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1.  Three-dimensional electron microscopy reveals the evolution of glomerular barrier injury 
Scientific Reports  2016;6:35068.
Glomeruli are highly sophisticated filters and glomerular disease is the leading cause of kidney failure. Morphological change in glomerular podocytes and the underlying basement membrane are frequently observed in disease, irrespective of the underlying molecular etiology. Standard electron microscopy techniques have enabled the identification and classification of glomerular diseases based on two-dimensional information, however complex three-dimensional ultrastructural relationships between cells and their extracellular matrix cannot be easily resolved with this approach. We employed serial block face-scanning electron microscopy to investigate Alport syndrome, the commonest monogenic glomerular disease, and compared findings to other genetic mouse models of glomerular disease (Myo1e−/−, Ptpro−/−). These analyses revealed the evolution of basement membrane and cellular defects through the progression of glomerular injury. Specifically we identified sub-podocyte expansions of the basement membrane with both cellular and matrix gene defects and found a corresponding reduction in podocyte foot process number. Furthermore, we discovered novel podocyte protrusions invading into the glomerular basement membrane in disease and these occurred frequently in expanded regions of basement membrane. These findings provide new insights into mechanisms of glomerular barrier dysfunction and suggest that common cell-matrix-adhesion pathways are involved in the progression of disease regardless of the primary insult.
doi:10.1038/srep35068
PMCID: PMC5057164  PMID: 27725732
2.  A flexible, multilayered protein scaffold maintains the slit in between glomerular podocytes 
JCI insight  2016;1(9):e86177.
Vertebrate life critically depends on renal filtration and excretion of low molecular weight waste products. This process is controlled by a specialized cell-cell contact between podocyte foot processes: the slit diaphragm (SD). Using a comprehensive set of targeted KO mice of key SD molecules, we provided genetic, functional, and high-resolution ultrastructural data highlighting a concept of a flexible, dynamic, and multilayered architecture of the SD. Our data indicate that the mammalian SD is composed of NEPHRIN and NEPH1 molecules, while NEPH2 and NEPH3 do not participate in podocyte intercellular junction formation. Unexpectedly, homo- and heteromeric NEPHRIN/NEPH1 complexes are rarely observed. Instead, single NEPH1 molecules appear to form the lower part of the junction close to the glomerular basement membrane with a width of 23 nm, while single NEPHRIN molecules form an adjacent junction more apically with a width of 45 nm. In both cases, the molecules are quasiperiodically spaced 7 nm apart. These structural findings, in combination with the flexibility inherent to the repetitive Ig folds of NEPHRIN and NEPH1, indicate that the SD likely represents a highly dynamic cell-cell contact that forms an adjustable, nonclogging barrier within the renal filtration apparatus.
doi:10.1172/jci.insight.86177
PMCID: PMC4943462  PMID: 27430022
3.  A flexible, multilayered protein scaffold maintains the slit in between glomerular podocytes 
JCI Insight  null;1(9):e86177.
Vertebrate life critically depends on renal filtration and excretion of low molecular weight waste products. This process is controlled by a specialized cell-cell contact between podocyte foot processes: the slit diaphragm (SD). Using a comprehensive set of targeted KO mice of key SD molecules, we provided genetic, functional, and high-resolution ultrastructural data highlighting a concept of a flexible, dynamic, and multilayered architecture of the SD. Our data indicate that the mammalian SD is composed of NEPHRIN and NEPH1 molecules, while NEPH2 and NEPH3 do not participate in podocyte intercellular junction formation. Unexpectedly, homo- and heteromeric NEPHRIN/NEPH1 complexes are rarely observed. Instead, single NEPH1 molecules appear to form the lower part of the junction close to the glomerular basement membrane with a width of 23 nm, while single NEPHRIN molecules form an adjacent junction more apically with a width of 45 nm. In both cases, the molecules are quasiperiodically spaced 7 nm apart. These structural findings, in combination with the flexibility inherent to the repetitive Ig folds of NEPHRIN and NEPH1, indicate that the SD likely represents a highly dynamic cell-cell contact that forms an adjustable, nonclogging barrier within the renal filtration apparatus.
