Tubular CPT1A deletion minimally affects aging and chronic kidney injury.

Kidney tubules use fatty acid oxidation (FAO) to support their high energetic requirements. Carnitine palmitoyltransferase 1A (CPT1A) is the rate-limiting enzyme for FAO, and it is necessary to transport long-chain fatty acids into mitochondria. To define the role of tubular CPT1A in aging and injury, we generated mice with tubule-specific deletion of Cpt1a (Cpt1aCKO mice), and the mice were either aged for 2 years or injured by aristolochic acid or unilateral ureteral obstruction. Surprisingly, Cpt1aCKO mice had no significant differences in kidney function or fibrosis compared with wild-type mice after aging or chronic injury. Primary tubule cells from aged Cpt1aCKO mice had a modest decrease in palmitate oxidation but retained the ability to metabolize long-chain fatty acids. Very-long-chain fatty acids, exclusively oxidized by peroxisomes, were reduced in kidneys lacking tubular CPT1A, consistent with increased peroxisomal activity. Single-nuclear RNA-Seq showed significantly increased expression of peroxisomal FAO enzymes in proximal tubules of mice lacking tubular CPT1A. These data suggest that peroxisomal FAO may compensate in the absence of CPT1A, and future genetic studies are needed to confirm the role of peroxisomal ß-oxidation when mitochondrial FAO is impaired.

A deletion in the N-terminal polymerizing domain of laminin ß2 is a new mouse model of chronic nephrotic syndrome.

The importance of the glomerular basement membrane (GBM) in glomerular filtration is underscored by the manifestations of Alport and Pierson syndromes, caused by defects in type IV collagen α3α4α5 and the laminin ß2 chain, respectively. Lamb2 null mice, which model the most severe form of Pierson syndrome, exhibit proteinuria prior to podocyte foot process effacement and are therefore useful for studying GBM permselectivity. We hypothesize that some LAMB2 missense mutations that cause mild forms of Pierson syndrome induce GBM destabilization with delayed effects on podocytes. While generating a CRISPR/Cas9-mediated analogue of a human LAMB2 missense mutation in mice, we identified a 44-amino acid deletion (LAMB2-Del44) within the laminin N-terminal domain, a domain mediating laminin polymerization. Laminin heterotrimers containing LAMB2-Del44 exhibited a 90% reduction in polymerization in vitro that was partially rescued by type IV collagen and nidogen. Del44 mice showed albuminuria at 1.8-6.0 g/g creatinine (ACR) at one to two months, plateauing at an average 200 g/g ACR at 3.7 months, when GBM thickening and hallmarks of nephrotic syndrome were first observed. Despite the massive albuminuria, some Del44 mice survived for up to 15 months. Blood urea nitrogen was modestly elevated at seven-nine months. Eight to nine-month-old Del44 mice exhibited glomerulosclerosis and interstitial fibrosis. Similar to Lamb2-/- mice, proteinuria preceded foot process effacement. Foot processes were widened but not effaced at one-two months despite the high ACRs. At three months some individual foot processes were still observed amid widespread effacement. Thus, our chronic model of nephrotic syndrome may prove useful to study filtration mechanisms, long-term proteinuria with preserved kidney function, and to test therapeutics.

Endothelial cell-specific collagen type IV-α3 expression does not rescue Alport syndrome in Col4a3-/- mice.

The glomerular basement membrane (GBM) is a critical component of the kidney's blood filtration barrier. Alport syndrome, a hereditary disease leading to kidney failure, is caused by the loss or dysfunction of the GBM's major collagen type IV (COL4) isoform α3α4α5. The constituent COL4 α-chains assemble into heterotrimers in the endoplasmic reticulum before secretion into the extracellular space. If any one of the α3-, α4-, or α5-chains is lost due to mutation of one of the genes, then the entire heterotrimer is lost. Patients with Alport syndrome typically have mutations in the X-linked COL4A5 gene or uncommonly have the autosomal recessive form of the disease due to COL4A3 or COL4A4 mutations. Treatment for Alport syndrome is currently limited to angiotensin-converting enzyme inhibition or angiotensin receptor blockers. Experimental approaches in Alport mice have demonstrated that induced expression of COL4A3, either widely or specifically in podocytes of Col4a3-/- mice, can abrogate disease progression even after establishment of the abnormal GBM. While targeting podocytes in vivo for gene therapy is a significant challenge, the more accessible glomerular endothelium could be amenable for mutant gene repair. In the present study, we expressed COL4A3 in Col4a3-/- Alport mice using an endothelial cell-specific inducible transgenic system, but collagen-α3α4α5(IV) was not detected in the GBM or elsewhere, and the Alport phenotype was not rescued. Our results suggest that endothelial cells do not express the Col4a3/a4/a5 genes and should not be viewed as a target for gene therapy.

