Activation of NFAT signaling in podocytes causes glomerulosclerosis.

Mutant forms of TRPC6 can activate NFAT-dependent transcription in vitro via calcium influx and activation of calcineurin. The same TRPC6 mutants can cause FSGS, but whether this involves an NFAT-dependent mechanism is unknown. Here, we generated mice that allow conditional induction of NFATc1. Mice with NFAT activation in nascent podocytes in utero developed proteinuria and glomerulosclerosis postnatally, resembling FSGS. NFAT activation in adult mice also caused progressive proteinuria and FSGS. Ultrastructural studies revealed podocyte foot process effacement and deposition of extracellular matrix. NFAT activation did not initially affect expression of podocin, synaptopodin, and nephrin but reduced their expression as glomerular injury progressed. In contrast, we observed upregulation of Wnt6 and Fzd9 in the mutant glomeruli before the onset of significant proteinuria, suggesting a potential role for Wnt signaling in the pathogenesis of NFAT-induced podocyte injury and FSGS. These results provide in vivo evidence for the involvement of NFAT signaling in podocytes, proteinuria, and glomerulosclerosis. Furthermore, this study suggests that NFAT activation may be a key intermediate step in the pathogenesis of mutant TRPC6-mediated FSGS and that suppression of NFAT activity may contribute to the antiproteinuric effects of calcineurin inhibitors.

Proteinuria precedes podocyte abnormalities inLamb2-/- mice, implicating the glomerular basement membrane as an albumin barrier.

Primary defects in either podocytes or the glomerular basement membrane (GBM) cause proteinuria, a fact that complicates defining the barrier to albumin. Laminin beta2 (LAMB2) is a GBM component required for proper functioning of the glomerular filtration barrier. To investigate the GBM's role in glomerular filtration, we characterized GBM and overlying podocyte architecture in relation to development and progression of proteinuria in Lamb2-/- mice, which model Pierson syndrome, a rare congenital nephrotic syndrome. We found ectopic deposition of several laminins and mislocalization of anionic sites in the GBM, which together suggest that the Lamb2-/- GBM is severely disorganized, although it is ultrastructurally intact. Importantly, albuminuria was detectable shortly after birth and preceded podocyte foot process effacement and loss of slit diaphragms by at least 7 days. Expression and localization of slit diaphragm and foot process-associated proteins appeared normal at early stages. GBM permeability to the electron-dense tracer ferritin was dramatically elevated in Lamb2-/- mice, even before widespread foot process effacement. Increased ferritin permeability was not observed in nephrotic CD2-associated protein-null (Cd2ap-/-) mice, which have a primary podocyte defect. Together these data show that the GBM serves as a barrier to protein in vivo and that the glomerular slit diaphragm alone is not sufficient to prevent the passage of albumin into the urinary space.

Glomerular filtration is normal in the absence of both agrin and perlecan-heparan sulfate from the glomerular basement membrane.

BACKGROUND: For several decades, it has been thought that the glomerular basement membrane (GBM) provides a charge-selective barrier for glomerular filtration. However, recent evidence has presented challenges to this concept: selective removal of heparan sulfate (HS) moieties that impart a negative charge to the GBM causes little if any increase in proteinuria. Removal of agrin, the major GBM HS-proteoglycan (HSPG), from the GBM causes a profound reduction in the glomerular anionic charge without changing the excretion of a negatively charged tracer. Perlecan is another HSPG present in the GBM, as well as in the mesangium and Bowman's capsule, that could potentially contribute to a charge barrier in the absence of agrin. METHODS: Here we studied the nature of the glomerular filtration barrier to albumin in mice lacking the HS chains of perlecan either alone or in combination with podocyte-specific loss of agrin. RESULTS: The results show significant reductions in anionic sites within the GBM in perlecan-HS and in perlecan-HS/agrin double mutants. Podocyte and overall glomerular architecture were normal, and renal function was normal up to 15 months of age with no measurable proteinuria. Moreover, excretion of a negatively charged Ficoll tracer was unchanged as compared to control mice. CONCLUSIONS: These findings cast further doubt upon a critical role for the GBM in charge selectivity.

