demonstrated that BCX treatment reduced hepatic steatosis severity and hepatic total cholesterol levels in both wide type and BCO1^–/–/BCO2^–/– double knock out (KO) mice. Whether the protective effect of BCX is seen in single BCO2^–/– KO mice is unclear.MethodsIn the present study, male BCO2^–/– KO mice at 1 and 5 months of age were assigned to two groups by age and weight-matching as follows: (I) –BCX control diet alone (AIN-93 purified diets); (II) +BCX 10 mg (supplemented with 10 mg of BCX/kg of diet) for 3 months. At 4 and 8 months of age, hepatic steatosis and inflammatory foci were evaluated by histopathology. Retinoids and BCX concentrations in liver tissue were analyzed by high-performance liquid chromatography (HPLC). Hepatic protein expressions of SIRT1, acetylated and total FoxO1, PGC1α, and PPARα were determined by the Western blot analysis. Real-time PCR for gene expressions (MCAD, SCD1, FAS, TNFα, and IL-1β gene expression relative to β-actin) was conducted in the liver.ResultsSteatosis was detected at 8 months but not at 4 months of age. Moreover, BCX supplementation significantly reduced the severity of steatosis in the livers of BCO2 KO mice, which was associated with changes in hepatic SIRT1 acetylation of FOXO1, PGC1α protein expression and PPARα protein expression in BCO2^–/– KO mice. HPLC analysis showed that hepatic BCX was detected in BCX supplemented groups, but there were no differences in the hepatic levels of retinol and retinyl palmitate (RP) among all groups.ConclusionsThe present study provided experimental evidence that BCX intervention can reduce liver steatosis independent of BCO2.     

