Experimental long-term diabetes mellitus alters the transcriptome and biomechanical properties of the rat urinary bladder.

Diabetes mellitus (DM) is the leading cause of chronic kidney disease and diabetic nephropathy is widely studied. In contrast, the pathobiology of diabetic urinary bladder disease is less understood despite dysfunctional voiding being common in DM. We hypothesised that diabetic cystopathy has a characteristic molecular signature. We therefore studied bladders of hyperglycaemic and polyuric rats with streptozotocin (STZ)-induced DM. Sixteen weeks after induction of DM, as assessed by RNA arrays, wide-ranging changes of gene expression occurred in DM bladders over and above those induced in bladders of non-hyperglycaemic rats with sucrose-induced polyuria. The altered transcripts included those coding for extracellular matrix regulators and neural molecules. Changes in key genes deregulated in DM rat bladders were also detected in db/db mouse bladders. In DM rat bladders there was reduced birefringent collagen between detrusor muscle bundles, and atomic force microscopy showed a significant reduction in tissue stiffness; neither change was found in bladders of sucrose-treated rats. Thus, altered extracellular matrix with reduced tissue rigidity may contribute to voiding dysfunction in people with long-term DM. These results serve as an informative stepping stone towards understanding the complex pathobiology of diabetic cystopathy.

In vivo three-dimensional photoacoustic imaging of the renal vasculature in preclinical rodent models.

Noninvasive imaging of the kidney vasculature in preclinical murine models is important for the assessment of renal development, studying diseases and evaluating new therapies but is challenging to achieve using existing imaging modalities. Photoacoustic imaging is a promising new technique that is particularly well suited to visualizing the vasculature and could provide an alternative to existing preclinical imaging methods for studying renal vascular anatomy and function. To investigate this, an all-optical Fabry-Perot-based photoacoustic scanner was used to image the abdominal region of mice. High-resolution three-dimensional, noninvasive, label-free photoacoustic images of the mouse kidney and renal vasculature were acquired in vivo. The scanner was also used to visualize and quantify differences in the vascular architecture of the kidney in vivo due to polycystic kidney disease. This study suggests that photoacoustic imaging could be utilized as a novel preclinical imaging tool for studying the biology of renal disease.

Vascular Endothelial Growth Factor C for Polycystic Kidney Diseases.

Polycystic kidney diseases (PKD) are genetic disorders characterized by progressive epithelial cyst growth leading to destruction of normally functioning renal tissue. Current therapies have focused on the cyst epithelium, and little is known about how the blood and lymphatic microvasculature modulates cystogenesis. Hypomorphic Pkd1(nl/nl) mice were examined, showing that cystogenesis was associated with a disorganized pericystic network of vessels expressing platelet/endothelial cell adhesion molecule 1 and vascular endothelial growth factor receptor 3 (VEGFR3). The major ligand for VEGFR3 is VEGFC, and there were lower levels of Vegfc mRNA within the kidneys during the early stages of cystogenesis in 7-day-old Pkd1(nl/nl) mice. Seven-day-old mice were treated with exogenous VEGFC for 2 weeks on the premise that this would remodel both the VEGFR3(+) pericystic vascular network and larger renal lymphatics that may also affect the severity of PKD. Treatment with VEGFC enhanced VEGFR3 phosphorylation in the kidney, normalized the pattern of the pericystic network of vessels, and widened the large lymphatics in Pkd1(nl/nl) mice. These effects were associated with significant reductions in cystic disease, BUN and serum creatinine levels. Furthermore, VEGFC administration reduced M2 macrophage pericystic infiltrate, which has been implicated in the progression of PKD. VEGFC administration also improved cystic disease in Cys1(cpk/cpk) mice, a model of autosomal recessive PKD, leading to a modest but significant increase in lifespan. Overall, this study highlights VEGFC as a potential new treatment for some aspects of PKD, with the possibility for synergy with current epithelially targeted approaches.

Genetic Background is a Key Determinant of Glomerular Extracellular Matrix Composition and Organization.

Glomerular disease often features altered histologic patterns of extracellular matrix (ECM). Despite this, the potential complexities of the glomerular ECM in both health and disease are poorly understood. To explore whether genetic background and sex determine glomerular ECM composition, we investigated two mouse strains, FVB and B6, using RNA microarrays of isolated glomeruli combined with proteomic glomerular ECM analyses. These studies, undertaken in healthy young adult animals, revealed unique strain- and sex-dependent glomerular ECM signatures, which correlated with variations in levels of albuminuria and known predisposition to progressive nephropathy. Among the variation, we observed changes in netrin 4, fibroblast growth factor 2, tenascin C, collagen 1, meprin 1-α, and meprin 1-ß. Differences in protein abundance were validated by quantitative immunohistochemistry and Western blot analysis, and the collective differences were not explained by mutations in known ECM or glomerular disease genes. Within the distinct signatures, we discovered a core set of structural ECM proteins that form multiple protein-protein interactions and are conserved from mouse to man. Furthermore, we found striking ultrastructural changes in glomerular basement membranes in FVB mice. Pathway analysis of merged transcriptomic and proteomic datasets identified potential ECM regulatory pathways involving inhibition of matrix metalloproteases, liver X receptor/retinoid X receptor, nuclear factor erythroid 2-related factor 2, notch, and cyclin-dependent kinase 5. These pathways may therefore alter ECM and confer susceptibility to disease.

Application of a hemolysis assay for analysis of complement activation by perfluorocarbon nanoparticles.