Genetic and high resolution ultrastructural data suggest that the podocyte slit diaphragm is a highly dynamic cell-cell contact that forms an adjustable, non-clogging barrier within the renal filtration apparatus.
doi:10.1172/jci.insight.86177
PMCID: PMC4943462  PMID: 27430022
4.  A role for genetic susceptibility in sporadic focal segmental glomerulosclerosis 
The Journal of Clinical Investigation  null;126(3):1067-1078.
Focal segmental glomerulosclerosis (FSGS) is a syndrome that involves kidney podocyte dysfunction and causes chronic kidney disease. Multiple factors including chemical toxicity, inflammation, and infection underlie FSGS; however, highly penetrant disease genes have been identified in a small fraction of patients with a family history of FSGS. Variants of apolipoprotein L1 (APOL1) have been linked to FSGS in African Americans with HIV or hypertension, supporting the proposal that genetic factors enhance FSGS susceptibility. Here, we used sequencing to investigate whether genetics plays a role in the majority of FSGS cases that are identified as primary or sporadic FSGS and have no known cause. Given the limited number of biopsy-proven cases with ethnically matched controls, we devised an analytic strategy to identify and rank potential candidate genes and used an animal model for validation. Nine candidate FSGS susceptibility genes were identified in our patient cohort, and three were validated using a high-throughput mouse method that we developed. Specifically, we introduced a podocyte-specific, doxycycline-inducible transactivator into a murine embryonic stem cell line with an FSGS-susceptible genetic background that allows shRNA-mediated targeting of candidate genes in the adult kidney. Our analysis supports a broader role for genetic susceptibility of both sporadic and familial cases of FSGS and provides a tool to rapidly evaluate candidate FSGS-associated genes.
doi:10.1172/JCI82592
PMCID: PMC4767358  PMID: 26901816
5.  Fatty Acid Transport Protein 1 can compensate for Fatty Acid Transport Protein 4 in the developing mouse epidermis 
SUMMARY
Fatty acid transport protein (FATP) 4 is one of a family of six FATPs that facilitate long- and very long-chain fatty acid uptake. Mice lacking FATP4 are born with tight, thick skin and a defective barrier; they die neonatally due to dehydration and restricted movements. Mutations in SLC27A4, the gene encoding FATP4, cause ichthyosis prematurity syndrome (IPS), characterized by premature birth, respiratory distress, and edematous skin with severe ichthyotic scaling. Symptoms of surviving patients become mild, though atopic manifestations are common. We previously showed that suprabasal keratinocyte expression of a Fatp4 transgene in Fatp4 mutant skin rescues the lethality and ameliorates the skin phenotype. Here we tested the hypothesis that FATP1, the closest FATP4 homolog, can compensate for the lack of FATP4 in our mouse model of IPS, as it might do postnatally in IPS patients. Transgenic expression of FATP1 in suprabasal keratinocytes rescued the phenotype of Fatp4 mutants, and FATP1 sorted to the same intracellular organelles as endogenous FATP4. Thus, FATP1 and FATP4 likely have overlapping substrate specificities, enzymatic activities, and biological functions. These results suggest that increasing expression of FATP1 in suprabasal keratinocytes could normalize the skin of IPS patients and perhaps prevent the atopic manifestations.
doi:10.1038/jid.2014.378
PMCID: PMC4289464  PMID: 25184958
Fatty acids; fatty acid transport; skin barrier; epidermis
6.  DLG1 influences distal ureter maturation via a non-epithelial cell autonomous mechanism involving reduced retinoic acid signaling, Ret expression, and apoptosis 
Developmental biology  2014;390(2):160-169.
