Kumar, Rajiv

We used immunohistochemical techniques to examine the distribution of 1 alpha, 25-dihydroxyvitamin D3 receptors (VDR) in developing rat and mouse kidneys and murine metanephric organ culture. In vivo, the patterns of expression in the two species were similar despite the slight difference in gestational periods (rat, 22 days; mouse, 19 days). Starting at gestational day 15, epitopes for VDR were found in cells of branching ureteral buds and in surrounding mesenchyme and at later developmental stages in glomerular visceral and parietal epithelial cells and proximal and distal tubules (DT). Epitopes for the 28-kDa calcium-binding protein (calbindin D28k) were found exclusively in DTs starting at gestational day 19. The pattern of VDR expression during in vitro nephrogenesis in serum-free murine metanephric organ culture paralleled that seen in vivo. At the time of explantation into organ culture (gestational day 13), VDR epitopes were not detected. By 3 days of in vitro development, VDR expression was identical to that found in gestational day 15 metanephroi in vivo. VDR expression after 5 days of in vitro development mirrored the pattern of gestational day 17 metanephroi in vivo. No calbindin D28k epitopes were seen at any in vitro developmental stage studied. We demonstrate for the first time that VDR are present in specific areas of the developing rat and mouse kidney early in gestation. Calbindin D28k appears later in developing rat and mouse kidney and is distributed differently than the VDR. Metanephric organ culture may be a useful model for studying the regulation and function of VDR during early renal development.

A number of peptide hormones have been shown to undergo receptor-mediated endocytosis (RME). RME involves the internalization of receptor-ligand complexes followed by delivery to an intracellular compartment, the endosome, from which ligands or receptors can be delivered to lysosomes or other cellular destinations. Vasopressin, a peptide hormone that plays a role in kidney and vascular physiology, has recently been demonstrated to undergo RME in LLC-PK1 and A10 cells, which express V2- and V1-type vasopressin receptors, respectively. Fluorescent vasopressin analogues are internalized by RME from the basolateral surface of polarized LLC-PK1 cells. The precise role of RME in vasopressin action is uncertain, but it is likely that it is involved in the desensitization of target cells by altering the number of cell surface vasopressin receptors. Alterations in the rate of RME may alter the response of the cell to vasopressin. Fluorescent and biotinylated vasopressins are useful tools for the study of this process.

In an effort to extend our studies on Ca2+ pumps to animal models, we developed a new monoclonal antibody (5F10) prepared against the human erythrocyte Ca2+-Mg2+-adenosinetriphosphatase (ATPase) that recognizes a protein of approximately 140 kDa in rat kidney homogenates. Enzyme-linked immunosorbent assays show that monoclonal antibody 5F10 binds purified Ca2+-Mg2+-ATPase and rat kidney membrane extracts in a concentration-dependent manner. In paraffin-embedded tissue sections, antibody 5F10 binds to an epitope in the basolateral membranes of rat kidney distal convoluted tubule principal cells. The antibody does not bind to intercalated cells. The latter cells were characterized by the presence of large amounts of carbonic anhydrase C. Polyclonal antibodies directed against chick intestinal 28-kDa vitamin D-dependent calcium binding protein (28-kDa CaBP) also bind epitopes in distal convoluted tubule cells, connecting tubules, and portions of collecting duct but not intercalated cells. Western blot and 45Ca blot analysis of renal cytosolic proteins showed that the polyclonal 28-kDa CaBP-directed antibody detects a protein which also binds calcium. Western blot analysis with monoclonal antibody 5F10 shows binding to both the authentic purified erythrocyte Ca2+ pump (approximately 138 kDa) and to tryptic fragments of this pump. Antibody JA3, previously used for staining of human kidney tubules, reacts with a different set of tryptic fragments, showing that the two antibodies are directed against different regions or conformational determinants on the pump molecule. We show that Ca2+-Mg2+-ATPase and 28-kDa CaBP are present in the principal cells of the distal convoluted tubule of the rat and are absent in intercalated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscle wasting and weakness occur frequently in patients with chronic renal failure. The mechanism(s) by which these abnormalities occur is unclear. We hypothesized that such findings were due to defective muscle protein synthesis. We measured synthetic rates of mixed muscle proteins, myosin heavy chain, and mitochondrial proteins in serial muscle biopsy samples during a continuous infusion ofl[1-13C]leucine from 12 patients with chronic renal failure and 10 healthy control subjects under identical study conditions. Patients with chronic renal failure have significantly lower synthetic rates of mixed muscle proteins and myosin heavy chain (27 and 37% reductions, respectively, P < 0.05 and P < 0.02). Significant declines in the synthetic rates of muscle mitochondrial protein (27%) ( P < 0.05), muscle cytochrome c-oxidase activity (42%) ( P < 0.007), and citrate synthase (27%) ( P < 0.007) were also observed in patients with chronic renal failure. The synthetic rates of muscle proteins and activity of mitochondrial enzymes were negatively correlated to the severity of renal failure. These results indicate that in chronic renal failure there is a decrease in the synthesis of muscle contractile and mitochondrial proteins and a decrease in muscle mitochondrial oxidative enzymes. Reduced synthetic rate of several muscle proteins is the likely biochemical basis of muscle loss and muscle weakness in people with chronic renal failure.

