Myosin 1e is a component of the glomerular slit diaphragm complex that regulates actin reorganization during cell-cell contact formation in podocytes.

Glomerular visceral epithelial cells, also known as podocytes, are critical to both normal kidney function and the development of kidney disease. Podocyte actin cytoskeleton and their highly specialized cell-cell junctions (also called slit diaphragm complexes) play key roles in controlling glomerular filtration. Myosin 1e (myo1e) is an actin-based molecular motor that is expressed in renal glomeruli. Disruption of the Myo1e gene in mice and humans promotes podocyte injury and results in the loss of the integrity of the glomerular filtration barrier. Here, we have used biochemical and microscopic approaches to determine whether myo1e is associated with the slit diaphragm complexes in glomerular podocytes. Myo1e was consistently enriched in the slit diaphragm fraction during subcellular fractionation of renal glomeruli and colocalized with the slit diaphragm markers in mouse kidney. Live cell imaging studies showed that myo1e was recruited to the newly formed cell-cell junctions in cultured podocytes, where it colocalized with the actin filament cables aligned with the nascent contacts. Myo1e-null podocytes expressing FSGS-associated myo1e mutant (A159P) did not efficiently assemble actin cables along new cell-cell junctions. We have mapped domains in myo1e that were critical for its localization to cell-cell junctions and determined that the SH3 domain of myo1e tail interacts with ZO-1, a component of the slit diaphragm complex and tight junctions. These findings suggest that myo1e represents a component of the slit diaphragm complex and may contribute to regulating junctional integrity in kidney podocytes.

Bone formation near direct current electrodes with and without motion.

The osteogenesis induced in the medullary canal of rabbits by the implantation of moving and stationary wire electrodes was studied with and without the simultaneous application of 20-microA constant direct cathodic current. After 3 weeks, the formation of new trabecular bone in the canal was studied and measured microscopically. Electrically stimulated osteogenesis was not observed at stationary electrodes. As in previous studies with this model, a movable electrode alone stimulated new bone formation whose area was 7-10% of the canal area. The amount of this bone was not statistically increased by the addition of cathode current. Movable, electrically active cathodes were associated, however, with fluid-filled spaces incorporated within the new trabecular bone. When mechanical stimuli were controlled, we were not able to demonstrate that the direct current stainless steel cathode acts either as an inducer or a substantial enhancer of medullary osteogenesis.

Electromagnetic effects on bone formation at implants in the medullary canal in rabbits.

This experiment was aimed at illuminating the relationship between electromagnetic and mechanical stimuli of bone formation when present simultaneously. Movable and stationary intramedullary wire implants were studied in rabbits treated with a pulsing electromagnetic field (PEMF) 4 h/day for 3 weeks, and were compared with identical control animals without PEMF. Trabecular bone formed routinely at spontaneously movable implants, but not at stationary ones. On average, PEMF-treated movable implants in the femur induced 44% more bone than untreated movable implants. Also, in the PEMF-treated femora, a 22% enlargement of the area of the medullary canal was observed compared with no-field controls. In the tibia, these effects were weak or nonexistent. The PEMF used did not induce bone at stationary implants, suggesting that under these conditions it is not a primary trigger in vivo.

Bone growth after osteotomy and internal fixation in young rabbits.

The factors leading to overgrowth following fixation of long-bone fractures in children have never been clearly understood. The amount of trauma and the type of fixation may play a role. A rabbit model was used to investigate the influence of a femoral osteotomy and plate fixation on subsequent growth. Unilateral midshaft femoral osteotomy was performed in 6-week-old rabbits, and the bone was fixed internally with a plate and screws. End-to-end reduction was performed either at full length or with a segment removed. Bone length measurements at the end of growth revealed no significant difference in growth between the control femur and the femur that had undergone osteotomy and plate fixation. Shortened plated femora also showed no tendency to grow longer or faster than full-length fixed femora or controls. Interestingly, in the ipsilateral tibia a small but statistically significant length increase, equivalent to about 2% increase in additional growth, was observed, whereas technetium-99 methylene diphosphonate uptake was reduced in the tibial physes. In the context of the rabbit experimental model chosen, these results suggest that significant femoral over-growth does not occur following femoral osteotomy and plate fixation.

Mechanical factors in electrode-induced osteogenesis.

Previous experiments have suggested that mechanical activity of electrode implants may contribute to the observed bone formation that has been attributed to the electrical currents. This was tested by implanting movable and stationary wires in the medullary canal of the rabbit femora or tibiae. The moving implants uniformly showed significant medullary bone formation at the wire, whereas the stationary ones did not. This bone persisted for as long as 8 weeks and was mostly resorbed by 12 weeks as the implants became fixed. The new trabecular bone closely resembled that typically seen at electrically active implants. Implant motion may have a more important role in electrically induced osteogenesis than previously thought.