EphA2 stimulates VCAM-1 expression through calcium-dependent NFAT1 activity.

Endothelial cell activation by proinflammatory stimuli drives leukocyte recruitment through enhanced expression of counter-receptors such as vascular cell adhesion molecule-1 (VCAM-1). We previously demonstrated that activation of the receptor tyrosine kinase EphA2 with its ligand ephrin-A1 induces VCAM-1 expression. Here, we sought to characterize the proinflammatory signaling pathways involved. Analysis of over-represented transcription factors in ephrin-A1-induced genes identified multiple potential transcriptional regulators, including the Rel family members nuclear factor-κB (NF-κB/p65) and nuclear factor of activated T-cells (NFAT). While ephrin-A1 failed to induce endothelial NF-κB activation, NF-κB inhibitors prevented ephrin-A1-induced VCAM-1 expression, suggesting basal NF-κB activity is required. In contrast, ephrin-A1 induced a robust EphA2-dependent increase in NFAT activation, and mutation of the NF-κB/NFAT-binding sites in the VCAM-1 promoter blunted ephrin-A1-induced promoter activity. NFAT activation classically occurs through calcium-dependent calcineurin activation, and inhibiting NFAT signaling with calcineurin inhibitors (cyclosporine A, FK506) or direct NFAT inhibitors (A-285222) was sufficient to block ephrin-A1-induced VCAM-1 expression. Consistent with robust NFAT activation, ephrin-A1-induced an EphA2-dependent calcium influx in endothelial cells that was required for ephrin-A1-induced NFAT activation and VCAM-1 expression. This work provides the first data showing EphA2-dependent calcium influx and NFAT activation and identifies NFAT as a novel EphA2-dependent proinflammatory pathway in endothelial activation.

Alport syndrome and Pierson syndrome: Diseases of the glomerular basement membrane.

The glomerular basement membrane (GBM) is an important component of the kidney's glomerular filtration barrier. Like all basement membranes, the GBM contains type IV collagen, laminin, nidogen, and heparan sulfate proteoglycan. It is flanked by the podocytes and glomerular endothelial cells that both synthesize it and adhere to it. Mutations that affect the GBM's collagen α3α4α5(IV) components cause Alport syndrome (kidney disease with variable ear and eye defects) and its variants, including thin basement membrane nephropathy. Mutations in LAMB2 that impact the synthesis or function of laminin α5ß2γ1 (LM-521) cause Pierson syndrome (congenital nephrotic syndrome with eye and neurological defects) and its less severe variants, including isolated congenital nephrotic syndrome. The very different types of kidney diseases that result from mutations in collagen IV vs. laminin are likely due to very different pathogenic mechanisms. A better understanding of these mechanisms should lead to targeted therapeutic approaches that can help people with these rare but important diseases.

Pathogenicity of a Human Laminin ß2 Mutation Revealed in Models of Alport Syndrome.

Pierson syndrome is a congenital nephrotic syndrome with eye and neurologic defects caused by mutations in laminin ß2 (LAMB2), a major component of the glomerular basement membrane (GBM). Pathogenic missense mutations in human LAMB2 cluster in or near the laminin amino-terminal (LN) domain, a domain required for extracellular polymerization of laminin trimers and basement membrane scaffolding. Here, we investigated an LN domain missense mutation, LAMB2-S80R, which was discovered in a patient with Pierson syndrome and unusually late onset of proteinuria. Biochemical data indicated that this mutation impairs laminin polymerization, which we hypothesized to be the cause of the patient's nephrotic syndrome. Testing this hypothesis in genetically altered mice showed that the corresponding amino acid change (LAMB2-S83R) alone is not pathogenic. However, expression of LAMB2-S83R significantly increased the rate of progression to kidney failure in a Col4a3-/- mouse model of autosomal recessive Alport syndrome and increased proteinuria in Col4a5+/- females that exhibit a mild form of X-linked Alport syndrome due to mosaic deposition of collagen α3α4α5(IV) in the GBM. Collectively, these data show the pathogenicity of LAMB2-S80R and provide the first evidence of genetic modification of Alport phenotypes by variation in another GBM component. This finding could help explain the wide range of Alport syndrome onset and severity observed in patients with Alport syndrome, even for family members who share the same COL4 mutation. Our results also show the complexities of using model organisms to investigate genetic variants suspected of being pathogenic in humans.

