[Show abstract][Hide abstract] ABSTRACT: New treatment paradigms that slow or reverse progression of chronic kidney disease(CKD) are needed to relieve significant patient and healthcare burdens. We have shown that a population of selected renal cells (SRCs) stabilized disease progression in a mass reduction model of CKD. Here, we further define the cellular composition of SRCs and apply this novel therapeutic approach to the ZSF1 rat, a model of severe progressive nephropathy secondary to diabetes, obesity, dyslipidemia, and hypertension. Injection of syngeneic SRCs into the ZSF1 renal cortex elicited a regenerative response that significantly improved survival and stabilized disease progression to renal structure and function beyond 1 year post-treatment. Functional improvements included normalization of multiple nephron structures and functions including, glomerular filtration, tubular protein handling, electrolyte balance and the ability to concentrate urine. Improvement to blood pressure, including reduced levels of circulating renin were also observed. These functional improvements following SRC treatment were accompanied by significant reductions in glomerular sclerosis, tubular degeneration and interstitial inflammation and fibrosis. Collectively, these data support the utility of a novel renal cell-based approach for slowing renal disease progression associated with diabetic nephropathy in the setting of metabolic syndrome, one of the most common causes of end stage renal disease.
[Show abstract][Hide abstract] ABSTRACT: Dedifferentiation and proliferation of resident tubular epithelial cells is a mechanism of action potentially contributing to repair and regeneration in kidneys presenting with ischemic or chronic disease. To more efficiently develop cell and tissue engineering technologies for the kidney, we have developed molecular assays to evaluate the acquisition of a pluripotent state associated with stem/progenitor cell phenotype during induction of a regenerative response within the kidneys of rats with chronic kidney disease (CKD) following therapeutic intervention. Intrarenal delivery of selected bioactive renal cells leads to significant upregulation of pluripotency-associated SOX2 mRNA within the diseased kidney tissue from 1 to 24 weeks after treatment. The overall regenerative response index was assessed by quantitative composite expression of CD24, NODAL and LEFTY1 proteins, which were induced within 1 week of cell treatment and peaked at 12 weeks after treatment, reaching statistical significance (p < 0.05) compared to untreated CKD controls. Molecular assays that incorporate the assessment of SOX2 and the regenerative response index may prove to be valuable tools for the detection and monitoring of the tissue response after the delivery of regenerative treatments for CKD, thereby significantly shortening the developmental timelines associated with such therapies.
[Show abstract][Hide abstract] ABSTRACT: Chronic kidney disease (CKD) is a global health problem; the growing gap between the number of patients awaiting transplant and organs actually transplanted highlights the need for new treatments to restore renal function. Regenerative medicine is a promising approach from which treatments for organ-level disorders (e.g., neurogenic bladder) have emerged and translated to clinics. Regenerative templates, composed of biodegradable material and autologous cells, isolated and expanded ex vivo, stimulate native-like organ tissue regeneration after implantation. A critical step for extending this strategy from bladder to kidney is the ability to isolate, characterize, and expand functional renal cells with therapeutic potential from diseased tissue. In this study, we developed methods that yield distinct subpopulations of primary kidney cells that are compatible with process development and scale-up. These methods were translated to rodent, large mammal, and human kidneys, and then to rodent and human tissues with advanced CKD. Comparative in vitro studies demonstrated that phenotype and key functional attributes were retained consistently in ex vivo cultures regardless of species or disease state, suggesting that autologous sourcing of cells that contribute to in situ kidney regeneration after injury is feasible, even with biopsies from patients with advanced CKD.
Tissue Engineering Part C Methods 03/2011; 17(3):261-73. DOI:10.1089/ten.TEC.2010.0399 · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Established chronic kidney disease (CKD) may be identified by severely impaired renal filtration that ultimately leads to the need for dialysis or kidney transplant. Dialysis addresses only some of the sequelae of CKD, and a significant gap persists between patients needing transplant and available organs, providing impetus for development of new CKD treatment modalities. Some postulate that CKD develops from a progressive imbalance between tissue damage and the kidney's intrinsic repair and regeneration processes. In this study we evaluated the effect of kidney cells, delivered orthotopically by intraparenchymal injection to rodents 4-7 wk after CKD was established by two-step 5/6 renal mass reduction (NX), on the regeneration of kidney function and architecture as assessed by physiological, tissue, and molecular markers. A proof of concept for the model, cell delivery, and systemic effect was demonstrated with a heterogeneous population of renal cells (UNFX) that contained cells from all major compartments of the kidney. Tubular cells are known contributors to kidney regeneration in situ following acute injury. Initially tested as a control, a tubular cell-enriched subpopulation of UNFX (B2) surprisingly outperformed UNFX. Two independent studies (3 and 6 mo in duration) with B2 confirmed that B2 significantly extended survival and improved renal filtration (serum creatinine and blood urea nitrogen). The specificity of B2 effects was verified by direct comparison to cell-free vehicle controls and an equivalent dose of non-B2 cells. Quantitative histological evaluation of kidneys at 6 mo after treatment confirmed that B2 treatment reduced severity of kidney tissue pathology. Treatment-associated reduction of transforming growth factor (TGF)-β1, plasminogen activator inhibitor (PAI)-1, and fibronectin (FN) provided evidence that B2 cells attenuated canonical pathways of profibrotic extracellular matrix production.
