Search

This article presents analysis, design details, and measurement result of a 224-Gb/s four-level pulse amplitude modulation (PAM-4) transmitter (TX) consisting of a 7-bit voltage digital-to-analog converter (DAC) driver, digital 8-tap feed-forward equalizer (FFE), and a 28-GHz inductively peaked clock distribution network. The TX DAC uses quarter-rate clocking with a 4:1 pulse-based data serialization architecture. Design techniques for generating and distributing low-jitter CMOS clocks up to 29 GHz, timing closure in the serializer, 112-Gbaud 4:1 data MUX using 1-UI pulse generator, and bandwidth/return loss/group delay optimized output pad network using a 9th-order LC filter are described. Fabricated in the Intel 10-nm FinFET process technology, the TX demonstrates random jitter (RJ) of 65 fs $_{{rms}}$ with nominal output swing of 1.0 $V_{{ppd}}$ at 224 Gb/s achieving 1.88-pJ/b energy efficiency including an on-die LC phase-locked loop (PLL). To the best of authors' knowledge, this TX achieved the highest data rate with the lowest RJ for CMOS SerDes TXs reported to date.

The minor type IV collagen chain, which is a significant component of the glomerular basement membrane in healthy individuals, is known to assemble into large structures (supercoils) that may contribute to the mechanical stability of the collagen network and the glomerular basement membrane as a whole. The absence of the minor chain, as in Alport syndrome, leads to glomerular capillary demise and eventually to kidney failure. An important consideration in this problem is that the glomerular capillary wall must be strong enough to withstand the filtration pressure and porous enough to permit filtration at reasonable pressures. In this work, we propose a coupled feedback loop driven by filtration demand and tensional homeostasis of the podocytes forming the outer portion of the glomerular capillary wall. Briefly, the deposition of new collagen increases the stiffness of basement membrane, helping to stress shield the podocytes, but the new collagen also decreases the permeability of the basement membrane, requiring an increase in capillary transmural pressure drop to maintain filtration; the resulting increased pressure outweighs the increased glomerular basement membrane stiffness and puts a net greater stress demand on the podocytes. This idea is explored by developing a multiscale simulation of the capillary wall, in which a macroscopic (µm scale) continuum model is connected to a set of microscopic (nm scale) fiber network models representing the collagen network and the podocyte cytoskeleton. The model considers two cases: healthy remodeling, in which the presence of the minor chain allows the collagen volume fraction to be increased by thickening fibers, and Alport syndrome remodeling, in which the absence of the minor chain allows collagen volume fraction to be increased only by adding new fibers to the network. The permeability of the network is calculated based on previous models of flow through a fiber network, and it is updated for different fiber radii and volume fractions. The analysis shows that the minor chain allows a homeostatic balance to be achieved in terms of both filtration and cell tension. Absent the minor chain, there is a fundamental change in the relation between the two effects, and the system becomes unstable. This result suggests that mechanobiological or mechanoregulatory therapies may be possible for Alport syndrome and other minor chain collagen diseases of the kidney.

The mechanical support of basement membranes, such as the lens capsule, is believed to arise from one of their main constituents - collagen IV. The basement membranes of the lens, kidney, and ear normally contain two different types of collagen IV networks, referred to as the major and minor chain networks. In Alport syndrome, a mutation in one of the minor chain COL4 genes leads to the absence of the minor chain network, causing life-threatening disturbances. We hypothesized that the absence of the minor chain network increases basement membrane distensibility, as measured in wild-type (n = 25) and Alport syndrome (n = 21) mice using the lens capsule as a model. Osmotic swelling experiments revealed direction-dependent changes. As a reflection of lens capsule properties, Alport lenses strained significantly more than wild-type lenses in the anterior-posterior direction, i.e. along their thickness, but not in the equatorial direction (p = 0.03 and p = 0.08, respectively). This is consistent with clinical data: Alport patients develop conical protrusions on the anterior and posterior lenticular poles. There was no evidence of significant change in total amount of collagen between Alport and wild-type lenses (p = 0.6). The observed differences in distensibility could indicate that the major chain network alone cannot fully compensate for the absence of the more highly cross-linked minor chain network, which is believed to be stronger, more stable, and resistant to deformation. The addition of mechanical information on Alport syndrome to the currently available biological data provides a fuller picture into the progression of the disease.

The importance of tissue remodelling is widely accepted, but the mechanism by which the remodelling process occurs remains poorly understood. At the tissue scale, the concept of tensional homeostasis, in which there exists a target stress for a cell and remodelling functions to move the cell stress towards that target, is an important foundation for much theoretical work. We present here a theoretical model of a cell in parallel with a network to study what factors of the remodelling process help the cell move towards mechanical stability. The cell-network system was deformed and kept at constant stress. Remodelling was modelled by simulating strain-dependent degradation of collagen fibres and four different cases of collagen addition. The model did not lead to complete tensional homeostasis in the range of conditions studied, but it showed how different expressions for deposition and removal of collagen in a fibre network can interact to modulate the cell's ability to shield itself from an imposed stress by remodelling the surroundings. This study also showed how delicate the balance between deposition and removal rates is and how sensitive the remodelling process is to small changes in the remodelling rules.

The activation of endogenous Oct4 transcription is a key step in the reprogramming of somatic cells into induced pluripotent stem (iPS) cells but until now it has been difficult to analyze this critical event in the reprogramming process. We have generated a transgenic mouse that expresses the tamoxifen-inducible Cre recombinase MerCreMer under the control of the endogenous Oct4 locus, enabling lineage tracing of Oct4 expression in cells in vivo or in vitro, during either reprogramming or differentiation. Using this novel resource, we have determined the timing and outcome of endogenous Oct4 induction during fibroblast reprogramming. We show that both the initiation of this key reprogramming step and the ability of cells activating endogenous Oct4 expression to complete reprogramming are not influenced by the presence of exogenous c-Myc, although the overall efficiency of the process is increased by c-Myc. Oct4 lineage tracing reveals that new reprogramming events continue to initiate over a period of 3 weeks. Furthermore, the analysis of mixed colonies, where only a subset of daughter cells induce endogenous Oct4 expression, indicates the role of unknown, stochastic events in the progression of reprogramming from the initial events to a pluripotent state. Our transgenic mouse model and cells derived from it provide powerful and precise new tools for the study of iPS cell reprogramming mechanisms and have wider implications for the investigation of the role of Oct4 during development.

