Human parietal epithelial cells (PECs) and proteinuria in lupus nephritis: a role for ClC-5, megalin, and cubilin?

BACKGROUND: Parietal epithelial cells are a heterogeneous population of cells located on Bowman's capsule. These cells are known to internalize albumin with a still undetermined mechanism, although albumin has been shown to induce phenotypic changes in parietal epithelial cells. Proximal tubular cells are the main actors in albumin handling via the macromolecular complex composed by ClC-5, megalin, and cubilin. This study investigated the role of ClC-5, megalin, and cubilin in the parietal epithelial cells of kidney biopsies from proteinuric lupus nephritis patients and control subjects and identified phenotypical changes occurring in the pathological milieu. METHODS: Immunohistochemistry and immunofluorescence analyses for ClC-5, megalin, cubilin, ANXA3, podocalyxin, CD24, CD44, HSA, and LTA marker were performed on 23 kidney biopsies from patients with Lupus Nephritis and 9 control biopsies (obtained from nephrectomies for renal cancer). RESULTS: Two sub-populations of hypertrophic parietal epithelial cells ANXA3+/Podocalyxin-/CD44-, both expressing ClC-5, megalin, and cubilin and located at the tubular pole, were identified and characterized: the first one, CD24+/HSA-/LTA- had characteristics of human adult parietal epithelial multipotent progenitors, the second one, CD24-/LTA+/HSA+ committed to become phenotypically proximal tubular cells. The number of glomeruli presenting hypertrophic parietal epithelial cells positive for ClC-5, megalin, and cubilin were significantly higher in lupus nephritis patients than in controls. CONCLUSIONS: Our results may provide further insight into the role of hypertrophic parietal epithelial cells located at the tubular pole and their possible involvement in protein endocytosis in lupus nephritis patients. These data also suggest that the presence of hypertrophic parietal epithelial cells in Bowman's capsule represents a potential resource for responding to protein overload observed in other glomerulonephritis.

Emerging Perspectives on the Rare Tubulopathy Dent Disease: Is Glomerular Damage a Direct Consequence of ClC-5 Dysfunction?

Dent disease (DD1) is a rare tubulopathy caused by mutations in the CLCN5 gene. Glomerulosclerosis was recently reported in DD1 patients and ClC-5 protein was shown to be expressed in human podocytes. Nephrin and actin cytoskeleton play a key role for podocyte functions and podocyte endocytosis seems to be crucial for slit diaphragm regulation. The aim of this study was to analyze whether ClC-5 loss in podocytes might be a direct consequence of the glomerular damage in DD1 patients. Three DD1 kidney biopsies presenting focal global glomerulosclerosis and four control biopsies were analyzed by immunofluorescence (IF) for nephrin and podocalyxin, and by immunohistochemistry (IHC) for ClC-5. ClC-5 resulted as down-regulated in DD1 vs. control (CTRL) biopsies in both tubular and glomerular compartments (p < 0.01). A significant down-regulation of nephrin (p < 0.01) in DD1 vs. CTRL was demonstrated. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Caspase9) gene editing of CLCN5 in conditionally immortalized human podocytes was used to obtain clones with the stop codon mutation p.(R34Efs*14). We showed that ClC-5 and nephrin expression, analyzed by quantitative Reverse Transcription/Polymerase Chain Reaction (qRT/PCR) and In-Cell Western (ICW), was significantly downregulated in mutant clones compared to the wild type ones. In addition, F-actin staining with fluorescent phalloidin revealed actin derangements. Our results indicate that ClC-5 loss might alter podocyte function either through cytoskeleton disorganization or through impairment of nephrin recycling.

Genotype Phenotype Correlation in Dent Disease 2 and Review of the Literature: OCRL Gene Pleiotropism or Extreme Phenotypic Variability of Lowe Syndrome?

