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.     

Thioredoxin-Interacting Protein Deficiency Protects against Diabetic Nephropathy

Expression of thioredoxin-interacting protein (TxNIP), an endogenous inhibitor of the thiol oxidoreductase thioredoxin, is augmented by high glucose (HG) and promotes oxidative stress. We previously reported that TxNIP-deficient mesangial cells showed protection from HG-induced reactive oxygen species, mitogen-activated protein kinase phosphorylation, and collagen expression. Here, we investigated the potential role of TxNIP in the pathogenesis of diabetic nephropathy (DN) in vivo. Wild-type (WT) control, TxNIP^−/−, and TxNIP^+/− mice were rendered equally diabetic with low-dose streptozotocin. In contrast to effects in WT mice, diabetes did not increase albuminuria, proteinuria, serum cystatin C, or serum creatinine levels in TxNIP^−/− mice. Whereas morphometric studies of kidneys revealed a thickened glomerular basement membrane and effaced podocytes in the diabetic WT mice, these changes were absent in the diabetic TxNIP^−/− mice. Immunohistochemical analysis revealed significant increases in the levels of glomerular TGF-β1, collagen IV, and fibrosis only in WT diabetic mice. Additionally, only WT diabetic mice showed significant increases in oxidative stress (nitrotyrosine, urinary 8-hydroxy-2-deoxy-guanosine) and inflammation (IL-1β mRNA, F4/80 immunohistochemistry). Expression levels of Nox4-encoded mRNA and protein increased only in the diabetic WT animals. A significant loss of podocytes, assessed by Wilms’ tumor 1 and nephrin staining and urinary nephrin concentration, was found in diabetic WT but not TxNIP^−/− mice. Furthermore, in cultured human podocytes exposed to HG, TxNIP knockdown with siRNA abolished the increased mitochondrial O₂⁻ generation and apoptosis. These data indicate that TxNIP has a critical role in the progression of DN and may be a promising therapeutic target.     

Characterization of a Novel Model of Lumbar Ligamentum Flavum Hypertrophy in Bipedal Standing Mice

ObjectiveTo explore the main causes of hypertrophied ligamentum flavum (HLF) and the possibility of using bipedal standing mouse model to simulate the pathological changes in human HLF.MethodsThirty‐two 8‐week‐old C57BL/6 male mice were randomly assigned to the experimental group (n = 16) and control group (n = 16). In the experimental group, mice were induced to adopt a bipedal standing posture by their hydrophobia. The experimental mice were maintained bipedal standing for 8 h a day with an interval of 2 h to consume food and water. The control mice were placed in a similar environment without bipedal standing. Eight 18‐month‐old C57BL/6 male mice were compared to evaluate the LF degeneration due to aging factor. Three‐dimensional (3D) reconstruction and finite element models were carried out to analyze the stress and strain distribution of the mouse LF in sprawling and bipedal standing postures. Hematoxylin and Eosin (HE), Verhoeff‐Van Gieson (VVG), and immunohistochemistry (IHC) staining were used to evaluate the LF degeneration of mice and humans. RT‐qPCR and immunofluorescence analysis were used to evaluate the expressions of fibrosis‐related factors and inflammatory cytokines of COL1A1, COL3A1, α‐SMA, MMP2, IL‐1β, and COX‐2.ResultsThe von Mises stress (8.85 × 10⁻² MPa) and maximum principal strain (6.64 × 10⁻¹) in LF were increased 4944 and 7703 times, respectively, in bipedal standing mice. HE staining showed that the mouse LF area was greater in the bipedal standing 10‐week‐old group ([10.01 ± 2.93] × 10⁴ μm²) than that in the control group ([3.76 ± 1.87] × 10⁴ μm²) and 18‐month‐old aged group ([6.09 ± 2.70] × 10⁴ μm²). VVG staining showed that the HLF of mice (3.23 ± 0.58) and humans (2.23 ± 0.31) had a similar loss of elastic fibers and an increase in collagen fibers. The cell density was higher during the process of HLF in mice (39.63 ± 4.81) and humans (23.25 ± 2.05). IHC staining showed that the number of α‐SMA positive cells were significantly increased in HLF of mice (1.63 ± 0.74) and humans (3.50 ± 1.85). The expressions of inflammatory cytokines and fibrosis‐related factors of COL1A1, COL3A1, α‐SMA, MMP2, IL‐1β, and COX‐2 were consistently higher in bipedal standing group than the control group.ConclusionOur study suggests that 3D finite element models can help analyze the abnormal stress and strain distributions of LF in modeling mice. Mechanical stress is the main cause of hypertrophied ligamentum flavum compared to aging. The bipedal standing mice model can reflect the pathological characteristics of human HLF. The bipedal standing mice model can provide a standardized condition to elucidate the molecular mechanisms of mechanical stress‐induced HLF in vivo.     

Liver-Specific Disruption of the Murine Glucagon Receptor Produces α-Cell Hyperplasia: Evidence for a Circulating α-Cell Growth Factor

Glucagon is a critical regulator of glucose homeostasis; however, mechanisms regulating glucagon action and α-cell function and number are incompletely understood. To elucidate the role of the hepatic glucagon receptor (Gcgr) in glucagon action, we generated mice with hepatocyte-specific deletion of the glucagon receptor. Gcgr^Hep−/− mice exhibited reductions in fasting blood glucose and improvements in insulin sensitivity and glucose tolerance compared with wild-type controls, similar in magnitude to changes observed in Gcgr^−/− mice. Despite preservation of islet Gcgr signaling, Gcgr^Hep−/− mice developed hyperglucagonemia and α-cell hyperplasia. To investigate mechanisms by which signaling through the Gcgr regulates α-cell mass, wild-type islets were transplanted into Gcgr^−/− or Gcgr^Hep−/− mice. Wild-type islets beneath the renal capsule of Gcgr^−/− or Gcgr^Hep−/− mice exhibited an increased rate of α-cell proliferation and expansion of α-cell area, consistent with changes exhibited by endogenous α-cells in Gcgr^−/− and Gcgr^Hep−/− pancreata. These results suggest that a circulating factor generated after disruption of hepatic Gcgr signaling can increase α-cell proliferation independent of direct pancreatic input. Identification of novel factors regulating α-cell proliferation and mass may facilitate the generation and expansion of α-cells for transdifferentiation into β-cells and the treatment of diabetes.The islets of Langerhans comprise distinct populations of differentiated endocrine cells whose functions are critical for maintenance of metabolic homeostasis. Considerable progress has been made in understanding the control of β-cell growth, function, and survival (1). Moreover, advances in understanding the developmental and adaptive control of β-cell formation coupled with insights gleaned from studies of adult and embryonic pancreatic endocrine stem cells have yielded new information regarding the cellular origin and formation of differentiated adult β-cells.In contrast, much less is known about mechanisms governing the development, growth, and survival of the glucagon-secreting α-cell (2). Glucagon secretion is stimulated by exercise or hypoglycemia; conversely, glucagon secretion is suppressed during conditions of fuel abundance. However, development of diabetes is often associated with failure to suppress glucagon secretion in the fed state (3,4); hence, therapeutic efforts to suppress α-cell function for the treatment of type 2 diabetes are ongoing (4). Moreover, α-cell mass appears dynamic in the context of diabetes, with expansion of α-cell mass noted in the diabetic primate (5) and human pancreas (6).The observation that functionally differentiated β-cells can arise from α-cell precursors (7–9) has engendered additional interest in the control of α-cell growth. α-Cell hyperplasia is frequently observed in settings of partial or complete glucagon deficiency (10,11) or resistance to glucagon action (12). Mice with targeted disruption of Pcsk2 exhibit impaired generation of bioactive glucagon, mild hypoglycemia, and marked α-cell proliferation, findings that are rapidly reversed by glucagon administration (13). Similarly, transgenic expression of Pax4 in pancreatic endocrine cells results in relative glucagon deficiency and compensatory α-islet cell proliferation; exogenous glucagon administration in this setting also reduces α-cell proliferation (7). Both transient genetic reduction of glucagon receptor (Gcgr) expression in normoglycemic or diabetic mice using antisense oligonucleotides or complete genetic germline disruption of GCGR signaling are associated with α-cell hyperplasia (10,14). Collectively, these findings raise the possibility that α-cell transdifferentiation toward a β-cell phenotype may represent an alternative strategy for replenishment of β-cell mass in vivo.Despite evidence linking reduction in GCGR signaling to α-cell hyperplasia, the precise tissues and signals important for stimulation of α-cell proliferation remain unknown. Because levels of GLP-1, a potent stimulator of islet cell proliferation, are extremely high in mice with partial or complete attenuation of Gcgr signaling (10,14), we analyzed α-cell mass in Gcgr^−/−:Glp1r^−/− mice (15). Although elimination of the Glp1r in Gcgr^−/− mice reversed improvements in β-cell function, fasting glycemia, and inhibition of gastric emptying, Gcgr^−/−:Glp1r^−/− mice continued to exhibit marked islet and α-cell hyperplasia. Similarly, mice with liver-specific disruption of Gsα exhibit α-cell hyperplasia despite genetic elimination of the Glp1r (16). Hence, although GLP-1 controls α-cell function, the Glp1r is not required for development of α-cell hyperplasia in Gcgr^−/− mice.To elucidate mechanisms and tissues through which reduction in GCGR signaling promotes α-cell hyperplasia, we have assessed the importance of the liver and the tissue environment. Surprisingly, selective elimination of the hepatic Gcgr in Gcgr^Hep−/− mice was sufficient to recapitulate the phenotype of α-cell hyperplasia in the endogenous pancreas, suggesting that reduction of GCGR signaling in liver originates one or more signals that promote α-cell proliferation. Remarkably, transplantation of Gcgr^+/+ islets under the kidney capsule of Gcgr^−/− or Gcgr^Hep−/− mice resulted in stimulation of α-cell proliferation and hyperplasia in transplanted islets, implying the existence of one or more circulating factors capable of promoting α-cell hyperplasia independent of the normal pancreatic microenvironment.     