Summary
The absence of Discs-large 1 (DLG1), the mouse ortholog of the Drosophila discs-large tumor suppressor, results in congenital hydronephrosis characterized by urinary tract abnormalities, reduced ureteric bud branching, and delayed disconnection of the ureter from the common nephric duct (CND). To define the specific cellular requirements for Dlg1 expression during urogenital development, we used a floxed Dlg1 allele and Pax2-Cre, Pax3-Cre, Six2-Cre, and HoxB7-Cre transgenes to generate cell type-restricted Dlg1 mutants. In addition, we used RetGFP knockin and retinoic acid response element-lacZ transgenic mice to determine the effects of Dlg1 mutation on the respective morphogenetic signaling pathways. Mutation of Dlg1 in urothelium and collecting ducts (via HoxB7-Cre or Pax2-Cre) and in nephron precursors (via Pax2-Cre and Six2-Cre) resulted in no apparent abnormalities in ureteric bud branching or in distal ureter maturation, and no hydronephrosis. Mutation in nephrons, ureteric smooth muscle, and mesenchyme surrounding the lower urinary tract (via the Pax3-Cre transgene) resulted in congenital hydronephrosis accompanied by reduced branching, abnormal distal ureter maturation and insertion, and smooth muscle orientation defects, phenotypes very similar to those in Dlg1 null mice. Dlg1 null mice showed reduced Ret expression and apoptosis during ureter maturation and evidence of reduced retinoic acid signaling in the kidney. Taken together, these results suggest that Dlg1 expression in ureter and CND-associated mesenchymal cells is essential for ensuring distal ureter maturation by facilitating retinoic acid signaling, Ret expression, and apoptosis of the urothelium.
doi:10.1016/j.ydbio.2014.03.014
PMCID: PMC4038003  PMID: 24699546
Urogenital system; PDZ domain; hydronephrosis
7.  Pathology versus molecular genetics: (re)defining the spectrum of Alport syndrome 
Kidney international  2014;86(6):1081-1083.
Next generation sequencing applied to families with glomerular disease has been instrumental in identifying new genes and pathways involved in podocyte homeostasis. Malone et al. performed sequencing on 70 families with FSGS and discovered that 10% had variants in surprising “old” genes, COL4A3 and COL4A4, which are involved in Alport syndrome and thin basement membrane nephropathy. These data show that a subset of renal manifestations associated with COL4A3 or COL4A4 variants cannot be distinguished from FSGS by clinical data or by histopathology. Thus, a diagnosis of FSGS may sometimes fall within the spectrum of Alport syndrome.
doi:10.1038/ki.2014.326
PMCID: PMC4246419  PMID: 25427084
Alport syndrome; glomerular disease; genetics; FSGS
8.  Nanoscale protein architecture of the kidney glomerular basement membrane 
eLife  2013;2:e01149.
In multicellular organisms, proteins of the extracellular matrix (ECM) play structural and functional roles in essentially all organs, so understanding ECM protein organization in health and disease remains an important goal. Here, we used sub-diffraction resolution stochastic optical reconstruction microscopy (STORM) to resolve the in situ molecular organization of proteins within the kidney glomerular basement membrane (GBM), an essential mediator of glomerular ultrafiltration. Using multichannel STORM and STORM-electron microscopy correlation, we constructed a molecular reference frame that revealed a laminar organization of ECM proteins within the GBM. Separate analyses of domains near the N- and C-termini of agrin, laminin, and collagen IV in mouse and human GBM revealed a highly oriented macromolecular organization. Our analysis also revealed disruptions in this GBM architecture in a mouse model of Alport syndrome. These results provide the first nanoscopic glimpse into the organization of a complex ECM.
DOI: http://dx.doi.org/10.7554/eLife.01149.001
eLife Digest
The blood that flows through the body must be continually filtered to remove waste products and to ensure that it contains optimal levels of water and salts. Filtration is performed inside the kidneys by tufts of small blood vessels called glomeruli. These glomerular capillaries allow water and waste products to pass from the blood into the urine, while holding back proteins and blood cells. The wall of a glomerular capillary consists of two layers of cells flanking a third layer called the glomerular basement membrane. If any of these layers malfunctions, it becomes possible for proteins to pass into the urine. This is a clear sign of kidney disease.
The basement membrane is composed of proteins secreted by the two layers of cells, but little was known about how these proteins are organized. Now, Suleiman et al. have adapted a new form of high-resolution optical microscopy called STORM to study the structure of the glomerular basement membrane in both mouse and human kidney tissue. By combining data from STORM and electron microscopy, Suleiman et al. showed that the proteins in the glomerular basement membranes of both species are arranged similarly to form a distinctive layered structure. This suggests that the organization of the basement membrane plays a critical role in its function.