This review provides current information about glomerular disorders that arise directly from inherited abnormalities in extracellular matrix proteins intrinsic to the glomerular basement membrane (Alport syndrome, thin basement membrane nephropathy, HANAC syndrome and Pierson syndrome). The authors also discuss disorders involving genetic defects in cellular proteins that result in structural defects in glomerular basement membranes (MYH9-related disorders, nail-patella syndrome).

Background. There have been isolated case reports of arterial disease in males with Alport syndrome (AS), a systemic disorder of Type IV collagen. In this paper, we describe five new cases of AS associated with significant aortic disease including dissection and aneurysm. Methods. We present brief clinical descriptions of five males with AS and aortic disease. We performed immunohistochemical analysis of the expression of the α5 chain of Type IV collagen in skin basement membranes from a previously reported family with AS and associated aortic disease and in the aortic media of male mice with X-linked Alport syndrome (XLAS) due to a nonsense mutation in the COL4A5 gene. Results. Three of the five patients exhibited aneurysm and dissection of the thoracic aorta, occurring at 25–32 years of age, while one had aortic dilatation and another had aortic insufficiency. All five men required renal replacement therapy by age 20. Immunohistochemistry of skin biopsy specimens in previously reported male siblings with aortic disease confirmed that they had XLAS. We further found that the α5 chain of Type IV collagen is abnormally absent from aortic media of transgenic mice with XLAS. Conclusions. Early onset aortic disease may be an unusual feature of AS. Screening of men with AS for aortic abnormalities may be clinically indicated in some families.

Target-derived factors organize synaptogenesis by promoting differentiation of nerve terminals at synaptic sites. Several candidate organizing molecules have been identified based on their bioactivities in vitro, but little is known about their roles in vivo. Here, we show that three sets of organizers act sequentially to pattern motor nerve terminals: FGFs, beta2 laminins, and collagen alpha(IV) chains. FGFs of the 7/10/22 subfamily and broadly distributed collagen IV chains (alpha1/2) promote clustering of synaptic vesicles as nerve terminals form. beta2 laminins concentrated at synaptic sites are dispensable for embryonic development of nerve terminals but are required for their postnatal maturation. Synapse-specific collagen IV chains (alpha3-6) accumulate only after synapses are mature and are required for synaptic maintenance. Thus, multiple target-derived signals permit discrete control of the formation, maturation, and maintenance of presynaptic specializations.

A variety of macromolecules accumulate in the glomerular mesangium in many different diseases, but the physics of the transport of these molecules within the mesangial matrix has not been extensively studied. We present a computational model of convection and diffusion within the porous mesangial matrix and apply this model to the specific instance of immunoglobulin A (IgA) transport in IgA nephropathy. We examine the influence of physiological factors including glomerular basement membrane (GBM) thickness and mesangial matrix density on the total accumulation of IgA. Our results suggest that IgA accumulation can be understood by relating convection and diffusion, thus demonstrating the importance of intrinsic glomerular factors.

Background/Aim: The discoidin domain receptors (DDRs) DDR1 and DDR2 are cardinal members of a receptor tyrosine kinase subfamily, activated by collagens. They are candidate effectors in tissue injury and fibrosis. We investigated the DDR expression in normal and remnant rat kidneys. Methods: The DDR expression in kidney and other tissues was examined by indirect immunofluorescence, immunoblotting, and ribonuclease protection assays. The expression patterns in remnant and control kidneys were compared at 2-, 4-, and 8-week time points, following induction of injury. Results: DDR1 is expressed in basolateral membranes of select nephron segments, from the connecting tubule to the renal papilla. DDR2 is expressed in apical membranes of select nephron segments, from the loop of Henle to the macula densa. The DDR1 protein expression is upregulated within the glomeruli of remnant kidneys. The distribution of DDR2 in remnant kidneys is similar to that in controls. The DDR mRNA levels in remnant and control kidneys were not significantly different, at any time point. Conclusions: The DDR1 localization in the rat kidney is consistent with roles in cell-matrix interactions. Upregulation within glomeruli of remnant kidneys suggests the possibility of additional roles in kidney injury. The DDR2 localization in adult rat kidneys is inconsistent with roles in cell-matrix interactions.

Little is known about mechanisms regulating gene expression for the a chains of basement membrane type IV collagen, arranged head-to-head in transcription units COL4A1–COL4A2, COL4A3–COL4A4, and COL4A5–COL4A6, and implicated broadly in genetic diseases. To investigate these mechanisms, we generated transgenic mouse lines bearing 5 0 -flanking sequences of COL4A5 and COL4A6, cloned upstream of a lacZ reporter gene. A 3.8-kb fragment upstream of COL4A6 directs reporter gene expression in the esophagus, stomach, and duodenum, whereas a 13.8-kb fragment directs expression in the esophagus only. A 10.6-kb fragment upstream of COL4A5 directs expression in the esophagus. Coupled with evidence of long-range conservation between human and mouse non-coding sequences, described herein, our findings provide the first indication that highly specialized patterns characteristic of COL4A5–COL4A6 expression in vivo arise from effects of distributed cis-acting regulatory elements on a bidirectional proximal promoter, itself transcriptionally competent. 2003 Elsevier Inc. All rights reserved.