Podocyte-specific deletion of dicer alters cytoskeletal dynamics and causes glomerular disease.

MicroRNAs (miRNAs) regulate gene expression by binding the 3' untranslated region of mRNAs. To define their role in glomerular function, miRNA biogenesis was disrupted in mouse podocytes using a conditional Dicer allele. Mutant mice developed proteinuria by 3 wk after birth and progressed rapidly to end-stage kidney disease. Podocyte pathology included effacement, vacuolization, and hypertrophy with crescent formation. Despite normal expression of WT1, podocytes underwent dedifferentiation, exemplified by cytoskeletal disruption with early transcriptional downregulation of synaptopodin. These abnormalities differed from Cd2ap(-/-) mice, indicating they were not a general consequence of glomerular disease. Glomerular labeling of ezrin, moesin, and gelsolin was altered at 3 wk, but expression of nestin and alpha-actinin was unchanged. Abnormal cell proliferation or apoptosis was not responsible for the glomerular injury. Mutant podocytes were incapable of synthesizing mature miRNA, as revealed by their loss of miR-30a. In contrast, expression of glomerular endothelial and mesangial cell miRNAs (miR-126 and miR-145, respectively) was unchanged. These findings demonstrate a critical role for miRNA in glomerular function and suggest a pathway that may participate in the pathogenesis of kidney diseases of podocyte origin. The unique architecture of podocytes may make them especially susceptible to cytoskeletal alterations initiated by aberrant miRNA dynamics.

Roles of neurotransmitter in synapse formation: development of neuromuscular junctions lacking choline acetyltransferase.

Activity-dependent and -independent signals collaborate to regulate synaptogenesis, but their relative contributions are unclear. Here, we describe the formation of neuromuscular synapses at which neurotransmission is completely and specifically blocked by mutation of the neurotransmitter-synthesizing enzyme choline acetyltransferase. Nerve terminals differentiate extensively in the absence of neurotransmitter, but neurotransmission plays multiple roles in synaptic differentiation. These include influences on the numbers of pre- and postsynaptic partners, the distribution of synapses in the target field, the number of synaptic sites per target cell, and the number of axons per synaptic site. Neurotransmission also regulates the formation or stability of transient acetylcholine receptor-rich processes (myopodia) that may initiate nerve-muscle contact. At subsequent stages, neurotransmission delays some steps in synaptic maturation but accelerates others. Thus, neurotransmission affects synaptogenesis from early stages and coordinates rather than drives synaptic maturation.

Inhibiting axon degeneration and synapse loss attenuates apoptosis and disease progression in a mouse model of motoneuron disease.

Apoptosis is a hallmark of motoneuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) [1]. In a widely used mouse model of motoneuron disease (progressive motor neuronopathy or pmn) [2-4], transgenic expression of the anti-apoptotic bcl-2 gene [5] or treatment with glial cell-derived neurotrophic factor [6] prevents the apoptosis of the motoneuron soma; however, they were unable to affect the life span of the animals. The goal of the present work was to determine whether the pmn phenotype could be rescued by means of a gene that inhibits axon degeneration. For this reason, the pmn mice were crossed with mice bearing the dominant Wlds ("slow Wallerian degeneration") mutation, which slows axon degeneration and synapse loss [7-9]. We show here that the Wlds gene product attenuates symptoms, extends life span, prevents axon degeneration, rescues motoneuron number and size, and delays retrograde transport deficits in pmn/pmn mice. These results suggest new pathogenic mechanisms and therapeutic avenues for motoneuron diseases.

Disruption of glomerular basement membrane charge through podocyte-specific mutation of agrin does not alter glomerular permselectivity.