Purinergic Inhibition of ENaC Produces Aldosterone Escape

The mechanisms underlying “aldosterone escape,” which refers to the excretion of sodium (Na⁺) during high Na⁺ intake despite inappropriately increased levels of mineralocorticoids, are incompletely understood. Because local purinergic tone in the aldosterone-sensitive distal nephron downregulates epithelial Na⁺ channel (ENaC) activity, we tested whether this mechanism mediates aldosterone escape. Here, urinary ATP concentration increased with dietary Na⁺ intake in mice. Physiologic concentrations of ATP decreased ENaC activity in a dosage-dependent manner. P2Y₂^−/− mice, which lack the purinergic receptor, had significantly less increased Na⁺ excretion than wild-type mice in response to high-Na⁺ intake. Exogenous deoxycorticosterone acetate and deletion of the P2Y₂ receptor each modestly increased the resistance of ENaC to changes in Na⁺ intake; together, they markedly increased resistance. Under the latter condition, ENaC could not respond to changes in Na⁺ intake. In contrast, as a result of aldosterone escape, wild-type mice had increased Na⁺ excretion in response to high-Na⁺ intake regardless of the presence of high deoxycorticosterone acetate. These data suggest that control of ENaC by purinergic signaling is necessary for aldosterone escape.Renal sodium (Na⁺) excretion influences BP by affecting systemic Na⁺ balance. Consequently, negative feedback in response to changes in Na⁺ balance, perceived as changes in effective circulating volume (ECV) and BP, control renal Na⁺ handling. The fine-tuning of Na⁺ balance occurs in the aldosterone-sensitive distal nephron (ASDN), including the connecting tubule (CNT) and the collecting duct (CD). Here, Na⁺ reabsorption is highest when ECV and BP are low. Activity of the epithelial Na⁺ channel (ENaC) is limiting for discretionary Na⁺ reabsorption across the ASDN.^1–7 As ECV declines, activity of the renin-angiotensin-aldosterone system (RAAS) increases with the mineralocorticoid aldosterone, stimulating channel activity to decrease Na⁺ excretion in correction of falling ECV. Aldosterone increases the number and activity of ENaC in the apical membrane.^8–11 Under normal conditions, the opposite is also true; aldosterone and, thus, ENaC activity decline in response to elevations in BP.The thiazide-sensitive Na-Cl co-transporter (NCC) in the distal convoluted tubule (DCT) is also a target of aldosterone with the mineralocorticoid increasing NCC activity and possibly transporter abundance in the apical membrane of DCT cells.^12–16 This increase in NCC activity decreases Na⁺ excretion. Thus, aldosterone promotes distal nephron Na⁺ reabsorption by increasing the activity of ENaC and NCC.The loss of negative-feedback regulation of renal Na⁺ excretion mediated by ENaC and NCC leads to hypertension. For instance, gain-of-function mutations in ENaC cause the channel to be hyperactive irrespective of mineralocorticoid status and systemic Na⁺ balance, thus leading to inappropriate Na⁺ retention and hypertension; conversely, loss-of-function mutations in ENaC can cause decreases in BP and renal salt wasting.^{2,3,5,7,17–19}The aldosterone-promoted decrease in renal Na⁺ excretion is overridden in some circumstances.^20,21 This disruption of aldosterone action is termed aldosterone escape and results in avid Na⁺ excretion during high Na⁺ intake despite elevated mineralocorticoid levels. Aldosterone escape seems to be a protective mechanism to allow appropriate response to positive Na⁺ balance despite inappropriate mineralocorticoid levels. Such escape is important clinically, for example, in primary aldosteronism, in which it may ameliorate, to some degree, the hypertensive effects of high circulating levels of aldosterone.^22–24 The mechanism allowing aldosterone escape is uncertain.Aldosterone escape is known to be dependent on increases in renal vascular perfusion pressure rather than systemic factors, including changes in the levels of circulating hormones, such as renin and aldosterone, or renal nerve activity.²⁵ This led to the idea that aldosterone escape is a manifestation of a pressure natriuresis response.^13,25,26 This is associated with declining Na⁺ reabsorption in the distal nephron despite elevated levels of aldosterone and occurs independent of changes in GFR.^26,27 Indeed, early micropuncture measurements demonstrated increases in urine flow and Na⁺ delivery to the CD during escape.^26,27 Details about the specific site(s) along the distal nephron involved in decreased Na⁺ reabsorption during aldosterone escape and the exact transport proteins involved in this escape, though, largely remain obscure.A study by Wang et al.¹³ revealed that during aldosterone escape, NCC levels in the DCT decrease. This response was selective because the levels of other apical membrane transport proteins, including ENaC, were unaffected. This finding supports the idea that NCC is, at least, one target for regulatory processes that mediate aldosterone escape, whereby decreasing NCC abundance facilitates Na⁺ excretion despite high levels of mineralocorticoid. The cellular mechanism underpinning declines in NCC levels during aldosterone escape is unknown. Similarly, it is unclear whether other transport proteins are involved in aldosterone escape because Na⁺ reabsorption is a manifestation not only of the number of transport proteins in the apical membrane but also of their activity.Because ENaC is critical to aldosterone regulation of Na⁺ excretion, particularly in response to changes in Na⁺ balance, evidenced by the hypertension associated with gain of ENaC function,^3,5,7 we sought to investigate the role of this channel in aldosterone escape. We recently demonstrated that local purinergic tone in the ASDN exerts paracrine downregulation of ENaC activity specifically by affecting channel open probability.²⁸ This paracrine pathway is physiologically relevant because mice lacking the purinergic receptor P2Y₂, responsible for the bulk of paracrine regulation in this system, have facilitated Na⁺ reabsorption in the distal nephron and mild increases in BP.²⁹ Increases in NKCC2 and ENaC activity contribute to this elevated BP.¹¹ It does not seem a coincidence that aldosterone and P2Y₂ signaling target the same final effector proteins, possibly allowing one to compensate for the loss of the other. This idea suggests that purinergic regulation of ENaC may contribute to aldosterone escape, a possibility supported by another recent finding from our laboratories: Elevated BP in P2Y₂^−/− mice is not salt sensitive in the presence of normal-feedback regulation by RAAS but becomes salt sensitive and associated with inappropriately active ENaC when negative-feedback regulation by aldosterone and local purinergic signaling both are disrupted.^11,29To test the role of ENaC and its regulation by mineralocorticoids and purinergic signaling in aldosterone escape and to understand better the mechanism underpinning escape, we quantified the actions of mineralocorticoid on renal Na⁺ excretion, ENaC activity, and urinary ATP levels in wild-type (WT) and P2Y₂^−/− mice stressed with different dietary Na⁺ regimens. We found that urinary [ATP] increases with dietary Na⁺ intake such that physiologic [ATP] decrease ENaC activity, resulting in increased Na⁺ excretion. Increased Na⁺ excretion is greater in WT compared with P2Y₂^−/− mice and associated with an inability of ENaC, particularly when mineralocorticoid is clamped at high levels, to respond appropriately to changes in dietary Na⁺ intake in the latter animals. Because ENaC activity normally is sensitive to changes in Na⁺ balance even when mineralocorticoids are clamped at high levels, these results show that control of ENaC by purinergic signaling is necessary for complete aldosterone escape, consistent with the loss of aldosterone-escape in P2Y₂^−/− mice and their pronounced hypertension relative to normal mice in the presence of elevated mineralocorticoids and high Na⁺ intake.     

