Keith A. Hruska

Protein transduction: unrestricted delivery into all cells?

Several proteins can traverse biological membranes through protein transduction. Small sections of these proteins (10-16 residues long) are responsible for this. Linking these domains covalently to compounds, peptides, antisense peptide nucleic acids or 40-nm iron beads, or as in-frame fusions with full-length proteins, lets them enter any cell type in a receptor- and transporter-independent fashion. Moreover, several of these fusions, introduced into mice, were delivered to all tissues, even crossing the blood-brain barrier. These domains thus might let us address new questions and even help in the treatment of human disease.

Bone Morphogenetic Proteins in Vascular Calcification

Vascular calcification is a common problem among the elderly and those with chronic kidney disease (CKD) and diabetes. The process of tunica media vascular calcification in CKD appears to involve a phenotypic change in the vascular smooth muscle cell (VSMC) resulting in cell-mediated mineralization of the extracellular matrix. The bone morphogenetic proteins (BMPs) are important regulators in orthotopic bone formation, and their localization at sites of vascular calcification raises the question of their role. In this review, we will discuss the actions of the BMPs in vascular calcification. Although the role of BMP-2 in vascular calcification is not proven, it has been the most studied member of the BMP family in this disease process. The role of BMP-2 may be through inducing osteoblastic differentiation of VSMCs through induction of MSX-2, or by inducing apoptosis of VSMCs, a process thought critical in the initiation of vascular calcification. Additionally, BMP-2 may be related to loss of regulation of the matrix Gla protein. A second BMP, BMP-7, less studied than BMP-2 may have opposing actions in vascular calcification. In postnatal life, BMP-7 is expressed primarily in the kidney, and expression is diminished by renal injury. BMP-7 is an important regulator of skeletal remodeling and the VSMC phenotype. BMP-7 restores skeletal anabolic balance in animal models of CKD with disordered skeletal modeling, also reducing serum phosphate in the process. BMP-7 also reverses vascular calcification in CKD, and reduction in vascular calcification is due, in part, to reduced serum phosphate, an important inducer of VSMC-mediated vascular mineralization and in part to direct actions on the VSMC.

Metabolism in immunoreactive parathyroid hormone in the dog. The role of the kidney and the effects of chronic renal disease.

The role of the kidney in the metabolism of parathyroid hormone (PTH) was examined in the dog. Studies were performed in awake normal and uremic dogs after administration of bovine parathyroid hormone (b-PTH) or synthetic amino terminal tetratricontapeptide of b-PTH (syn b-PTH 1-34). The renal clearance of immunoreactive PTH was determined from the product of renal plasma flow and the percent extraction of PTH immunoreactivity by the kidney. Blood levels of circulating immunoreactive PTH were determined by radioimmunoassay. The normal dog kidney extracted 20 plus or minus 1% of the immunoreactive b-PTH delivered to it, and renal clearance (RC) of immunoreactivity was 60 ml/min. When RC was compared to an estimate of total metabolic clearance (MCR) of immunoreactivity, it accounted for 61% of the total. Both MCR and RC were markedly decreased in dogs with chronic renal disease. However, the percent extraction of immunoreactive PTH was unchanged in chronic renal disease, and the observed decrease in RC was due to changes in renal plasma flow. The largest portion of the reduction in total MCR was accounted for by the decrease in RC, and there was no compensation for the decrease in RC by extrarenal sites of PTH metabolism.

Hyperphosphatemia of chronic kidney disease

Observational studies have determined hyperphosphatemia to be a cardiovascular risk factor in chronic kidney disease. Mechanistic studies have elucidated that hyperphosphatemia is a direct stimulus to vascular calcification, which is one cause of morbid cardiovascular events contributing to the excess mortality of chronic kidney disease. This review describes the pathobiology of hyperphosphatemia that develops as a consequence of positive phosphate balance in chronic kidney disease and the mechanisms by which hyperphosphatemia acts on neointimal vascular cells that are stimulated to mineralize in chronic kidney disease. The characterization of hyperphosphatemia of chronic kidney disease as a distinct syndrome in clinical medicine with unique disordered skeletal remodeling, heterotopic mineralization and cardiovascular morbidity is presented.

The Peripheral Metabolism of Parathyroid Hormone

AFTER the radioimmunoassay for parathyroid hormone (PTH) was introduced by Berson et al. in 1963,1 initial enthusiasm for its clinical application was tempered because widely differing results were...

BMP-7 Is an Efficacious Treatment of Vascular Calcification in a Murine Model of Atherosclerosis and Chronic Renal Failure

Chronic renal failure is complicated by high cardiovascular mortality. One key contributor to this mortality is vascular calcification, for which no therapy currently exists. Bone morphogenetic protein 7 is an essential renal morphogen that maintains renal tubular differentiation in the adult and is downregulated in renal failure. Several studies have demonstrated its efficacy in treating various renal diseases in rodents, and it was hypothesized that it would also be an effective treatment of vascular calcification in this setting. Uremia was imposed on LDL receptor null mice (a model of atherosclerosis), which were then treated with bone morphogenetic protein 7 for 15 wk. Uremic animals had increased vascular calcification by histology and chemical analysis. Calcification in treated animals was similar to or less than non-uremic control animals. Cells exhibiting an osteoblast-like phenotype in the vessel wall may be important in the etiology of vascular calcification. Expression of osteocalcin was assessed as a marker of osteoblastic function, and it is shown that it is increased in untreated uremic animals but downregulated to levels similar to non-uremic control animals with treatment. The data are compatible with bone morphogenetic protein 7 deficiency as a pathophysiologic factor in chronic renal failure, and they demonstrate its efficacy as a potential treatment of vascular calcification.