Dent disease is a rare X-linked renal tubulopathy due to CLCN5 and OCRL (DD2) mutations. OCRL mutations also cause Lowe syndrome (LS) involving the eyes, brain and kidney. DD2 is frequently described as a mild form of LS because some patients may present with extra-renal symptoms (ESs). Since DD2 is a rare disease and there are a low number of reported cases, it is still unclear whether it has a clinical picture distinct from LS. We retrospectively analyzed the phenotype and genotype of our cohort of 35 DD2 males and reviewed all published DD2 cases. We analyzed the distribution of mutations along the OCRL gene and evaluated the type and frequency of ES according to the type of mutation and localization in OCRL protein domains. The frequency of patients with at least one ES was 39%. Muscle findings are the most common ES (52%), while ocular findings are less common (11%). Analysis of the distribution of mutations revealed (1) truncating mutations map in the PH and linker domain, while missense mutations map in the 5-phosphatase domain, and only occasionally in the ASH-RhoGAP module; (2) five OCRL mutations cause both DD2 and LS phenotypes; (3) codon 318 is a DD2 mutational hot spot; (4) a correlation was found between the presence of ES and the position of the mutations along OCRL domains. DD2 is distinct from LS. The mutation site and the mutation type largely determine the DD2 phenotype.

Downregulation of megalin, cubilin, ClC-5 and podocin in Fabry nephropathy: potential implications in the decreased effectiveness of enzyme replacement therapy.

Fabry disease is an X-linked disorder due to mutations in α-galactosidase A, resulting in the accumulation of enzyme substrates and cell malfunction. Kidney involvement is frequent, affecting all native kidney cell types. Podocyte damage results in proteinuria and chronic kidney disease. End-stage kidney disease is the rule in middle-aged males and some females with the classic phenotype. In podocytes and kidney proximal tubular cells, megalin is one of the molecules involved in enzyme replacement therapy (ERT) cellular absorption. After podocyte damage, podocin concentration is decreased and contributes to progressive proteinuria. We report in a male and a female patient the decreased expression of megalin, cubilin, ClC-5 and podocin compared to controls and chronic kidney disease (CKD) biopsies. Moreover, the decrease in ClC-5, a molecule engaged in endosomal-lysosomal acidification, could also affect ERT. These findings may partially explain some of the dysfunctions described in Fabry nephropathy and could highlight possible alterations in the pharmacokinetics of the delivered enzyme.

From protein uptake to Dent disease: An overview of the CLCN5 gene.

Proteinuria is a well-known risk factor, not only for renal disorders, but also for several other problems such as cardiovascular diseases and overall mortality. In the kidney, the chloride channel Cl-/H+ exchanger ClC-5 encoded by the CLCN5 gene is actively involved in preventing protein loss. This action becomes evident in patients suffering from the rare proximal tubulopathy Dent disease because they carry a defective ClC-5 due to CLCN5 mutations. In fact, proteinuria is the distinctive clinical sign of Dent disease, and mainly involves the loss of low-molecular-weight proteins. The identification of CLCN5 disease-causing mutations has greatly improved our understanding of ClC-5 function and of the ClC-5-related physiological processes in the kidney. This review outlines current knowledge regarding the CLCN5 gene and its protein product, providing an update on ClC-5 function in tubular and glomerular cells, and focusing on its relationship with proteinuria and Dent disease.

CLCN5 5'UTR isoforms in human kidneys: differential expression analysis between controls and patients with glomerulonephritis.

ClC-5, the electrogenic chloride/proton exchanger strongly expressed in renal proximal tubules, belongs to the endocytic macromolecular complex responsible for albumin and low-molecular-weight protein uptake. ClC-5 was found to be overexpressed in glomeruli of glomerulonephritis and in cultured human podocytes under albumin overload. The transcriptional regulation of human ClC-5 is not fully understood. Three functional promoters of various strengths and 11 different 5' untranslated region (5'UTR) isoforms of CLCN5 messenger RNA (mRNA) were detected in the human kidney (variants 1-11). The aim of this study was to investigate the expression pattern of CLCN5 5'UTR variants and the CLCN5 common translated region in glomerulonephritis. The 5'UTR ends and the translated region of CLCN5 mRNA were analyzed using quantitative relative real-time PCR or quantitative comparative endpoint PCR with GAPDH as housekeeping gene in 8 normal kidneys and 12 renal biopsies from patients with glomerulonephritis. The expression profile for all variants in normal and glomerulonephritis biopsies was similar, and variant 3 and alternative variant 4 were the most abundantly expressed in both sets. In glomerulonephritis biopsies, isoforms under the control of a weak promoter (variants 4, 6 and 7) showed an increased expression leading to an increase in the CLCN5 translated region, underscoring their importance in kidney pathophysiology. Since weak promoters can be turned on by different stimuli, these data support the hypothesis that proteinuria could be one of the stimuli capable of starting a signaling pathway that induces an increase in CLCN5 transcription.

