Raymonde F. Gagnon

Erythropoietin Delivery by Genetically Engineered Bone Marrow Stromal Cells for Correction of Anemia in Mice with Chronic Renal Failure

The goal of this research was to develop a strategy to couple stem cell and gene therapy for in vivo delivery of erythropoietin (Epo) for treatment of anemia of ESRD. It was shown previously that autologous bone marrow stromal cells (MSCs) can be genetically engineered to secrete pharmacologic amounts of Epo in normal mice. Therefore, whether anemia in mice with mild to moderate chronic renal failure (CRF) can be improved with Epo gene-modified MSCs (Epo+MSCs) within a subcutaneous implant was examined. A cohort of C57BL/6 mice were rendered anemic by right kidney electrocoagulation and left nephrectomy. In these CRF mice, the hematocrit (Hct) dropped from a prenephrectomy baseline of approximately 55% to 40% after induction of renal failure. MSCs from C57BL/6 donor mice were genetically engineered to secrete murine Epo at a rate of 3 to 4 units of Epo/10(6) cells per 24 h, embedded in a collagen-based matrix, and implanted subcutaneously in anemic CRF mice. It was observed that Hct increased after administration of Epo+MSCs, according to cell dose. Implants of 3 million Epo+MSCs per mouse had no effect on Hct, whereas 10 million led to a supraphysiologic effect. The Hct of CRF mice that received 4.5 or 7.5 million Epo+MSCs rose to a peak 54+/-4.0 or 63+/-5.5%, respectively, at 3 wk after implantation and remained above 48 or 54% for >19 wk. Moreover, mice that had CRF and received Epo+MSCs showed significantly greater swimming exercise capacity. In conclusion, these results demonstrate that subcutaneous implantation of Epo-secreting genetically engineered MSCs can correct anemia that occurs in a murine model of CRF.

Characterization of a mouse model of chronic uremia

SummaryA mouse model of renal failure, which is induced by the sequential electrocoagulation of the right renal cortex and left nephrectomy, was examined for the capacity to reproduce the characteristics of chronic uremia. Assessment was conducted six weeks after the second surgical procedure in 13 week old female C57BL/6 inbred mice with renal failure and in normal and sham-operated controls. The surgery, which was well tolerated, was free of local and systemic signs of inflammation or infection. Growth was significantly delayed in all animals post surgery however renal failure mice presented the most severe growth retardation. Biochemical analysis of plasma revealed multiple abnormalities with commensurate elevations of urea and creatinine. In addition to the expected hyperphosphatemia, hyperkalemia and acidosis, a significant increase in cholesterol was present. Furthermore, in contrast to controls, renal failure mice produced large volumes of urine which contained significant levels of protein. Renal failure mice presented profound hematological changes in the red cell series in which anemia was evident. Changes in plasma biochemistry and in bone histology revealed the presence of severe secondary hyperparathyroidism. It was therefore concluded that the described mouse model of chronic renal failure presented characteristics consistent with those observed clinically in end-stage renal disease.

A reproducible model for chronic renal failure in the mouse

SummaryChronic renal failure was induced in mice using bilateral electrocoagulation of the renal cortex. Thermal injury via an angled point cautery was applied to the fully exteriorized right kidney. Following a 10-day interval the left kidney was injured in a similar fashion. The incidence of local complications was negligible and because of the limited blood loss, very small laboratory animals can be used. By restricting trauma to varying proportions of the visible cortex graded levels of injury were obtained. Thus this model can produce a uniform pattern of renal failure and will therefore facilitate the acquisition of information on the biological consequences of chronic uraemia.

Erythrocytosis after renal transplantation represents an abnormality of insulin-like growth factor-I and its binding proteins.