The technique was used to demonstrate that proteins were not organized in the glomerular basement membrane in tissue samples taken from mice suffering from Alport syndrome, a genetic disorder of the kidneys. In addition to suggesting that the disorganization of basement membranes may play an important role in disease, this work also provides a method for investigating the structure of the basement membrane in diverse types of tissue.
DOI: http://dx.doi.org/10.7554/eLife.01149.002
doi:10.7554/eLife.01149
PMCID: PMC3790497  PMID: 24137544
super-resolution microscopy; extracellular matrix; kidney; basement membrane; Human; Mouse
10.  The 2014 International Workshop on Alport Syndrome 
Kidney international  2014;86(4):679-684.
Alport syndrome, historically referred to as hereditary glomerulonephritis with sensorineural deafness and anterior lenticonus, is a genetic disease of collagen α3α4α5(IV) resulting in renal failure. The collagen α3α4α5(IV) heterotrimer forms a network that is a major component of the kidney glomerular basement membrane (GBM) and basement membranes in the cochlea and eye. Alport syndrome, estimated to affect 1 in 5,000 to 10,000 individuals, is caused by mutations in any one of the three genes that encode the α chain components of the collagen α3α4α5(IV) heterotrimer: COL4A3, COL4A4, and COL4A5. Although angiotensin converting enzyme inhibition is effective in Alport syndrome patients for slowing progression to end-stage renal disease, it is neither a cure nor an adequate long-term protector. The 2014 International Workshop on Alport Syndrome, held in Oxford, UK from January 3–5, was organized by individuals and families living with Alport syndrome, in concert with international experts in the clinical, genetic, and basic science aspects of the disease. Stakeholders from diverse communities—patient families, physicians, geneticists, researchers, Pharma, and funding organizations—were brought together so that they could meet and learn from each other and establish strategies and collaborations for the future, with the overall aim of discovering much needed new treatments to prolong kidney function.
doi:10.1038/ki.2014.229
PMCID: PMC4182137  PMID: 24988067
11.  A mouse collagen4α4 mutation causing Alport glomerulosclerosis with abnormal collagen α3α4α5(IV) trimers 
Kidney international  2014;85(6):1461-1468.
A spontaneous mutation termed bilateral wasting kidneys (bwk) was identified in a colony of NONcNZO recombinant inbred mice. These mice exhibit a rapid increase of urinary albumin at an early age associated with glomerulosclerosis, interstitial nephritis, and tubular atrophy. The mutation was mapped to a location on Chromosome 1 containing the Col4a3 and Col4a4 genes, for which mutations in the human orthologs cause the hereditary nephritis Alport syndrome. DNA sequencing identified a G to A mutation in the conserved GT splice donor of Col4a4 intron 30, resulting in skipping of exon 30 but maintaining the mRNA reading frame. Protein analyses showed that mutant collagen α3α4α5(IV) trimers were secreted and incorporated into the glomerular basement membrane (GBM), but levels were low, and GBM lesions typical of Alport syndrome were observed. Moving the mutation into the more renal damage-prone DBA/2J and 129S1/SvImJ backgrounds revealed differences in albuminuria and its rate of increase, suggesting an interaction between the Col4a4 mutation and modifier genes. This novel mouse model of Alport syndrome is the only one shown to accumulate abnormal collagen α3α4α5(IV) in the GBM, as also found in a subset of Alport patients. These mice will be valuable for testing potential therapies, for understanding abnormal collagen IV structure and assembly, for gaining better insights into the mechanisms leading to Alport syndrome and to the variability in the age of onset and associated phenotypes.
doi:10.1038/ki.2013.493
PMCID: PMC4040157  PMID: 24522496
12.  The Glomerular Basement Membrane as a Barrier to Albumin 
Nature reviews. Nephrology  2013;9(8):10.1038/nrneph.2013.109.