Deletion of tumor progression locus 2 attenuates alcohol-induced hepatic inflammation

Background

The pathogenesis of alcoholic liver disease (ALD) involves the interaction of several inflammatory signaling pathways. Tumor progression locus 2 (TPL2), also known as Cancer Osaka Thyroid (COT) and MAP3K8, is a serine-threonine kinase that functions as a critical regulator of inflammatory pathways by up-regulating production of inflammatory cytokines. The present study aims to fill the gap in knowledge regarding the involvement of TPL2 in the mechanism of alcohol-induced hepatic inflammation.

Methods

Male TPL2^−/− knockout (TPL2KO) mice and TPL2^+/+ wild-type (WT) mice were group pair-fed with Lieber-DeCarli liquid ethanol diet (EtOH diet, 27% energy from EtOH) or control diet (ctrl diet) for 4 weeks. Both histological and molecular biomarkers involved in the induction of hepatic inflammation by alcohol consumption were examined.

Results

Consumption of the EtOH diet in WT mice lead to a significant induction of TPL2 mRNA expression as compared with WT mice fed ctrl diet. A significant induction in inflammatory foci and steatosis was also observed in WT mice fed EtOH diet. The deletion of TPL2 significantly reduced inflammatory foci in the liver of mice consuming both ctrl and EtOH diets as compared to their respective WT controls. This reduction was associated with suppression of hepatic inflammatory gene expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) and macrophage marker F4/80. In addition, histological analysis of livers revealed that TPL2 deletion resulted in reduced steatosis in both ctrl (significant) and EtOH (non-significant) diet-fed mice as compared to their respective WT controls.

Conclusions

The demonstration that TPL2 deletion attenuates alcohol-induced hepatic inflammation provides evidence of a novel role for TPL2 in the pathogenesis of ALD.     

AMP-Activated Protein Kinase α1 Protects Against Diet-Induced Insulin Resistance and Obesity

AMP-activated protein kinase (AMPK) is an essential sensor of cellular energy status. Defects in the α2 catalytic subunit of AMPK (AMPKα1) are associated with metabolic syndrome. The current study investigated the role AMPKα1 in the pathogenesis of obesity and inflammation using male AMPKα1-deficent (AMPKα1^−/−) mice and their wild-type (WT) littermates. After being fed a high-fat diet (HFD), global AMPKα1^−/− mice gained more body weight and greater adiposity and exhibited systemic insulin resistance and metabolic dysfunction with increased severity in their adipose tissues compared with their WT littermates. Interestingly, upon HFD feeding, irradiated WT mice that received the bone marrow of AMPKα1^−/− mice showed increased insulin resistance but not obesity, whereas irradiated AMPKα1^−/− mice with WT bone marrow had a phenotype of metabolic dysregulation that was similar to that of global AMPKα1^−/− mice. AMPKα1 deficiency in macrophages markedly increased the macrophage proinflammatory status. In addition, AMPKα1 knockdown enhanced adipocyte lipid accumulation and exacerbated the inflammatory response and insulin resistance. Together, these data show that AMPKα1 protects mice from diet-induced obesity and insulin resistance, demonstrating that AMPKα1 is a promising therapeutic target in the treatment of the metabolic syndrome.AMP-activated protein kinase (AMPK) is a major cellular energy sensor and plays a major role in regulating metabolic homeostasis (1,2). In mammals, AMPK is a heterotrimeric complex with a catalytic subunit (α1 or α2) and two regulatory subunits (β1 or β1 and γ1, γ2, or γ3) (1,2). AMPKα2 is the predominant catalytic form of AMPK in the liver, muscle, and hypothalamus. There is evidence that AMPKα2 is important for the regulation of systemic insulin sensitivity and metabolic homeostasis. In the hypothalamus, AMPKα2 signals regulate food intake and body weight gain (3). Mice globally deficient in AMPKα2 display different metabolic phenotypes when fed different diets (4,5). A lack of AMPKα2 activity in skeletal muscle exacerbates glucose intolerance and the insulin resistance that is caused by high-fat diets (HFDs) (6). In addition, AMPKα2 is required for the effects of many physiologic regulators or pharmaceutical modalities that maintain insulin sensitivity and metabolic homeostasis (7–10).Mice deficient in AMPKα1 had an increased inflammatory response in an experimental autoimmune encephalomyelitis model (11). AMPKα1 deficiency elevated the levels of reactive oxygen species and oxidized proteins, thereafter shortening the erythrocyte life span in mice (12). Macrophage AMPKα1 has been characterized as a key regulator of inflammatory function (13,14). Its role in protecting against diet-induced metabolic disorders has been hypothesized but not demonstrated (14). The activation of AMPK in adipocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) suppresses adipocyte differentiation and diet-induced obesity (15). However, the activation of AMPK is able to reduce isoproterenol-induced lipolysis; this result is supported by a decrease in adipocyte size and adipose mass in globally deficient in AMPKα1 (AMPKα1^−/−) mice (16). To define the physiologic role of AMPKα1 in energy homeostasis, we administered an HFD to AMPKα1^−/− mice and then evaluated diet-induced obesity and insulin resistance. We also used bone marrow (BM) transplantation (BMT) to characterize the specific roles of AMPKα1 in macrophages and adipocytes in the regulation of the diet-induced inflammatory response, adiposity, and systemic insulin resistance.     

