Wednesday, April 14, 2010

Kidney diseases and bone phosphate reserve.

PIDAY

 

Rickets

 

Old English term for Rickets was “Wrickken“ as described by Glisson F, Bate G and Regemorter A on 1651 as a common disease present in children. Near 350 years have been elapsed since the first monograph publication on wrickken and many spectacular advances in our understanding of Vitamin D dependent metabolism have enriched our knowledge wallet.

Last century was plenty of relevant discoveries from isolation of nuclear receptor for Vitamin D, identification of metabolic pathways of vitamin D synthesis, activation and degradation, until to isolation of genes coding for enzymes and co-factors as well’s hormonal factors involved into vitamin D cycle.

We can define rickets the defect in bone mineralization leading to formation of a normal bone matrix whithout deposition of calcium salts. Anatomically speaking this alteration in bone structure is called “osteomalacia”, whereas clinically propension to multiple fractures, short stature, bone deformities and renal alterations are often found. Initially the defect was ascribed to a loss of vitamin D expecially in young adults presenting lower exposure to sunlight; later it was clear that different forms of rickets are present; some involving abnormalities in vitamin D metabolism, other involving renal cells alterations.

It’s quite surprising that the horthologues of oncogenes designed such as “Interruptor-1” and “Interruptor-2” have been demonstrated to be involved into phosphate homeostasis. Int-1 is Wnt family growth factors, whereas Int-2 is FGFs family growth factor; both are very important in molecular embriology influencing cellular condensation and diffusion. Phosphate ions with carbon and azote ions, can be considered the base for live organisms in the earth, forming the backbone of all biomolecular structures. It is surprising that a so higly conserved and sophysticated biochemical pathway has been created to conserve adequate levels of phosphate in our organisms.

Interestingly, it is not at present clear the relation between bone loss and kidney stone formation. Excessive bone reabsorption clearly leads to hypercalciuria and hyperphsophaturia for example by excessive production of parathyroid hormone or vitamin D3. Excessive increase in urinary calcium excretion from bone can also be observed in osteogenesis imperfecta and in McCune-Albright syndrome; however, nephrolithiasis is very rare in these disorders.

On the other side, many studies have reported lower bone density in renal calcium stone formers compared to controls. The mechnisms underlying these findings is not clear, but may involve hyper responsiveness to calcitriol or to parathormone or bone abnormalities. However, low bone density in calcum stome formers has not been associated with increased bone resorption or with any gene mutations or polymorphisms.

According to my opinion, whereas osteoporotic bone disease can be truly considered an alteration of bone cells, osteomalacia (rickets) is better considered a bone symptom of a kidney disease. In the first situation we have altered bone turnover due to alterations of bone regulatory factors, in the second we have bone disease as secondary and sporadic manifestation of primary kidney disease.

Among the causes of defects of inadequate mineralization of bone (osteomalacia) and defective mineralization of cartilage (rickets) are renal phosphate wasting disorders that produce hypophosphatemia.

Kidney stones

Urinary PH determines the solubility of various substances in urine, Low PH decreases uric acid solubility but prevents calcium-phosphate crystal formation in contrast to a PH > 7 that augments urate solubility but precipitates calcium-phosphate salts. Urinary PH depends on the proton load in the diet and on the ability of kidney to buffer free proton in the urine. Renal acidosis is due to a defect of the renal tubule in secreting protons while buffers are normally produced, resulting in urinary PH > 5.5 and in metabolic acidosis. Patients with renal acidosis frequently have hypercalciuria and hyperphosphaturia. This may be due to calcium and phosphate release from bone because of proton buffering by bone.

Nephrolithiasis and nephrocalcinosis are frequent in these disorders. Distal renal acidosis is due to mutations in the chloride-bicarbonate exchanger or in proton ATP ase subunits.