Collagen IV networks in the glomerular basement membrane (GBM) are essential for the maintenance and regulation of blood filtration in the kidneys. The GBM contains two different types of collagen IV networks: [α1(IV)]2α2(IV) and α3(IV)α4(IV)α5(IV), the latter of which has a higher number of supercoils (two or more collagens coiling around each other). To investigate the effects of supercoiling on the mechanical and permeability properties of collagen IV networks, we generated model collagen IV networks in the GBM and reconnected them to create different levels of supercoiling. We found that supercoiling greatly increases the stiffness of collagen IV networks but only minimally decreases the permeability. Also, doubling the amount of supercoils in a network had a bigger effect than doubling the stiffness of the supercoils. Our results suggest that the formation of supercoils is a specialized mechanism by the GBM that provides with a network stiff and strong enough to withstand the high hydrostatic pressures of filtration, yet porous enough that filtration is not hindered. Clinically, understanding the effects of supercoiling gives us insight into the mechanisms of GBM failure in some disease states where the normal collagen IV structure is disrupted.

Tissue-specific expression patterns of the paired type IV collagen genes COL4A5 and COL4A6 form the basis for organ involvement in X-linked Alport syndrome, a disorder in which these genes are mutated. We investigated the proximal promoter region of COL4A5 and COL4A6 using glomerular visceral epithelial cells, in which COL4A5 alone is transcribed; keratinocytes, in which the genes are co-transcribed; and additional model cell lines. By RNase protection assays, the intergenic region is 292 base pairs. Transcription start sites for two 5' splice variants of COL4A6 are 1 kilobase apart. Transient transfections with reporter gene constructs revealed that the minimal promoters for COL4A5 and COL4A6 are within 100 base pairs of their respective transcription start sites and are functionally distinct. In further transfection, gel shift and footprinting assays, we defined a bidirectional positive regulatory element, which functions in several cell types, but not in glomerular visceral epithelial cells selectively transcribing COL4A5. The existence of separate promoters for COL4A5 and COL4A6 permits fine control over their expression. Activation through the bidirectional element can bring about co-expression of the genes, exploiting their paired arrangement. Features of the proximal promoter region frame its roles in a hierarchy regulating type IV collagen gene expression.

Most patients with autosomal dominant polycystic kidney disease (ADPKD) harbor mutations truncating polycystin-1 (PC1) or polycystin-2 (PC2), products of the PKD1 and PKD2 genes, respectively. A third member of the polycystin family, polycystin-L (PCL), was recently shown to function as a Ca2+-modulated nonselective cation channel. More recently, PC2 was also shown to be a nonselective cation channel with comparable properties to PCL, though the membrane targeting of PC2 likely varies with cell types. Here we show that PC2 expressed heterologously in Xenopus oocytes is targeted to intracellular compartments. By contrast, a truncated form of mouse PC2 corresponding to a naturally occurring human mutation R742X is targeted predominantly to the plasma membrane where it mediates K+, Na+, and Ca2+ currents. Unlike PCL, the truncated form does not display Ca2+-activated transport activities, possibly due to loss of an EF-hand at the C-terminus. We propose that PC2 forms ion channels utilizing structural components which are preserved in the R742X form of the protein. Implications for epithelial cell signaling are discussed.

SummaryDeletions encompassing the 5′ termini of the paired type IV collagen genes COL4A5 and COL4A6 on chromosome Xq22 give rise to Alport syndrome (AS) and associated diffuse leiomyomatosis (DL), a syndrome of disseminated smooth-muscle tumors involving the esophagus, large airways, and female reproductive tract. In this study, we report isolation and characterization of two deletion junctions. The first, in a patient described elsewhere, arose by a nonhomologous recombination event fusing a LINE-1 (L1) repetitive element in intron 1 of COL4A5 to intron 2 of COL4A6, resulting in a 13.4-kb deletion. The second, in a previously undescribed family, arose by unequal homologous recombination between the same L1 and a colinear L1 element in intron 2 of COL4A6, resulting in a >40-kb deletion. L1 elements have contributed to the emergence of this locus as a site of frequent recombinations by diverse mechanisms. These give rise to AS-DL by disruption of type IV collagen and perhaps other as yet unidentified genes, evidenced by deletions as small as 13.4 kb.

COL4A5 mutations causing X-linked Alport syndrome (XLAS) are frequently associated with absence of the alpha3, alpha4,alpha5 and alpha6 chains of type IV collagen from basement membranes and increased amounts of the alpha1(IV) and alpha2(IV) chains in glomerular basement membrane. Although many COL4A5 mutations have been described in XLAS, the mechanisms by which these mutations influence the basement membrane appearance of chains other than alpha5(IV) remain poorly understood. In this study, we used dermal fibroblasts from eight normal individuals and nine males with XLAS to test the hypotheses that COL4A5 mutations increase transcription of COL4A1 and suppress transcription of COL4A6. Ribonuclease protection assays revealed that alpha1(IV), alpha5(IV) and alpha6(IV) transcripts were expressed in cultures of dermal fibroblasts. The mRNA levels for alpha1(IV) in eight of nine patients with XLAS were not increased compared to controls; one patient with a large COL4A5 deletion showed significant elevation of alpha1(IV) mRNA levels. No differences in steady-state mRNA levels for alpha6(IV) were found when XLAS fibroblasts were compared with controls, even though little or no alpha6(IV) protein was detectable at the dermal-epidermal junction by immunofluorescence study. This finding suggests that post-transcriptional events account for the absence of alpha6(IV) in the Alport dermal-epidermal junction.