Genetic Analyses in Dent Disease and Characterization of CLCN5 Mutations in Kidney Biopsies.

Dent disease (DD), an X-linked renal tubulopathy, is mainly caused by loss-of-function mutations in CLCN5 (DD1) and OCRL genes. CLCN5 encodes the ClC-5 antiporter that in proximal tubules (PT) participates in the receptor-mediated endocytosis of low molecular weight proteins. Few studies have analyzed the PT expression of ClC-5 and of megalin and cubilin receptors in DD1 kidney biopsies. About 25% of DD cases lack mutations in either CLCN5 or OCRL genes (DD3), and no other disease genes have been discovered so far. Sanger sequencing was used for CLCN5 gene analysis in 158 unrelated males clinically suspected of having DD. The tubular expression of ClC-5, megalin, and cubilin was assessed by immunolabeling in 10 DD1 kidney biopsies. Whole exome sequencing (WES) was performed in eight DD3 patients. Twenty-three novel CLCN5 mutations were identified. ClC-5, megalin, and cubilin were significantly lower in DD1 than in control biopsies. The tubular expression of ClC-5 when detected was irrespective of the type of mutation. In four DD3 patients, WES revealed 12 potentially pathogenic variants in three novel genes (SLC17A1, SLC9A3, and PDZK1), and in three genes known to be associated with monogenic forms of renal proximal tubulopathies (SLC3A, LRP2, and CUBN). The supposed third Dent disease-causing gene was not discovered.

Cell Death in the Kidney.

Apoptotic cell death is usually a response to the cell's microenvironment. In the kidney, apoptosis contributes to parenchymal cell loss in the course of acute and chronic renal injury, but does not trigger an inflammatory response. What distinguishes necrosis from apoptosis is the rupture of the plasma membrane, so necrotic cell death is accompanied by the release of unprocessed intracellular content, including cellular organelles, which are highly immunogenic proteins. The relative contribution of apoptosis and necrosis to injury varies, depending on the severity of the insult. Regulated cell death may result from immunologically silent apoptosis or from immunogenic necrosis. Recent advances have enhanced the most revolutionary concept of regulated necrosis. Several modalities of regulated necrosis have been described, such as necroptosis, ferroptosis, pyroptosis, and mitochondrial permeability transition-dependent regulated necrosis. We review the different modalities of apoptosis, necrosis, and regulated necrosis in kidney injury, focusing particularly on evidence implicating cell death in ectopic renal calcification. We also review the evidence for the role of cell death in kidney injury, which may pave the way for new therapeutic opportunities.

Human proximal tubular cells can form calcium phosphate deposits in osteogenic culture: role of cell death and osteoblast-like transdifferentiation.

Nephrocalcinosis is a clinicopathological entity characterized by microscopic calcium crystals in the renal parenchyma, within the tubular lumen or in the interstitium. Crystal binding to tubular cells may be the cause underlying nephrocalcinosis and nephrolithiasis. Pathological circumstances, such as acute cortical necrosis, may induce healthy cells to acquire a crystal-binding phenotype. The present study aimed to investigate whether human renal proximal tubular cells (HK-2 cells) can form calcium phosphate deposits under osteogenic conditions, and whether apoptosis and/or osteogenic-like processes are involved in cell calcification. HK-2 cells were cultured in standard or osteogenic medium for 1, 5, and 15 days. Von Kossa staining and ESEM were used to analyze crystal deposition. Apoptosis was investigated, analyzing caspase activation by in-cell Western assay, membrane translocation of phosphotidylserine by annexin V-FITC/propidium iodide staining, and DNA fragmentation by TUNEL assay. qRT/PCR, immunolabeling and cytochemistry were performed to assess osteogenic activation (Runx2, Osteonectin, Osteopontin and ALP), and early genes of apoptosis (BAX, Bcl-2). HK-2 cell mineralization was successfully induced on adding osteogenic medium. Calcium phosphate deposition increased in a time-dependent manner, and calcified cell aggregates exhibited characteristic signs of apoptosis. At 15 days, calcifying HK-2 cells revealed osteogenic markers, such as Runx2, ALP, osteonectin and osteopontin. Monitoring the processes at 1, 5, and 15 days showed apoptosis starting already after 5 days of osteogenic induction, when the first small calcium phosphate crystals began to appear on areas where cell aggregates were in apoptotic conditions. The cell death process proved caspase-dependent. The importance of apoptosis was reinforced by the time-dependent increase in BAX expression, starting from day 1. These findings strongly support the hypothesis that apoptosis triggered HK-2 calcification even before any calcium phosphate crystal deposition or acquisition of an osteogenic phenotype.