Previous studies using microdissected nephron segments reported that the exclusive site of renal 25-hydroxyvitamin D3-24-hydroxylase (24OHase) activity is the renal proximal convoluted tubule (PCT). We now report the presence of 24OHase mRNA, protein, and activity in cells that are devoid of markers of proximal tubules but express characteristics highly specific for the distal tubule. 24OHase mRNA was undetectable in vehicle-treated mouse distal convoluted tubule (DCT) cells but was markedly induced when DCT cells were treated with 1,25 dihydroxyvitamin D3[1,25(OH)2D3]. 24OHase protein and activity were also identified in DCT cells by Western blot analysis and HPLC, respectively. 8-Bromo-cAMP (1 mM) or parathyroid hormone [PTH-(1—34); 10 nM] was found to potentiate the effect of 1,25(OH)2D3on 24OHase mRNA. The stimulatory effect of cAMP or PTH on 24OHase expression in DCT cells suggests differential regulation of 24OHase expression in the PCT and DCT. In the presence of cAMP and 1,25(OH)2D3, a four- to sixfold induction in vitamin D receptor (VDR) mRNA was observed. VDR protein, as determined by Western blot analysis, was also enhanced in the presence of cAMP. Transient transfection analysis in DCT cells with rat 24OHase promoter deletion constructs demonstrated that cAMP enhanced 1,25(OH)2D3-induced 24OHase transcription but this enhancement was not mediated by cAMP response elements (CREs) in the 24OHase promoter. We conclude that 1) although the PCT is the major site of localization of 24OHase, 24OHase mRNA and activity can also be localized in the distal nephron; 2) both PTH and cAMP modulate the induction of 24OHase expression by 1,25(OH)2D3in DCT cells in a manner different from that reported in the PCT; and 3) in DCT cells, upregulation of VDR levels by cAMP, and not an effect on CREs in the 24OHase promoter, is one mechanism involved in the cAMP-mediated modulation of 24OHase transcription.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is required for normal glucose-stimulated insulin release from pancreatic beta-cells. Biochemical characterization techniques have demonstrated the presence of the 1,25(OH)2D3 receptor (VDR) in homogenates of whole pancreas. Autoradiographic studies using radiolabeled 1,25(OH)2D3 suggest that the VDR is localized to beta-cells but are inconclusive. We used immunohistochemical techniques to stain serial sections from both human and rat pancreas with polyclonal antibodies to human VDR, chick calbindin D28k, insulin, glucagon, and somatostatin. VDR was present in the islet cells and also at low levels in acinar cells of the human and rat pancreas. Calbindin D28k was distributed in a manner similar to the VDR in pancreatic islets but was not present in acini. These results show for the first time that VDR and calbindin D28k are present in human pancreatic tissue. VDR and calbindin D28k are focally distributed throughout pancreatic islet cell types in humans and rats; VDR is also present in the exocrine pancreas. These findings suggest that 1,25(OH)2D3 may influence both endocrine and exocrine pancreatic function.

Abstract Downstream regulatory element antagonist modulator (DREAM) is an EF-hand Ca2+-binding protein that also binds to a specific DNA sequence, downstream regulatory elements (DRE), and thereby regulates transcription in a calcium-dependent fashion. DREAM binds to DRE in the absence of Ca2+ but detaches from DRE under Ca2+ stimulation, allowing gene expression. The Ca2+ binding properties of DREAM and the consequences of the binding on protein structure are key to understanding the function of DREAM. Here we describe the application of hydrogen–deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis to investigate the Ca2+ binding properties and the subsequent conformational changes of full-length DREAM. We demonstrate that all EF-hands undergo large conformation changes upon calcium binding even though the EF-1 hand is not capable of binding to Ca2+. Moreover, EF-2 is a lower-affinity site compared to EF-3 and -4 hands. Comparison of HDX profiles between wild-type DREAM and two EF-1 mutated constructs illustrates that the conformational changes in the EF-1 hand are induced by long-range structural interactions. HDX analyses also reveal a conformational change in an N-terminal leucine-charged residue-rich domain (LCD) remote from Ca2+-binding EF-hands. This LCD domain is responsible for the direct interaction between DREAM and cAMP response element-binding protein (CREB) and regulates the recruitment of the co-activator, CREB-binding protein. These long-range interactions strongly suggest how conformational changes transmit the Ca2+ signal to CREB-mediated gene transcription.