Nanoparticles offer new options for medical diagnosis and therapeutics with their capacity to specifically target cells and tissues with imaging agents and/or drug payloads. The unique physical aspects of nanoparticles present new challenges for this promising technology. Studies indicate that nanoparticles often elicit moderate to severe complement activation. Using human in vitro assays that corroborated the mouse in vivo results we previously presented mechanistic studies that define the pathway and key components involved in modulating complement interactions with several gadolinium-functionalized perfluorocarbon nanoparticles (PFOB). Here we employ a modified in vitro hemolysis-based assay developed in conjunction with the mouse in vivo model to broaden our analysis to include PFOBs of varying size, charge and surface chemistry and examine the variations in nanoparticle-mediated complement activity between individuals. This approach may provide the tools for an in-depth structure-activity relationship study that will guide the eventual development of biocompatible nanoparticles. FROM THE CLINICAL EDITOR: Unique physical aspects of nanoparticles may lead to moderate to severe complement activation in vivo, which represents a challenge to clinical applicability. In order to guide the eventual development of biocompatible nanoparticles, this team of authors report a modified in vitro hemolysis-based assay developed in conjunction with their previously presented mouse model to enable in-depth structure-activity relationship studies.

Targeted glomerular angiopoietin-1 therapy for early diabetic kidney disease.

Vascular growth factors play an important role in maintaining the structure and integrity of the glomerular filtration barrier. In healthy adult glomeruli, the proendothelial survival factors vascular endothelial growth factor-A (VEGF-A) and angiopoietin-1 are constitutively expressed in glomerular podocyte epithelia. We demonstrate that this milieu of vascular growth factors is altered in streptozotocin-induced type 1 diabetic mice, with decreased angiopoietin-1 levels, VEGF-A upregulation, decreased soluble VEGF receptor-1 (VEGFR1), and increased VEGFR2 phosphorylation. This was accompanied by marked albuminuria, nephromegaly, hyperfiltration, glomerular ultrastructural alterations, and aberrant angiogenesis. We subsequently hypothesized that restoration of angiopoietin-1 expression within glomeruli might ameliorate manifestations of early diabetic glomerulopathy. Podocyte-specific inducible repletion of angiopoietin-1 in diabetic mice caused a 70% reduction of albuminuria and prevented diabetes-induced glomerular endothelial cell proliferation; hyperfiltration and renal morphology were unchanged. Furthermore, angiopoietin-1 repletion in diabetic mice increased Tie-2 phosphorylation, elevated soluble VEGFR1, and was paralleled by a decrease in VEGFR2 phosphorylation and increased endothelial nitric oxide synthase Ser(1177) phosphorylation. Diabetes-induced nephrin phosphorylation was also reduced in mice with angiopoietin-1 repletion. In conclusion, targeted angiopoietin-1 therapy shows promise as a renoprotective tool in the early stages of diabetic kidney disease.

Albuminuria is associated with too few glomeruli and too much testosterone.

Normally, the glomerular filtration barrier almost completely excludes circulating albumin from entering the urine. Genetic variation and both pre- and postnatal environmental factors may affect albuminuria in humans. Here we determine whether glomerular gene expression in mouse strains with naturally occurring variations in albuminuria would allow identification of proteins deregulated in relatively 'leaky' glomeruli. Albuminuria increased in female B6 to male B6 to female FVB/N to male FVB/N mice, whereas the number of glomeruli/kidney was the exact opposite. Testosterone administration led to increased albuminuria in female B6 but not female FVB/N mice. A common set of 39 genes, many expressed in podocytes, were significantly differentially expressed in each of the four comparisons: male versus female B6 mice, male versus female FVB/N mice, male FVB/N versus male B6 mice, and female FVB/N versus female B6 mice. The transcripts encoded proteins involved in oxidation/reduction reactions, ion transport, and enzymes involved in detoxification. These proteins may represent novel biomarkers and even therapeutic targets for early kidney and cardiovascular disease.

Angiogenesis and autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the growth of multiple cysts that in many cases result in end-stage renal disease. Current strategies to reduce cyst progression in ADPKD focus on modulating cell turnover, fluid secretion, and vasopressin signalling; but an alternative approach may be to target pathways providing "general support" for cyst growth, such as surrounding blood vessels. This could be achieved by altering the expression of growth factors involved in vascular network formation, such as the vascular endothelial growth factor (VEGF) and angiopoietin families. We highlight the evidence that blood vessels and vascular growth factors play a role in ADPKD progression. Recent experiments manipulating VEGF in ADPKD are described, and we discuss how alternative strategies to manipulate angiogenesis may be used in the future as a novel treatment for ADPKD.

Variable antibody-dependent activation of complement by functionalized phospholipid nanoparticle surfaces.

A wide variety of nanomaterials are currently being developed for use in the detection and treatment of human diseases. However, there is no systematic way to measure and predict the action of such materials in biological contexts. Lipid-encapsulated nanoparticles (NPs) are a class of nanomaterials that includes the liposomes, the most widely used and clinically proven type of NPs. Liposomes can, however, activate the complement system, an important branch of innate immunity, resulting in undesirable consequences. Here, we describe the complement response to lipid-encapsulated NPs that are functionalized on the surface with various lipid-anchored gadolinium chelates. We developed a quantitative approach to examine the interaction of NPs with the complement system using in vitro assays and correlating these results with those obtained in an in vivo mouse model. Our results indicate that surface functionalization of NPs with certain chemical structures elicits swift complement activation that is initiated by a natural IgM antibody and propagated via the classical pathway. The intensity of the response is dependent on the chemical structures of the lipid-anchored chelates and not zeta potential effects alone. Moreover, the extent of complement activation may be tempered by complement inhibiting regulatory proteins that bind to the surface of NPs. These findings represent a step forward in the understanding of the interactions between nanomaterials and the host innate immune response and provide the basis for a systematic structure-activity relationship study to establish guidelines that are critical to the future development of biocompatible nanotherapeutics.