Uric acid

Uric acid stones are less frequent than calcium stones and they represent 5 to 10% of nephrolithiasis. Uric acid is the final breakdown product of purine metabolism and it also derives from amino acid catabolism. Two-third of uric acid production are eliminated by the kidneys in humans. Uric acid is filtered at the glomerulus and then it is almost completely reabsorbed in the initial part of the proximal tubule by at least two transporters, called URAT1 , coded by gene SLC22A12) and GLUT9 ( coded by gene SLC2A9). A member of the ATP binding cassette family (ABCG2) is expressed in proximal tubular cells and secretes urate into the urine.

Two factors increase the risk of uric acid stone formation: low PH and hyperuricosuria. Genetic disorders can increase uric acid production or alter urate tubular transport. An increase in uric acid production induces hyperuricemia, as in the case of hypoxantine-guanine- phosphorybosyl transferase or in glucose-6-phosphatase deficiencies, and in phosporybosyl pyrophosphatase synthetase over-activity.

In contrast mutations in urate transporters result in hypouricemia and hyperuricosuria. Hence loss-of-function mutations in the URAT1 transporter decrease urate reabsorption in the proximal tubule. Functional experiments demonstrated that SLC9A2, is expressed at the apical and basolateral sides of proximal tubular cells, it transport urate, and that polymorphisms decrease urate reuptake increasing uric acid secretion in urine.

Inactivating mutations in tha ABCG2 gene have been identified as cause of gout increasing uric acid concentration in plasma.

Oxalate

Elevatd urinary oxalate excretion is critical for the growth of renal stones, Oxalate comes from the diet and it is produced by the liver and arythrocytes from glyoxalate. It is filtered freely at the glomerulus and probably reabsorbed ad then secreted in the proximal tubule. In the intestine, oxalate is also absorbed and secreted, but absorption exceeds secretion.

Enzymatic defects (primary hyperoxalurias) can induce oxalate overproduction.

The gene SLCA26A6 encodes an oxalate-chloride exchanger that is expressed in the intestine and in the renal proximal tubule. The disruption of this gene in mice results in an increase in oxalate plasma concentration, hyperoxaluria and renal calcium oxalate stone formation. The role of SLC26A6 is probably to secrete oxalate in feces. Its role in proximal tubule is not clear. Mutations in this gene have not been identified in humans.

Claudins

The selecive permeability of the intercellular unctions to calcium and magnesium ions is due to expression of claudin-16 , also known such as paracellin. This protein acts as a specific gate for clacium and magnesium. Mutations in claudin 16 abolished the permeability of the intercellular pathway to calcium, and are a rare cause of hypercalciuria, hypomagnesemia and calcium renal stones.

Variants of the gne enchoding claudin 14 have recently been asociated with kidney stones and low bone mineral density in a genome wide association study performed in patients from the Netherlands, Iceland, and Denmark, Claudin 14 is a tight junction protein expressed in the proximal tubule and in the loop of Henle. The mechanism by which these variants are associated with renal stones is unclear since they were not associated with calcium or phosphate concentration in plasma or urine, but only associted with serum PTH and bone markers.

OSTEOMALACIA

Vitamin D metabolism abnormalities

Rickets-Vitamin-D-dependent-type-I 1ahydroxylaseD3 (Ch12q14)

Rickets-Vitamin-D-dependent-type-II              Vit.-D receptor (Ch12q13-14)

Rickets-PseudoVitamin-D-deficiency              1ahydroxylaseD3(Ch12q13.3) point mutation

Kidney Proximal tubular defect

Rickets hypophosphatemic with Hypercalciuria       NTP2c gene (HHRH)(NPT2 genes Ch5q35.1-q35.3)

Autosomal dominant hypophosphatemic Rickets    FGF23 gene (ADHR) OMIM 193100 of the young (Ch.12p13.3)

X Linked Hypophosphatemic Richets                       PHEX gene (HYP) like OMIM adult female (Ch. Xq22.1)

Tumor Induced Osteomalacia                                 FRP4 gene(OHO) (Ch.7q14.1)