EphA2 Expression Regulates Inflammation and Fibroproliferative Remodeling in Atherosclerosis.

BACKGROUND: Atherosclerotic plaque formation results from chronic inflammation and fibroproliferative remodeling in the vascular wall. We previously demonstrated that both human and mouse atherosclerotic plaques show elevated expression of EphA2, a guidance molecule involved in cell-cell interactions and tumorigenesis. METHODS: Here, we assessed the role of EphA2 in atherosclerosis by deleting EphA2 in a mouse model of atherosclerosis (Apoe-/-) and by assessing EphA2 function in multiple vascular cell culture models. After 8 to 16 weeks on a Western diet, male and female mice were assessed for atherosclerotic burden in the large vessels, and plasma lipid levels were analyzed. RESULTS: Despite enhanced weight gain and plasma lipid levels compared with Apoe-/- controls, EphA2-/-Apoe-/- knockout mice show diminished atherosclerotic plaque formation, characterized by reduced proinflammatory gene expression and plaque macrophage content. Although plaque macrophages express EphA2, EphA2 deletion does not affect macrophage phenotype, inflammatory responses, and lipid uptake, and bone marrow chimeras suggest that hematopoietic EphA2 deletion does not affect plaque formation. In contrast, endothelial EphA2 knockdown significantly reduces monocyte firm adhesion under flow. In addition, EphA2-/-Apoe-/- mice show reduced progression to advanced atherosclerotic plaques with diminished smooth muscle and collagen content. Consistent with this phenotype, EphA2 shows enhanced expression after smooth muscle transition to a synthetic phenotype, and EphA2 depletion reduces smooth muscle proliferation, mitogenic signaling, and extracellular matrix deposition both in atherosclerotic plaques and in vascular smooth muscle cells in culture. CONCLUSIONS: Together, these data identify a novel role for EphA2 in atherosclerosis, regulating both plaque inflammation and progression to advanced atherosclerotic lesions. Cell culture studies suggest that endothelial EphA2 contributes to atherosclerotic inflammation by promoting monocyte firm adhesion, whereas smooth muscle EphA2 expression may regulate the progression to advanced atherosclerosis by regulating smooth muscle proliferation and extracellular matrix deposition.

Muscular dystrophy meets protein biochemistry, the mother of invention.

Muscular dystrophies result from a defect in the linkage between the muscle fiber cytoskeleton and the basement membrane (BM). Congenital muscular dystrophy type MDC1A is caused by mutations in laminin α2 that either reduce its expression or impair its ability to polymerize within the muscle fiber BM. Defects in this BM lead to muscle fiber damage from the force of contraction. In this issue of the JCI, McKee and colleagues use a laminin polymerization-competent, designer chimeric BM protein in vivo to restore function of a polymerization-defective laminin, leading to normalized muscle structure and strength in a mouse model of MDC1A. Delivery of such a protein to patients could ameliorate many aspects of their disease.

Functional assessment of a novel COL4A5 splice region variant and immunostaining of plucked hair follicles as an alternative method of diagnosis in X-linked Alport syndrome.

BACKGROUND: Many COL4A5 splice region variants have been described in patients with X-linked Alport syndrome, but few have been confirmed by functional analysis to actually cause defective splicing. We sought to demonstrate that a novel COL4A5 splice region variant in a family with Alport syndrome is pathogenic using functional studies. We also describe an alternative method of diagnosis. METHODS: Targeted next-generation sequencing results of an individual with Alport syndrome were analyzed and the results confirmed by Sanger sequencing in family members. A splicing reporter minigene assay was used to examine the variant's effect on splicing in transfected cells. Plucked hair follicles from patients and controls were examined for collagen IV proteins using immunofluorescence microscopy. RESULTS: A novel splice region mutation in COL4A5, c.1780-6T>G, was identified and segregated with disease in this family. This variant caused frequent skipping of exon 25, resulting in a frameshift and truncation of collagen α5(IV) protein. We also developed and validated a new approach to characterize the expression of collagen α5(IV) protein in the basement membranes of plucked hair follicles. Using this approach we demonstrated reduced collagen α5(IV) protein in affected male and female individuals in this family, supporting frequent failure of normal splicing. CONCLUSIONS: Differing normal to abnormal transcript ratios in affected individuals carrying splice region variants may contribute to variable disease severity observed in Alport families. Examination of plucked hair follicles in suspected X-linked Alport syndrome patients may offer a less invasive alternative method of diagnosis and serve as a pathogenicity test for COL4A5 variants of uncertain significance.