The glomerular basement membrane (GBM) is the central, non-cellular layer of the glomerular filtration barrier that is situated between the two cellular components – fenestrated endothelial cells and interdigitated podocyte foot processes. The GBM is composed primarily of four extracellular matrix macromolecules – laminin-521, type IV collagen α3α4α5, the heparan sulfate proteoglycan agrin, and nidogen – that produce an interwoven meshwork thought to impart both size- and charge-selective properties. Although the composition and biochemical nature of the GBM have been known for a long time, the functional importance of the GBM vs. podocytes and endothelial cells for establishing the glomerular filtration barrier to albumin is still debated. Together with mouse genetics studies, the discoveries of four human mutations in GBM components in two inherited kidney disorders, Alport syndrome and Pierson syndrome, support essential roles for the GBM in glomerular permselectivity. Here we explain in detail the proposed mechanisms whereby the GBM can serve as the major albumin barrier and discuss possible approaches to circumvent GBM defects associated with loss of permselectivity.
doi:10.1038/nrneph.2013.109
PMCID: PMC3839671  PMID: 23774818
14.  Albumin-associated free fatty acids induce macropinocytosis in podocytes 
The Journal of Clinical Investigation  2015;125(6):2307-2316.
Podocytes are specialized epithelial cells in the kidney glomerulus that play important structural and functional roles in maintaining the filtration barrier. Nephrotic syndrome results from a breakdown of the kidney filtration barrier and is associated with proteinuria, hyperlipidemia, and edema. Additionally, podocytes undergo changes in morphology and internalize plasma proteins in response to this disorder. Here, we used fluid-phase tracers in murine models and determined that podocytes actively internalize fluid from the plasma and that the rate of internalization is increased when the filtration barrier is disrupted. In cultured podocytes, the presence of free fatty acids (FFAs) associated with serum albumin stimulated macropinocytosis through a pathway that involves FFA receptors, the Gβ/Gγ complex, and RAC1. Moreover, mice with elevated levels of plasma FFAs as the result of a high-fat diet were more susceptible to Adriamycin-induced proteinuria than were animals on standard chow. Together, these results support a model in which podocytes sense the disruption of the filtration barrier via FFAs bound to albumin and respond by enhancing fluid-phase uptake. The response to FFAs may function in the development of nephrotic syndrome by amplifying the effects of proteinuria.
doi:10.1172/JCI79641
PMCID: PMC4518691  PMID: 25915582
Cell Biology; Nephrology
15.  Organogenesis of the kidney glomerulus 
Organogenesis  2011;7(2):75-82.
The glomerular basement membrane (GBM) is a crucial component of the kidney's filtration barrier that separates the vasculature from the urinary space. During glomerulogenesis, the GBM is formed from fusion of two distinct basement membranes, one synthesized by the glomerular epithelial cell (podocyte) and the other by the glomerular endothelial cell. The main components of the GBM are laminin-521 (α5β2γ1), collagen α3α4α5(IV), nidogen and the heparan sulfate proteoglycan, agrin. By studying mice lacking specific GBM components, we have shown that during glomerulogenesis, laminin is the only one that is required for GBM integrity and in turn, the GBM is required for completion of glomerulogenesis and glomerular vascularization. In addition, our results from laminin β2-null mice suggest that laminin-521, and thus the GBM, contribute to the establishment and maintenance of the glomerular filtration barrier to plasma albumin. In contrast, mutations that affect GBM collagen IV or agrin do not impair glomerular development or cause immediate leakage of plasma proteins. However, collagen IV mutation, which causes Alport syndrome and ESRD in humans, leads to gradual damage to the GBM that eventually leads to albuminuria and renal failure. These results highlight the importance of the GBM for establishing and maintaining a perfectly functioning, highly selective glomerular filter.
doi:10.4161/org.7.2.15275
PMCID: PMC3142441  PMID: 21519194
laminin; collagen IV; nephrotic syndrome; alport syndrome; podocyte; mesangial cell; glomerulogenesis
16.  Epidermal hyperproliferation in mice lacking fatty acid transport protein 4 (FATP4) involves ectopic EGF receptor and STAT3 signaling 
Developmental biology  2010;344(2):707-719.
