Different laboratories recently reported incongruous results describing the quantification of albumin filtration using two-photon microscopy. We investigated the factors that influence the glomerular sieving coefficient for albumin (GSC A ) in an effort to explain these discordant reports and to develop standard operating procedures for determining GSC A . Multiple factors influenced GSC A , including the kidney depth of image acquisition (10-20 mm was appropriate), the selection of fluorophore (probes emitting longer wavelengths were superior), the selection of plasma regions for fluorescence measurements, the size and molecular dispersion characteristics of dextran polymers if used, dietary status, and the genetic strain of rat. Fasting reduced the GSC A in Simonsen Munich Wistar rats from 0.03560.005 to 0.01660.004 (P,0.01). Frömter Munich Wistar rats had a much lower GSC A in both the fed and the fasted states. Finally, we documented extensive albumin transcytosis with vesicular and tubular delivery to and fusion with the basolateral membrane in S1 proximal tubule cells. In summary, these results help explain the previously conflicting microscopy and micropuncture data describing albumin filtration and highlight the dynamic nature of glomerular albumin permeability. Over the past several years, the roles the glomerular filtration barrier and the proximal tubule play in the development of albuminuria have been debated. 1,2 The present textbook model, in which albumin filtration across a normal glomerulus is thought to be minimal, has recently been challenged. A wide array of genetic, molecular, biochemical, and imaging studies are consistent with proximal tubule cells (PTCs) having a role in reclaiming filtered proteins, including albumin, and thus minimizing proteinuria. These studies include the following: knockout mice lacking Na+-H+ exchanger isoform 3 or chloride channel-5; 3 megalin-cubilin complex defects; 4 Rab 38-mediated tubular proteinuria and albuminuria; 5 statin-mediated inhibition of guanosine triphosphatase prenylation with reduced proximal tubule (PT) endocytosis; 4,6-8 mice lacking FcRn, the IgG and albumin receptor; 9,10 and selective PTC injury using D-serine 11 or the selective expression of diphtheria toxin receptor on PTC. 12 In a preliminary communication, Menzel and colleagues showed that PT reabsorption and transcytosis of podocyte produced and secreted albumin labeled with V5 and hemagglutinin tags. 13 Finally, recently published data 14 using advanced scanning electron microscope imaging techniques indicate that the podocyte slit diaphragm has pores that are much larger than previously determined and are of the size required for albumin filtration.The development of in vivo two-photon microscopy has enabled the direct visualization and quantitation of fluorescent compounds as they filter through the glomerulus and are endocytosed by

Mutations in 11β-hydroxysteroid dehydrogenase type 2 gene () cause an extraordinarily rare autosomal recessive disorder, apparent mineralocorticoid excess (AME). AME is a form of low renin hypertension that is potentially fatal if untreated. Mutations in the gene result either in severe AME or a milder phenotype (type 2 AME). To date, ∼40 causative mutations have been identified. As part of the International Consortium for Rare Steroid Disorders, we have diagnosed and followed the largest single worldwide cohort of 36 AME patients. Here, we present the genotype and clinical phenotype of these patients, prominently from consanguineous marriages in the Middle East, who display profound hypertension and hypokalemic alkalosis. To correlate mutations with phenotypic severity, we constructed a computational model of the HSD11B2 protein. Having used a similar strategy for the in silico evaluation of 150 mutations of, the disease-causing gene in congenital adrenal hyperplasia, we now provide a full structural explanation for the clinical severity of AME resulting from each known missense mutation. We find that mutations that allow the formation of an inactive dimer, alter substrate/coenzyme binding, or impair structural stability of HSD11B2 yield severe AME. In contrast, mutations that cause an indirect disruption of substrate binding or mildly alter intramolecular interactions result in type 2 AME. A simple in silico evaluation of novel missense mutations could help predict the often-diverse phenotypes of an extremely rare monogenic disorder.

Stretch activation (SA) is a fundamental property of all muscle types that increases power output and efficiency, yet its mechanism is unknown. Recently, studies have implicated troponin isoforms as important in the SA mechanism. The highly stretch-activated Drosophila IFMs express two isoforms of the Ca2+-binding subunit of troponin (TnC). TnC1 (TnC-F2 in Lethocerus IFM) has two calcium binding sites, while an unusual isoform, TnC4 (TnC-F1 in Lethocerus IFM), has only one binding site. We investigated the roles of these two TnC isoforms in Drosophila IFM by targeting RNAi to each isoform. IFMs with TnC4 expression (normally ~90 % of total TnC) replaced by TnC1 did not generate isometric tension, power or display SA. However, TnC4 knockdown resulted in sarcomere ultrastructure disarray, which could explain the lack of mechanical function and thus make interpretation of the influence of TnC4 on SA difficult. Elimination of TnC1 expression (normally ~10 % of total TnC) by RNAi resulted in normal muscle structure. In these IFMs, fiber power generation, isometric tension, stretch-activated force and calcium sensitivity were statistically identical to wild type. When TnC1 RNAi was driven by an IFM specific driver, there was no decrease in flight ability or wing beat frequency, which supports our mechanical findings suggesting that TnC1 is not essential for the mechanical function of Drosophila IFM. This finding contrasts with previous work in Lethocerus IFM showing TnC1 is essential for maximum isometric force generation. We propose that differences in TnC1 function in Lethocerus and Drosophila contribute to the ~40-fold difference in IFM isometric tension generated between these species.

The cornea's acoustic properties (speed-of-sound, backscatter, attenuation) are related to its state of hydration. Our aim was to determine these properties as a function of corneal hydration using high frequency ultrasound. Bovine corneas were suspended in a Dexsol-equivalent corneal preservation medium at 33°C and then immersed successively in 75%, 50%, 25% medium and distilled water. Using a 38-MHz focused ultrasound transducer, we measured speed-of-sound and corneal thickness (n=8) and stromal backscatter (n=6) after 45-minutes immersion in each medium. Corneal speed-ofsound was modeled as a function of corneal thickness. We found the mean speed-of-sound to be 1605.4±2.9 m/s in normotensive medium. The maximum observed speed-of-sound was 1616 m/s. As we decreased medium tonicity, the cornea swelled and the speed-of-sound decreased, reaching 1563.0±2.2 m/s in water. Average corneal thickness increased from 969±93 μm in 100% medium to 1579±104 μm in water. Going from 100% medium to water, stromal backscatter (midband-fit) increased from -60.0±0.8 dBr to -52.5±3.5 dBr, spectral slope increased from -0.119±0.021 to -0.005 ±0.030 dB/MHz and attenuation coefficient decreased from 0.927±0.434 to 0.010±0.581 dB/cmMHz. The observed correlation between acoustic backscatter and attenuation with the speed-ofsound offers a potential means for more accurate determination of speed-of-sound, and hence thickness, in edematous corneas.