BACKGROUND Secondary erythrocytosis is classically defined by an increase in erythropoietin (EPO) production. Despite increased levels of EPO often seen in secondary erythrocytosis, some of these forms such as that seen after renal transplantation remain undefined. Our group has recently investigated the in vivo function of insulin-like growth factor-I (IGF-I) in erythropoiesis both in humans and in a murine model of chronic renal failure. These data, and the recently recognized role of IGF-I in polycythemia vera, suggested that IGF-I might be involved in secondary erythrocytosis. METHODS Renal transplant recipients who developed erythrocytosis after transplantation were compared to normal individuals and to renal transplant recipients without erythrocytosis. We measured fasting serum EPO and IGF-I in all three groups. Because binding proteins may modify IGF-I function, IGF-I-binding proteins (IGFBP) 1 and 3, major binding proteins of IGF-I, were also measured. RESULTS Renal transplant recipients have significantly elevated serum of IGF-I and IGFBP3 compared to normal individuals. When transplant recipients with and without posttransplant erythrocytosis were compared, similar levels of IGF-I were found; however, the group with erythrocytosis had significantly elevated IGFBP1 and IGFBP3. No other significant differences including EPO levels were found between the groups. CONCLUSIONS Erythrocytosis after renal transplantation represents an anomaly of both IGF-I and its major binding proteins. Further studies are under way to better define this dysregulation and determine whether IGF-I can play a more generalized role in secondary forms of erythropoiesis.

Cytotoxicity of commercial peritoneal dialysis solutions towards peritoneal cells of chronically uremic mice

A gradual loss of cell viability was observed during in vitro incubation of peritoneal cells from chronically uremic mice in commercial peritoneal dialysis solutions. The magnitude of this cytotoxicity toward peritoneal cells harvested from uremic mice and controls was comparable. Resident peritoneal cells were always found to be more susceptible than thioglycolate-elicited peritoneal populations of either macrophages or polymorphonuclear cells. In order to elucidate the factors contributing to this phenomenon, resident peritoneal cells recovered from normal mice were incubated in vitro for 1 h in various solutions of known pH and osmolarity consisting of buffered and unbuffered commercial peritoneal dialysis solutions. The results clearly show that the major part of the cytotoxicity is attributable to the low pH of the solutions. Once pH was corrected, the hyperosmolarity of these solutions had no effect on cell viability; however, a small but significant cytotoxicity remained. Factors other than those addressed in this study probably account for the observed residual cytotoxicity.

Mesenchymal stromal cells genetically engineered to overexpress IGF-I enhance cell-based gene therapy of renal failure-induced anemia

We previously demonstrated that erythropoietin (EPO)-secreting mesenchymal stromal cells (MSC) can be used for the long-term correction of renal failure-induced anemia. The present study provides evidence that coimplantation of insulin-like growth factor I (IGF-I)-overexpressing MSC (MSC-IGF) improves MSC-based gene therapy of anemia by providing paracrine support to EPO-secreting MSC (MSC-EPO) within a subcutaneous implant. IGF-I receptor RNA expression in murine MSC was demonstrated by RT-PCR. Functional protein expression was confirmed by immunoblots and MSC responsiveness to IGF-I stimulation in vitro. IGF-I was also shown to improve MSC survival following staurosporin-induced apoptosis in vitro. A cohort of C57Bl/6 mice was rendered anemic by right kidney electrocoagulation and left nephrectomy. MSC-EPO were subsequently admixed in a bovine collagen matrix and implanted, in combination with MSC-IGF or MSC null, by subcutaneous injection in renal failure mice. In mice receiving MSC-EPO coimplanted with MSC-IGF, hematocrit elevation was greater and enhanced compared with control mice; heart function was also improved. MSC-IGF coimplantation, therefore, represents a promising new strategy for enhancing MSC survival within implanted matrices and for improving cell-based gene therapy of renal anemia.

The Activity of Rifampin and Analogs against Staphylococcus epidermidis Biofilms in a CAPD Environment Model

Rifampin has been noted to exhibit exceptional antimicrobial activity against Staphylococcus epidermidis biofilms as compared to commonly used antibiotics. To further explore this unique effect of rifampin, we evaluated the antimicrobial activity of three commercially available preparations of rifampin, two rifampin analogs (CGP29861 and rifapentine) and the parent compound rifamycin SV. These were tested against standardized S. epidermidis biofilms in various milieus. All six members of the rifamycin group tested demonstrated marked antimicrobial activity but with minor foci of resisters when tested in a peptone water environment. The microscopy of the exposed biofilms showed profound lysis and morphological distortion of the remaining cells. The synergistic elimination of the foci of resistance was achieved in an environment of fresh peritoneal dialysis (PD) solution or by the addition of vancomycin. Neither vancomycin nor fresh PD solution demonstrated significant antimicrobial activity when tested alone with biofilm preparations. Spent PD fluid markedly antagonized the activity of the rifamycins with the exception of the rifampin analogs, an effect primarily of pH. The synergistic effect of vancomycin with the rifamycins was not affected either by protein content or pH, leaving the antagonistic properties of spent PD fluid unexplained. The variable activity of the different members of the rifamycin group underlines the importance of structural differences in determining their interaction with bacterial biofilms. Further precision of the nature of these structural interactions is seen to have considerable potential for therapeutic advancement of catheter-associated sepsis.

