Two-photon microscopy reveals stationary podocytes in living zebrafish larvae.

Podocytes are an essential component of the glomerular filtration barrier and cover the outer aspect of glomerular capillaries. They form a complex actin-based cytoskeleton in vivo and show prominent motility in vitro, but whether podocytes are stationary or mobile in vivo is debated. To address this question, the pronephros of translucent zebrafish larvae (casper) expressing enhanced green fluorescent protein (eGFP) specifically in podocytes (wt1a:eGFP larvae) was observed by intravital two-photon microscopy over extended periods of time. Podocyte cell bodies and the interdigitating branching pattern of major processes could be resolved with a resolution of approximately 1 µm in the xy-plane. Time-lapse imaging of zebrafish larvae at 5-7 days after fertilization demonstrated that podocytes neither migrated nor changed the branching pattern of their major processes over a time period of up to 23 hours. In summary, we show by extended intravital two-photon microscopy that podocytes are stationary cells in the intact glomerulus of a translucent zebrafish with fluorescently-labeled podocytes.

OPN deficiency results in severe glomerulosclerosis in uninephrectomized mice.

Osteopontin (OPN) expression has been reported to be elevated in experimental models of renal injury such as arterial hypertension or diabetic nephropathy finally leading to focal segmental glomerulosclerosis (FSGS). FSGS is characterized by glomerular matrix deposition and loss or damage of podocytes that represent the main constituents of the glomerular filtration barrier. To evaluate the role of OPN in the kidney we investigated WT and OPN knockout mice (OPN-/-) without treatment, after uninephrectomy (UNX), as well as after UNX and desoxycorticosterone acetate (DOCA)-salt treatment with respect to urine parameters, glomerular morphology, and expression of podocyte markers. OPN-/- mice showed normal urine parameters while a thickening of the glomerular basement membrane was evident. Intriguingly, following UNX, OPN-/- mice exhibited prominent FSGS, proteinuria, and glomerular matrix deposition. Electron microscopy revealed bulgings of the glomerular basement membrane and occasionally an effacement of podocytes. After UNX and DOCA-salt treatment, severe glomerular lesions as well as proteinuria and albuminuria were seen in WT and OPN-/- mice. Moreover, we found a reduction of specific markers such as Wilm's tumor-1, podocin, and synaptopodin in both experimental groups indicating a loss of podocytes. Podocyte damage was accompanied by increased number of Ki-67-positive cells in the parietal epithelium of Bowman's capsule. We conclude that OPN plays a crucial role in adaptation of podocytes following renal ablation and is renoprotective when glomerular mechanical load is increased.

Epidermal growth factor receptor promotes glomerular injury and renal failure in rapidly progressive crescentic glomerulonephritis.

Rapidly progressive glomerulonephritis (RPGN) is a life-threatening clinical syndrome and a morphological manifestation of severe glomerular injury that is marked by a proliferative histological pattern ('crescents') with accumulation of T cells and macrophages and proliferation of intrinsic glomerular cells. We show de novo induction of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in intrinsic glomerular epithelial cells (podocytes) from both mice and humans with RPGN. HB-EGF induction increases phosphorylation of the epidermal growth factor receptor (EGFR, also known as ErbB1) in mice with RPGN. In HB-EGF-deficient mice, EGFR activation in glomeruli is absent and the course of RPGN is improved. Autocrine HB-EGF induces a phenotypic switch in podocytes in vitro. Conditional deletion of the Egfr gene from podocytes of mice alleviates the severity of RPGN. Likewise, pharmacological blockade of EGFR also improves the course of RPGN, even when started 4 d after the induction of experimental RPGN. This suggests that targeting the HB-EGF-EGFR pathway could also be beneficial in treatment of human RPGN.

Non-muscle myosin IIA is required for the development of the zebrafish glomerulus.

Mutations in the MYH9 gene, coding for the non-muscle myosin heavy chain IIA (NMHC-IIA), are responsible for syndromes characterized by macrothrombocytopenia associated with deafness, cataracts, and severe glomerular disease. Electron microscopy of renal biopsies from these patients found glomerular abnormalities characterized by alterations in mesangial cells, podocytes, and thickening of the glomerular basement membrane. Knockout of NMHC-IIA in mice is lethal, and therefore little is known about the glomerular-related functions of Myh9. Here, we use zebrafish as a model to study the role and function of zNMHC-IIA in the glomerulus. Knockdown of zNMHC-IIA resulted in malformation of the glomerular capillary tuft characterized by few and dilated capillaries of the pronephros. In zNMHC-IIA morphants, endothelial cells failed to develop fenestrations, mesangial cells were absent or reduced, and the glomerular basement membrane appeared nonuniformly thickened. Knockdown of zNMHC-IIA did not impair the formation of podocyte foot processes or slit diaphragms; however, podocyte processes were less uniform in these morphants compared to controls. In vivo clearance of fluorescent dextran indicated that the glomerular barrier function was not compromised by zNMHC-IIA knockdown; however, glomerular filtration was significantly reduced. Thus, our results demonstrate an important role of zNMHC-IIA for the proper formation and function of the glomerulus in zebrafish.

Hydroxyapatite grafting promotes new bone formation and osseointegration of smooth titanium implants.

Titanium is the ideal metal for intra-osseous dental implants. It permits the natural formation of an oxide layer on its surface and thereby it prevents the release of potentially toxic molecules. New formation of bone around implants, partially placed into the bone marrow cavity, is a gradual process that runs from the endosteum to the surface of the implant. Deposition of hydroxyapatite crystals on collagen type I fibrils is initiated by acidic proteins and leads to bone mineralization. This study analyzed the effects of hydroxyapatite upon peri-implant bone formation after insertion of smooth titanium implants. Screw-shaped smooth titanium implants of 3.75 mm thickness and 8.5 mm length were inserted into the metaphysis of rabbit tibia, either together with bovine hydroxyapatite into the right tibia or in controls without hydroxyapatite into the left tibia. Polyfluorochrome tracers (alizarin complex, calcein, tetracycline) were injected subcutaneously at different time intervals after implantation to evaluate the time frame of bone new formation over a period of 8 weeks. All samples were processed for histology and analyzed by fluorescence and polarizing microscopy. Our results showed a higher quantity of mature type I collagen fibers around implants and an acceleration of bone formation in the presence of hydroxyapatite. Mainly immature organic matrix was formed at the surface of implants in controls. The presence of hydroxyapatite seems to promote the maturation of collagen fibers surrounding the titanium implants and to support osteoconduction. Moreover, new formation of bone was faster in all samples where implants were inserted together with hydroxyapatite.