Caspase-independent programmed cell death triggers Ca2PO4 deposition in an in vitro model of nephrocalcinosis.

Nephrocalcinosis involves the deposition of microscopic crystals in the tubular lumen or interstitium. While the clinical, biochemical, and genetic aspects of the diseases causing nephrocalcinosis have been elucidated, little is known about the cellular events in this calcification process. We previously reported a phenomenon involving the spontaneous formation of Ca2PO4 nodules in primary papillary renal cells from a patient with medullary nephrocalcinosis harboring a rare glial cell-derived neurotrophic factor (GDNF) gene variant. We also demonstrated that cultivating GDNF-silenced human kidney-2 (HK-2) cells in osteogenic conditions for 15 days triggered Ca2PO4 deposits. Given the reportedly close relationship between cell death and pathological calcification, aim of the present study was to investigate whether apoptosis is involved in the calcification of GDNF-silenced HK-2 cells under osteogenic conditions. Silenced and control cells were cultured in standard and osteogenic medium for 1, 5, and 15 days, and any Ca2PO4 deposition was identified by means of von Kossa staining and environmental SEM (ESEM) analyses. Based on the results of annexin V and propidium iodide (PI) analysis, and terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling (TUNEL) assay, the silenced cells in the osteogenic medium showed a significant increase in the percentage of cells in the late phase of apoptosis and an increased Ca2PO4 deposition at 15 days. The results of quantitative real-time PCR (qRT-PCR) of BAX and BCL2, and in-cell Western analysis of caspases indicated that the cell death process was independent of caspase-3, -6, -7, and -9 activation, however. Using this model, we provide evidence of caspase-independent cell death triggering the calcification process in GDNF-silenced HK-2 cells.

Albumin uptake in human podocytes: a possible role for the cubilin-amnionless (CUBAM) complex.

Albumin re-uptake is a receptor-mediated pathway located in renal proximal tubuli. There is increasing evidence of glomerular protein handling by podocytes, but little is known about the mechanism behind this process. In this study, we found that human podocytes in vitro are committed to internalizing albumin through a receptor-mediated mechanism even after exposure to low doses of albumin. We show that these cells express cubilin, megalin, ClC-5, amnionless and Dab2, which are partners in the tubular machinery. Exposing human podocytes to albumin overload prompted an increase in CUBILIN, AMNIONLESS and CLCN5 gene expression. Inhibiting cubilin led to a reduction in albumin uptake, highlighting its importance in this mechanism. We demonstrated that human podocytes are committed to performing endocytosis via a receptor-mediated mechanism even in the presence of low doses of albumin. We also disclosed that protein overload first acts on the expression of the cubilin-amnionless (CUBAM) complex in these cells, then involves the ClC-5 channel, providing the first evidence for a possible role of the CUBAM complex in albumin endocytosis in human podocytes.

Spontaneous calcification process in primary renal cells from a medullary sponge kidney patient harbouring a GDNF mutation.