Fatty acid transport protein (FATP) 4 is one of a family of six FATPs that facilitate long- and very long-chain fatty acid uptake. Mice lacking FATP4 are born with tight, thick skin and a defective epidermal barrier; they die neonatally due to dehydration and restricted movements. Both the skin phenotype and the lethality are rescued by transgene-driven expression of FATP4 solely in suprabasal keratinocytes. Here we show that Fatp4 mutants exhibit epidermal hyperplasia resulting from an increased number of proliferating suprabasal cells. In addition, barrier formation initiates precociously but never progresses to completion. To investigate possible mechanisms whereby Fatp4 influences skin development, we identified misregulated genes in Fatp4 mutants. Remarkably, three members of the epidermal growth factor (EGF) family (Ereg, Areg, and Epgn) showed increased expression that was associated with elevated epidermal activation of the EGF receptor (EGFR) and STAT3, a downstream effector of EGFR signaling. Both Tyrphostin AG1478, an EGFR tyrosine kinase inhibitor, and curcumin, an inhibitor of both STAT3 and EGFR, attenuated STAT3 activation/nuclear translocation, reduced skin thickening, and partially suppressed the barrier abnormalities. These data identify FATP4 activity as negatively influencing EGFR activation and the resulting STAT3 signaling during normal skin development. These findings have important implications for understanding the pathogenesis of ichthyosis prematurity syndrome, a disease recently shown to be caused by FATP4 mutations.
doi:10.1016/j.ydbio.2010.05.503
PMCID: PMC2914132  PMID: 20513444
Epiregulin; amphiregulin; epithelial mitogen; PPAR; skin barrier; epidermal hyperplasia
17.  Glomerular filtration is normal in the absence of both agrin and perlecan–heparan sulfate from the glomerular basement membrane 
Nephrology Dialysis Transplantation  2009;24(7):2044-2051.
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.
doi:10.1093/ndt/gfn758
PMCID: PMC2721481  PMID: 19144998
charge selectivity; Ficoll; glomerular basement membrane; glomerular filtration; perlecan
18.  Update on the glomerular filtration barrier 
Purpose of the review
The nephrology community lacks a unified view of protein sieving through the glomerular capillary wall (GCW). The GCW consists of three distinct but closely interacting layers: the fenestrated endothelium, with its glycocalyx; the podocytes, with their interdigitated foot processes and slit diaphragms; and the intervening glomerular basement membrane (GBM). Proteinuria is associated with abnormalities in any one layer, suggesting that each contributes to the glomerular filtration barrier (GFB). Proteinuria can also be induced in the context of a normal GCW. Here we review some classic studies as well as some newer concepts and present competing hypotheses about the GFB.
Recent findings
Two almost forgotten concepts have recently emerged. One group has challenged the exquisite selectivity of the GFB to albumin and suggested that proteinuria is the result of abnormal tubular uptake. There has also been a reemphasis on diffusion through the GBM as the driving force behind macromolecular filtration. New evidence suggests that the endothelial glycocalyx is an important charge-selective barrier.
Summary
We suggest viewing the GFB as a dynamic rather than as a rigid barrier, requiring three healthy layers and a hemodynamic steady state. Multiple challenges to studying the endothelium, the tubular handling of albumin, and the role of hemodynamic forces will require new tools, new hypotheses, and open minds.