BAMBI Elimination Enhances Alternative TGF-β Signaling and Glomerular Dysfunction in Diabetic Mice

BMP, activin, membrane-bound inhibitor (BAMBI) acts as a pseudo-receptor for the transforming growth factor (TGF)-β type I receptor family and a negative modulator of TGF-β kinase signaling, and BAMBI^−/− mice show mild endothelial dysfunction. Because diabetic glomerular disease is associated with TGF-β overexpression and microvascular alterations, we examined the effect of diabetes on glomerular BAMBI mRNA levels. In isolated glomeruli from biopsies of patients with diabetic nephropathy and in glomeruli from mice with type 2 diabetes, BAMBI was downregulated. We then examined the effects of BAMBI deletion on streptozotocin-induced diabetic glomerulopathy in mice. BAMBI^−/− mice developed more albuminuria, with a widening of foot processes, than BAMBI^+/+ mice, along with increased activation of alternative TGF-β pathways such as extracellular signal–related kinase (ERK)1/2 and Smad1/5 in glomeruli and cortices of BAMBI^−/− mice. Vegfr2 and Angpt1, genes controlling glomerular endothelial stability, were downmodulated in glomeruli from BAMBI^−/− mice with diabetes. Incubation of glomeruli from nondiabetic BAMBI^+/+ or BAMBI^−/− mice with TGF-β resulted in the downregulation of Vegfr2 and Angpt1, effects that were more pronounced in BAMBI^−/− mice and were prevented by a MEK inhibitor. The downregulation of Vegfr2 in diabetes was localized to glomerular endothelial cells using a histone yellow reporter under the Vegfr2 promoter. Thus, BAMBI modulates the effects of diabetes on glomerular permselectivity in association with altered ERK1/2 and Smad1/5 signaling. Future therapeutic interventions with inhibitors of alternative TGF-β signaling may therefore be of interest in diabetic nephropathy.     

Curtailing Endothelial TGF-β Signaling Is Sufficient to Reduce Endothelial-Mesenchymal Transition and Fibrosis in CKD

Excessive TGF-β signaling in epithelial cells, pericytes, or fibroblasts has been implicated in CKD. This list has recently been joined by endothelial cells (ECs) undergoing mesenchymal transition. Although several studies focused on the effects of ablating epithelial or fibroblast TGF-β signaling on development of fibrosis, there is a lack of information on ablating TGF-β signaling in the endothelium because this ablation causes embryonic lethality. We generated endothelium-specific heterozygous TGF-β receptor knockout (TβRII^endo+/−) mice to explore whether curtailed TGF-β signaling significantly modifies nephrosclerosis. These mice developed normally, but showed enhanced angiogenic potential compared with TβRII^endo+/+ mice under basal conditions. After induction of folic acid nephropathy or unilateral ureteral obstruction, TβRII^endo+/− mice exhibited less tubulointerstitial fibrosis, enhanced preservation of renal microvasculature, improvement in renal blood flow, and less tissue hypoxia than TβRII^endo+/+ counterparts. In addition, partial deletion of TβRII in the endothelium reduced endothelial-to-mesenchymal transition (EndoMT). TGF-β–induced canonical Smad2 signaling was reduced in TβRII^+/− ECs; however, activin receptor-like kinase 1 (ALK1)–mediated Smad1/5 phosphorylation in TβRII^+/− ECs remained unaffected. Furthermore, the S-endoglin/L-endoglin mRNA expression ratio was significantly lower in TβRII^+/− ECs compared with TβRII^+/+ ECs. These observations support the hypothesis that EndoMT contributes to renal fibrosis and curtailing endothelial TGF-β signals favors Smad1/5 proangiogenic programs and dictates increased angiogenic responses. Our data implicate endothelial TGF-β signaling and EndoMT in regulating angiogenic and fibrotic responses to injury.     

The Extracellular Matrix Protein MAGP1 Supports Thermogenesis and Protects Against Obesity and Diabetes Through Regulation of TGF-β

Microfibril-associated glycoprotein 1 (MAGP1) is a component of extracellular matrix microfibrils. Here we show that MAGP1 expression is significantly altered in obese humans, and inactivation of the MAGP1 gene (Mfap2^−/−) in mice results in adipocyte hypertrophy and predisposition to metabolic dysfunction. Impaired thermoregulation was evident in Mfap2^−/− mice prior to changes in adiposity, suggesting a causative role for MAGP1 in the increased adiposity and predisposition to diabetes. By 5 weeks of age, Mfap2^−/− mice were maladaptive to cold challenge, uncoupling protein-1 expression was attenuated in the brown adipose tissue, and there was reduced browning of the subcutaneous white adipose tissue. Levels of transforming growth factor-β (TGF-β) activity were elevated in Mfap2^−/− adipose tissue, and the treatment of Mfap2^−/− mice with a TGF-β–neutralizing antibody improved their body temperature and prevented the increased adiposity phenotype. Together, these findings indicate that the regulation of TGF-β by MAGP1 is protective against the effects of metabolic stress, and its absence predisposes individuals to metabolic dysfunction.     