Medullary nephrocalcinosis is a hallmark of medullary sponge kidney (MSK). We had the opportunity to study a spontaneous calcification process in vitro by utilizing the renal cells of a patient with MSK who was heterozygous for the c.-27 + 18G>A variant in the GDNF gene encoding glial cell-derived neurotrophic factor. The cells were obtained by collagenase digestion of papillary tissues from the MSK patient and from two patients who had no MSK or nephrocalcinosis. These cells were typed by immunocytochemistry, and the presence of mineral deposits was studied using von Kossa staining, scanning electron microscopy analysis and an ALP assay. Osteoblastic lineage markers were studied using immunocytochemistry and RT-PCR. Staminality markers were also analysed using flow cytometry, magnetic cell separation technology, immunocytochemistry and RT-PCR. Starting from p2, MSK and control cells formed nodules with a behaviour similar to that of calcifying pericytes; however, Ca2PO4 was only found in the MSK cultures. The MSK cells had morphologies and immunophenotypes resembling those of pericytes or stromal stem cells and were positive for vimentin, ZO1, αSMA and CD146. In addition, the MSK cells expressed osteocalcin and osteonectin, indicating an osteoblast-like phenotype. In contrast to the control cells, GDNF was down-regulated in the MSK cells. Stable GDNF knockdown was established in the HK2 cell line and was found to promote Ca2PO4 deposition when the cells were incubated with calcifying medium by regulating the osteonectin/osteopontin ratio in favour of osteonectin. Our data indicate that the human papilla may be a perivascular niche in which pericyte/stromal-like cells can undergo osteogenic differentiation under particular conditions and suggest that GDNF down-regulation may have influenced the observed phenomenon.

Further insights about the beneficial effects of n-3 fatty acids in the early molecular events of renal fibrosis in vitro.

BACKGROUND: Accumulating experimental and clinical evidence reveals beneficial effects of n-3 polyunsaturated fatty acids (PUFAs) in kidney disease by modulating inflammation and fibrosis mechanisms that lead to renal failure. METHODS: EPA, DHA (n-3 PUFAs) and AA (n-6 PUFA) effects, compared to those of AngII, on renal fibrotic processes at the extracellular matrix (ECM) level were verified in human mesangial cells in vitro, by means of RT-PCR, mitogenic assay and Western-blot analysis. RESULTS: Unlike AngII, EPA and DHA enhanced the expression of MMP2 and DN, a TGFbeta inhibitor, while decreasing mitogenic factors such as PDGF and bFGF, and cell proliferation. Moreover, n-3 PUFAs elicited Bax expression in AngII-treated cells and downregulated COX-2--an enzyme involved in the inflammatory cascade. The mechanism of action could implicate PPARgamma activation, as this transcription factor was shown to translocate to the nucleus upon n-3 PUFA treatment. CONCLUSIONS: These results complement our previous reports demonstrating that EPA and DHA prevent ECM accumulation and inflammation that typify the fibrotic process, providing new insights into the cellular and molecular mechanisms underlying their beneficial effects. We confirm that n-3 PUFAs could effectively counteract kidney fibrosis development providing a rationale for their use in clinical settings.

EPA and DHA suppress AngII- and arachidonic acid-induced expression of profibrotic genes in human mesangial cells.

BACKGROUND: There is some evidence suggesting a close relationship between polyunsaturated fatty acids (PUFAs) and renal inflammation and fibrosis, which are crucial stages in chronic kidney disease. METHODS: To verify the role of PUFAs in renal fibrosis processes, we investigated the effects of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) on the gene expression of TGFbeta, fibronectin (FN), connective tissue growth factor (CTGF) and type IV collagen (COLIV) in human mesangial cells, in the absence or presence of angiotensin II (AngII), using reverse transcriptase PCR. RESULTS: The addition of AA to mesangial cell cultures induced a significant up-regulation of TGFbeta, FN, CTGF and COLIV expression, similar to that induced by AngII, while EPA and DHA had no stimulatory effects. The coincubation of cells with AngII and AA potentiated AngII-induced gene expression; on the contrary, the coexposure of cells to EPA or DHA suppressed the AngII- and AA-induced up-regulation of TGFbeta, FN, CTGF and COLIV. CONCLUSION: We conclude that the PUFAs have different effects, dependent on their chemical structure, on the AngII-TGFbeta system, a major regulator of the renal fibrotic process. Our in vitro results may provide new therapeutic options toward interrupting the irreversible process of renal fibrosis and ameliorating chronic renal injury.

Effects of unsaturated free fatty acids on adhesion and on gene expression of extracellular matrix macromolecules in human osteoblast-like cell cultures.