Hyperhomocysteinemia and Hyperglycemia Induce and Potentiate Endothelial Dysfunction via μ-Calpain Activation

Plasma homocysteine (Hcy) levels are positively correlated with cardiovascular mortality in diabetes. However, the joint effect of hyperhomocysteinemia (HHcy) and hyperglycemia (HG) on endothelial dysfunction (ED) and the underlying mechanisms have not been studied. Mild (22 µmol/L) and moderate (88 µmol/L) HHcy were induced in cystathionine β-synthase wild-type (Cbs^+/+) and heterozygous-deficient (Cbs^−/+) mice by a high-methionine (HM) diet. HG was induced by consecutive injection of streptozotocin. We found that HG worsened HHcy and elevated Hcy levels to 53 and 173 µmol/L in Cbs^+/+ and Cbs^−/+ mice fed an HM diet, respectively. Both mild and moderate HHcy aggravated HG-impaired endothelium-dependent vascular relaxation to acetylcholine, which was completely abolished by endothelial nitric oxide synthase (eNOS) inhibitor N^G-nitro-L-arginine methyl ester. HHcy potentiated HG-induced calpain activation in aortic endothelial cells isolated from Cbs mice. Calpain inhibitors rescued HHcy- and HHcy/HG-induced ED in vivo and ex vivo. Moderate HHcy- and HG-induced μ-calpain activation was potentiated by a combination of HHcy and HG in the mouse aorta. μ-Calpain small interfering RNA (μ-calpsiRNA) prevented HHcy/HG-induced ED in the mouse aorta and calpain activation in human aortic endothelial cells (HAECs) treated with DL-Hcy (500 µmol/L) and d-glucose (25 mmol) for 48 h. In addition, HHcy accelerated HG-induced superoxide production as determined by dihydroethidium and 3-nitrotyrosin staining and urinary 8-isoprostane/creatinine assay. Antioxidants rescued HHcy/HG-induced ED in mouse aortas and calpain activation in cultured HAECs. Finally, HHcy potentiated HG-suppressed nitric oxide production and eNOS activity in HAECs, which were prevented by calpain inhibitors or μ-calpsiRNA. HHcy aggravated HG-increased phosphorylation of eNOS at threonine 497/495 (eNOS-pThr497/495) in the mouse aorta and HAECs. HHcy/HG-induced eNOS-pThr497/495 was reversed by µ-calpsiRNA and adenoviral transduced dominant negative protein kinase C (PKC)β₂ in HAECs. HHcy and HG induced ED, which was potentiated by the combination of HHcy and HG via μ-calpain/PKCβ₂ activation–induced eNOS-pThr497/495 and eNOS inactivation.     

MicroRNA-21 in Glomerular Injury

TGF-β₁ is a pleotropic growth factor that mediates glomerulosclerosis and podocyte apoptosis, hallmarks of glomerular diseases. The expression of microRNA-21 (miR-21) is regulated by TGF-β₁, and miR-21 inhibits apoptosis in cancer cells. TGF-β₁–transgenic mice exhibit accelerated podocyte loss and glomerulosclerosis. We determined that miR-21 expression increases rapidly in cultured murine podocytes after exposure to TGF-β₁ and is higher in kidneys of TGF-β₁–transgenic mice than wild-type mice. miR-21–deficient TGF-β₁–transgenic mice showed increased proteinuria and glomerular extracellular matrix deposition and fewer podocytes per glomerular tuft compared with miR-21 wild-type TGF-β₁–transgenic littermates. Similarly, miR-21 expression was increased in streptozotocin-induced diabetic mice, and loss of miR-21 in these mice was associated with increased albuminuria, podocyte depletion, and mesangial expansion. In cultured podocytes, inhibition of miR-21 was accompanied by increases in the rate of cell death, TGF-β/Smad3-signaling activity, and expression of known proapoptotic miR-21 target genes p53, Pdcd4, Smad7, Tgfbr2, and Timp3. In American-Indian patients with diabetic nephropathy (n=48), albumin-to-creatinine ratio was positively associated with miR-21 expression in glomerular fractions (r=0.6; P<0.001) but not tubulointerstitial fractions (P=0.80). These findings suggest that miR-21 ameliorates TGF-β1 and hyperglycemia-induced glomerular injury through repression of proapoptotic signals, thereby inhibiting podocyte loss. This finding is in contrast to observations in murine models of tubulointerstitial kidney injury but consistent with findings in cancer models. The aggravation of glomerular disease in miR-21–deficient mice and the positive association with albumin-to-creatinine ratio in patients with diabetic nephropathy support miR-21 as a feedback inhibitor of TGF-β signaling and functions.     