Feasibility of Repairing Glomerular Basement Membrane Defects in Alport Syndrome

Alport syndrome is a hereditary glomerular disease that leads to kidney failure. It is caused by mutations affecting one of three chains of the collagen α3α4α5(IV) heterotrimer, which forms the major collagen IV network of the glomerular basement membrane (GBM). In the absence of the α3α4α5(IV) network, the α1α1α2(IV) network substitutes, but it is insufficient to maintain normal kidney function. Inhibition of angiotensin-converting enzyme slows progression to kidney failure in patients with Alport syndrome but is not a cure. Restoration of the normal collagen α3α4α5(IV) network in the GBM, by either cell- or gene-based therapy, is an attractive and logical approach toward a cure, but whether or not the abnormal GBM can be repaired once it has formed and is functioning is unknown. Using a mouse model of Alport syndrome and an inducible transgene system, we found that secretion of α3α4α5(IV) heterotrimers by podocytes into a preformed, abnormal, filtering Alport GBM is effective at restoring the missing collagen IV network, slowing kidney disease progression, and extending life span. This proof-of-principle study demonstrates the plasticity of the mature GBM and validates the pursuit of therapeutic approaches aimed at normalizing the GBM to prolong kidney function.The glomerular basement membrane (GBM) is the extracellular matrix component of the glomerular filtration barrier that lies between podocytes and endothelial cells. It is composed primarily of collagen α3α4α5(IV), laminin-521 (α5β2γ1), nidogen, and agrin.¹ Alport syndrome is a hereditary nephritis caused by mutation of any one of the three GBM collagen IV chain genes (COL4A3, COL4A4, or COL4A5).² The major molecular features usually observed in Alport GBM are (1) the absence of the collagen α3α4α5(IV) network, which is made by podocytes,^3,4 and (2) compensation by increased levels of the α1α1α2(IV) network, which is normally a minor GBM component found on the subendothelial aspect.⁵ Compensation by α1α1α2(IV) keeps the GBM intact and functioning for years, but eventually GBM splitting and thickening imparts a basket-weave appearance.² A progression of hematuria, proteinuria, and glomerulosclerosis leads to ESRD, usually by adolescence or young adulthood. Angiotensin-converting enzyme (ACE) inhibition slows progression to proteinuria and ESRD,⁶ but there is no cure for Alport syndrome.Mouse and dog Alport syndrome models, which demonstrate GBM lesions and a disease progression similar to that in human Alport syndrome,^7,8 have been instrumental for gaining insights into disease pathogenesis and for testing potential therapies. The efficacy of ACE inhibition in human patients was predicted by the ameliorative effects of treating Alport dogs and mice with ACE inhibitors.^9,10 The ease with which Alport mice can be manipulated has made them the preferred choice for preclinical investigations since the first models were generated by mutating Col4a3.^11,12Several approaches have been used in attempts to cure Alport syndrome in mice, or to at least slow progression of the disease and lengthen lifespan. These include efforts to prevent the development of GBM lesions and glomerulosclerosis^13–15; to attenuate the glomerular and tubulointerstitial inflammation that inevitably occurs^16–18; and to transplant or infuse various types of stem cells, with or without irradiation, with the goal of replacing mutant podocytes with exogenous cells capable of expressing the missing collagen α3α4α5(IV).^19–22 Despite claims of success at replacing podocytes and/or restoring GBM composition,^19–21 these results stirred controversy²³ and have not been convincingly replicated. The recent finding that injection of amniotic fluid stem cells is beneficial in Alport mice without any effect on GBM composition or glomerular cell identity²⁴ suggests that exogenous cells do not need to home to glomeruli, cross the GBM, and replace or fuse with mutant podocytes (if such events are even possible) to be ameliorative. Nevertheless, should breakthroughs occur such that a large percentage of podocytes can be replaced or genetically altered to repair the mutation, perhaps via a renal perfusion approach developed for gene therapy,²⁵ it is important to know whether or not the missing collagen α3α4α5(IV) network can be incorporated into an existing defective Alport GBM and restore function.Previously a transgenic approach was used to restore the missing collagen α3(IV) chain in Col4a3^−/− mice using regulatory elements of the human COL4A3 gene to drive expression in a developmentally correct fashion.²⁶ In contrast, here we used a very different transgenic approach to express COL4A3 on the Col4a3^−/− background via a doxycycline-inducible system. A cDNA encoding full-length COL4A3 was placed under the control of the (TetO)₇/CMV promoter (Figure 1A). Eight (TetO)₇/CMV-Col4a3 (T-Col4a3) transgenic lines were generated, and each was bred to the Rosa26-reverse tetracycline trans-activator (rtTA) line,²⁷ induced with doxycycline during gestation, and tested for expression in multiple tissues with an anti-COL4A3 antibody. Several lines showed novel expression in intestine and spleen that was not present in controls (data not shown), and one line was deemed the best expressor and was used for subsequent studies.Open in a separate windowFigure 1.NEFTA and Rosa26-rtTA drive expression of T-Col4a3 and deposition of transgene-derived protein into the GBM. (A) The (TetO)₇/CMV-Col4a3 (T-Col4a3) transgene is induced by a doxycycline (Dox)-bound rtTA. (B) To achieve rtTA expression in podocytes, the nephrin (Nphs1) promoter was placed upstream of the rtTA-3G cDNA and the SV40 large T-antigen poly A signal (SV40pA) to generate the NEFTA transgene. (C–E) Kidney sections from a 10-day-old NEFTA; T-HISGFP; T-hLAMA5 mouse induced with doxycycline prenatally shows green fluorescent protein (GFP) fluorescence in podocyte nuclei and continuous staining for human laminin α5 in the GBM (C). Immunostaining for Wilms tumor 1 (D) identifies podocyte nuclei, all of which are GFP-positive (green/yellow in the merge in E). (F–H) Kidney sections of 23-week-old Col4a3^+/− (F), Col4a3^−/−; NEFTA; T-Col4a3 (G), and Col4a3^−/−; Rosa26-rtTA; T-Col4a3 (H) mice fed doxycycline from birth were stained for COL4A3. Original magnifications: C, ×200; D and E, ×600; F–H, ×400. g, glomerulus.Three different rtTA lines were used to drive expression of T-Col4a3 in Col4a3^−/− mice. The extent and level of expression were assayed using antibodies to COL4A3 (Figure 1) and to the α3α4α5(IV) hexamer (Figure 2), staining for which indicates α3α4α5(IV) heterotrimer assembly and network formation. The Nphs2 (podocin)–rtTA driver²⁸ was tried first, but it drove expression of T-Col4a3 and of the (TetO)₇/CMV-histone 2B-GFP fusion transgene²⁹ (T-H2BGFP) in only a subset of podocytes (not shown). We therefore generated a new podocyte driver using the 4.1 kb Nphs1 (nephrin) promoter³⁰ to drive expression of a more sensitive rtTA,³¹ trade-named rtTA-3G. Microinjection of the Nphs1-rtTA-3G transgene (hereafter called NEFTA) (Figure 1B) yielded 36 transgenic founders. Expression in a subset was assayed by breeding to T-H2BGFP and to a (TetO₇)/CMV-human laminin α5 (T-hLAMA5) transgene³² and inducing with doxycycline. Several good expressors were identified by nuclear green fluorescent protein fluorescence and linear human laminin α5 GBM immunostaining (Figure 1C), and the NEFTA line (line 8) that appeared to have the highest and most specific doxycycline-dependent expression in all podocytes (identified by anti–Wilms tumor 1 staining) (Figure 1, D and E) was used for further studies. The third rtTA driver used was the widely expressed Rosa26-rtTA.Open in a separate windowFigure 2.Podocyte-specific expression of T-Col4a3 from postnatal day (P) 0 and P21 attenuates Alport