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

Glomerular injury is often characterized by the effacement of podocytes, loss of slit diaphragms, and proteinuria. Renal ischemia or the loss of blood flow to the kidneys has been widely associated with tubular and endothelial injury but rarely has been shown to induce podocyte damage and disruption of the slit diaphragm. In this study, we have used an in vivo rat ischemic model to demonstrate that renal ischemia induces podocyte effacement with loss of slit diaphragm and proteinuria. Biochemical analysis of the ischemic glomerulus shows that ischemia induces rapid loss of interaction between slit diaphragm junctional proteins Neph1 and ZO-1. To further understand the effect of ischemia on molecular interactions between slit diaphragm proteins, a cell culture model was employed to study the binding between Neph1 and ZO-1. Under physiologic conditions, Neph1 co-localized with ZO-1 at cell-cell contacts in cultured human podocytes. Induction of injury by ATP depletion resulted in rapid loss of Neph1 and ZO-1 binding and redistribution of Neph1 and ZO-1 proteins from cell membrane to the cytoplasm. Recovery resulted in increased Neph1 tyrosine phosphorylation, restoring Neph1 and ZO-1 binding and their localization at the cell membrane. We further demonstrate that tyrosine phosphorylation of Neph1 mediated by Fyn results in significantly increased Neph1 and ZO-1 binding, suggesting a critical role for Neph1 tyrosine phosphorylation in reorganizing the Neph1-ZO-1 complex. This study documents that renal ischemia induces dynamic changes in the molecular interactions between slit diaphragm proteins, leading to podocyte damage and proteinuria.

Evidence from multiple studies supports the concept that both glomerular filtration and proximal tubule (PT) reclamation affect urinary albumin excretion rate. To better understand these roles of glomerular filtration and PT uptake, we investigated these processes in two distinct animal models. In a rat model of acute exogenous albumin overload, we quantified glomerular sieving coefficients (GSC) and PT uptake of Texas Red-labeled rat serum albumin using two-photon intravital microscopy. No change in GSC was observed, but a significant decrease in PT albumin uptake was quantified. In a second model, loss of endogenous albumin was induced in rats by podocyte-specific transgenic expression of diphtheria toxin receptor. In these albumin-deficient rats, exposure to diphtheria toxin induced an increase in albumin GSC and albumin filtration, resulting in increased exposure of the PTs to endogenous albumin. In this case, PT albumin reabsorption was markedly increased. Analysis of known albumin receptors and assessment of cortical protein expression in the albumin overload model, conducted to identify potential proteins and pathways affected by acute protein overload, revealed changes in the expression levels of calreticulin, disabled homolog 2, NRF2, angiopoietin-2, and proteins involved in ATP synthesis. Taken together, these results suggest that a regulated PT cell albumin uptake system can respond rapidly to different physiologic conditions to minimize alterations in serum albumin level. 27: 482-494, 201627: 482-494, . doi: 10.1681 While the clinical relevance of proteinuria, and especially albuminuria, has been well documented, the quantitative and mechanistic significance of different components to albumin excretion remains an area of considerable excitement and debate. 1 Recent data from several laboratories utilizing different approaches have delineated a role of proximal tubules (PTs) in regulation of albumin reabsorption and reclamation. 2 Furthermore, albumin transcytosis has been visualized in PT cells 3 and FcRn has been shown to mediate the transcytosis of albumin across PTCs, 4 as it is known to do for many other cell types. 2 Therefore, the present studies were conducted to begin differentiating and quantifying glomerular and PT contributions to albuminuria under physiologic and disease conditions. This understanding is important because before appropriate therapies can be developed to modulate albuminuria, the structural, functional and mechanistic characterization need to be better understood and interrelated. J Am Soc Nephrol

Acute ischemic kidney injury results in marked increases in local and systemic cytokine levels. IL-1alpha, IL-6, and TNF-alpha orchestrate various inflammatory reactions influencing endothelial permeability by altering cell-to-cell and cell-to-extracellular matrix attachments. To explore the role of actin and the regulatory proteins RhoA and cofilin in this process, microvascular endothelial cells (MS1) were exposed to individual cytokines or a cytokine cocktail. Within minutes, a marked, time-dependent redistribution of the actin cytoskeleton occurred with the formation of long, dense F-actin basal stress fibers. The concentration of F-actin, normalized to nuclear staining, significantly increased compared with untreated cells (up 20%, P < or = 0.05). Western blot analysis of MS1 lysates incubated with the cytokine cocktail for 4 h showed an increase in phosphorylated/inactive cofilin (up 25 +/- 15%, P < or = 0.05) and RhoA activation (up to 227 +/- 26% increase, P < or = 0.05) compared with untreated cells. Decreasing RhoA levels using small interfering RNA blocked the effect of cytokines on stress fiber organization. Treatment with Y-27632, an inhibitor of the RhoA effector p160-ROCK, decreased levels of phosphorylated cofilin and reduced stress fiber fluorescence by 22%. In cells treated with Y-27632 followed by treatment with the cytokine cocktail, stress fiber levels were similar to control cells and cofilin phosphorylation was 55% of control levels. Taken together, these studies demonstrate cytokine stimulation of RhoA, which in turn leads to cofilin phosphorylation and formation of numerous basal actin stress fibers. These results suggest cytokines signal through the Rho-ROCK pathway, but also through another pathway to affect actin dynamics.