Glomerular charge selectivity has been attributed to anionic heparan sulfate proteoglycans (HSPGs) in the glomerular basement membrane (GBM). Agrin is the predominant GBM-HSPG, but evidence that it contributes to the charge barrier is lacking, because newborn agrin-deficient mice die from neuromuscular defects. To study agrin in adult kidney, a new conditional allele was used to generate podocyte-specific knockouts. Mutants were viable and displayed no renal histopathology up to 9 months of age. Perlecan, a HSPG normally confined to the mesangium in mature glomeruli, did not appear in the mutant GBM, which lacked heparan sulfate. Moreover, GBM agrin was found to be derived primarily from podocytes. Polyethyleneimine labeling of fetal kidneys revealed anionic sites along both laminae rarae of the GBM that became most prominent along the subepithelial aspect at maturity; labeling was greatly reduced along the subepithelial aspect in agrin-deficient and conditional knockout mice. Despite this severe charge disruption, the glomerular filtration barrier was not compromised, even when challenged with bovine serum albumin overload. We conclude that agrin is not required for establishment or maintenance of GBM architecture. Although agrin contributes significantly to the anionic charge to the GBM, both it and its charge are not needed for glomerular permselectivity. This calls into question whether charge selectivity is a feature of the GBM.

Transgenic isolation of skeletal muscle and kidney defects in laminin beta2 mutant mice: implications for Pierson syndrome.

Pierson syndrome is a recently defined disease usually lethal within the first postnatal months and caused by mutations in the gene encoding laminin beta2 (LAMB2). The hallmarks of Pierson syndrome are congenital nephrotic syndrome accompanied by ocular abnormalities, including microcoria (small pupils), with muscular and neurological developmental defects also present. Lamb2(-/-) mice are a model for Pierson syndrome; they exhibit defects in the kidney glomerular barrier, in the development and organization of the neuromuscular junction, and in the retina. Lamb2(-/-) mice fail to thrive and die very small at 3 weeks of age, but to what extent the kidney and neuromuscular defects each contribute to this severe phenotype has been obscure, though highly relevant to understanding Pierson syndrome. To investigate this, we generated transgenic mouse lines expressing rat laminin beta2 either in muscle or in glomerular epithelial cells (podocytes) and crossed them onto the Lamb2(-/-) background. Rat beta2 was confined in skeletal muscle to synapses and myotendinous junctions, and in kidney to the glomerular basement membrane. In transgenic Lamb2(-/-) mice, beta2 deposition in only glomeruli prevented proteinuria but did not ameliorate the severe phenotype. By contrast, beta2 expression in only muscle restored synaptic architecture and led to greatly improved health, but the mice died from kidney disease at 1 month. Rescue of both glomeruli and synapses was associated with normal weight gain, fertility and lifespan. We conclude that muscle defects in Lamb2(-/-) mice are responsible for the severe failure to thrive phenotype, and that renal replacement therapy alone will be an inadequate treatment for Pierson syndrome.

Discs-large homolog 1 regulates smooth muscle orientation in the mouse ureter.

Discs-large homolog 1 (DLGH1) is a mouse ortholog of the Drosophila discs-large (DLG) tumor suppressor protein, a founding member of the PDZ and MAGUK protein families. DLG proteins play important roles in regulating cell proliferation, epithelial cell polarity, and synapse formation and function. Here, we generated a null allele of Dlgh1 and studied its role in urogenital development. Dlgh1(-/-) mice developed severe urinary tract abnormalities, including congenital hydronephrosis, which is the leading cause of renal failure in infants and children. DLGH1 is expressed in the developing ureter; in its absence, the stromal cells that normally lie between the urothelial and smooth muscle layers were missing. Moreover, in ureteric smooth muscle, the circular smooth muscle cells were misaligned in a longitudinal orientation. These abnormalities in the ureter led to severely impaired ureteric peristalsis. Similar smooth muscle defects are observed frequently in patients with ureteropelvic junction obstruction, a common form of hydronephrosis. Our results suggest that (i) besides its well documented role in regulating epithelial polarity, Dlgh1 also regulates smooth muscle orientation, and (ii) human DLG1 mutations may contribute to hereditary forms of hydronephrosis.