glomerulosclerosis. Genotypes: Col4a3^+/−, control; Col4a3^−/−, nonrescued Alport; NEFTA-P0-R23W, rescued Alport induced at P0 for 23 weeks; NEFTA-P21-R20W, rescued Alport induced at P21 for 20 weeks. (A) Immunostaining for assembled collagen α3α4α5(IV) NC1 hexamers. Basement membranes of glomeruli (G) and tubules (T) are positive in the control (a) and negative in the mutant (b). Only GBM is positive in the podocyte-specific rescue mice (c and d). (B) Double staining for COL4A2 (green) and COL4A4 (red), as indicated. COL4A4 is only detected in control (e) and rescued (g and h) GBMs. COL4A2 is normally present primarily in the mesangium (a and e), but is increased in the mutant GBM (b and f). More COL4A2 is present in the P21 rescue GBM (d and h) versus P0 rescue (c and g). Arrows indicate GBM. (C) Periodic acid-Schiff staining of paraffin sections shows some glomerulosclerosis (gs), a crescentic glomerulus (cg), and protein casts (P) in the mutant (b) versus control (a), but more normal kidney architecture in the rescues (c and d). (D) Ultrastructural analysis of the glomerular capillary wall shows the normal ribbon-like GBM (arrowheads) in most segments of control (a) and rescues (c and d); areas of GBM thickening (arrows) are present diffusely in the mutant (b) but only segmentally in the rescues (c and d). rbc, red blood cells within the glomerular capillaries. Original magnifications: A and B, ×400; C, ×200; D, ×7500.Cohorts of Col4a3^−/−;NEFTA;T-Col4a3 and Col4a3^−/−;Rosa26-rtTA;T-Col4a3 mice and control littermates lacking one or more of these loci were generated to test whether postnatal induction of COL4A3 expression, either in podocytes (via NEFTA) or in all cells (via Rosa26-rtTA), would normalize the composition of the Alport GBM and prevent or slow kidney disease. But as proof of concept, induction was first begun at or before birth in a subset of mice by feeding the dams doxycycline chow so that COL4A3 expression would be induced in the still developing kidney. With this approach, and with doxycycline administered continuously after birth, most GBMs should always contain the proper collagen α3α4α5(IV) network because most mouse glomeruli form after birth. Indeed, immunostaining showed that this was what occurred: With NEFTA as driver, the GBM contained COL4A3 (Figure 1G), which drove assembly of a collagen α3α4α5(IV) network (Figure 2Ac); with Rosa26-rtTA as driver, the GBM and many tubular basement membranes contained COL4A3, although GBM staining was weak compared with tubular basement membranes (Figure 1H), indicating that the Rosa26 promoter is probably weak in podocytes. For this reason, we focused on the Col4a3^−/−;NEFTA;T-Col4a3 mice for functional analyses.As alluded to above, the data in Figure 2A show that the COL4A3 encoded by the T-Col4a3 transgene is capable of assembling with COL4A4 and COL4A5 to form α3α4α5(IV) heterotrimers, and that expression only from podocytes is sufficient; this finding agrees with data showing that podocytes, but not endothelial cells, make collagen α3α4α5(IV).^3,4 Many GBM segments of Col4a3^−/−;NEFTA;T-Col4a3 mice induced at birth were ultrastructurally normal even at 23 weeks of age compared with Col4a3^−/− littermates (Figure 2Db,c), and the mice were still alive with either no or low levels of albuminuria long after their Col4a3^−/− littermates reached ESRD (Figure 3 and data not shown).Open in a separate windowFigure 3.Survival of doxycycline-fed Col4a3^−/−; NEFTA; T-Col4a3 mice versus nonrescued Col4a3^−/− mice correlates with albuminuria and GBM contour. (A and B) SDS-PAGE analysis of 1 µl of urine from control (C), mutant (M), and rescued (R) mice induced at embryonic day 14 (E14), birth (P0), or 21 days (P21). Ages at urine collection are indicated in weeks (W). Littermates are indicated by brackets below the gels. BSA standards at 1 and 5 µg are shown for each. (C) Survival curve showing age at ESRD for 19 Col4a3^−/− and 14 Col4a3^−/−; NEFTA; T-Col4a3 rescued (NEFTA-R) mice induced with doxycycline at various ages, E11-P21. All mice expressing T-Col4a3 survived longer than the longest-surviving nonexpressor. (D–F) Scanning electron microscopic analysis of decellularized GBM shows it to be rough and blebbed in the Col4a3^−/− at 12 weeks (D), smooth in the control at 40 weeks (E), and slightly blebbed yet smooth in the 40-week-old rescued mouse fed doxycycline from P21 (F). Original magnifications: main images, ×6000; insets, ×40,000.With proof that the transgene-encoded COL4A3 is functional and expressed as anticipated, we next induced its expression at weaning and continuously thereafter by feeding doxycycline to Col4a3^−/−;NEFTA;T-Col4a3 mice starting at 3 weeks, when all glomeruli have GBMs with abnormal composition (i.e., containing collagen α1α1α2[IV]) that are functioning in glomerular ultrafiltration. After induction, we collected urine every three to four weeks to monitor the integrity of the filtration barrier. SDS-PAGE analysis of urine showed that Alport mice with transgene induction beginning at 3 weeks (or earlier) remained nonalbuminuric for months, even at ages when their littermates without transgene expression had easily detectable urinary albumin (Figure 3). That albuminuria is a sign of progressive disease in Alport syndrome⁶ was reflected by the fact that mice with the higher levels of urinary albumin died of ESRD by 4–8 months of age, while most of their Col4a3^−/− littermates expressing the Col4a3 transgene (beginning prenatally, at birth, or at 3 weeks) remained alive, either with no or low levels of urinary albumin (Figure 3). The survival data for these mice showed that no Alport mice without transgene expression survived beyond 32 weeks, whereas all transgene-expressing Alport mice survived longer. As of this writing, two such mice induced with doxycycline at 3 weeks are still alive, though with some albuminuria at 15 and 16 months of age, respectively.Immunostaining showed that the transgenic “rescued” Col4a3^−/− mice did have a GBM with a collagen α3α4α5(IV) network, but some segments appeared thickened and had significant accumulation of collagen α1 and α2(IV) chains, although less than observed in nontransgenic Alport mice (Figure 2, A and B). Ultrastructural analysis showed some segmental GBM thickening in the rescued Alport mice, but in many cases podocyte foot process architecture was maintained (Figure 2D). Consistent with this, light microscopy showed that glomerulosclerosis and tubular protein cast formation were attenuated in rescued Alport mice, in agreement with the lack or low level of urinary albumin and longer lifespan compared with Alport mice not expressing the transgene (Figures 2C and ​and33).These results show that once formed and functioning in filtration, the Alport GBM’s composition can still be changed and partially normalized by incorporation of the missing collagen α3α4α5(IV) network. We also found segmental GBM splitting and thickening even after postnatal collagen α3α4α5(IV) network assembly, yet kidney function decline was attenuated and lifespan was lengthened. Interestingly, scanning electron microscopy showed that decellularized Col4a3^−/− GBM had a rough, blebby surface compared with the smooth surface of control GBM (Figure 3, D and E); the P21 rescued GBM exhibited some blebs, consistent with the transmission electron microscopy (Figure 2D), but an overall smooth surface (Figure 3F) that we speculate was conferred by incorporation of the collagen α3α4α5(IV) network. Together, these data suggest that a split, thickened GBM can be compatible with long-term kidney function, despite the ultrastructural abnormalities. This study validates the utility of therapeutic approaches aimed at normalizing the composition of the Alport GBM. Importantly, our results suggest that even imperfect molecular and structural repair of the GBM can significantly delay the onset of proteinuria and extend the time to ESRD.     