PMCID: PMC2895306  PMID: 19374010
Proteinuria; Glomerular filtration barrier; Permselectivity; Gel permeation; Podocytes
19.  The Pax3-Cre Transgene Exhibits a Rostrocaudal Gradient of Expression in the Skeletal Muscle Lineage 
Summary
Pax3-Cre (P3Pro-Cre) transgenic mice have been used for conditional gene deletion and/or lineage tracing in derivatives of neural crest, neural tube, metanephric mesenchyme, and ureteric mesenchyme. However, the extent of its expression in skeletal muscle has not been reported. We investigated the expression of P3Pro-Cre in the skeletal muscle lineage using the R26R reporter and found an unexpected rostrocaudal gradient of expression. By X-gal staining, head, neck, forelimb, diaphragm, and most of the chest wall muscles did not show evidence of Cre expression, whereas all muscle groups posterior of the diaphragm stained blue. Intercostal muscles exhibited a rostrocaudal gradient of staining. The consistency of this expression pattern was demonstrated by using P3Pro-Cre to mutate a conditional dystroglycan allele. The result was loss of dystroglycan from caudal muscles, which exhibited the histological signs of muscle fiber injury and regeneration characteristic of muscular dystrophy. The lack of dystroglycan in regenerating myofibers suggests that the P3Pro-Cre transgene is active in satellite cells and/or in their precursors. In contrast, rostral muscles, including feeding and breathing muscles, maintained dystroglycan expression and were spared from disease. Accordingly, the mutants were viable for over a year. Its unique gradient of activity makes the P3Pro-Cre transgene a previously unappreciated yet powerful tool for manipulating gene expression in skeletal muscle and its precursors.
doi:10.1002/dvg.20447
PMCID: PMC2759890  PMID: 18942111
Pax3; Cre; Dystroglycan; Muscular dystrophy
20.  Laminin α5 influences the architecture of the mouse small intestinal mucosa 
Journal of cell science  2008;121(Pt 15):2493-2502.
Summary
The mammalian intestine displays two distinct patterns of mucosal organization. The small intestine contains mucosal epithelial invaginations called crypts of Lieberkühn that are continuous with evaginations into the lumen called villi. The colon also contains crypts, but its epithelial surface is lined by flat surface cuffs. The epithelial cells of both organs communicate with the underlying mesenchyme through a basement membrane that is composed of a variety of extracellular matrix proteins, including members of the laminin family. The basement membranes of the small intestine and colon contain distinct laminin subtypes; notably, the villus basement membrane is rich in laminin α5. Here we show that diminution of laminin α5 in a mouse model led to a compensatory deposition of colonic laminins that resulted in a transformation from a small intestinal to a colonic mucosal architecture. The alteration in mucosal architecture was associated with reduced levels of nuclear p27Kip1, a cell cycle regulator, and altered intestinal epithelial cell proliferation, migration, and differentiation. Our results suggest that laminin α5 plays a crucial role in establishing and maintaining the specific mucosal pattern of the mouse small intestine.
doi:10.1242/jcs.025528
PMCID: PMC2587097  PMID: 18628307
basement membrane; small intestine; intestinal epithelial cell; Lutheran; p27Kip1
21.  A Hypomorphic Mutation in the Mouse Laminin α5 Gene (Lama5) Causes Polycystic Kidney Disease 
Extracellular matrix abnormalities have been found in both human and animal models of polycystic kidney disease (PKD). We have produced a new mouse PKD model through insertion of a PGKneo cassette in an intron of the gene encoding laminin α5, a major tubular and glomerular basement membrane component important for glomerulogenesis and ureteric bud branching. Lama5neo represents a hypomorphic allele due to aberrant splicing. Lama5neo/neo mice exhibit PKD, proteinuria, and death from renal failure by 4 weeks of age. This contrasts with mice totally lacking laminin α5, which die in utero with multiple developmental defects. At 2 days of age, Lama5neo/neo mice exhibited mild proteinuria and microscopic cystic transformation. By 2 weeks, cysts were grossly apparent in cortex and medulla, involving both nephron and collecting duct segments. Tubular basement membranes appeared to form normally, and early cyst basement membranes showed normal ultrastructure but developed marked thickening as cysts enlarged. Overall, laminin α5 protein levels were severely reduced due to mRNA frameshift caused by exon skipping. This was accompanied by aberrant accumulation of laminin-332 (α3β3γ2; formerly called laminin-5) in some cysts, as also observed in human PKD. This constitutes the first evidence that a primary defect in an extracellular matrix component can cause PKD.
doi:10.1681/ASN.2005121298
PMCID: PMC1482806  PMID: 16790509
BM, basement membrane; E, embryonic day; GBM, glomerular BM; P, postnatal day; PKD, polycystic kidney disease; PC1, polycystin-1
22.  Transgenic isolation of skeletal muscle and kidney defects in laminin beta2 mutant mice: Implications for Pierson syndrome 
Development (Cambridge, England)  2006;133(5):967-975.
