Carrie L. Phillips

β1 integrin expression by podocytes is required to maintain glomerular structural integrity

Integrins are transmembrane heteromeric receptors that mediate interactions between cells and extracellular matrix (ECM). beta1, the most abundantly expressed integrin subunit, binds at least 12 alpha subunits. beta1 containing integrins are highly expressed in the glomerulus of the kidney; however their role in glomerular morphogenesis and maintenance of glomerular filtration barrier integrity is poorly understood. To study these questions we selectively deleted beta1 integrin in the podocyte by crossing beta1(flox/flox) mice with podocyte specific podocin-cre mice (pod-Cre), which express cre at the time of glomerular capillary formation. We demonstrate that podocyte abnormalities are visualized during glomerulogenesis of the pod-Cre;beta1(flox/flox) mice and proteinuria is present at birth, despite a grossly normal glomerular basement membrane. Following the advent of glomerular filtration there is progressive podocyte loss and the mice develop capillary loop and mesangium degeneration with little evidence of glomerulosclerosis. By 3 weeks of age the mice develop severe end stage renal failure characterized by both tubulointerstitial and glomerular pathology. Thus, expression of beta1 containing integrins by the podocyte is critical for maintaining the structural integrity of the glomerulus.

Guiding Principles of Specimen Preservation for Confocal Fluorescence Microscopy

Traditionally, biologists have been confined to transmission electron microscopy (TEM) and light microscopy (LM) in order to correlate biochemical and molecular data with morphology. Electron microscopy (EM) provides fine ultrastructural detail but is limited to the study of cellular structures that react with electron dense stains deposited in fixed specimens. Immunogold labeling permits the study of non–electron-dense material, but EM sections must still be very thin to avoid problems with the penetration of the labeled antibodies and to reduce scattering of the electron beam.

Soluble Thrombomodulin Protects Ischemic Kidneys

Altered coagulation and inflammation contribute to the pathogenesis of ischemic renal injury. Thrombomodulin is a necessary factor in the anticoagulant protein C pathway and has inherent anti-inflammatory properties. We studied the effect of soluble thrombomodulin (sTM) in a hypoperfusion model of ischemic kidney injury. To markedly reduce infrarenal aortic blood flow and femoral arterial pressures, we clamped the suprarenal aorta of rats, occluding them 90%, for 60 min. Reversible acute kidney injury (AKI) occurred at 24 h in rats subjected to hypoperfusion. Histologic analysis at 24 h revealed acute tubular necrosis (ATN), and intravital two-photon microscopy showed flow abnormalities in the microvasculature and defects of endothelial permeability. Pretreatment with rat sTM markedly reduced both I-R-induced renal dysfunction and tubular histologic injury scores. sTM also significantly improved microvascular erythrocyte flow rates, reduced microvascular endothelial leukocyte rolling and attachment, and minimized endothelial permeability to infused fluorescence dextrans, assessed by intravital quantitative multiphoton microscopy. Furthermore, sTM administered 2 h after reperfusion protected against ischemia-induced renal dysfunction at 24 h and improved survival. By using an sTM variant, we also determined that the protective effects of sTM were independent of its ability to generate activated protein C. These data suggest that sTM may have therapeutic potential for ischemic AKI.

Renal Cysts of inv/inv Mice Resemble Early Infantile Nephronophthisis

Cystic kidney disease has been linked to mutations in the Invs gene in mice with inversion of embryonic turning (inv/inv) and the INVS (NPHP2) gene in infants with nephronophthisis type 2 (NPHP2). The inv mouse model features multiorgan defects including renal cysts, altered left-right laterality, and hepatobiliary duct malformations transmitted in an autosomal recessive manner. Affected mice usually die of renal and liver failure by postnatal day 7. Although cardiopulmonary and liver anomalies have been carefully detailed, renal cysts have yet to be fully characterized in inv/inv. By use of three-dimensional visualization by two-photon microscopy, this study provides the first comprehensive analysis of in situ cyst formation and progression in inv/inv kidneys. At embryonic day 15, there is dilatation of Bowmans capsule followed temporally by corticomedullary cysts involving collecting ducts, proximal tubules, and thick ascending limbs. Collecting ducts of newborn inv/inv mice are uniformly and diffusely cystic from medulla to cortex, with normal diameters found only at their most proximal tips. Proximal tubules form fusiform cysts that alternate with segments of normal or narrowed caliber along torturous convolutions. Because defective cilia have been linked to situs inversus and cystogenesis, we examined inv/inv cilia by scanning and transmission electron microscopy. The former detected monocilia of expected length in cystic collecting ducts and proximal tubules; the latter demonstrated the usual 9 + 2 pattern in respiratory cilia. The inv mutant mouse has renal cysts resembling infantile NPHP2 and will provide broader insight into the role cilia play in renal cystogenesis.