Abstract Hypercalcemia occurs in up to 4% of the population in association with malignancy, primary hyperparathyroidism, ingestion of excessive calcium and/or vitamin D, ectopic production of 1,25-dihydroxyvitamin D [1,25(OH)2D], and impaired degradation of 1,25(OH)2D. The ingestion of excessive amounts of vitamin D3 (or vitamin D2) results in hypercalcemia and hypercalciuria due to the formation of supraphysiological amounts of 25-hydroxyvitamin D [25(OH)D] that bind to the vitamin D receptor, albeit with lower affinity than the active form of the vitamin, 1,25(OH)2D, and the formation of 5,6-trans 25(OH)D, which binds to the vitamin D receptor more tightly than 25(OH)D. In patients with granulomatous disease such as sarcoidosis or tuberculosis and tumors such as lymphomas, hypercalcemia occurs as a result of the activity of ectopic 25(OH)D-1-hydroxylase (CYP27B1) expressed in macrophages or tumor cells and the formation of excessive amounts of 1,25(OH)2D. Recent work has identified a novel cause of non-PTH-mediated hypercalcemia that occurs when the degradation of 1,25(OH)2D is impaired as a result of mutations of the 1,25(OH)2D-24-hydroxylase cytochrome P450 (CYP24A1). Patients with biallelic and, in some instances, monoallelic mutations of the CYP24A1 gene have elevated serum calcium concentrations associated with elevated serum 1,25(OH)2D, suppressed PTH concentrations, hypercalciuria, nephrocalcinosis, nephrolithiasis, and on occasion, reduced bone density. Of interest, first-time calcium renal stone formers have elevated 1,25(OH)2D and evidence of impaired 24-hydroxylase-mediated 1,25(OH)2D degradation. We will describe the biochemical processes associated with the synthesis and degradation of various vitamin D metabolites, the clinical features of the vitamin D-mediated hypercalcemia, their biochemical diagnosis, and treatment.

Abstract BACKGROUND Patients have been described with loss-of-function CYP24A1 (cytochrome P450, family 24, subfamily A, polypeptide 1) mutations that cause a high ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D [25(OH)D/24,25(OH)2D], increased serum 1,25-dihydroxyvitamin D, and resulting hypercalcemia, hypercalciuria and nephrolithiasis. A 25(OH)D/24,25(OH)2D ratio that can identify patients who are candidates for confirmatory CYP24A1 genetic testing would be valuable. We validated an LC-MS/MS assay for 24,25(OH)2D (D3 and D2) and determined a 25(OH)D/24,25(OH)2D cutoff to identify candidates for confirmatory genetic testing. METHODS After addition of isotope-labeled internal standard, serum samples were extracted by solid-phase extraction, derivatized with 4-phenyl-1,2,4,-triazoline-3,5-dione, and quantified by LC-MS/MS. We measured 25(OH)D/24,25(OH)2D in 91 healthy patients and 34 patients with clinically suspected CYP24A1-mediated hypercalcemia. RESULTS The limits of detection and quantification were 0.03 (0.2) and 0.1 (0.24) nmol/L, respectively, for 24,25(OH)2D3, and 0.1 (0.23) and 0.5 (1.16) nmol/L for 24,25(OH)2D2. Intra- and interassay imprecision was 4%–15% across the analytical measurement range of 0.1–25 ng/mL (0.2–60 nmol/L). No interference was observed with 25(OH)D and 1,25(OH)2D. 25(OH)D/24,25(OH)2D of 7–35 was observed in healthy patients, whereas in 2 patients with CYP24A1 mutations, 25(OH)D/24,25(OH)2D was significantly increased (99–467; P &lt; 0.001). A 25(OH)D/24,25(OH)2D ratio ≥99 identified patients who were candidates for CYP24A1 genetic testing. CONCLUSIONS Increased 25(OH)D/24,25(OH)2D supports the diagnosis of reduced CYP24A1 activity due to mutations in CYP24A1. Measurement of 25(OH)D/24,25(OH)2D should be considered a part of the clinical workup in patients with hypercalcemia of otherwise unknown etiology.

AbstractSecondary hyperparathyroidism almost universally accompanies end‐stage renal disease (ESRD). In some, but not all studies, elevated serum parathyroid hormone (PTH) concentrations are associated with increased fracture rates, cardiovascular disease, and mortality in ESRD. The serum concentration of PTH required for optimal bone health and reduced cardiovascular risk in such patients remains elusive. Recent clinical trials have failed to show substantial changes in morbidity and mortality following reductions of elevated serum PTH concentrations. In this review, we will assess some of the difficulties in evaluating elevated serum PTH concentrations, and their association with skeletal fractures and mortality in ESRD patients. We are of the opinion that in the context of ESRD, elevated PTH concentrations occur in conjunction with other comorbid conditions such as diabetes mellitus, malnutrition, hypertension, volume excess, preexisting heart disease, all of which have prevented establishing a precise association between elevated serum PTH concentrations and global or skeletal outcomes. Age, gender, and racial variability among groups make interpretation exceptionally difficult. Analysis of prevalent ESRD populations with secondary hyperparathyroidism should take all these factors into account. We suggest that future clinical trials which examine the usefulness of reductions in serum PTH concentrations be conducted in age, sex, and racially balanced groups, without or with minimal coexisting confounding disease. Furthermore, trials in such populations should have as their primary outcome a reduction in fractures rather than an alteration in mortality.