PKD1, the gene that is mutated in approximately 85% of autosomal dominant polycystic kidney disease (ADPKD) cases in humans, has recently been identified (Eur. PKD Consortium. Cell 77: 881-894, 1994; also, erratum in Cell 78: 1994). The longest open-reading frame of PKD1 encodes polycystin, a novel approximately 460-kDa protein that contains a series of NH2-terminal adhesive domains (J. Hughes, C. J. Ward, B. Peral, R. Aspinwall, K. Clark, J. San Millan, V. Gamble, and P. C. Harris. Nat. Genet. 10: 151-160, 1995; and Int. PKD Consortium. Cell 81: 289–298, 1995) and several putative transmembrane segments. To extend studies of polycystin to an experimentally accessible animal, we have isolated a cDNA clone encoding the 3' end of Pkd1, the mouse homologue of PKD1, and raised a specific antibody to recombinant murine polycystin. This antibody was used to determine the subcellular localization and tissue distribution of the protein by Western analysis and immunocytochemistry. In the mouse, polycystin is an approximately 400-kDa molecule that is predominantly found in membrane fractions of tissue and cell extracts. It is expressed in many tissues including kidney, liver, pancreas, heart, intestine, lung, and brain. Renal expression, which is confined to tubular epithelia, is highest in late fetal and early neonatal life and drops 20-fold by the third postnatal week, maintaining this level into adulthood. Thus the temporal profile of polycystin expression coincides with kidney tubule differentiation and maturation.

The basement membrane is a specialized part of the extra-cellular matrix. It is usually characterized as a scaffold for epithelial cells but in some tissues it serves other, mechanical, roles [1]. The mechanical properties of the basement membrane are mainly determined by one of its main constituents — type IV collagen. Unlike the well-known fibrous type I collagen, collagen IV assembles into planar networks (Fig. 1) [2]. The α1(IV) and α2(IV) collagen IV chains assemble into the so-called major chain network, present in all basement membranes. The α3(IV), α4(IV), α5(IV) collagen IV chains form the minor chain network which is found only in the adult basement membranes of the kidney glomerular capillaries (GBM), ocular lens (LBM), cochlea, and the testes [3]. The minor chains have a higher number of cysteine residues, allowing them to form a higher number of lateral interactions. In the minor chain network, the greater potential to interact laterally manifests in the formation of super-coils, which are rarely observed in the major chain network [4]. Increasing the number of cross-links in a polymeric material is known to increase material stiffness; therefore, it is believed that the minor chain network confers basement membranes with additional strength and stability [5]. In the hereditary disease Alport syndrome, a mutation causes the absence of the minor chain network. The GBM and LBM of Alport patients appear weakened and unable to meet their mechanical demands, further supporting this theory [6]. The objective of this study was to evaluate the importance of cross-linking in the minor chains for the mechanical properties of type IV collagen networks, specifically in the GBM and LBM where the absence of the minor chains has an observed mechanical effect.Copyright © 2013 by ASME

Transport in the kidney is critically important in normal function and in disease. Solute transport occurs in many locations within the kidney, including convection within the capillaries, filtration across the basement membrane, and convection/diffusion within the mesangium (Figure 1). Models of transport in the kidney have traditionally focused on ultrafiltration [1], which is the predominant pathway for fluid and solutes passing into the urinary space. However, the mesangium is often the site of the first symptoms in disease [2], suggesting that mesangial transport is significant, at least in some cases.Copyright © 2013 by ASME

Alport post-transplant nephritis (APTN) is an aggressive form of anti-glomerular basement membrane disease that targets the allograft in transplanted patients with X-linked Alport syndrome. Alloantibodies develop against the NC1 domain of α5(IV) collagen, which occurs in normal kidneys, including renal allografts, forming distinct α345(IV) and α1256(IV) networks. Here, we studied the roles of these networks as antigens inciting alloimmunity and as targets of nephritogenic alloantibodies in APTN. We found that patients with APTN, but not those without nephritis, produce two kinds of alloantibodies against allogeneic collagen IV. Some alloantibodies targeted alloepitopes within α5NC1 monomers, shared by α345NC1 and α1256NC1 hexamers. Other alloantibodies specifically targeted alloepitopes that depended on the quaternary structure of α345NC1 hexamers. In Col4a5-null mice, immunization with native forms of allogeneic collagen IV exclusively elicited antibodies to quaternary α345NC1 alloepitopes, whereas alloimmunogens lacking native quaternary structure elicited antibodies to shared α5NC1 alloepitopes. These results imply that quaternary epitopes within α345NC1 hexamers may initiate alloimmune responses after transplant in X-linked Alport patients. Thus, α345NC1 hexamers are the culprit alloantigen and primary target of all alloantibodies mediating APTN, whereas α1256NC1 hexamers become secondary targets of anti-α5NC1 alloantibodies. Reliable detection of alloantibodies by immunoassays using α345NC1 hexamers may improve outcomes by facilitating early, accurate diagnosis.

This chapter discusses recent advances in the understanding of genetic kidney diseases that primarily involve the glomerulus. The chapter is organized according to the site or mechanism of the primary abnormality: abnormalities of extracellular matrix, storage diseases affecting the glomerulus, primary defects in the slit diaphragm, cytoskeletal defects and defective transcriptional regulation.

Polycystin, the product of autosomal dominant polycystic kidney disease (ADPKD) 1 gene (PKD1) is the cardinal member of a novel class of proteins. As a first step towards elucidating the function of polycystin and the pathogenesis of ADPKD, three types of information were collected in the current study: the subcellular localization of polycystin, the spatial and temporal distribution of the protein within normal tissues and the effects of ADPKD mutations on the pattern of expression in affected tissues. Antisera directed against a synthetic peptide and two recombinant proteins of different domains of polycystin revealed the presence of an approximately 400-kD protein (polycystin) in the membrane fractions of normal fetal, adult, and ADPKD kidneys. Immunohistological studies localized polycystin to renal tubular epithelia, hepatic bile ductules, and pancreatic ducts, all sites of cystic changes in ADPKD, as well as in tissues such as skin that are not known to be affected in ADPKD. By electron microscopy, polycystin was predominantly associated with plasma membranes. Polycystin was significantly less abundant in adult than in fetal epithelia. In contrast, polycystin was overexpressed in most, but not all, cysts in ADPKD kidneys.