Hyperphosphatemic Familial Tumoral Calcinosis          KLOTHO (HFTC) OMIM 21900

Hypeostosis-hyperphosphatemia syndrome                  GALNT3 (HHS) OMIM 610233

Renal Tubular Acidosis type II                              NPT2a gene (Fanconi’s Syndrome) (Ch5q35.1-q35.3)

 

Distal tubular defect

Renal-Tubular-Acidosis-type-I          Basolateral- Cl/HCO3   (RTA Distal)                                               Proton-ATPase

Hyperkalemic RTA type IV    Hyporeninemic Hyperaldosteronism

Bartter’s syndrome              Na-K-2Cl transporter mutation

(Henle loop hyperca)           K channel-calcium sensing receptor

                                       Chloride channel (CICKa-b)-Barttrin

Gitelman’s syndrome          Na-Cl transporter thiazide sensitive

(distal hyperca)

Dent’s disease                   Voltage gated Chloride channel

INHERITED BONE DISEASES

Autosomal dominant Aplasia of                             FGF10 gene

Lacrimal and Salivary glands

Autosomal dominant Cerebellar                             FGF14 gene

Ataxia

Craniosynostosis disorders                  FGFR type 2 (Pro250X)

Achondroplasia                                        FGFR type 3 (Ch4p16.3)

Hypochondroplasia                                  FGFR type 3

Spondyloepiphyseal dysplasia *          FGFR type 3 stop codon

Stickler syndrome *                            FGFR type 3 Lys650Glu

*thanatophoric dysplasia type I and type II.

Pfeiffer syndrome                              FGFR type 1 Pro252Arg

Apert syndrome                                 FGFR type 2 Pro253Arg

Muenke craniosynostosis                   FGFR type 3 Pro 250Arg

Crouzon syndrome                            FGFR type 2 Cys342Arg

Jackson-Weiss syndrome                   FGFR type 2 Cys342Arg

Phosphate wasting is either inherited as X-linked hypophosphatemic rickets or autosomal dominant hypophosphatemic rickets, or acquired, as can occur in patients with a variety of benign mesenchimal tumors such as hemangiopericytomas, fibromas, angiosarcomas. Osteomalacia induced by tumors is invariably curable if the tumor can be found and resected, indicating that it may have an humoral basis. However the pathogenesis of rachitic syndromes requires also defective mineralization coupled with phosphate wasting.

Phosphatonins have been demonstrated to be able to impair the action of kidney 1a hydroxylasis, activating 24-hydroxylasis, and mediating the parathyroid action on many cell types, including kidney proximal cells.

However no clear action at bone forming units has been at present demonstrated by “phosphatonins” per se.

NPT2 and NHERF

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We are understanding the mechanisms exerted by epithelial scaffold proteins in regulation of renal phosphate handling. These PDZ-containing proteins are able to form macromolecular complexes with true Na/Pi channels.

Called Na+/H+ Exchangers Regulatory Factor (NHERFs), they are known to be present on apical microvillar structure (NHERF1) or at the base of microvilli in the vescicle rich domains (NHERF2). They are ancillary cytoplasmic proteins, responsive to hormonal stimulation such as parathyroid hormone, and directing the localization of ion channel proteins (NPT2) at specific sites of cellular membrane. Their action has been demonstrated for microvillar apical membrane of proximal kidney tubular cells, but they probably, as PDZ-containing proteins, are ubiquitariously active in regulating the activity, internalization and recycling, of true receptors.

Their action is strictly dependent from transmembrane potential and finally from different concentration of sodium and potassium ions outside and inside cells respectively. As we know cell life is linked to the presence of this different concentration, allowing the formation of true concentration gradient across a lipid bilayer.

Moreover they are co-responsive of the presence of a difference in proton concentration (i.e.PH) across plasmamembrane. The PH difference across lipid bilayer between inner and extracellular fluid account for a different solubility or organic and inorganic salts as well’s of cathalization of enzymatic reactions possible only at a given PH.