Klotho and Phosphate Are Modulators of Pathologic Uremic Cardiac Remodeling

Cardiac dysfunction in CKD is characterized by aberrant cardiac remodeling with hypertrophy and fibrosis. CKD is a state of severe systemic Klotho deficiency, and restoration of Klotho attenuates vascular calcification associated with CKD. We examined the role of Klotho in cardiac remodeling in models of Klotho deficiency—genetic Klotho hypomorphism, high dietary phosphate intake, aging, and CKD. Klotho-deficient mice exhibited cardiac dysfunction and hypertrophy before 12 weeks of age followed by fibrosis. In wild-type mice, the induction of CKD led to severe cardiovascular changes not observed in control mice. Notably, non-CKD mice fed a high-phosphate diet had lower Klotho levels and greatly accelerated cardiac remodeling associated with normal aging compared with those on a normal diet. Chronic elevation of circulating Klotho because of global overexpression alleviated the cardiac remodeling induced by either high-phosphate diet or CKD. Regardless of the cause of Klotho deficiency, the extent of cardiac hypertrophy and fibrosis correlated tightly with plasma phosphate concentration and inversely with plasma Klotho concentration, even when adjusted for all other covariables. High-fibroblast growth factor–23 concentration positively correlated with cardiac remodeling in a Klotho-deficient state but not a Klotho-replete state. In vitro, Klotho inhibited TGF-β1–, angiotensin II–, or high phosphate–induced fibrosis and abolished TGF-β1– or angiotensin II–induced hypertrophy of cardiomyocytes. In conclusion, Klotho deficiency is a novel intermediate mediator of pathologic cardiac remodeling, and fibroblast growth factor–23 may contribute to cardiac remodeling in concert with Klotho deficiency in CKD, phosphotoxicity, and aging.     

Effect of Plasma Uric Acid on Antioxidant Capacity,Oxidative Stress,and Insulin Sensitivity in Obese Subjects

Oxidative stress is purported to be involved in the pathogenesis of obesity-associated insulin resistance. We evaluated whether alterations in levels of circulating uric acid (UA), a systemic antioxidant, affects the following: 1) systemic (plasma and saliva) nonenzymatic antioxidant capacity (NEAC); 2) markers of systemic (urinary 8-iso-prostaglandin-F_2α) and muscle (carbonylated protein content) oxidative stress; and 3) whole-body insulin sensitivity (percentage increase in glucose uptake during a hyperinsulinemic-euglycemic clamp procedure). Thirty-one obese subjects (BMI 37.1 ± 0.7 kg/m²) with either high serum UA (HUA; 7.1 ± 0.4 mg/dL; n = 15) or normal serum UA (NUA; 4.5 ± 0.2 mg/dL; n = 16) levels were studied; 13 subjects with HUA levels were studied again after reduction of serum UA levels to 0 by infusing a recombinant urate oxidase. HUA subjects had 20–90% greater NEAC, but lower insulin sensitivity (40%) and levels of markers of oxidative stress (30%) than subjects in the NUA group (all P < 0.05). Acute UA reduction caused a 45–95% decrease in NEAC and a 25–40% increase in levels of systemic and muscle markers of oxidative stress (all P < 0.05), but did not affect insulin sensitivity (from 168 ± 25% to 156 ± 17%, P = NS). These results demonstrate that circulating UA is a major antioxidant and might help protect against free-radical oxidative damage. However, oxidative stress is not a major determinant of insulin action in vivo.     

Renal Production,Uptake, and Handling of Circulating αKlotho

αKlotho is a multifunctional protein highly expressed in the kidney. Soluble αKlotho is released through cleavage of the extracellular domain from membrane αKlotho by secretases to function as an endocrine/paracrine substance. The role of the kidney in circulating αKlotho production and handling is incompletely understood, however. Here, we found higher αKlotho concentration in suprarenal compared with infrarenal inferior vena cava in both rats and humans. In rats, serum αKlotho concentration dropped precipitously after bilateral nephrectomy or upon treatment with inhibitors of αKlotho extracellular domain shedding. Furthermore, the serum half-life of exogenous αKlotho in anephric rats was four- to five-fold longer than that in normal rats, and exogenously injected labeled recombinant αKlotho was detected in the kidney and in urine of rats. Both in vivo (micropuncture) and in vitro (proximal tubule cell line) studies showed that αKlotho traffics from the basal to the apical side of the proximal tubule via transcytosis. Thus, we conclude that the kidney has dual roles in αKlotho homeostasis, producing and releasing αKlotho into the circulation and clearing αKlotho from the blood into the urinary lumen.     

Thrombospondin-1 Activation of Signal-Regulatory Protein-α Stimulates Reactive Oxygen Species Production and Promotes Renal Ischemia Reperfusion Injury