A model is developed and analyzed for type IV collagen turnover in the kidney glomerular basement membrane (GBM), which is the primary structural element in the glomerular capillary wall. The model incorporates strain dependence in both deposition and removal of the GBM, leading to an equilibrium tissue strain at which deposition and removal are balanced. The GBM thickening decreases tissue strain per unit of transcapillary pressure drop according to the law of Laplace, but increases the transcapillary pressure drop required to maintain glomerular filtration. The model results are in agreement with the observed GBM alterations in Alport syndrome and thin basement membrane disease, and the model-predicted linear relation between the inverse capillary radius and inverse capillary thickness at equilibrium is consistent with published data on different mammals. In addition, the model predicts a minimum achievable strain in the GBM based on the geometry, properties, and mechanical environment; that is, an infinitely thick GBM would still experience a finite strain. Although the model assumptions would be invalid for an extremely thick GBM, the minimum achievable strain could be significant in diseases, such as Alport syndrome, characterized by focal GBM thickening. Finally, an examination of reasonable values for the model parameters suggests that the oncotic pressure drop—the osmotic pressure difference between the plasma and the filtrate due to large molecules—plays an important role in setting the GBM strain and, thus, leakage of protein into the urine may be protective against some GBM damage.

The ocular lens capsule (LC) is a specialized basement membrane which completely surrounds the lens. The LC serves as an attachment point for lens epithelial and fiber cells, controls lens solute and water transport, and makes accommodation possible [1]. It is primarily composed of type IV collagen (65% of dry weight), laminin, nidogen, and proteoglycans, of which type IV collagen is the main-tension resisting element [1,2]. Collagen IV monomers organize into polygonal planar networks resembling chicken wire (Fig.1) [3]. There are six different collagen IV monomers, labeled α1(IV) to α6(IV) each produced by a separate gene – COL4A1 to COL4A6. Monomers form triple helical protomers in a highly selective manner. In nature, only three monomer combinations have been discovered: the [α1(IV)]2α2(IV) protomer, referred to as the major chain, is found in all basement membranes; the α3(IV)α4(IV)α5(IV) protomer (minor chain) is found only in few basement membranes including the LC; the [α5(IV)]2α6(IV) protomer is very rare and will not be discussed further. Protomers of the same type assemble with one another to form separate networks which are known to have some differences [4]. For example, the minor chain network is more cross-linked than the major chain network. In a hereditary disease called Alport syndrome, the minor chain network is completely missing in males due to a mutation in the COL4A5 gene (located on the X chromosome) which prevents production of the α5(IV) monomer. Male Alport syndrome patients have significant ocular manifestations such as anterior lenticonus (protrusion of the lens), cataract, and even lens rupture [5] and they exhibit significant thinning of the LC. Because 1) the minor network is more cross-linked than the major network, 2) its absence affects lens shape, and 3) the LC displays pathological disruptions when it is missing, we theorize that its presence confers additional mechanical strength to the LC. Therefore, the objective of this study is to assess the contribution of the minor chain network to the mechanics of the LC.Copyright © 2012 by ASME

Animal models have provided important insights into human renal diseases that arise from mutations in genes that encode or regulate the synthesis of glomerular basement membrane proteins. This chapter describes several well-characterized animal models of type IV collagen disorders (Alport syndrome, HANAC syndrome), a laminin disorder (Pierson syndrome), nail-patella syndrome and HERNS syndrome. These models can be exploited in studies of the pathogenesis and treatment of such disorders.

Animal models have provided important insights into human renal diseases that arise from mutations in genes that encode or regulate the synthesis of glomerular basement membrane proteins. This chapter

Elevation of intracellular cAMP levels in Necturus gallbladder epithelium (NGB) induces an apical membrane Cl- conductance (GaCl). Its characteristics (i.e., magnitude, anion selectivity, and block) were studied with intracellular microelectrode techniques. Under control conditions, the apical membrane conductance (Ga) was 0.17 mS.cm-2, primarily ascribable to GaK. With elevation of cell cAMP to maximum levels, Ga increased to 6.7 mS.cm-2 and became anion selective, with the permeability sequence SCN- > NO3- > I- > Br- > Cl- > SO4(2-) approximately gluconate approximately cyclamate. GaCl was not affected by the putative Cl- channel blockers Cu2+, DIDS, DNDS, DPC, furosemide, IAA-94, MK-196, NPPB, SITS, verapamil, and glibenclamide. To characterize the cAMP-activated Cl- channels, patch-clamp studies were conducted on the apical membrane of enzyme-treated gallbladders or on dissociated cells from tissues exposed to both theophylline and forskolin. Two kinds of Cl- channels were found. With approximately 100 mM Cl- in both bath and pipette, the most frequent channel had a linear current-voltage relationship with a slope conductance of approximately 10 pS. The less frequent channel was outward rectifying with slope conductances of approximately 10 and 20 pS at -40 and 40 mV, respectively. The Cl- channels colocalized with apical maxi-K+ channels in 70% of the patches. The open probability (Po) of both kinds of Cl- channels was variable from patch to patch (0.3 on average) and insensitive to [Ca2+], membrane voltage, and pH. The channel density (approximately 0.3/patch) was one to two orders of magnitude less than that required to account for GaCl. However, addition of 250 U/ml protein kinase A plus 1 mM ATP to the cytosolic side of excised patches increased the density of the linear 10-pS Cl- channels more than 10-fold to four per patch and the mean Po to 0.5, close to expectations from GaCl. The permeability sequence and blocker insensitivity of the PKA-activated channels were identical to those of the apical membrane. These data strongly suggest that 10-pS Cl- channels are responsible for the cAMP-induced increase in apical membrane conductance of NGB epithelium.