Finally, these apparent unuseful PDZ-containing proteins should give the life to an inhert lipid bilayer; making it responsive to extracellular hormonal signals and so orchestrating the action of an uncohordinated lipid structure.

Their presence at specific sites of plasma membrane explaines the localization and activity of the products of SCL34A1 and SCL34A3 genes located on chromosome 5q35.1-35.3 and coding for (NPT2a) Na/Pi exchanger type IIa and (NPT2c) type IIc respectively. The sodium-phosphate cotransporter NTP-2c is responsible for the bulk of phosphate reabsorption in proximal renal tubules and its alteration is the cause of hypophosphatemic rickets with hypercalciuria. The putative “phosphatonin” should directly inhibit renal sodium-phosphate cotransporter.NPT2c is the primary hormone – regulated renal phosphate transporter, localized at the apical membrane of cells of proximal tubule in each nephron. It accounts for 80% of sodium dependent phosphate reabsorption.Interestingly dietary phosphate load causes a significant down-regulation of NPT-2c.

A distinct apical membrane sodium-phosphate cotransporter called NTP-2a is present in nephrons, sharing homologies with the former responsible for Proximal Renal Tubular Acidosis also called Fanconi’s Syndrome.It is quite consequential that the phosphaturic action exerted by FGF23 at kidney level is done by a down-regulation or block of the action of NTP-2 gene products mediating the reabsorption of phosphate ions.

Hyp mice experiments, showing a 50% reduction in NPT2, and an increase in FGF23 can be a perfect example of what happen in phosphate wasting syndromes.

FGF23

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A paper of Shimada et al. on PNAS (2001) identifies a member of the fibroblast growth factor family, FGF23, as the humoral factor that is secreted by tumors to cause tumor-induced osteomalacia. They cloned a cDNA from a hemangiopericytoma that caused hypophosphatemic osteomalacia and found clones identical to FGF23, which was recently identified by positional cloning as the gene responsible for autosomal dominant hypophosphatemic rickets. When injected into mice the recombinant FGF23 produces mild phosphaturia and hypophosphatemia; interestingly Chinese Hamster Ovary (CHO) cells - FGF23, when grown as tumor in nude mice, fully reproduced the human syndrome of severe hypophosphatemia, growth retardation and rickets in the growth plates, deformities in the skeleton, reduced mineralized matrix and seems of unmineralized osteoid in bone. FGF 23 was expressed at high levels in the tumor from which it was cloned, and as recently reported by another group it is also expressed at high levels in other tumors associated with acquired osteomalacia.

Its expression in bone reach the highest level, in regions of active bone formation, a strong hybridization signal can be seen in osteoblasts lining bone surfaces. Newly formed osteocytes and osteoprogenitor cells are also labeled. In other tissues it has been detected in particular in parathyroid, thymus, brain, heart, and vascular system. If in the past some contraddictory results have been obtained it was due to difficulties in metods of measurements. In particular it is necessary to evaluate if we have to measure the entire FGF23 or only its biologically active portion, its C terminal (Ct) part.

With a 72 aa Ct domain not shared with other family members FGF23 is the largest member of the FGF family. Insight into its functions are provided by demostration that mutations caused hypophosphatemic rickets. Moreover it may seems more soluble that other family members lacking the heparing-binding motif presented in other FGFs.

Whyte KE demonstrated that four unrelated families had a missense mutations in one or two closely spaced arginine residues at position 176 and 179 that cosegregates with rickets, with two families sharing the same mutation. This clustering of missense mutations is a disorder with a dominant inheritance and strongly suggest a “gain of function mutation”.

Shimada et al demonstrated that arginine 179 and S180 is a processing site in FGF23, because they found in CHO cells expressing FGF23, in addition to the mature protein a fragmented protein beginning with S180. It is believed that mutations of the flanking arginine at position 179 could confer a gain of function on FGF23 by blocking its degradation at cleavage site between the unique Ct domain and the FGF-homologue regions.