The Mechanism of Phosphorus as a Cardiovascular Risk Factor in CKD

Hyperphosphatemia and vascular calcification have emerged as cardiovascular risk factors among those with chronic kidney disease. This study examined the mechanism by which phosphorous stimulates vascular calcification, as well as how controlling hyperphosphatemia affects established calcification. In primary cultures of vascular smooth muscle cells derived from atherosclerotic human aortas, activation of osteoblastic events, including increased expression of bone morphogenetic protein 2 (BMP-2) and the transcription factor RUNX2, which normally play roles in skeletal morphogenesis, was observed. These changes, however, did not lead to matrix mineralization until the phosphorus concentration of the media was increased; phosphorus stimulated expression of osterix, a second critical osteoblast transcription factor. Knockdown of osterix with small interference RNA (siRNA) or antagonism of BMP-2 with noggin prevented matrix mineralization in vitro. Similarly, vascular BMP-2 and RUNX2 were upregulated in atherosclerotic mice, but significant mineralization occurred only after the induction of renal dysfunction, which led to hyperphosphatemia and increased aortic expression of osterix. Administration of oral phosphate binders or intraperitoneal BMP-7 decreased expression of osterix and aortic mineralization. It is concluded that, in chronic kidney disease, hyperphosphatemia stimulates an osteoblastic transcriptional program in the vasculature, which is mediated by osterix activation in cells of the vascular tunica media and neointima.

Vitamin D Receptor Activators Can Protect against Vascular Calcification

An apparent conflict exists between observational studies that suggest that vitamin D receptor (VDR) activators provide a survival advantage for patients with ESRD and other studies that suggest that they cause vascular calcification. In an effort to explain this discrepancy, we studied the effects of the VDR activators calcitriol and paricalcitol on aortic calcification in a mouse model of chronic kidney disease (CKD)-stimulated atherosclerotic cardiovascular mineralization. At dosages sufficient to correct secondary hyperparathyroidism, calcitriol and paricalcitol were protective against aortic calcification, but higher dosages stimulated aortic calcification. At protective dosages, the VDR activators reduced osteoblastic gene expression in the aorta, which is normally increased in CKD, perhaps explaining this inhibition of aortic calcification. Interpreting the results obtained using this model, however, is complicated by the adynamic bone disorder; both calcitriol and paricalcitol stimulated osteoblast surfaces and rates of bone formation. Therefore, the skeletal actions of the VDR activators may have contributed to their protection against aortic calcification. We conclude that low, clinically relevant dosages of calcitriol and paricalcitol may protect against CKD-stimulated vascular calcification.

Wnt-Dependent β-Catenin Signaling Is Activated after Unilateral Ureteral Obstruction, and Recombinant Secreted Frizzled-Related Protein 4 Alters the Progression of Renal Fibrosis

beta-Catenin functions as a transducer of Wnt signals to the nucleus, where it interacts with the T cell factor (TCF) family of DNA binding proteins to regulate gene expression. On the basis of the genes regulated by beta-catenin and TCF in various biologic settings, two predicted functions of beta-catenin/TCF-dependent transcription are to mediate the loss of epithelial polarity and to promote fibroblast activities, such as the increased synthesis of fibronectin during chronic renal disease. These predictions were tested by determination of the expression and function of an inhibitor of Wnt signaling, secreted frizzled-related protein 4 (sFRP4), during renal tubular epithelial injury initiated by unilateral ureteral obstruction (UUO). Despite increased sFRP4 gene expression in perivascular regions of injured kidneys, total sFRP4 protein levels decreased after injury. The decreased sFRP4 protein levels after UUO accompanied increased Wnt-dependent beta-catenin signaling in tubular epithelial and interstitial cells, along with increased expression of markers of fibrosis. Administration of recombinant sFRP4 protein caused a reduction in tubular epithelial beta-catenin signaling and suppressed the progression of renal fibrosis, as evidenced by a partial maintenance of E-cadherin mRNA expression and a reduction in the amount of fibronectin and alpha-smooth muscle actin proteins. Furthermore, recombinant sFRP4 reduced the number of myofibroblasts, a central mediator of fibrosis. It is concluded that beta-catenin signaling is activated in tubular epithelial and interstitial cells after renal injury, and recombinant sFRP4 can interfere with epithelial de-differentiation and with fibroblast differentiation and function during progression of renal fibrosis.

Low Turnover Osteodystrophy and Vascular Calcification Are Amenable to Skeletal Anabolism in an Animal Model of Chronic Kidney Disease and the Metabolic Syndrome

LDL receptor (LDLR)-null mice fed high-fat/cholesterol diets, a model of the metabolic syndrome, have vascular calcification (VC) worsened by chronic kidney disease (CKD) and ameliorated by bone morphogenetic protein-7 (BMP-7), an efficacious agent in treating animal models of renal osteodystrophy. Here, LDLR-/- high-fat-fed mice without CKD were shown to have significant reductions in bone formation rates, associated with increased VC and hyperphosphatemia. Superimposing CKD resulted in a low turnover osteodystrophy, whereas VC worsened and hyperphosphatemia persisted. BMP-7 treatment corrected the hyperphosphatemia, corrected the osteodystrophy, and prevented VC, compatible with skeletal phosphate deposition leading to reduced plasma phosphate and removal of a major stimulus to VC. A pathologic link between abnormal bone mineralization and VC through the serum phosphorus was supported by the partial effectiveness of directly reducing the serum phosphate by a phosphate binder that had no skeletal action. Thus, in this model of the metabolic syndrome with CKD, a reduction in bone-forming potential of osteogenic cells leads to low bone turnover rates, producing hyperphosphatemia and VC, processes ameliorated by the skeletal anabolic agent BMP-7, in part through deposition of phosphate and increased bone formation.