Ischemia reperfusion injury (IRI) causes tissue and organ injury, in part, through alterations in tissue blood flow and the production of reactive oxygen species. The cell surface receptor signal-regulatory protein-α (SIRP-α) is expressed on inflammatory cells and suppresses phagocytosis, but the function of SIRP-α in IRI has not been determined. We reported previously that the matricellular protein thrombospondin-1 is upregulated in IRI. Here, we report a novel interaction between thrombospondin-1 and SIRP-α on nonphagocytic cells. In cell-free experiments, thrombospondin-1 bound SIRP-α. In vascular smooth muscle cells and renal tubular epithelial cells, treatment with thrombospondin-1 led to phosphorylation of SIRP-α and downstream activation of Src homology domain 2–containing phosphatase-1. Thrombospondin-1 also stimulated phosphorylation of p47^phox (an organizer subunit for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1/2) and increased production of superoxide, both of which were abrogated by knockdown or antibody blockade of SIRP-α. In rodent aortic rings, treatment with thrombospondin-1 increased the production of superoxide and inhibited nitric oxide–mediated vasodilation in a SIRP-α–dependent manner. Renal IRI upregulated the thrombospondin-1–SIRP-α signaling axis and was associated with increased superoxide production and cell death. A SIRP-α antibody that blocks thrombospondin-1 activation of SIRP-α mitigated the effects of renal IRI, increasing blood flow, suppressing production of reactive oxygen species, and preserving cellular architecture. A role for CD47 in SIRP-α activation in these pathways is also described. Overall, these results suggest that thrombospondin-1 binding to SIRP-α on nonphagocytic cells activates NADPH oxidase, limits vasodilation, and promotes renal IRI.Thrombospondin-1 (TSP1) is a secreted matricellular protein produced by platelets, endothelial and vascular smooth muscle cells (VSMCs), and nonvascular cells.¹ TSP1 transduces signals from the extracellular to cellular components of tissues through binding to cell surface receptors, including the integrins, CD36 and CD47.² We and others have shown that TSP1 levels are increased in plasma and in conditions associated with decreased blood flow, such as ischemia reperfusion injury (IRI),³ atherosclerosis,⁴ pulmonary hypertension,^5,6 and sickle cell anemia.⁷Signal regulatory protein-α (SIRP-α) is a cell surface receptor expressed on phagocytic and neuronal cells and activated through interactions with the cell surface protein CD47, by growth factors or integrin signaling.^8,9 SIRP-α controls cell responses through the recruitment and phosphorylation of Src homology domain 2–containing phosphatase-1 (SHP1) and -2 (SHP2).¹⁰ SIRP-α is classified as an inhibitory cell receptor, and SIRP-α–mediated signaling suppresses macrophage phagocytosis.¹¹ However, little is known about the role of SIRP-α in vascular cells and IRI.Loss of nitric oxide (NO) signaling, including decreased NO bioavailability, is a major contributor to cardiovascular disease.¹² NO reacts rapidly with the reactive oxygen species (ROS) superoxide anion (O₂^·−) which dramatically limits its biologic effect.¹³ This interaction becomes important after ischemia reperfusion, where pathologic ROS production, including O₂^·−, is increased. We have shown that TSP1 inhibits NO signaling⁵ and limits blood flow,^14–16 but the exact mechanisms are still unclear.Our data demonstrate that TSP1 stimulates phosphorylation of nonphagocytic SIRP-α and stimulates NADPH oxidase (Nox)–mediated O₂^·− production and that SIRP-α phosphorylation is absent upon CD47 deletion. In arteries, TSP1 inhibits NO-mediated vasodilation through SIRP-α–dependent stimulation of ROS. IRI upregulates renal TSP1–SIRP-α signaling, increases pathologic ROS production, and promotes cell death. Disruption of TSP1–SIRP-α signaling inhibits O₂^·− production, promotes vasodilation, improves blood flow, and limits IRI.     