FGF23 may be the long-sought “phosphatonin”, the phosphaturic factor normally accounting for phosphate homeostasis, independently from parathyroid hormone, so independently from calcium levels. It has been postulated that the main help to FGF23 secretion is the product between the concentration of Calcium and Phosphorus. So acquiring a relevance in regulation of ectopic calcifications such as those present in vascular system with aging.It may be that FGF23 is also secreted by one or more normal tissues as a phosphate regulating hormone, and that the blood level of FGF23 in blood is determined in part by the rate of cleavage by Subtilisin-like proprotein convertase (SPC) at R179/S180. Two fragments are present an entire protein of 32 kDa and C terminal segment of 12 kDa.On 2004 it was demonstrated the complete action of FGF23 on NTP2 renal cotransporter, giving the final answer to identification of FGF23 such as phosphatonin.

Quite recently a longitudinal study demonstrated that in dyalitic patients measurements of FGF23 plasma levels can be useful in assessing normo-phosphatemic patients who should benefits of therapeutic strategies devoted to manage phosphorus balance, considering that hyperphosphatemic hemodyalitic patients show an increased risk of death. This study suggests that hyperphosphatemia in these patients is only partially assessment of risk associated with abnormal phosphorus metabolism. However, measurement of FGF23 could represent a new biomarker in assessing the risk of death in patients with early kidney disease (Wolf M. NEJM August 7, 2008).

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PHEX

The other piece of the hypophosphatemic puzzle is the X-linked hypophosphatemic rickets, the most common disorder of renal phosphate transporter.

PHEX belongs to the M13 family of MA clan of Zn-metalloendopeptidases. The prototypic member of this group of type of integral membrane glycoproteins is Neutral Endopeptidase (NEP). These proteins have a short cytoplasmic N-terminal region, a single transmembrane domain, and a large extracellular C-terminal domain with a zinc binding motif.

Other members of this group include:

. Endothelin Converting Enzyme-1 (ECE-1alfa, ECE-1beta and ECE-2)

. ECE-like enzyme/distress induced neuronal endopeptidase (ECEL1/DINE)

. Soluble Endopeptidase/NEP-like enzyme-1/Neprilysin-2 (NL1/NEP2)

. Membrane Matallo Endopeptidase-like 2 (MMEL-2)

. Kell Blood Group Protein antigen (KELL)

Neprilysin is aslo calle common acute lymphoblastic leukemia antigen (CALLA), or CD10, NEP, or Enkephilinase.

The M13 zinc metallo endopeptidases are integrally involved in several essential elements of cellular regulation and physiology as well’s in diseases including renal function defects, bone mineral loss disorder, cardiovascular diseases, arthritis nd inflammatory disorders.In particular PHEX gene is similar to those of NEP family in several important aspects: numebrs of small exons (22 exons characterized for PHEX with a sequence of 749 aminoacids), higly conserved aminoacid Zinc binding motif (HEFTH fof PHEX, HEITH for NEP).

Informations concerning the structure and nature of the PHEX gene product catalytic site was acquired from the analysis of 99 families affected by X Linked Hypophosphatemic Richets (HYP) and compter generated physichemical data and site-directed mutagenesis studies published for M13 and M3 metallo peptidases.

Interestingly full lenght FGF23 and MEFE do not appear to be PHEX substrates. Remarkably PHEX protects full-leght MEPE from proteolysis, notably by catepsin B cleavage in vitro. In addition, osteocalcin is not degraded by PHEX and inhibits PHEX cleavage of PTHrP.

PTHrP is one of the very few naturally occurring substrates cleaved by PHEX.

Osteocalcin is not cleaved by PHEX, the negatively charged Gla residues in osteocalcin are thought to interact with higly conserved charged present in PHEX. Similar charged region is present in MEPE and ASARM proteins.