As an alternative to purely mechanical methods, optical tracking of passive osmotic swelling was used to assess mechanical properties of the porcine lens capsule. A simple model was developed accounting for the permeability of the lens fiber cells and capsule to water, the concentration of fixed charges in the fiber cells, and the capsule’s resistance to the swelling of fiber cells. Fitting the model solution to experimental data provided an estimate of the elastic modulus of the lens capsule under the assumption of linear isotropic elasticity. The calculated elastic modulus at a fixed charge density of 20 mol m−3 was 2.0±0.5 MPa (mean±95% confidence interval; n=15) for 0.1% saline solution, 0.64±0.3 MPa(n=10) for 0.2% saline solution, and 0.28±0.5 MPa(n=6) for 0.5% saline solution. These values are comparable to previously reported moduli of elasticity for the porcine lens capsule at small strains (

Basement membranes are planar extracellular matrices ubiquitous within tissues and serve roles in the organization, support and regulation of resident cell populations. The ocular lens capsule is experimentally accessible accounting for its wide use as a model in studies of basement membrane mechanics [1–3]. Optical tracking of passive osmotic swelling, unlike previously employed methods of determining the elasticity of the lens capsule, involves minimal manipulation of the lens, which is desirable when using smaller animal models, such as the mouse.

Background. Female carriers of X-linked Alport syndrome (XLAS) demonstrate variability in clinical phenotype that, unlike males, cannot be correlated with genotype. X-inactivation, the method by which females (XX) silence transcription from one X chromosome in order to achieve gene dosage parity with males (XY), likely modifies the carrier phenotype, but this hypothesis has not been tested directly. Methods. Using a genetically defined mouse model of XLAS, we generated two groups of Alport female (Col4a5+/−) carriers that differed only in the X-controlling element (Xce) allele regulating X-inactivation. We followed the groups as far as 6 months of age comparing survival and surrogate outcome measures of urine protein and plasma urea nitrogen. Results. Preferential inactivation of the mutant Col4a5 gene improved survival and surrogate outcome measures of urine protein and plasma urea nitrogen. In studies of surviving mice, we found that X-inactivation in kidney, measured by allele-specific mRNA expression assays, correlated with surrogate outcomes. Conclusions. Our findings establish X-inactivation as a major modifier of the carrier phenotype in X-linked Alport syndrome. Thus, X-inactivation patterns may offer prognostic information and point to possible treatment strategies for symptomatic carriers.

Benign prostatic hyperplasia (BPH) is one of the most common disorders and the most common cause of lower urinary tract symptoms (LUTS) in elderly men [1]. Transurethral resection of the prostate (TURP) is considered the gold standard treatment; however, laser prostatectomy has several advantages with regard to reduced catheterization and morbidity particularly in high-risk patients [2]. Of the many lasers that can be used in the laser prostatectomy, the GreenLight potassium-titanyl-phosphate (KTP) laser is one of the most commonly used [3]. The optimum outcome of this laser procedure is ablation with minimal or no coagulation. Our objective was to experimentally and theoretically characterize the KTP laser-tissue interactions in order to understand the laser and tissue parameters that lead to an optimal outcome.

The glomerular basement membrane of the kidney, responsible for performing ultrafiltration blood plasma, is largely comprised of type-IV collagen and laminin. Type-IV collagen self-assembles into a heterotrimer composed of three distinct domains (fig. 1A): (1) the globular non-collagenous NCl domain of ∼10 nm in diameter, (2) the non-collagenous 7S domain ∼30 nm in length and ∼3nm in diameter, and (3) the collagenous triple helix of ∼370 nm in length and ∼3 nm in diameter composed of a repeating Gly-X-Y subunit [1]. The heterotrimers associate with remarkable specificity from six genetically distinct α-chains, α1(IV) to α6(IV) forming α1α1α2, α3α4α5, and α5α5α6 heterotrimers [2]. In the healthy glomerulus, α1α1α2 ([α1]2α2) is the predominate collagen while significant α3α4α5 is present; α5α5α6 exists only in negligible quantities [2].

1. The patch clamp technique was used to study the effects of internal and external pH on the Ca(2+)- and voltage-activated maxi K+ channel present in the apical membrane of Necturus gall-bladder epithelial cells. 2. When the pH of the solution bathing the cytosolic side of inside-out patches (pHi) was lowered from 7.9 to 6.9, with internal free Ca2+ concentration ([Ca2+]i) buffered below saturation levels for the channel gating sites, channel open probability (Po) decreased. At saturating Ca2+ concentrations, Po was near 1.0, and unaffected by pHi. The results are consistent with a competitive interaction between Ca2+ and H+ at regulatory binding sites. Kinetic analysis assuming competitive binding yields a Hill coefficient for H+ of 1.3. 3. At sub-maximal [Ca2+]i, changing the pH of the solution bathing the extracellular surface of the patch (pHo) between 8 and 7, had no effect on maxi K+ channel Po, but lowering pHo to 6 or 5 significantly reduced Po. At saturating [Ca2+]i, Po was independent of pHo. 4. There were no effects of either pHi or pHo on single-channel conductance. 5. Inasmuch as reductions in either pHo or pHi decrease maxi K+ channel Po, changes in maxi K+ channel activity account in part for the reduction of apical membrane K+ conductance elicited by acidification of the bathing medium. The dominant effect of pH on maxi K+ channels is on the cytosolic surface of the membrane. 6. The change in Po elicited by small changes in [H+]i (delta Po/delta [H+]i) is -7.6 microM-1, compared to delta Po/delta [Ca2+]i = 2.6 microM-1, both at Vm = -30 mV and at physiological intracellular [H+] and [Ca2+]. This implies that [H+]i and [Ca2+]i have opposite effects on channel Po at physiological levels and underlines the importance of pHi in channel gating.