Calorie Restriction Modulates Renal Expression of Sterol Regulatory Element Binding Proteins, Lipid Accumulation, and Age-Related Renal Disease

Sterol regulatory element binding proteins (SREBP) are major regulators of fatty acid and cholesterol synthesis. This study found that age-related renal matrix deposition and proteinuria were associated with increased renal expression of SREBP-1 and SREBP-2 and increased renal accumulation of triglyceride and cholesterol. Because calorie restriction (CR) modulates age-related renal disease, it then was determined whether the effects of CR are mediated partially by modulation of renal lipid metabolism. Compared with ad libitum (AL)-fed 24-month-old (24 m) F344BN rats, CR resulted in significant decreases in extracellular matrix accumulation (periodic acid-Schiff staining and immunofluorescence of type IV collagen and fibronectin) and proteinuria. A significant decrease was also observed in the renal expression of growth factors (connective tissue growth factor and vascular endothelial growth factor) and matrix metalloproteinase inhibitor (plasminogen activator inhibitor-1). These structural and functional changes were associated with significant decreases in renal nuclear SREBP-1 (5.2 in 24 m AL versus 3.3 densitometry units in 24 m CR; P < 0.01) and SREBP-2 (7.1 in 24 m AL versus 4.1 densitometry units in 24 m CR; P < 0.01) protein abundance and renal triglyceride and cholesterol contents. It is interesting that serum leptin level was significantly increased as a function of aging, and CR resulted in significant reduction in serum leptin level. Because it was shown previously that increased renal expression of SREBP-1a per se caused renal lipid accumulation, glomerulosclerosis, and proteinuria, the results suggest that CR modulates age-related renal disease in part by modulation of renal SREBP expression and renal lipid accumulation.

Expression of Cytolethal Distending Toxin and Hemolysin Is Not Required for Pustule Formation by Haemophilus ducreyi in Human Volunteers

ABSTRACT Haemophilus ducreyi makes cytolethal distending toxin (CDT) and hemolysin. In a previous human challenge trial, an isogenic hemolysin-deficient mutant caused pustules with a rate similar to that of its parent. To test whether CDT was required for pustule formation, six human subjects were inoculated with a CDT mutant and parent at multiple sites. The pustule formation rates were similar at both parent and mutant sites. A CDT and hemolysin double mutant was constructed and tested in five additional subjects. The pustule formation rates were similar for the parent and double mutant. These results indicate that neither the expression of CDT, nor that of hemolysin, nor both are required for pustule formation by H. ducreyi in humans.

Three-dimensional imaging of embryonic mouse kidney by two-photon microscopy

Developing mammalian embryonic kidney becomes progressively more elaborate as the ureteric bud branches into undifferentiated mesenchyme. Morphological perturbations of nephrogenesis, such as those seen in inherited renal diseases or induced in transgenic animals, require careful and often tedious documentation by multiple methodologies. We have applied a relatively quick and simple approach combining two-photon microscopy and advanced three-dimensional (3-D) imaging techniques to visualize and evaluate these complex events. As compared with laser confocal microscopy, two-photon microscopy offers superior optical sectioning deep into biological tissues, permitting analysis of large, heterogeneous, 3-D structures such as developing mouse kidney. Embryonic and newborn mouse kidneys were fluorescently labeled with lectins, phalloidin, or antibody. Three-dimensional image volumes were then collected. The resulting volume data sets were processed using a novel 3-D visualization technique. Reconstructed image volumes demonstrate the dichotomous branching of ureteric bud as it progresses from a simple, symmetrical structure into an elaborate, asymmetrical collecting system of multiple branches. Detailed morphology of in situ cysts was elucidated in a transgene-induced mouse model of polycystic kidney disease. We expect this integration of two-photon microscopy with advanced 3-D image analysis will provide a powerful tool for illuminating a variety of complex developmental processes in multiple dimensions.

Three‐dimensional imaging of human skin and mucosa by two‐photon laser scanning microscopy

Background: Various structural components of human skin biopsy specimens are difficult to visualize using conventional histologic approaches.

Acute tubulointerstitial nephritis attributable to indinavir therapy.

Indinavir sulfate has been reported to cause asymptomatic crystalluria and nephrolithiasis in patients with human immunodeficiency virus (HIV) infection. Patients taking indinavir may present with asymptomatic crystalluria, nephrolithiasis with frank renal colic and obstruction, flank pain in the absence of nephrolithiasis, and dysuria or urgency. Asymptomatic crystalluria has been described as benign. Discontinuation of the drug has not been recommended in the absence of nephrolithiasis. We report two HIV-positive patients receiving indinavir who developed acute interstitial nephritis with foreign body giant cell reaction on renal biopsies. Both patients had asymptomatic crystalluria, although crystals were associated with clumps of white blood cells (WBCs) on urinalysis in one patient. Both cases show that the inflammatory response was significant enough to lead to tubular injury and acute renal impairment. Our findings suggest that asymptomatic crystalluria attributable to indinavir may illicit an inflammatory response with acute renal insufficiency, warranting monitoring of renal function, especially in patients with crystalluria.

A Non-Nephrotoxic Gentamicin Congener That Retains Antimicrobial Efficacy

Aminoglycoside antibiotics, although of major clinical importance in the treatment of serious Gram- negative infections and a potential therapeutic agent in the amelioration of diseases that are characterized by premature stop mutations, are associated with a high incidence of acute renal failure. With the use of HPLC techniques, the four components (congeners) of gentamicin, the most commonly used aminoglycoside, were isolated and characterized. Described here is a congener with minimal cytotoxicity in cell culture and animal studies that retained normal bactericidal properties in both Bacillus subtilis and a multidrug-resistant form of Klebsiella pneumoniae. Furthermore, in animal studies, this congener failed to induce the functional and pathologic changes that are characteristic of gentamicin nephrotoxicity that is seen with the native compound. Finally, internalization of this non-nephrotoxic component was unaltered, but the subcellular distribution was different from native gentamicin or the other three cytotoxic congeners. These studies have identified a component of the native gentamicin congener mixture that retains its bactericidal properties with minimal or no apparent nephrotoxicity.