The HLA-DRB1*15:01–Restricted Goodpasture’s T Cell Epitope Induces GN

Human anti-glomerular basement membrane (GBM) disease strongly associates with HLA-DRB1*15:01. The target autoantigen in this disease is the noncollagenous domain of the α3 chain of type IV collagen, α3(IV)NC1, but critical early T cell epitopes presented by this human MHC class II molecule are unknown. Here, by immunizing HLA-DRB1*15:01 transgenic mice with whole recombinant α3(IV)NC1 and with overlapping α3(IV)NC1 peptides, we defined a HLA-DRB1*15:01–restricted α3(IV)NC1 T cell epitope (α3_136–146) with four critical residues. This peptide was not immunogenic in HLA-DRB1*01:01 transgenic or C57BL/6 mice. The T cell epitope is naturally processed from α3(IV)NC1. CD4⁺ T cell clones, generated from HLA-DRB1*15:01 transgenic mice and specific for α3_136–146, transferred disease into naive HLA-DRB1*15:01 transgenic mice, evidenced by the development of necrotizing crescentic GN, albuminuria, renal impairment, and accumulation of CD4⁺ T cells and macrophages in glomeruli. Because Fcγ receptors are implicated in disease susceptibility, we crossed HLA transgenic mice onto an FcγRIIb-deficient background. Immunization with either α3_136–146 or α3(IV)NC1 induced GN in HLA-DRB1*15:01 transgenic FcγRIIb-deficient mice, but HLA-DRB1*01:01 transgenic FcγRIIb-deficient mice were unaffected. Taken together, these results demonstrate that the HLA-DRB1*15:01–restricted T cell epitope α3_136–146 can induce T cell responses and injury in anti-GBM GN.Goodpasture’s disease, also known as anti-glomerular basement membrane (GBM) disease, is characterized by rapidly progressive GN with crescentic and necrotizing glomerular lesions, and in some patients, pulmonary hemorrhage. It is caused by autoreactivity to the noncollagenous domain of the α3 chain of type IV collagen, α3(IV)NC1, expressed in the GBM.^1,2 The diagnosis of anti-GBM disease is made by detecting serum α3(IV)NC1-reactive IgG autoantibodies, with linear IgG deposition in glomeruli.³ Treatment includes immunosuppressive agents and plasmapheresis to remove pathogenic autoantibodies.³ Cellular effectors also seem to play a role, based on studies in human anti-GBM disease⁴ and in experimental autoimmune anti-GBM GN.^5–9The MHC class II allele HLA-DRB1*15:01 is a key susceptibility element in anti-GBM disease.¹⁰ It confers a markedly increased relative risk for anti-GBM GN (odds ratio, 8.5; 95% confidence interval, 5.5–13.1). Most other HLA-DR alleles do not predispose people to developing anti-GBM disease and may even be protective (e.g., HLA-DRB1*01:01 [odds ratio, 0.6; 95% confidence interval, 0.3–1.0]). The strong HLA-DRB1*15:01 association and the accuracy of diagnostic criteria allow meaningful studies on how the MHC II molecule confers risk in mechanistic terms.Key human B cell epitopes in anti-GBM disease have been defined. Autoantibodies bind to the conformational α3(IV)NC1 epitopes “E_A” α3_17–31 and “E_B” α3_127–141 (all amino acid numbering, including other cited epitopes, follows Netzer et al.).¹¹ Studies examining T cell epitopes in human anti-GBM disease are less common, although an important study showed reactivity to two peptides, α3_68–89 and α3_129–148 (close to the E_B autoantibody epitope), in all six patients studied.¹² However, in the Wistar Kyoto (WKY) rat, T cell reactivity occurs in several different areas.^13–15 T cell–mediated disease can be induced by α3_14–26 and α3_11–25^15–17 and there is evidence of epitope spreading to involve B cell epitopes.^16,18Our hypothesis was based on studies in humans with anti-GBM GN showing strong MHC II association and suggesting key epitopes. We hypothesized that either the regions in or around α3_68–89 or α3_129–148 (or both) would be immunogenic in the context of HLA-DRB1*15:01 but not other MHC II molecules, and could be defined as nephritogenic CD4⁺ T cell epitopes. Therefore, HLA-DRB1*15:01 would permit binding of one or both of these peptides at an appropriate avidity, allowing naïve autoreactive CD4⁺ cells to escape negative selection in the thymus, thus explaining why people who express HLA-DRB1*15:01 are more susceptible to anti-GBM GN. We studied mice that were entirely deficient in mouse MHC II,^19,20 but that coexpressed the essentially nonpolymorphic HLA-DRA1*01:01, with either HLA-DRB1*15:01 or DRB1*01:01. HLA transgenic mice, especially those lacking all murine MHC II elements,¹⁹ have been powerful tools in studying the pathogenesis of autoimmune disease,^20,21 because their CD4⁺ T cell repertoire is shaped by the presence of human (but not mouse) MHC II molecules. We found that in anti-GBM GN, α3_136–146 is an immunodominant and nephritogenic CD4⁺ T epitope in DRB1*15:01 Tg mice, but not in DRB1*01:01 Tg or C57BL/6 mice. Importantly, CD4⁺ responses to this epitope induce severe anti-GBM GN.     

Mesenchymal Stem Cells From Infants Born to Obese Mothers Exhibit Greater Potential for Adipogenesis: The Healthy Start BabyBUMP Project

Maternal obesity increases the risk for pediatric obesity; however, the molecular mechanisms in human infants remain poorly understood. We hypothesized that mesenchymal stem cells (MSCs) from infants born to obese mothers would demonstrate greater potential for adipogenesis and less potential for myogenesis, driven by differences in β-catenin, a regulator of MSC commitment. MSCs were cultured from the umbilical cords of infants born to normal-weight (prepregnancy [pp] BMI 21.1 ± 0.3 kg/m²; n = 15; NW-MSCs) and obese mothers (ppBMI 34.6 ± 1.0 kg/m²; n = 14; Ob-MSCs). Upon differentiation, Ob-MSCs exhibit evidence of greater adipogenesis (+30% Oil Red O stain [ORO], +50% peroxisome proliferator–activated receptor (PPAR)-γ protein; P < 0.05) compared with NW-MSCs. In undifferentiated cells, total β-catenin protein content was 10% lower and phosphorylated Thr41Ser45/total β-catenin was 25% higher (P < 0.05) in Ob-MSCs versus NW-MSCs (P < 0.05). Coupled with 25% lower inhibitory phosphorylation of GSK-3β in Ob-MSCs (P < 0.05), these data suggest greater β-catenin degradation in Ob-MSCs. Lithium chloride inhibition of GSK-3β increased nuclear β-catenin content and normalized nuclear PPAR-γ in Ob-MSCs. Last, ORO in adipogenic differentiating cells was positively correlated with the percent fat mass in infants (r = 0.475; P < 0.05). These results suggest that altered GSK-3β/β-catenin signaling in MSCs of infants exposed to maternal obesity may have important consequences for MSC lineage commitment, fetal fat accrual, and offspring obesity risk.     