Summary
Pierson syndrome is a recently defined disease usually lethal within the first postnatal months and caused by mutations in the gene encoding laminin β2 (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 β2 either in muscle or in glomerular epithelial cells (podocytes) and crossed them onto the Lamb2−/− background. Rat β2 was confined in skeletal muscle to synapses and myotendinous junctions, and in kidney to the glomerular basement membrane. In transgenic Lamb2−/− mice, β2 deposition only in glomeruli prevented proteinuria but did not ameliorate the severe phenotype. In contrast, β2 expression only in 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.
doi:10.1242/dev.02270
PMCID: PMC1363729  PMID: 16452099
23.  Homozygous and compound heterozygous mutations in ZMPSTE24 cause the laminopathy restrictive dermopathy 
Restrictive dermopathy is a lethal human genetic disorder characterized by very tight, thin, easily eroded skin, rocker bottom feet, and joint contractures. This disease was recently reported to be associated with a single heterozygous mutation in ZMPSTE24 and hypothesized to be a digenic disorder (Navarro et al., Lamin A and ZMPSTE24 (FACE-1) defects cause nuclear disorganization and identify restrictive dermopathy as a lethal neonatal laminopathy. Hum Mol Genet 13:2493-2503, 2004). ZMPSTE24 encodes an enzyme necessary for the correct processing and maturation of lamin A, an intermediate filament component of the nuclear envelope. Here we present four unrelated patients with homozygous mutations in ZMPSTE24 and a fifth patient with compound heterozygous mutations in ZMPSTE24. Two of the three different mutations we found are novel, and all are single base insertions that result in mRNA frameshifts. As a consequence of the presumed lack of ZMPSTE24 activity, prelamin A, the unprocessed toxic form of lamin A, was detected in the nuclei of both cultured cells and tissue from restrictive dermopathy patients, but not in control nuclei. Abnormally aggregated lamin A/C was also observed. These results indicate that restrictive dermopathy is an autosomal recessive laminopathy caused by inactivating ZMPSTE24 mutations that result in defective processing and nuclear accumulation of prelamin A.
doi:10.1111/j.0022-202X.2005.23846.x
PMCID: PMC1360172  PMID: 16297189
STE24 protein; lamin A; nuclear envelope; FATP4 protein; RD, restrictive dermopathy
24.  Rac1 Activation in Podocytes Induces Rapid Foot Process Effacement and Proteinuria 
Molecular and Cellular Biology  2013;33(23):4755-4764.
The kidney's vital filtration function depends on the structural integrity of the glomerulus, the proximal portion of the nephron. Within the glomerulus, the architecturally complex podocyte forms the final cellular barrier to filtration. Injury to the podocyte results in a morphological change called foot process effacement, which is a ubiquitous feature of proteinuric diseases. The exact mechanism underlying foot process effacement is not known, but recently it has been proposed that this change might reflect activation of the Rac1 GTPase. To test this hypothesis, we generated a podocyte-specific, inducible transgenic mouse line that expressed constitutively active Rac1. When the Rac1 transgene was induced, we observed a rapid onset of proteinuria with focal foot process effacement. Using superresolution imaging, we verified that the induced transgene was expressed in damaged podocytes with altered foot process morphology. This work sheds new light on the complex balance of Rho GTPase signaling that is required for proper regulation of the podocyte cytoskeleton.
doi:10.1128/MCB.00730-13
PMCID: PMC3838009  PMID: 24061480
25.  Proteinuria precedes podocyte abnormalities inLamb2–/– mice, implicating the glomerular basement membrane as an albumin barrier  
The Journal of Clinical Investigation  2006;116(8):2272-2279.
Primary defects in either podocytes or the glomerular basement membrane (GBM) cause proteinuria, a fact that complicates defining the barrier to albumin. Laminin β2 (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.
doi:10.1172/JCI28414
PMCID: PMC1523402  PMID: 16886065

Results 1-25 (57)