In the intact MEPE and PHEX may be associated through the interaction with the MEPE C-terminal ASARM motif. This interaction may not necessarily lead to proteolysis. MEPE-PHEX interaction may therefore prevent proteolytic cleavage and release of ASARM peptide by protecting MEPE from localized matrix proteases. PHEX is localized on plasma membrane surface of osteoblasts, with its extracellular long C terminal region ideally situated in extracellular matrix for protein-protein interactions.

Several PHEX mutations has been detected in patients affected by X Linked Hypophosphatemic Richets (HYP) results in sequestration of disease causing PHEX in endoplasmic reticulum and subsequent failure to targeting to plasmamembrane.

PHEX play a major role in mineralization and it is expressed predominantly in bones and teeth.

The bone expression is localized into osteoblasts, osteocytes (not pre-osteoblasts); in teeth it is present in odontoblasts.

Interestingly loss of function of PHEX results in a defective mineralization. Its action on kidney is expressed modulating renal phosphate handling but not directly, suggesting a secondary involvement in regulation of a circulating systemic factor.

Finally it is reasonable to speculate that PHEX may well function as a small peptide protease and also as a matix-protein ligand.

SPC Subtilisin-like proprotein convertase

The SPC are a family of serine proteases, involved in processing of a wide variety of polypeptides including neuropeptides, growth factors, receptors, blood coagulation factors. Their substrates are cleaved at C terminal side where a specific sequence is present . SPC are present at Golgi apparatus and at trans Golgi network, where they act also on FGF23.

Matrix Extracellular phosphoglycoprotein (MEPE)

MEPE was first cloned from a tumor reseacted from a patients with OHO. It belong to a family of proteins that ahve recently been named Shorth Integrin Binding Ligand Interacting Glycoprotein (SIBLINGs). Between them we have:

. Osteopontin

. Matrix extracellular phosphoprotein (MEPE)

. Dentin Matrix Protein 1

. Bone Sialoprotein

. Dentin Sialo Phosphoprotein

. Enamelin

All mapping on chromosome 4q21 and sharing many properties. All these proteins have a links with bone/dentin mineralization and phosphate/calcium salts.

The structure of these proteins contains RGD motif tipical of integrin ligands, glycosylation pattern very similar between them, phosphorylation pattern again similar, and a so called ASARM motif.

ASRM (Acidic Serine Aspartate Rich MEPE associated motif) described a region of these protein able to block the mineralization. However if it is bounded to other extracellular matrix components it may be required a a nucleator of mineralization itself. The ASARM peptide is very stable and it is resistant to know proteases. Free ASARM peptides may also contribute to inhibition of renal phosphate uptake. This mechanism of action is likely to be steric and exacerbating the effect of NTP2 exchanger protein probably with the aids of FGF23.

The normal action of MEPE is to act such as mineralization inhibitor, due to the presence of ASARM fragment normally released from the entire protein by the cathepsin C cleavage.

Another action of MEPE is a dose dependent inhibition of BMP-2 menediated mineralization in a murine osteoblast cell line in vitro, another effect linked to ASARM presence.

Mice models

From studies in hypophosphatemic mice called “hyp mice” and “gyro mice” it was identified the cause of reduced phosphate reabsorption in a gene located in mouse chromosome X coding for PHEX a metalloproteinase enzyme. This proteins was defective in these mice; in hyp mouse the defect was localized primary in kidney, whereas in gyro mouse the defect was located also in the inner ear and so clinically associated with circling behaviour.

KO mice for Na-Pi cotransporter gene called Npt2, showed Pi renal wasting comparable to inactivation of PHEX gene, but in Npt2 KO mice calcitriol responds appropriately to hypophosphatemic challenges, intestinal absorption of both Pi and Ca ensues, and rickets and osteomalcia are absent.