Neonatal Fc Receptor Promotes Immune Complex–Mediated Glomerular Disease

The neonatal Fc receptor (FcRn) is a major regulator of IgG and albumin homeostasis systemically and in the kidneys. We investigated the role of FcRn in the development of immune complex–mediated glomerular disease in mice. C57Bl/6 mice immunized with the noncollagenous domain of the α3 chain of type IV collagen (α3NC1) developed albuminuria associated with granular capillary loop deposition of exogenous antigen, mouse IgG, C3 and C5b-9, and podocyte injury. High-resolution imaging showed abundant IgG deposition in the expanded glomerular basement membrane, especially in regions corresponding to subepithelial electron dense deposits. FcRn-null and -humanized mice immunized with α3NC1 developed no albuminuria and had lower levels of serum IgG anti-α3NC1 antibodies and reduced glomerular deposition of IgG, antigen, and complement. Our results show that FcRn promotes the formation of subepithelial immune complexes and subsequent glomerular pathology leading to proteinuria, potentially by maintaining higher serum levels of pathogenic IgG antibodies. Therefore, reducing pathogenic IgG levels by pharmacologic inhibition of FcRn may provide a novel approach for the treatment of immune complex–mediated glomerular diseases. As proof of concept, we showed that a peptide inhibiting the interaction between human FcRn and human IgG accelerated the degradation of human IgG anti-α3NC1 autoantibodies injected into FCRN-humanized mice as effectively as genetic ablation of FcRn, thus preventing the glomerular deposition of immune complexes containing human IgG.The MHC class I–like neonatal Fc receptor (FcRn), a heterodimer comprising a heavy chain and β₂-microglobulin light chain, is the major regulator of IgG and albumin homeostasis.¹ Perinatally, FcRn mediates the transfer of IgG from mother to offspring, across the placenta in primates and trans-intestinally in suckling rodents. Throughout life, FcRn protects IgG and albumin from catabolism, explaining the unusually long t_1/2 and high serum levels of these proteins. IgG and albumin taken up by cells by pinocytosis bind strongly to FcRn at pH 6.0–6.5 in endosomes. FcRn-bound ligands are then recycled to the plasma membrane, where they dissociate at pH 7.4, whereas IgG and albumin not bound to FcRn are targeted to lysosomes for degradation. FcRn is thought to promote some autoimmune diseases because it protects pathogenic IgG from degradation. For instance, Fcrn^−/− mice are resistant to passive transfer of arthritis by K/BxN sera and autoimmune skin pathology induced by antibodies targeting autoantigens at the dermal–epidermal junction, although this protection can be overcome by excess autoantibodies.^2–4In kidneys, FcRn is expressed in podocytes and proximal tubular epithelial cells.⁵ Overall, renal FcRn reclaims albumin but facilitates elimination of IgG.⁶ Tubular FcRn mediates IgG transcytosis.⁷ Podocytes use FcRn to clear IgG from the glomerular basement membrane (GBM).⁸ IgG accumulates in the glomeruli of aged Fcrn^−/− mice due to impaired clearance of IgG from the GBM, and saturating this clearance mechanism by excess ligand potentiates the pathogenicity of nephrotoxic sera in wild-type mice. Podocyte FcRn has been postulated to be involved in the clearance of immune complexes (ICs) present in pathologic conditions such as membranous nephropathy.⁵ Expression of FcRn in human podocytes is increased in various immune-mediated glomerular diseases.⁹ Given its role in IgG and albumin handling in the kidneys and systemically, FcRn can be expected to influence the development of immune-mediated kidney diseases at multiple levels. This conjecture awaits experimental verification.To determine the role of FcRn in IgG-mediated glomerular disease, we asked how FcRn deficiency alters the course of disease in mice immunized with the NC1 domain of α3 type IV collagen (α3NC1). We chose this antigen because of its reported ability to induce disease in C57Bl/6 (B6) mice,¹⁰ corroborated in pilot studies (Supplemental Figure 1). Fcrn^−/− mice are hypoalbuminemic due to impaired albumin recycling,¹¹ and also exhibit reduced urinary albumin excretion.¹² As a control for this potential confounder, we used FCRN-humanized mice, which have normal serum albumin because human FcRn recycles mouse albumin but not mouse IgG.¹³All mice immunized with α3NC1 developed circulating mouse IgG anti-α3NC1 antibodies, which reached the maximum titer about 6 weeks later and gradually declined thereafter. At all times, the levels of mouse IgG anti-α3NC1 antibodies in sera from Fcrn^−/− mice and FCRN-humanized mice were approximately 50%–70% lower than those in wild-type mouse sera (Figure 1A). The results were similar for mouse IgG1, IgG2b, and IgG2c anti-α3NC1 antibodies (Supplemental Figure 2). Wild-type B6 mice immunized with α3NC1 started developing progressive albuminuria 8–10 weeks later (Figure 1B). By week 14, the urinary albumin creatinine ratio increased approximately 100-fold, and hypoalbuminemia developed (Figure 1C). Urinary albumin excretion in Fcrn^−/− mice and FCRN-humanized mice immunized with α3NC1 was not significantly higher than in adjuvant-immunized control mice. No mice developed renal failure (Supplemental Figure 3).Open in a separate windowFigure 1.FcRn ablation reduces serum levels of mouse IgG anti-α3NC1 antibodies and prevents the development of albuminuria in α3NC1-immunized mice. (A) The left panel shows circulating mIgG anti-α3NC1 antibodies from C57Bl6 wild-type mice (○), Fcrn^−/− mice (□), FCRN-humanized (hFCRN) mice (◇), and the control CFA group (△), which are assayed by indirect ELISA in plates coated with α3NC1 (100 ng/well). Mouse sera are diluted 1:5000. The right panel shows the significance of circulating mIgG anti-α3NC1 antibody differences among groups at week 12, as assessed by one-way ANOVA followed by Bonferroni post tests for pairwise comparisons. (B) The left panel shows that the urinary albumin creatinine ratio (mean±SEM) time course is monitored in C57Bl6 wild-type mice (○), Fcrn^−/− mice (□), and hFCRN mice (◇) immunized with α3NC1 (n=5–8 mice in each group, from two separate experiments). Mice in the control group (△) are immunized with adjuvant alone (n=9). The right panel shows the urinary albumin creatinine ratio (mean±SEM) at 14 weeks, when mice are euthanized. The significance of differences among groups is assessed by one-way ANOVA followed by Bonferroni post tests for pairwise comparisons. (C) The left panel shows SDS-PAGE analysis of serum (0.5 µl/lane) and urine samples (2 µl/lane) from CFA-immunized control mice (a) and α3NC1-immunized wild-type mice (b), Fcrn^−/− mice (c), and hFCRN mice (d) collected at week 14. The right panel presents a densitometric analysis of the relative levels of albumin in mouse serum samples showing that α3NC1-immunized wild-type mice developed hypoalbuminemia. *P<0.05 by two-tailed t test versus CFA-immunized wild-type mice; **P<0.01; ***P<0.001. ns, not significant; WT, wild type.At 14 weeks after α3NC1 immunization, kidneys examined by light microscopy showed mild glomerular pathology, with few crescents and relatively little inflammation (Figure 2A), similar to α3NC1-immunized DBA/1 mice with comparable albuminuria.^14,15 Electron microscopy showed extensive subepithelial IC deposits surrounded by an expanded GBM and effacement of podocyte foot processes in α3NC1-immunized B6 mice, whereas Fcrn^−/− mice had fewer subepithelial deposits (Figure 2B, Supplemental Figure 4). Immunofluorescence staining showed granular capillary loop deposition of mouse IgG, exogenous antigen, C3, and C5b-9, more intense in wild-type mice than in Fcrn^−/− mice and FCRN-humanized mice (Figure 2, Ca–Cp, Supplemental Figure 5). A loss of nephrin staining, indicative of podocyte injury, occurred in α3NC1-immunized B6 mice but not in Fcrn^−/− mice or FCRN-humanized mice (Figure 2, Cq–Ct).Open in a separate windowFigure 2.FcRn deficiency reduces formation of pathogenic subepithelial ICs. (A) Light microscopic evaluation of kidneys from adjuvant-immunized control mice (a) and α3NC1-immunized wild-type mice (b) and Fcrn^−/− mice (c) revealed few pathogenic changes and the absence of glomerular inflammation (periodic acid–Schiff staining). (B) Transmission electron microscopy shows normal GBM (arrow) and podocyte foot processes in control mice (a), extensive subepithelial electron dense deposits (arrowhead), thickened GBM, and podocyte foot process effacement in α3NC1-immunized wild-type mice (b), and fewer IC deposits in the Fcrn^−/− mice (c). (C) Immunofluorescence analysis of kidneys from adjuvant-immunized control mice (a, e, i, m, and q) and α3NC1-immunized wild-type mice (b, f, j, n, and r), FcRn^−/− mice (c, g, k, o, and s), and hFCRN mice (d, h, l, p, and t) evaluate the deposition of mouse IgG (a–d), exogenous α3NC1 antigen stained by mAb RH34 (e–h), mouse C3c (i–l), C5b-9 (m–p), and nephrin staining (q–t) at 14 weeks. Wild-type mice exhibit linear-granular GBM deposition of mouse IgG and granular GBM deposition of exogenous antigen, C3, and C5b-9, which are attenuated in Fcrn^−/− mice