Intercalated Cell BK-α/β4 Channels Modulate Sodium and Potassium Handling During Potassium Adaptation

The large-conductance, calcium-activated potassium (BK) channels help eliminate potassium in mammals consuming potassium-rich diets. In the distal nephron, principal cells contain BK-α/β1 channels and intercalated cells contain BK-α/β4 channels. We studied whether BK-β4–deficient mice (Kcnmb4^−/−) have altered renal sodium and potassium clearances compared with wild-type mice when fed a regular or potassium-rich diet for ten days. We did not detect differences in urinary flow or fractional excretions of potassium (FE_K) or sodium (FE_Na) between Kcnmb4-deficient and wild-type mice fed a regular diet. However, a potassium-rich diet led to >4-fold increases in urinary flows for both groups of mice, although Kcnmb4-deficient mice exhibited less urinary flow, higher plasma potassium concentration, more fluid retention, and significantly lower FE_K and FE_Na than wild-type mice despite similar plasma aldosterone levels. Immunohistochemical analysis revealed increased basolateral Na-K-ATPase in principal cells of all potassium-adapted mice, but expression of Na-K-ATPase in intercalated cells was >10-fold lower. The size of intercalated cells reduced and luminal volume increased among potassium-adapted wild-type but not Kcnmb4-deficient mice. Paradoxically, this led to increased urinary fluid velocity in potassium-adapted Kcnmb4-deficient mice compared with wild-type mice. Taken together, these data suggest that BK-α/β4 channels in intercalated cells reduce cell size, increasing luminal volume to accommodate higher distal flow rates during potassium adaptation. These changes streamline flow across the principal cells, producing gradients more favorable for potassium secretion and less favorable for sodium reabsorption.A high-K diet is a natural diuretic,¹ causing decreased Na and Cl reabsorption in the thick ascending limb (TAL) because of medullary recycling and high interstitial K levels.² The decreased Na transport in the medullary TAL disrupts the concentrating mechanism, thereby increasing flow to the distal nephron.² The high deliveries of Na to the connecting tubules (CNT) and cortical collecting ducts (CCD) is exchanged for K, and the increased flow stimulates K secretion to maximize the amount of K secreted to Na absorbed. The renal outer medullary kidney K channel (ROMK) and the large conductance, calcium-activated K channels (BK) in the CNT and CCD serve to eliminate K during K adaptation.^3–6In the distal nephron, the CNT and CCD consist of two epithelial cell types: principal cells (PCs) and intercalated cells (ICs). The PCs mediate Na and water reabsorption and K secretion, and the ICs mediate acid/base transport. Under normal conditions, K secretion by the PCs is mediated primarily by the ROMK channel.⁷ However, flow-induced K secretion in the distal nephron is mediated by BK.^4,8,9BK are a complex of pore-forming α and accessory β subunits (BK-α/β). The PCs of the CNT contain BK-α/β1 and are well equipped with an abundance of basolateral Na-K-ATPase to secrete K in K-adapted (KA) conditions. However, the preponderance of BK-α reside in ICs^10,11 along with the ancillary subunit, BK-β4 (gene: Kcnmb4).¹² A study has indicated that BK of ICs are regulated by mitogen-activated protein kinase to prevent K reabsorption during demand for maximal K secretion.¹³ It has also been proposed that BK-α/β4 in ICs have a role in flow-mediated K secretion.If high flow induces BK-mediated K secretion, then BK-α/β4 of ICs must have a role. It has been shown that the shear stress produced by high flow causes a transient increase in intracellular Ca to levels that may activate BK. Indeed, ICs, which protrude into the lumens of the CNT and CCD, are particularly subjected to shear stress forces that may elevate intracellular Ca. However, a transient Ca activation of BK would not produce the sustained K transport required for long-term K adaptation.That BK-α/β4 of ICs may not produce sustained K secretion is also indicated by the paucity of Na-K-ATPase. K adaptation¹⁴ or mineralocorticoid treatment^15–17 increases the quantity of basolateral Na-K-ATPase of mammalian collecting ducts to maintain a favorable electrochemical driving force for K secretion. However, because ICs have considerably less Na-K-ATPase than PCs,^18–21 ICs may not have an adequate K source to sustain K secretion via BK-α/β4.²² Still, Na-K-ATPase has not been quantified in ICs in KA conditions. If the BK-α/β4 were directly involved in the increased K transport associated with K adaptation, then it would be expected that the Na-K-ATPase in ICs would increase summarily to PCs.²³To this end, we determined whether Kcnmb4^−/− have altered renal K and Na excretions compared with wild type (WT) under control and KA conditions. Evidence from KA Kcnmb4^−/− indicates that the role of BK-α/β4 in ICs is to reduce cell size, thereby increasing tubular fluid volumes to accommodate the higher distal flow rates of KA mice. By reducing the protrusion of ICs into the lumen and increasing tubular volume, flow will be more streamlined across the PCs and a more a favorable chemical gradient for K secretion and less favorable gradient for Na reabsorption will be produced.     