MEPE KO mice have increased bone mass, resistance to aging associated trabecular bone loss, increased mineralization apposition rate and a dramatically accelerated mineralization rate in ex vivo osteoblasts cultures.

Interestingly Vitamin D3 Receptor KO mice have markedly increased levels of mRNA for MEPE expression.

Comparison between these mice models illustrates that renal phosphate wasting can be dissocated from defective synthesis of calcitriol, implying that phosphatonins have at least two independent actions:

1. they inhibit the Pi reabsorption

2. they impair the synthesis of calcitriol

The different phenotypes in Npt2 KO mice and PHEX KO mice also raise the possibility that FGF23 has a direct affect on bone and cartilage that contribute, along with hypophasphatemia to a defect in mineralization.

HYP mice model hepl us in understanding a possible explanation; the HYP mouse model of PHEX inactivation responds to phosphate deprivation, with continued phosphaturia relative to wilde type mice.

Normally phosphate can be cleared from urine by low dietary Pi intake, protecting against Pi depletion. So that decreased FGF23 secretion could be in this view the humoral factor of this response, coupling with at yet unidentifed phosphate sensor, possibly in the intestinal mucosa, to regulate intestinal phosphate reabsorption. Moreover FGF23 may explaine only a local paracrine regulatory role perhaps even unrelated to Pi homeostasis, and only when it is inappropriately secreted into blood may exert a Pi wasting action.

In this scenario, the phosphate wasting in tumor induced osteomalacia would be analogous to the Pi wasting that occurs when tumors overexpress the PTH-related peptides, PTH-rP. PTH-rP is normally a local regulator of cell differentiation, but when overproduction gives it access to the circulation, it co-opts the PTH receptor in kidney to cause phosphaturia.

Conerning FGF23 it is the first FGFs for which mutations are associated with a disease, and althought the othr 22 FGFs share only 4 known receptors, it is likely that FGF23 has a different receptor, because cleavage of its unique C terminal domain inactivates it.

Tumor Induced Osteomalacia

In literature about 70 cases have been described with such rare form of hypophosphaturia, which occurs in association with coexisting tumor and resolve after its excision; with a possible relapsing episodes.

Hypophosphatemia is probably due to a diminished renal phosphate reabsorption and this phenomenon causes a decrease in 1a hydroxylation of vitamin D3.

Tumor are generally of mesenchymal origin ( such as hemangiopericytomas), but also prostate and breast cancer have been described. They are often small and difficult to locate. From Cai Q data on 1994 it was described a “unidentified soluble factor” heat labile that is devoted normally to control renal phosphate reabsorption.

Oncogenic osteomalacia has been linked to secretion of a Frizzled receptor protein (FRP4) containing cysteine rich ligand binding domain as well’s hydrophilic C terminal region. The normally bound receptor link Wnt proteins in tandem with LPR family co-receptors. The binding to Wnt proteins to frizzled receptors and LPR5/6 coreceptors in heterotrimeric complexes on the cell surfaces leads to stabilization of intrcellular catenin beta and a complex network of singalling cascade.

It has been demonstrated in many cancers both in vivo and in vitro, that this pathways account for bone osteolysis in cancer diffusion to bone tissues.

Moreover some rare inherited disorders are characterized by involvement of Wnt/LPR pathways alterations:

. osteoporosis pseudoglioma syndrome : congenital blindness and severe chilhood osteoporosis

. High Bone Mass syndrome: associated with phenotypical presence of inherited high bone mass level.

FRP4 is located on chromosome 7p14.1 and it is conprised of six encoding exons, spanning 10.8 kb of genomic sequence. The translated protein product consist of 346 aminoacids, of which the first 21 residues constitute the predicted signal peptide. Finally the molecular mass of FRP4 is approximately of 40 kDa, but it is glycosylated to form a mature peptide of 48 kDa. It is ubiquitariously expressed, but importantly on bone cells it is present indicating a possible auto and paracrine effect in the skeleton too.