and hFCRN mice and essentially absent in control mice. Compared with control mice, α3NC1-immunized wild-type mice but not Fcrn^−/− or hFCRN mice exhibit a loss of nephrin staining, indicative of podocyte injury. WT, wild type; EM, electron microscopy, PAS, periodic acid–Schiff. Original magnification, ×400 in A; ×2850 in B; ×200 in C.Because B6 mice immunized with bovine GBM NC1 hexamers have normal kidney function and histology despite linear GBM deposition of IgG autoantibodies binding to mouse α345(IV) collagen (Supplemental Figure 1), the question arises as to what causes proteinuria in α3NC1-immunized mice. Because the clinical presentation, morphology, and effector mechanisms depend on where ICs are localized in the capillary wall, we compared IgG distribution in α3NC1-immunized mice and mice injected with anti-α3NC1 antibodies modeling anti-GBM autoantibodies. The distribution and relative abundance of mouse IgG, as imaged by immunoperoxidase immunoelectron microscopy and stochastic optical reconstruction microscopy (STORM), a method for super-resolution fluorescence microscopy, were concordant. In α3NC1-immunized mice, IgG deposition was abundant in the areas of expanded GBM and especially in regions corresponding to the subepithelial dense deposits seen by routine electron microscopy. By contrast, in mice injected with α3NC1-specific anti-GBM mAb, the IgG was confined to an ultrastructurally normal GBM that lacked subepithelial deposits (Figure 3).Open in a separate windowFigure 3.Localization of IgG by high-resolution imaging. The localization of mouse IgG in glomerular capillary walls of wild-type mice immunized with α3NC1 (A, C–E), or intravenously injected with anti-mouse α3NC1 IgG mAb 8D1 (B, F–H) is determined by immunoperoxidase electron microscopy (A and B) and STORM imaging (C–H). In A, the GBM is irregularly thickened, and abundant electron dense peroxidase reaction product is present in discontinuous, subepithelial patterns beneath broadly effaced podocyte foot processes (arrows). In B, the peroxidase reaction product is diffusely present throughout the GBM (arrowhead), but less abundant compared with A. Electron dense deposits are absent, and podocyte foot process architecture appears normal. (C–E) By STORM imaging, anti-agrin (blue) identifies both normal and thickened areas of the GBM, both of which contain dense accumulations of mouse IgG throughout (red). The electron microscopy correlation in E shows GBM staining with respect to the podocytes and endothelial cells. (F–H) IgG mAb 8D1 (red) is present in the GBM, which shows no evidence of thickening. CL, capillary lumen; EM, electron microscopy En, endothelium;Po, podocyte.Subepithelial ICs, a hallmark of human membranous nephropathy (MN), form when IgG antibodies bind to podocyte antigens, such as phospholipase A2 receptor (PLA2R) and neutral endopeptidase (NEP), or to planted antigens, such as cationic BSA.^16–18 Subsequent expansion of the GBM, complement activation, and podocyte injury by C5b-9 cause proteinuria. Although it is unexpected, formation of subepithelial ICs in α3NC1-immunized mice may be explained by exogenous α3NC1 deposited in glomeruli acting as a planted antigen.¹⁹ Alternatively, anti-α3NC1 antibodies in complex with α3NC1 antigen may act as surrogate antipodocyte antibodies, because α3NC1-containing ICs bind to podocytes.²⁰ After four immunizations with α3NC1 monomers, B6 mice and DBA/1 mice eventually develop crescentic GN by 26 and 10 weeks, respectively.^10,14 The combination of subepithelial ICs and crescentic anti-GBM antibody GN was most recently described in a series of eight patients with circulating anti-α3NC1 autoantibodies but undetectable anti-PLA2R autoantibodies.²¹In contrast to wild-type B6 mice, congenic Fcrn^−/− mice and FCRN-humanized mice did not develop albuminuria after α3NC1 immunization. Their resistance to proteinuria was associated with lower serum titers of anti-α3NC1 IgG antibodies and reduced glomerular deposition of IgG, antigen, C3, and C5b-9. Because C5b-9 is an essential mediator of podocyte damage and proteinuria by subepithelial ICs,^22,23 reduced complement activation potentially explains the attenuated glomerular pathology in FcRn-deficient mice. The resistance of FCRN-humanized mice indicates that FcRn promotes IC-mediated glomerular disease due to its interaction with IgG rather than albumin. We propose that FcRn promotes the development of subepithelial ICs and subsequent glomerular injury primarily by maintaining higher serum levels of pathogenic IgG (Supplemental Figure 6). However, we cannot formally exclude a possible pathogenic role of podocyte FcRn, whose stimulation by ICs may induce maladaptive signaling.⁹ Future studies in mice with podocyte-specific ablation of FcRn would address this possibility.Our findings identify FcRn as a potential target for therapeutic intervention in IC-mediated glomerular diseases, typically treated with nonspecific immunosuppressants that are toxic and sometimes ineffective. More specific therapies include ablation of B cells by rituximab. In patients with idiopathic MN who respond to rituximab therapy, serum levels of anti-PLA2R IgG autoantibodies decline over a period of many months, and their disappearance is followed by resolution of proteinuria.²⁴ The slow decline in proteinuria is problematic for patients already suffering from complications of nephrotic syndrome, who would benefit from ancillary therapies that reduce pathogenic IgG antibodies more rapidly. This may be achieved by inhibiting FcRn.One implementation of this concept is therapy with high-dose intravenous Ig (HD-IVIG). HD-IVIG accelerates the degradation of IgG by saturating FcRn,²⁵ one of the mechanisms that explain the beneficial effects of HD-IVIG therapy in some autoimmune diseases.³ In pregnant women with circulating anti-NEP alloantibodies mediating antenatal MN, treatment with HD-IVIG reduces the titers of IgG alloantibodies by approximately 30% within 2–3 weeks.²⁶ However, HD-IVIG is inefficient, because large amounts of IgG (1–2 g/kg) cause relatively modest reductions in pathogenic IgG titers. Specific FcRn inhibitors recapitulate this activity of HD-IVIG more effectively at lower doses. By reducing pathogenic IgG levels, function-blocking anti-FcRn mAbs ameliorate experimental myasthenia gravis in rats,²⁷ and engineered IgG “Abdegs” that bind with high affinity to FcRn ameliorate arthritis transferred by K/BxN serum.²⁸To assess the translational potential of our findings, we asked whether pharmacologic blockade of human FcRn can reproduce the effects of genetic FcRn deficiency. To this end, FCRN-humanized and Fcrn^−/− mice were passively immunized with human IgG containing anti-α3NC1 (Goodpasture) autoantibodies. To inhibit human FcRn, we used a lysine analog of SYN1436 (Figure 4A),²⁹ a peptide that binds with subnanomolar affinity to human FcRn, thus preventing IgG binding.³⁰ In vivo, SYN1436 reduces IgG levels in cynomolgus monkeys by 80%.³⁰ Serum anti-α3NC1 autoantibodies in FCRN-humanized mice treated with anti-FcRn peptide, but not with control peptide, sharply decreased to the same levels as in Fcrn^−/− mice (Figure 4B), and were no longer detected after 4 days. In mice, human IgG elicits murine anti-human IgG antibodies, forming ICs that can deposit in glomeruli, as shown in active serum sickness models. Glomerular deposition of ICs containing human IgG was abolished in mice treated with anti-FcRn peptide, but not with control peptide (Figure 4C). Linear GBM deposition of human anti-GBM IgG was not observed, because the epitopes recognized by Goodpasture autoantibodies are completely inaccessible in the mouse GBM.³¹ These results provide proof of concept that therapies targeting human FcRn effectively lower serum levels of pathogenic human IgG autoantibodies, which could be beneficial in patients with IgG-mediated kidney diseases. Because FcRn also mediates the trans-placental transfer of IgG from mother to the fetus, FcRn inhibition may be particularly attractive for preventing antenatal MN caused by maternal anti-NEP alloantibodies.Open in a separate windowFigure 4.Pharmacologic blockade of human FcRn accelerates the catabolism of human IgG autoantibodies in FCRN-humanized mice. (A) Structure of a peptide that binds with high affinity to human FcRn, competitively inhibiting its interaction with human IgG (top). The control peptide (bottom) containing D-amino acids does not bind to human FcRn. Pen, Sar, and NMeLeu denote penicillamine, sarcosine, and N-methyl-leucine, respectively. (B) Serum level of human IgG anti-α3NC1 antibodies in FCRN-humanized mice treated with anti-FcRn peptide (▪) or control peptide (●) and in Fcrn^−/− (▲) mice sera (n=3 in each group) is analyzed by indirect ELISA in plates coated with α3NC1 (100 ng/well). Mouse sera are diluted 1:500. (C) Kidney deposition of human IgG (a and b) and mouse IgG (c and d) in FCRN-humanized mice treated with control peptide (a and c) or anti-FcRn peptide (b and d) is evaluated by direct immunofluorescence staining. Treatment with anti-FcRn peptide prevents the glomerular deposition of ICs containing human IgG.     