14-3-3η protein is associated with disease activity and osteoporosis in patients with rheumatoid arthritis

IntroductionThis study was designed to explore the potential association of serum 14-3-3η protein level with disease activity and bone mineral density (BMD) in Egyptian patients with rheumatoid arthritis (RA). Patients were recruited from the outpatient clinic at Mansoura University Hospital.Material and methodsOne hundred eighty-eight patients with RA and 192 matched controls were enrolled. The rheumatoid arthritis activity parameters were evaluated in RA patients. Bone mineral density was measured. Serum levels of 14-3-3η protein and IL-6 were estimated for all participants by enzyme-linked immunosorbent assays (ELISA).ResultsRheumatoid arthritis patients had a significantly higher median serum 14-3-3η protein level compared to matched controls (p ≤ 0.05). Serum level of 14-3-3η protein was significantly correlated with DAS28–ESR (p ≤ 0.05) and serum IL-6 level (p ≤ 0.05). The rheumatoid arthritis-osteoporosis group had significantly higher serum 14-3-3η protein than the RA-osteopenia group and RA-control group. Similarly, the difference of the serum 14-3-3η protein between the RA-osteopenia group and the RA-control group was significant. In the linear regression analysis, the strongest factors that were associated with BMD in RA patients were the serum level of 14-3-3η protein (p ≤ 0.05), IL-6 (p ≤ 0.05) and DAS28–ESR (p ≤ 0.05).ConclusionsSerum level of 14-3-3η protein was significantly elevated in RA patients compared to controls and is significantly correlated with parameters of activity disease. The RA-osteoporosis group had significantly higher serum 14-3-3η protein than the RA-osteopenia group and RA-control group. Serum 14-3-3η protein can be a promising biomarker to reflect RA activity and predict presence of osteoporosis in RA patients.     

SH2B1 in β-Cells Regulates Glucose Metabolism by Promoting β-Cell Survival and Islet Expansion

IGF-1 and insulin promote β-cell expansion by inhibiting β-cell death and stimulating β-cell proliferation, and the phosphatidylinositol (PI) 3-kinase/Akt pathway mediates insulin and IGF-1 action. Impaired β-cell expansion is a risk factor for type 2 diabetes. Here, we identified SH2B1, which is highly expressed in β-cells, as a novel regulator of β-cell expansion. Silencing of SH2B1 in INS-1 832/13 β-cells attenuated insulin- and IGF-1–stimulated activation of the PI 3-kinase/Akt pathway and increased streptozotocin (STZ)-induced apoptosis; conversely, overexpression of SH2B1 had the opposite effects. Activation of the PI 3-kinase/Akt pathway in β-cells was impaired in pancreas-specific SH2B1 knockout (PKO) mice fed a high-fat diet (HFD). HFD-fed PKO mice also had increased β-cell apoptosis, decreased β-cell proliferation, decreased β-cell mass, decreased pancreatic insulin content, impaired insulin secretion, and exacerbated glucose intolerance. Furthermore, PKO mice were more susceptible to STZ-induced β-cell destruction, insulin deficiency, and hyperglycemia. These data indicate that SH2B1 in β-cells is an important prosurvival and proproliferative protein and promotes compensatory β-cell expansion in the insulin-resistant state and in response to β-cell stress.     

Reduced Renal Calcium Excretion in the Absence of Sclerostin Expression: Evidence for a Novel Calcium-Regulating Bone Kidney Axis

The kidneys contribute to calcium homeostasis by adjusting the reabsorption and excretion of filtered calcium through processes that are regulated by parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D₃ (1α,25[OH]₂D₃). Most of the filtered calcium is reabsorbed in the proximal tubule, primarily by paracellular mechanisms that are not sensitive to calcium-regulating hormones in physiologically relevant ways. In the distal tubule, however, calcium is reabsorbed by channels and transporters, the activity or expression of which is highly regulated and increased by PTH and 1α,25(OH)₂D₃. Recent research suggests that other, heretofore unrecognized factors, such as the osteocyte-specific protein sclerostin, also regulate renal calcium excretion. Clues in this regard have come from the study of humans and mice with inactivating mutations of the sclerostin gene that both have increased skeletal density, which would necessitate an increase in intestinal absorption and/or renal reabsorption of calcium. Deletion of the sclerostin gene in mice significantly diminishes urinary calcium excretion and increases fractional renal calcium reabsorption. This is associated with increased circulating 1α,25(OH)₂D₃ levels, whereas sclerostin directly suppresses 1α-hydroxylase in immortalized proximal tubular cells. Thus, evidence is accumulating that sclerostin directly or indirectly reduces renal calcium reabsorption, suggesting the presence of a novel calcium-excreting bone-kidney axis.