Altered nitric oxide production mediates matrix-specific PAK2 and NF-κB activation by flow.

Shear stress generated by distinct blood flow patterns modulates endothelial cell phenotype to spatially restrict atherosclerotic plaque development. Signaling through p21-activated kinase (PAK) mediates several of the deleterious effects of shear stress, including enhanced NF-κB activation and proinflammatory gene expression. Whereas shear stress activates PAK in endothelial cells on a fibronectin matrix, basement membrane proteins limit shear-induced PAK activation and inflammation through a protein kinase A-dependent pathway; however, the mechanisms underlying this regulation were unknown. We show that basement membrane proteins limit membrane recruitment of PAK2, the dominant isoform in endothelial cells, by blocking its interaction with the adaptor protein Nck. This uncoupling response requires protein kinase A-dependent nitric oxide production and subsequent PAK2 phosphorylation on Ser-20 in the Nck-binding domain. Of importance, shear stress does not stimulate nitric oxide production in endothelial cells on fibronectin, resulting in enhanced PAK activation, NF-κB phosphorylation, ICAM-1 expression, and monocyte adhesion. These data demonstrate that differential flow-induced nitric oxide production regulates matrix-specific PAK signaling and describe a novel mechanism of nitric oxide-dependent NF-κB inhibition.

Ephs and ephrins resurface in inflammation, immunity, and atherosclerosis.

Despite significant advancements in treatment regimens, cardiovascular disease remains a worldwide leader of morbidity, mortality, and healthcare cost. A large percentage of cardiovascular disease is directly attributable to the process of atherosclerosis, a chronic inflammatory disease of the vessel wall. In the hunt for novel therapeutic targets in cardiovascular disease, neuronal guidance molecules are emerging as significant regulators of cardiovascular remodeling and inflammation. The Eph family of neuronal guidance molecules comprises the largest family of receptor tyrosine kinases in the mammalian genome. While best characterized in embryogenesis and carcinogenesis, Eph receptors and their ephrin ligands are becoming increasingly recognized as important players in chronic inflammatory diseases and immune function. Herein we discuss the current evidence for how Eph/ephrin interactions, particularly EphA2/ephrinA1 and EphB/ephrinB2, affect inflammation and cardiovascular disease.

Hyperglycemia and endothelial dysfunction in atherosclerosis: lessons from type 1 diabetes.

A clear relationship between diabetes and cardiovascular disease has been established for decades. Despite this, the mechanisms by which diabetes contributes to plaque formation remain in question. Some of this confusion derives from studies in type 2 diabetics where multiple components of metabolic syndrome show proatherosclerotic effects independent of underlying diabetes. However, the hyperglycemia that defines the diabetic condition independently affects atherogenesis in cell culture systems, animal models, and human patients. Endothelial cell biology plays a central role in atherosclerotic plaque formation regulating vessel permeability, inflammation, and thrombosis. The current paper highlights the mechanisms by which hyperglycemia affects endothelial cell biology to promote plaque formation.

EphA2 activation promotes the endothelial cell inflammatory response: a potential role in atherosclerosis.