The apical membrane of Necturus gallbladder epithelium contains a voltage-activated K+ conductance [Ga(V)]. Large-conductance (maxi) K+ channels underlie Ga(V) and account for 17% of the membrane conductance (Ga) under control conditions. We examined the Ba2+, tetraethylammonium (TEA+), and quinine sensitivities of Ga and single maxi K+ channels. Mucosal Ba2+ addition decreased resting Ga in a concentration-dependent manner (65% block at 5 mM) and decreased Ga(V) in a concentration- and voltage-dependent manner. Mucosal TEA+ addition also decreased control Ga (60% reduction at 5 mM). TEA+ block of Ga(V) was more potent and less voltage dependent that Ba2+ block. Maxi K+ channels were blocked by external Ba2+ at millimolar levels and by external TEA+ at submillimolar levels. At 0.3 mM, quinine (mucosal addition) hyperpolarized the cell membranes by 6 mV and reduced the fractional apical membrane resistance by 50%, suggesting activation of an apical membrane K+ conductance. At 1 mM, quinine both activated and blocked K(+)-conductive pathways. Quinine blocked maxi K+ channel currents at submillimolar concentrations. We conclude that 1) Ba2+ and TEA+ block maxi K+ channels and other K+ channels underlying resting Ga; 2) parallels between the Ba2+ and TEA+ sensitivities of Ga(V) and maxi K+ channels support a role for these channels in Ga(V); and 3) quinine has multiple effects on K(+)-conductive pathways in gallbladder epithelium, which are only partially explained by block of apical membrane maxi K+ channels.

A preparation has been developed that permits patch clamping of the basolateral membrane of Necturus gallbladder epithelial cells with a high success rate. The epithelium is separated from the underlying tissues mechanically, without enzymatic treatment. Its apical surface is attached to a plastic cover slip, and the basolateral surface, facing up, is cleaned with a suction pipette under microscopic observation. With this cleaning procedure, the success rate in obtaining gigaohm seals increases from less than 1% to approximately 10% of the attempts. The cells appear to retain their structural and functional integrity, as evidenced by electron-microscopic appearance and magnitude of cell membrane voltages. Major advantages of the preparation are that the basolateral membrane domain is preserved and that enzymatic treatment, which could potentially alter membrane proteins, is not necessary.

Aldosterone participates in the pathophysiology of several models of progressive chronic renal disease. Because of the causal connection between transforming growth factor-β1(TGF-β) and scarring in many such models, we hypothesized that aldosterone could evoke TGF-β in the kidney. Aldosterone infusion for 3 days in otherwise normal rats caused a more than twofold increase in TGF-β excretion without changes in systolic pressure or evidence of kidney damage. Concurrent treatment with amiloride did not alter this effect, indicating that aldosterone's stimulation of TGF-β was independent of its regulation of sodium or potassium transport. However, concurrent treatment with spironolactone did block the increase in TGF-β, indicating that the effect depends on the mineralocorticoid receptor. Renal mRNA for serum glucocorticoid kinase rose, but no change in TGF-β message occurred, suggesting posttranscriptional enhancement of renal TGF-β. In summary, aldosterone provokes renal TGF-β, and this action may contribute to aldosterone's fibrotic propensity.

Polycystin-1 (PC1), a 4,303-amino acid integral membrane protein of unknown function, interacts with polycystin-2 (PC2), a 968-amino acid alpha-type channel subunit. Mutations in their respective genes cause autosomal dominant polycystic kidney disease. Using a novel heterologous expression system and Ca(2+) and K(+) channels as functional biosensors, we found that full-length PC1 functioned as a constitutive activator of G(i/o)-type but not G(q)-type G-proteins and modulated the activity of Ca(2+) and K(+) channels via the release of Gbetagamma subunits. PC1 lacking the N-terminal 1811 residues replicated the effects of full-length PC1. These effects were independent of regulators of G-protein signaling proteins and were lost in PC1 mutants lacking a putative G-protein binding site. Co-expression with full-length PC2, but not a C-terminal truncation mutant, abrogated the effects of PC1. Our data provide the first experimental evidence that full-length PC1 acts as an untraditional G-protein-coupled receptor, activity of which is physically regulated by PC2. Thus, our study strongly suggests that mutations in PC1 or PC2 that distort the polycystin complex would initiate abnormal G-protein signaling in autosomal dominant polycystic kidney disease.

Importance Although vitamin E and memantine have been shown to have beneficial effects in moderately severe Alzheimer disease (AD), evidence is limited in mild to moderate AD. Objective To determine if vitamin E (alpha tocopherol), memantine, or both slow progression of mild to moderate AD in patients taking an acetylcholinesterase inhibitor. Design, Setting, and Participants Double-blind, placebo-controlled, parallel-group, randomized clinical trial involving 613 patients with mild to moderate AD initiated in August 2007 and concluded in September 2012 at 14 Veterans Affairs medical centers. Interventions Participants received either 2000 IU/d of alpha tocopherol (n = 152), 20 mg/d of memantine (n = 155), the combination (n = 154), or placebo (n = 152). Main Outcomes and Measures Alzheimer’s Disease Cooperative Study/Activities of Daily Living (ADCS-ADL) Inventory score (range, 0-78). Secondary outcomes included cognitive, neuropsychiatric, functional, and caregiver measures. Results Data from 561 participants were analyzed (alpha tocopherol = 140, memantine = 142, combination = 139, placebo = 140), with 52 excluded because of a lack of any follow-up data. Over the mean (SD) follow-up of 2.27 (1.22) years, ADCS-ADL Inventory scores declined by 3.15 units (95% CI, 0.92 to 5.39; adjusted P = .03) less in the alpha tocopherol group compared with the placebo group. In the memantine group, these scores declined 1.98 units less (95% CI, −0.24 to 4.20; adjusted P = .40) than the placebo group’s decline. This change in the alpha tocopherol group translates into a delay in clinical progression of 19% per year compared with placebo or a delay of approximately 6.2 months over the follow-up period. Caregiver time increased least in the alpha tocopherol group. All-cause mortality and safety analyses showed a difference only on the serious adverse event of “infections or infestations,” with greater frequencies in the memantine (31 events in 23 participants) and combination groups (44 events in 31 participants) compared with placebo (13 events in 11 participants). Conclusions and Relevance Among patients with mild to moderate AD, 2000 IU/d of alpha tocopherol compared with placebo resulted in slower functional decline. There were no significant differences in the groups receiving memantine alone or memantine plus alpha tocopherol. These findings suggest benefit of alpha tocopherol in mild to moderate AD by slowing functional decline and decreasing caregiver burden. Trial Registration clinicaltrials.gov Identifier:NCT00235716