The natural history of leukocyturia associated with indinavir treatment in HIV+ individuals.

Urinary complications observed during indinavir treatment of HIV disease are often attributed to indinavir crystalluria. In a prospective study of urinalysis during the first year of indinavir therapy, 5 of 54 asymptomatic HIV+ individuals presented severe leukocyturia (≧100 cells/HPF) usually accompanying indinavir crystalluria. The clinical course of these 5 individuals, successfully treated for HIV and monitored for an second follow-up year, suggests that recurrence of severe leukocyturia may be an indicator of renal damage, likely tubulointerstitial disease caused by indinavir crystalluria. This is in contrast to the remaining 49 subjects, including those presenting mild leukocyturia, who did not demonstrate any evidence of renal disease. Regular urinalysis is therefore recommended in the clinical management of indinavir-treated individuals to detect early renal damage secondary to indinavir crystalluria and to prevent further renal impairment.

Experimental Staphylococcus epidermidis implant infection in the mouse. Kinetics of rifampin and vancomycin action.

Staphylococcus epidermidis implant infections remain a therapeutic challenge; they frequently result in failure of conservative management and require removal of the implant. This resistance to antibiotic therapy has been ascribed to the presence of a protective bacterial biofilm at the surface of the implant. An animal model of implant associated infection has been developed in which preformed bacterial biofilm catheter segments are implanted intraperitoneally, resulting in a chronic standardized localized infection. The authors have determined the superior rapid action of rifampin (Cieba-Geigy) compared to vancomycin hydrochloride (Eli Lilly) and determined that the combination is superior to either used alone. No rifampin resistant surviving infection was noted, which indicates the significant contribution of mammalian host defenses. This animal model is an excellent vehicle for the study of Staphylococcus epidermidis implant associated infection and the evaluation of the relative efficacy of antibiotic regimens, singly and in combination.

Light microscopy of indinavir urinary crystals.

TO THE EDITOR: We read with interest the article by Kopp and colleagues [1]. The authors clearly show that indinavir, a protease inhibitor widely used in the treatment of HIV infection, forms distinctive urinary crystals. Indinavir is metabolized largely by the liver, but up to 10% of the compound is excreted intact in the urine [2]. The drug is generally well tolerated except for the formation of renal calculi, which has been reported with a frequency of 4% to 12% [3]. In a recent study [4], urinary calculi that were recovered from 29 patients with HIV infection consisted primarily of indinavir monohydrate and contained needle-shaped crystals. In the single reported case of indinavir nephropathy [5], needle-shaped crystals were noted in the collecting ducts on renal biopsy. In their article, Kopp and colleagues documented crystalluria in 20% of indinavir-treated patients, usually asymptomatic patients but also some with a spectrum of urologic disorders. They present five photographs showing the microscopic appearance of indinavir crystals under polarized light but none taken with regular brightfield microscopy. For this reason, we wish to report the findings of our examination of more than 200 urinary sediments from similarly treated patients with HIV infection. Indinavir crystals appear as colorless, polarizable, pointed needles. They may occur singly but most often are grouped in various formations, such as rosettes, fans, and starbursts. In our experience, the needles are frequently arranged in rectangular plates and sheaves (Figure 1); these distinctive groupings vary greatly in size, and large forms are common. Figure 1. Light microscopy of a fresh unstained preparation of a representative urinary sediment from an asymptomatic patient with HIV infection who was treated with indinavir sulfate. Although the clinical significance of the crystalluria remains to be determined, we believe that physicians managing patients with HIV infection should be aware of the presence of crystalluria in those receiving indinavir and of the distinctive appearance and nature of the crystals on routine light microscopy.