To investigate the possible role for unsaturated free fatty acids in osteoblast adhesion, the effects of two polyunsaturated fatty acids (PUFAs), arachidonic (AA) and eicosapentaenoic (EPA) acids, and of one monounsaturated fatty acid, oleic acid (OA), on adhesion to the substrate and on gene expression of three extracellular matrix macromolecules were investigated in an in vitro model system--cultured osteoblast-like human cells. AA, but neither EPA nor OA, diminished bone cell adhesion, whereas both EPA and OA, but not AA, increased gene expression of type I collagen and fibronectin via a transforming growth factor-beta-independent mechanism. These results extend previous evidence for unsaturated fatty acids in bone cell metabolism.

Arachidonic acid influences intracellular calcium handling in human osteoblasts.

The effect of arachidonic acid (AA) on intracellular Ca(2+) concentration ([Ca(2+)]i) in human osteoblasts MG63 was studied. AA caused a concentration-dependent increase in [Ca(2+)]i, mainly due to inward Ca(2+) transport from extracellular environment. Moreover, AA in Ca(2+) -free medium produced a small, transient increase of [Ca(2+)]i, indicating that AA may also trigger Ca(2+) release from intracellular stores. Because the [Ca(2+)]i response to AA was inhibited by the cyclooxygenase (COX) inhibitor indomethacin, we tested the effect of prostaglandins (PGs), products of COX pathway. PGs E1 and E2 caused an increase in [Ca(2+)]i, which, however, was far lower than that obtained with AA. The [Ca(2+)]i response to AA was not inhibited by nifedipine, suggesting that AA did not activate a voltage-dependent Ca(2+) channel. Our results indicate that AA could modulate [Ca(2+)]i in MG63 human osteoblasts, where it may influence Ca(2+) transport across both plasma and endoplasmic membranes. Furthermore, they suggest that osteoblast activity may be modulated by AA.

Relationship between plasma phospholipid polyunsaturated fatty acid composition and bone disease in renal transplantation.

To investigate the relationship between polyunsaturated fatty acid (PUFA) and bone metabolism in renal transplant patients, plasma phospholipid (PP) PUFA levels, biochemical markers of bone turnover and bone mineral density (BMD) were determined in 22 recipients of a first renal allograft at baseline and after a mean 24.4 month follow-up. A significant increase in PP n-3 PUFA content, in the [n-3 PUFA/ arachidonic acid] ratio and in BMD values was observed, as well as a close correlation between the increase in PP n-3 PUFA content and femoral neck BMD. Multivariate regression analysis showed that BMD improvement was positively related to PP n-3 PUFA variation and baseline PP eicosapentaenoic acid levels, and negatively to PP arachidonic acid modification. Tacrolimus- versus cyclosporine-treated patients demonstrated a significant increase in femoral neck BMD and PP n-3 PUFA content. This is the first longitudinal study showing a link between PP-PUFA composition and bone disease in renal transplantation.

Specific effect of arachidonic acid on inducible nitric oxide synthase mRNA expression in human osteoblastic cells.

A specific modulatory effect of PUFAs (polyunsaturated fatty acids) on gene expression of some cytokines involved in bone remodelling has been reported previously. In particular, although a direct action of AA (arachidonic acid) on bone cytokine gene expression has been shown in human osteoblastic cells, OA (oleic acid) and EPA (eicosapentaenoic acid) were ineffective. Since the NO (nitric oxide) system has also been shown to have an important modulatory activity on osteoblasts, osteoclasts and bone metabolism, in the present study we have investigated the effects of PUFAs on iNOS (inducible NO synthase) gene expression in a human osteoblast-like cell line. AA induced a significant increase in iNOS mRNA expression, whereas EPA and OA had no stimulatory effects but instead caused a significant inhibition of AA-induced iNOS gene expression. Blocking of the COX (cyclo-oxygenase) pathway did not inhibit AA-induced iNOS expression. AA action was inhibited instead by the addition of calphostin C and genistein, inhibitors of PKC (protein kinase C) and tyrosine kinases respectively. Experiments performed with specific anti-cytokine antibodies showed a significant decrease in iNOS expression in AA-treated osteoblastic cells, suggesting that both cytokine-dependent and -independent mechanisms account for the effects of AA on iNOS gene expression. In conclusion, our investigation clearly shows specific effects of PUFAs on iNOS expression in human osteoblast-like cells with a cytokine-dependent and -independent mechanism. These results might have clinical relevance and are of interest for understanding the reported beneficial effects of dietary PUFA manipulation on the prevention and/or treatment of primary and secondary bone disease.