Surveillance of γδ T Cells Predicts Cytomegalovirus Infection Resolution in Kidney Transplants

Cytomegalovirus (CMV) infection in solid-organ transplantation is associated with increased morbidity and mortality, particularly if a CMV mutant strain with antiviral resistance emerges. Monitoring CMV–specific T cell response could provide relevant information for patient care. We and others have shown the involvement of Vδ2^neg γδ T cells in controlling CMV infection. Here, we assessed if Vδ2^neg γδ T cell kinetics in peripheral blood predict CMV infection resolution and emergence of a mutant strain in high–risk recipients of kidney transplants, including 168 seronegative recipients receiving organs from seropositive donors (D+R−) and 104 seropositive recipients receiving antithymocyte globulins (R+/ATG). Vδ2^neg γδ T cell percentages were serially determined in patients grafted between 2003 and 2011. The growing phase of Vδ2^neg γδ T cells was monitored in each infected patient, and the expansion rate during this phase was estimated individually by a linear mixed model. A Vδ2^neg γδ T cell expansion rate of ˃0.06% per day predicted the growing phase. The time after infection at which an expansion rate of 0.06% per day occurred was correlated with the resolution of CMV DNAemia (r=0.91; P<0.001). At 49 days of antiviral treatment, Vδ2^neg γδ T cell expansion onset was associated with recovery, whereas absence of expansion was associated with recurrent disease and DNAemia. The appearance of antiviral–resistant mutant CMV strains was associated with delayed Vδ2^neg γδ T cell expansion (P<0.001). In conclusion, longitudinal surveillance of Vδ2^neg γδ T cells in recipients of kidney transplants may predict CMV infection resolution and antiviral drug resistance.     

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.     

Role of BH3-Only Molecules Bim and Puma in β-Cell Death in Pdx1 Deficiency

Mutations in pancreatic duodenal homeobox-1 (PDX1) are associated with diabetes in humans. Pdx1-haploinsufficient mice develop diabetes due to an increase in β-cell death leading to reduced β-cell mass. For definition of the molecular link between Pdx1 deficiency and β-cell death, Pdx1-haploinsufficient mice in which the genes for the BH3-only molecules Bim and Puma had been ablated were studied on a high-fat diet. Compared with Pdx1^+/− mice, animals haploinsufficient for both Pdx1 and Bim or Puma genes showed improved glucose tolerance, enhanced β-cell mass, and reduction in the number of TUNEL-positive cells in islets. These results suggest that Bim and Puma ablation improves β-cell survival in Pdx1^+/− mice. For exploration of the mechanisms responsible for these findings, Pdx1 gene expression was knocked down in mouse MIN6 insulinoma cells resulting in apoptotic cell death that was found to be associated with increased expression of BH3-only molecules Bim and Puma. If the upregulation of Bim and Puma that occurs during Pdx1 suppression was prevented, apoptotic β-cell death was reduced in vitro. These results suggest that Bim and Puma play an important role in β-cell apoptosis in Pdx1-deficient diabetes.     

Genetic Evidence for a Normal-Weight “Metabolically Obese” Phenotype Linking Insulin Resistance,Hypertension, Coronary Artery Disease,and Type 2 Diabetes

The mechanisms that predispose to hypertension, coronary artery disease (CAD), and type 2 diabetes (T2D) in individuals of normal weight are poorly understood. In contrast, in monogenic primary lipodystrophy—a reduction in subcutaneous adipose tissue—it is clear that it is adipose dysfunction that causes severe insulin resistance (IR), hypertension, CAD, and T2D. We aimed to test the hypothesis that common alleles associated with IR also influence the wider clinical and biochemical profile of monogenic IR. We selected 19 common genetic variants associated with fasting insulin–based measures of IR. We used hierarchical clustering and results from genome-wide association studies of eight nondisease outcomes of monogenic IR to group these variants. We analyzed genetic risk scores against disease outcomes, including 12,171 T2D cases, 40,365 CAD cases, and 69,828 individuals with blood pressure measurements. Hierarchical clustering identified 11 variants associated with a metabolic profile consistent with a common, subtle form of lipodystrophy. A genetic risk score consisting of these 11 IR risk alleles was associated with higher triglycerides (β = 0.018; P = 4 × 10⁻²⁹), lower HDL cholesterol (β = −0.020; P = 7 × 10⁻³⁷), greater hepatic steatosis (β = 0.021; P = 3 × 10⁻⁴), higher alanine transaminase (β = 0.002; P = 3 × 10⁻⁵), lower sex-hormone-binding globulin (β = −0.010; P = 9 × 10⁻¹³), and lower adiponectin (β = −0.015; P = 2 × 10⁻²⁶). The same risk alleles were associated with lower BMI (per-allele β = −0.008; P = 7 × 10⁻⁸) and increased visceral-to-subcutaneous adipose tissue ratio (β = −0.015; P = 6 × 10⁻⁷). Individuals carrying ≥17 fasting insulin–raising alleles (5.5% population) were slimmer (0.30 kg/m²) but at increased risk of T2D (odds ratio [OR] 1.46; per-allele P = 5 × 10⁻¹³), CAD (OR 1.12; per-allele P = 1 × 10⁻⁵), and increased blood pressure (systolic and diastolic blood pressure of 1.21 mmHg [per-allele P = 2 × 10⁻⁵] and 0.67 mmHg [per-allele P = 2 × 10⁻⁴], respectively) compared with individuals carrying ≤9 risk alleles (5.5% population). Our results provide genetic evidence for a link between the three diseases of the “metabolic syndrome” and point to reduced subcutaneous adiposity as a central mechanism.     

Small changes in bone structure of female α7 nicotinic acetylcholine receptor knockout mice

Background

Recently, analysis of bone from knockout mice identified muscarinic acetylcholine receptor subtype M3 (mAChR M3) and nicotinic acetylcholine receptor (nAChR) subunit α2 as positive regulator of bone mass accrual whereas of male mice deficient for α7-nAChR (α7KO) did not reveal impact in regulation of bone remodeling. Since female sex hormones are involved in fair coordination of osteoblast bone formation and osteoclast bone degradation we assigned the current study to analyze bone strength, composition and microarchitecture of female α7KO compared to their corresponding wild-type mice (α7WT).

Methods

Vertebrae and long bones of female 16-week-old α7KO (n = 10) and α7WT (n = 8) were extracted and analyzed by means of histological, radiological, biomechanical, cell- and molecular methods as well as time of flight secondary ion mass spectrometry (ToF-SIMS) and transmission electron microscopy (TEM).

Results

Bone of female α7KO revealed a significant increase in bending stiffness (p < 0.05) and cortical thickness (p < 0.05) compared to α7WT, whereas gene expression of osteoclast marker cathepsin K was declined. ToF-SIMS analysis detected a decrease in trabecular calcium content and an increase in C₄H₆N⁺ (p < 0.05) and C₄H₈N⁺ (p < 0.001) collagen fragments whereas a loss of osteoid was found by means of TEM.

Conclusions

Our results on female α7KO bone identified differences in bone strength and composition. In addition, we could demonstrate that α7-nAChRs are involved in regulation of bone remodelling. In contrast to mAChR M3 and nAChR subunit α2 the α7-nAChR favours reduction of bone strength thereby showing similar effects as α7β2-nAChR in male mice. nAChR are able to form heteropentameric receptors containing α- and β-subunits as well as the subunits α7 can be arranged as homopentameric cation channel. The different effects of homopentameric and heteropentameric α7-nAChR on bone need to be analysed in future studies as well as gender effects of cholinergic receptors on bone homeostasis.