Bacterial inhibition by electrical activation of percutaneous silver implants.

Percutaneous silver wire implants were looped through the dorsal skin of rats and inoculated with Staphylococcus aureus to test the effect on bacteria in the tract. The silver was activated with four brief daily applications of anodic microcurrent. Contralateral 316L stainless steel implants, identically inoculated, served as controls. Cultures from the silver tracts showed a marked reduction or elimination of bacteria, which persisted for the 3-week study period. In tracts with colonization established for 1 week, subsequent electrical activation of the silver also suppressed the bacteria. Inflammatory reactions at 3 weeks were mild at both the silver and stainless implants and no giant cells or toxicity were seen. This suggests that electrically activated silver may be useful in preventing or treating infection at percutaneous devices.

Evaluation of rifalazil in a combination treatment regimen as an alternative to isoniazid-rifampin therapy in a mouse tuberculosis model.

The newer rifamycin, rifalazil (RLZ) (previously known as KRM-1648), has been shown in prior experiments to be a highly potent drug against Mycobacterium tuberculosis. In this report, we studied the efficacy of RLZ in combination with pyrazinamide (PZA) and ethambutol (EMB) in a long-term in vivo experiment and compared their activity with the isoniazid (INH)-rifampin (RIF) combination which is presently used in the clinic. Combinations of RLZ with PZA alone or with both PZA and EMB were both found to have sterilizing activities comparable to that of the INH-RIF combination but significantly better activity with respect to relapse of infection. These results suggest that RLZ, or other agents with similar activity, could be combined with available agents to act as a potential alternative drug regimen to the currently used INH-RIF combination.

Evaluation of rifapentine in long-term treatment regimens for tuberculosis in mice.

Besides direct bactericidal activity, long-term effectiveness is one of the most important features to consider when developing new drugs for chemotherapy. In this study, we evaluated the ability of rifapentine (RFP), in monotherapy and combination therapy, to completely eradicate a Mycobacterium tuberculosis infection and to prevent relapse posttreatment in a Swiss mouse model. The combination of RFP, isoniazid (INH), and pyrazinamide (PZA) administered daily resulted in an apparent clearance of M. tuberculosis organisms in the lungs and spleens of infected mice after 10 weeks of treatment. However, 3 months after the cessation of therapy, bacterial regrowth occurred in mice treated for a 12-week period, indicating a relapse of infection. In intermittent treatment regimens of RFP in combination with INH and PZA, sterilization was achieved when mice were treated two to five times per week for 9 weeks. Bacterial growth was still observed in the once-weekly treatment group. Our results show that mouse models can predict important parameters for new drugs. We stress the necessity for long-term posttreatment observation in animal models for the routine evaluation of new drugs for antituberculosis chemotherapy.

Physeal response to absorbable polydioxanone bone pins in growing rabbits.

Absorbable fixation materials would seem especially useful for treating transphyseal fractures in growing children, but their degradation products may affect physeal growth. The histologic response of an open physis to placement of transphyseal, polydioxanone (PDS), bioabsorbable pins was studied in skeletally immature New Zealand White rabbits. A 1.3-mm PDS pin was inserted across the right femoral physis, and a 1.3-mm empty drill hole across the left femoral physis served as a control. The animals were sacrificed at 3, 6, 9, and 12 weeks postsurgery. Biplanar radiographs, bone length measurements, and histology sections of the physis and adjacent bone were made. Three independent observers graded the histologic response of the physis to the drilling and implant. There was no evidence of inflammation, foreign body reaction, or distortion of the growth plate during the entire growth period. This suggests bioabsorbable pins do not cause any appreciable inflammatory response or adverse effect on physeal function during active longitudinal growth of the bone.

Evaluation of rifalazil in long-term treatment regimens for tuberculosis in mice.

Previous experiments with rifalazil (RLZ) (also known as KRM-1648) in combination with isoniazid (INH) demonstrated its potential for short-course treatment of Mycobacterium tuberculosis infection. In this study we investigated the minimum RLZ-INH treatment time required to eradicate M. tuberculosis in a murine model. RLZ-INH treatment for 6 weeks or longer led to a nonculturable state. Groups of mice treated in parallel were killed following an observation period to evaluate regrowth. RLZ-INH treatment for a minimum of 10 weeks was necessary to maintain a nonculturable state through the observation period. Pyrazinamide (PZA) was added to this regimen to determine whether the treatment duration could be further reduced. In this model, the addition of PZA did not shorten the duration of RLZ-INH treatment required to eradicate M. tuberculosis from mice. The addition of PZA reduced the number of mice in which regrowth occurred, although the reduction was not statistically significant.