OBJECTIVE: Endothelial cell activation results in altered cell-cell interactions with adjacent endothelial cells and with infiltrating leukocytes. Eph receptors and their ephrin ligands regulate cell-cell interactions during tissue remodeling, and multiple proinflammatory mediators induce endothelial EphA receptor and ephrinA ligand expression. Therefore, we sought to elucidate the role of EphA receptors and ephrinA ligands in endothelial cell activation and atherosclerosis. METHODS AND RESULTS: Quantitative reverse transcription-polymerase chain reaction screening for EphA/ephrinA expression in atherosclerosis-prone macrovascular endothelium identified EphA2, EphA4, and ephrinA1 as the dominant isoforms. Endothelial activation with oxidized low-density lipoprotein and proinflammatory cytokines induced EphA2 and ephrinA1 expression and sustained EphA2 activation, whereas EphA4 expression was unaffected. Atherosclerotic plaques from mice and humans showed enhanced EphA2 and ephrinA1 expression colocalizing in the endothelial cell layer. EphA2 activation with recombinant Fc-ephrinA1 induced proinflammatory gene expression (eg vascular cell adhesion molecule-1, E-selectin) and stimulated monocyte adhesion, whereas inhibiting EphA2 (small interfering RNA, pharmacological inhibitors) abrogated both ephrinA1-induced and oxidized low-density lipoprotein-induced vascular cell adhesion molecule-1 expression. CONCLUSION: The current data suggest that enhanced EphA2 signaling during endothelial cell activation perpetuates proinflammatory gene expression. Coupled with EphA2 expression in mouse and human atherosclerotic plaques, these data implicate EphA2 as a novel proinflammatory mediator and potential regulator of atherosclerotic plaque development.

Podocytes require the engagement of cell surface heparan sulfate proteoglycans for adhesion to extracellular matrices.

Podocytes adhere to the glomerular basement membrane by cell surface receptors. Since in other cells these adhesions are enhanced by cell surface proteoglycans, we examined the contribution of these molecules and their glycosaminoglycan side chains to podocyte adhesion by developing immortalized podocyte cell lines with (control) or without (mutant) heparan sulfate glycosaminoglycan chains. In adhesion assays control podocytes attached, spread, and migrated more efficiently compared with mutants, indicating a requirement for heparan sulfate chains in these processes. The proteoglycan syndecan-4 is known to have direct effects on cell attachment, spreading, and cytoskeletal organization. We found it localized to focal adhesions in control podocytes coincident with stress fiber formation. In mutant cells, syndecan-4 was associated with smaller focal contacts and cortical actin organization. Analysis by flow cytometry showed that mutant cells had twice the amount of surface syndecan-4 of control cells. Protein kinase Cα, a signaling molecule bound to and activated by syndecan-4, showed a fourfold increase in membrane localization-activation than that seen in control cells. In vivo, the loss of heparan sulfate glycosaminoglycans in PEXTKO mice led to a loss of glomerular syndecan-4. Overall, our study provides further evidence for a dynamic role of cell surface heparan sulfate glycosaminoglycans in podocyte activity.

Matrix-specific protein kinase A signaling regulates p21-activated kinase activation by flow in endothelial cells.

RATIONALE: Atherosclerosis is initiated by blood flow patterns that activate inflammatory pathways in endothelial cells. Activation of inflammatory signaling by fluid shear stress is highly dependent on the composition of the subendothelial extracellular matrix. The basement membrane proteins laminin and collagen found in normal vessels suppress flow-induced p21 activated kinase (PAK) and nuclear factor (NF)-kappaB activation. By contrast, the provisional matrix proteins fibronectin and fibrinogen found in wounded or inflamed vessels support flow-induced PAK and NF-kappaB activation. PAK mediates both flow-induced permeability and matrix-specific activation of NF-kappaB. OBJECTIVE: To elucidate the mechanisms regulating matrix-specific PAK activation. METHODS AND RESULTS: We now show that matrix composition does not affect the upstream pathway by which flow activates PAK (integrin activation, Rac). Instead, basement membrane proteins enhance flow-induced protein kinase (PK)A activation, which suppresses PAK. Inhibiting PKA restored flow-induced PAK and NF-kappaB activation in cells on basement membrane proteins, whereas stimulating PKA inhibited flow-induced activation of inflammatory signaling in cells on fibronectin. PKA suppressed inflammatory signaling through PAK inhibition. Activating PKA by injection of the prostacyclin analog iloprost reduced PAK activation and inflammatory gene expression at sites of disturbed flow in vivo, whereas inhibiting PKA by PKA inhibitor (PKI) injection enhanced PAK activation and inflammatory gene expression. Inhibiting PAK prevented the enhancement of inflammatory gene expression by PKI. CONCLUSIONS: Basement membrane proteins inhibit inflammatory signaling in endothelial cells via PKA-dependent inhibition of PAK.