Mutations in polycystins-1 and -2 (PC1 and PC2) cause autosomal dominant polycystic kidney disease (ADPKD), which is characterized by progressive development of epithelial renal cysts, ultimately leading to renal failure. The functions of these polycystins remain elusive. Here we show that PC2 is a Ca(2+)-permeable cation channel with properties distinct from any known intracellular channels. Its kinetic behavior is characterized by frequent transitions between closed and open states over a wide voltage range. The activity of the PC2 channel is transiently increased by elevating cytosolic Ca(2+). Given the predominant endoplasmic reticulum (ER) location of PC2 and its unresponsiveness to the known modulators of mediating Ca(2+) release from the ER, inositol-trisphosphate (IP(3)) and ryanodine, these results suggest that PC2 represents a novel type of channel with properties distinct from those of the other Ca(2+)-release channels. Our data also show that the PC2 channel can be translocated to the plasma membranes by defined chemical chaperones and proteasome modulators, suggesting that in vivo, it may also function in the plasma membrane under specific conditions. The sensitivity of the PC2 channel to changes of intracellular Ca(2+) concentration is deficient in a mutant found in ADPKD patients. The dysfunction of such mutants may result in defective coupling of PC2 to intracellular Ca(2+) homeostasis associated with the pathogenesis of ADPKD.

Polycystic kidney diseases are genetic disorders in which the renal parenchyma is progressively replaced by fluid-filled cysts1. Two members of the polycystin family (polycystin-1 and -2) are mutated in autosomal dominant polycystic kidney disease (ADPKD)2,3,4,5, and polycystin-L is deleted in mice with renal and retinal defects6. Polycystins are membrane proteins that share significant sequence homology6,7, especially polycystin-2 and -L (50% identity and 71% similarity). The functions of the polycystins remain unknown. Here we show that polycystin-L is a calcium-modulated nonselective cation channel that is permeable to sodium, potassium and calcium ions. Patch-clamp experiments revealed single-channel activity with a unitary conductance of 137 pS. Channel activity was substantially increased when either the extracellular or intracellular calcium-ion concentration was raised, indicating that polycystin-L may act as a transducer of calcium-mediated signalling in vivo. Its large single-channel conductance and regulation by calcium ions distinguish it from other structurally related cation channels.

Using the patch-clamp technique, we have identified large-conductance (maxi) K+ channels in the apical membrane of Necturus gallbladder epithelium, and in dissociated gallbladder epithelial cells. These channels are more than tenfold selective for K+ over Na+, and exhibit unitary conductance of approximately 200 pS in symmetric 100 mM KCl. They are activated by elevation of internal Ca2+ levels and membrane depolarization. The properties of these channels could account for the previously observed voltage and Ca2+ sensitivities of the macroscopic apical membrane conductance (Ga). Ga was determined as a function of apical membrane voltage, using intracellular microelectrode techniques. Its value was 180 microS/cm2 at the control membrane voltage of -68 mV, and increased steeply with membrane depolarization, reaching 650 microS/cm2 at -25 mV. We have related maxi K+ channel properties and Ga quantitatively, relying on the premise that at any apical membrane voltage Ga comprises a leakage conductance and a conductance due to maxi K+ channels. Comparison between Ga and maxi K+ channels reveals that the latter are present at a surface density of 0.09/microns 2, are open approximately 15% of the time under control conditions, and account for 17% of control Ga. Depolarizing the apical membrane voltage leads to a steep increase in channel steady-state open probability. When correlated with patch-clamp studies examining the Ca2+ and voltage dependencies of single maxi K+ channels, results from intracellular microelectrode experiments indicate that maxi K+ channel activity in situ is higher than predicted from the measured apical membrane voltage and estimated bulk cytosolic Ca2+ activity. Mechanisms that could account for this finding are proposed.

Publisher Summary This chapter discusses various techniques used for the study of ion and water transport mechanisms in isolated Necturus gall bladder epithelium .With appropriate modifications, these methods can be applied to other epithelia as well as to non-epithelial preparations. The emphasis is on techniques, but this chapter also has attempted to provide orientation for the non-specialist on the rationale of the experimental approaches utilizing these methods. This chapter also illustrates the application of each technique in identifying and characterizing mechanisms of ion and water transport across individual cell membranes, and their regulation. Compared to other methods, electrophysiological techniques have major advantages in both accuracy and time resolution, and permit measurements in a nondestructive fashion. Conventional intracellular microelectrodes are used in epithelial transport studies to measure cell membrane voltages, electrical resistances, and ion selectivities. This chapter concludes with the brief discussion on electrophysiologic apparatus.

Diffuse leiomyomatosis is associated with the inherited kidney disease Alport syndrome, and characterized by visceral smooth muscle overgrowth within the respiratory, gastrointestinal and female reproductive tracts. Although partial deletions of the type IV collagen genes COL4A5 and COL4A6, paired head-to-head on chromosome Xq22, are known to cause diffuse leiomyomatosis, loss of function for type IV collagen does not explain smooth muscle overgrowth. To further clarify pathogenic mechanisms, we have characterized novel deletions in patients with Alport syndrome-diffuse leiomyomatosis or Alport syndrome alone. A 27.6-kb deletion, in a female with Alport syndrome-diffuse leiomyomatosis, is marked by the most proximal, i.e. most 5', COL4A5 breakpoint described to date. By comparing this deletion to others described here and previously, we have defined a minimal overlap region, only 4.2 kb in length and containing the COL4A5-COL4A6 proximal promoters, loss of which contributes to smooth muscle overgrowth. A novel deletion in a male with Alport syndrome alone is>1.4 Mb in length, encompassing COL4A5 and COL4A6 entirely, as well as neighboring genes. We postulate that loss of the 4.2-kb region in diffuse leiomyomatosis causes misregulation of neighboring genes, contributing to smooth muscle overgrowth. Deletion of the neighboring genes themselves may afford protection from this condition. © 2003 Wiley-Liss, Inc.