[Show abstract][Hide abstract] ABSTRACT: Background: The HIV-1 protease (PR) is translated as part of the GagPol precursor and is both necessary and sufficient for precursor processing. The PR is only active as a dimer; enzyme activation is initiated when the PR domains in two GagPol precursors dimerize. PR-mediated precursor processing is an absolute requirement for the production of fully infectious viral particles; mutations that produce imprecise cleavage at individual sites or that alter the order in which sites are cleaved result in the elaboration of aberrantly assembled virions that are markedly less infectious. Methods: Using an in vitro expression system in which full length GagPol is cleaved by its embedded PR, we examined the earliest events in precursor processing. Results: We demonstrate that the embedded PR is approximately 10,000-fold less sensitive to the available PR inhibitors. Further, we find that different concentrations of the active site inhibitor are required to inhibit the processing of different cleavage sites within GagPol. We also show that the order of cleavage of GagPol by the embedded PR is different than that seen with the mature PR added in trans. Mutational analyses indicate that the wild type specificity is dictated by the presence of a proline at the first amino acid of the PR. Substitution of this structurally rigid amino acid alters the pattern of cleavage and produces an enzyme that is hypersensitive to inhibition by an active site inhibitor (ritonavir). Conclusions: Overall, our results indicate that (1) inhibition of the earliest steps of precursor processing requires very high levels of PR inhibitors; (2) the specificity of the PR changes as processing proceeds; and (3) the first cleavages carried out by the activated PR within GagPol are intramolecular.
Infectious Diseases Society of America 2004 Annual Meeting; 10/2004
[Show abstract][Hide abstract] ABSTRACT: Processing of the GagPol polyprotein precursor of human immunodeficiency virus type 1 (HIV-1) is a critical step in viral assembly and replication. The HIV-1 protease (PR) is translated as part of GagPol and is both necessary and sufficient for precursor processing. The PR is active only as a dimer; enzyme activation is initiated when the PR domains in two GagPol precursors dimerize. The precise mechanism by which the PR becomes activated and the subsequent initial steps in precursor processing are not well understood. However, it is clear that processing is initiated by the PR domain that is embedded within the precursor itself. We have examined the earliest events in precursor processing using an in vitro assay in which full-length GagPol is cleaved by its embedded PR. We demonstrate that the embedded, immature PR is as much as 10,000-fold less sensitive to inhibition by an active-site PR inhibitor than is the mature, free enzyme. Further, we find that different concentrations of the active-site inhibitor are required to inhibit the processing of different cleavage sites within GagPol. Finally, our results indicate that the first cleavages carried out by the activated PR within GagPol are intramolecular. Overall, our data support a model of virus assembly in which the first cleavages occur in GagPol upstream of the PR. These intramolecular cleavages produce an extended form of PR that completes the final processing steps accompanying the final stages of particle assembly by an intermolecular mechanism.
Journal of Virology 09/2004; 78(16):8477-85. DOI:10.1128/JVI.78.16.8477-8485.2004 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As is the case for all retroviruses, the protease of HIV-1 is only functional as a homodimer; dimerization of two protease monomers results in the formation of the enzyme active site. This dimer structure is supported primarily by interactions between the first four amino-terminal and the last four carboxy-terminal amino acids. These eight amino acids form a beta-sheet in which hydrophobic residues are oriented towards the core of the molecule and polar residues are directed towards the solvent. Although the structure of the dimer interface has been determined, the forces that support dimerization have not been fully characterized. Here, we describe a tethered construct in which two protease monomers are joined by a 5 amino acid linker. We evaluate the relative role of each dimer interface residue in functional homo- and heterodimers. Our studies indicate that the hydrophobic residues of the dimer interface are particularly important in maintaining enzyme activity and that enzyme activity is more sensitive to substitutions of the C-terminal amino acids. Further, we demonstrate that the presence of the tether is able to compensate for mutations within the dimer interface that inactivate the enzyme.
[Show abstract][Hide abstract] ABSTRACT: Chronic kidney disease (CKD) is a global public health problem; U.S. patients on dialysis awaiting organ transplant more than doubled between 1991 and 2004. To avoid an immune response to implanted cells, functional autologous cells would be preferred components of regenerative medicine therapies for CKD; however, evidentiary support for autologous sourcing of therapeutically-relevant cells from CKD patients and large animal models is lacking. Fresh kidney tissue (CKD and non-CKD) was obtained from porcine and human subjects. CKD was confirmed by serology and histopathology. Tissue dissociation and cell isolation methods developed with non-CKD tissue were employed to isolate and propagate cells from CKD tissue and yielded tubular cells and erythropoietin (EPO)-expressing cells. Comparative in vitro studies demonstrated expression of megalin:cubilin and receptor-mediated uptake of fluorescein-conjugated albumin in tubular cell cultures from both CKD and non-CKD tissues. Cells expressing erythropoietin (EPO) were present in both CKD and non-CKD tissues and could be isolated and expanded with retention of oxygen-responsive, HIF1-α-driven EPO expression. Taken together, these results suggest that autologous sourcing of at least two therapeutically-relevant cell populations is feasible in advanced CKD.