Concerning phosphate metabolism the study of Berndt et al (2003) revealed that this peptide has the capacity of inhibit the sodium dependent phosphate reuptake in opossum kidney in vitro experiments. Moreover infusion of FRP4 in vivo in parathyroidectomized mice caused an increase in the fractional excretion of phosphate and subsequent hypophosphatemia, indicating a mechansim of action partially independent from PTH.

Hyperphosphatemic Familial Tumoral Calcinosis

Recent work regarding fibroblast growth factor 23 addressed the question of pathogenesis of familial tumoral calcinosis. A missense mutation in the gene encoding FGF23 cause autosomal dominant hypophophatemic rickets. In addition FGF23 is highly expressed by tumors causing oncogenic hypophophatemic osteomalacia. FGF23 is therefore a strong candidate for “phophatonin”, the factor implicated as a cause of the phophate wasting in patients with oncogenic hypophosphatemic osteomalacia. The mutations tht cause autosomal dominant hypophosphatemic rickets stabilizes FGF23, potentially elevating its concentration in serum and leading to renal phosphate wasting. The same would be true in oncogenic hypophosphatemic osteomalacia: i.e. a great production of FGF23 by tumor and so increased levels of plasma FGF.

The clinical response to octreotide therapy as described by Seufert J in a patients may suggest that secretion of fibroblast growth factor 23 by the tumor can be modulated throught the somatostatin receptor signaling pathway. This protein (FGF23) has been demonstrated to be a substrate for the endopeptidase PHEX and to inhibit the phosphate transport in kidney cells. Moreover in autosomal dominant form of hypophosphatemic rickets, mutations in FGF23 have been identified that render the molecule resistant to cleavage by PHEX. So that in these patients octreotide scanning may be useful in identifying such a small tumors or relapses.

 

References

Resnick M, Pridgen DB, Goodman HO. Genetic predisposition to formation of calcium oxalate renal calculi. N Engl J Med 1968;278:1313-8.

Tieder M, Arie R, Modai D et al. Elevated serum 1,25-dihydroxyvitamin D3 concentration in siblings with primary Fanconi’s syndrome. N Engl J Med 1988;319:845-9.

Cai Q, Hodgsan SF, Kao PC et al. Brief report:inhibition of renal phosphate transport by a tumor product in a patient with oncogenic osteomalacia. N Engl J Med 1994;330:1645-9.

Econs MJ, Drezner MK. Tumor induced osteomalacia – unveiling a new hormone. N Engl J Med 1994;330:1679-81.

Seufert J, Ebert K, Muller J et al. Octreotide therapy for tumor induced osteomalacia. N Engl J Med 2001;345:1883-8.

Prié D, Huart V, Bakouh N et al. Nephrolithiasis and osteoporosis associated with hypophosphatemia caused by mutations in the type 2a sodium-phosphate cotransporter. N Engl J Med 2002;347:983-91.

Kronenberg HM. NPT2a – The key to phosphate homeostasis. N Engl J Med 2002;347:1022-4.

Jonsson KB, Zahradnik R, Larsson T et al. Fibroblast growth factor 23 in oncogenic osteomalacia and X-linked hypophosphatemia. N Engl J Med 2003;348:1656-63.

Carpenter TO. Oncogenic osteomalacia – A complex dance of factors. N Engl J Med 2003;348:1705-8.

Hesse E, Rosenthal H, Bastian L. Radiofrequency ablation of a tumor causing oncogenic osteomalacia. N Engl J Med 2007;357:422-4.

Karim Z, Gérard B, Bakouh N et al. NHERF1 mutations and responsiveness of renal parathyroid hormone. N Engl J Med 2008;359:1128-35.

Gutiérrez OM, Mannstadt M, Isakova T et al. Fibroblast growth factor 23 and mortality among patients undergoing hemodialysis. N Engl J Med 2008;359:584-92.

Reilly BM, Hart PD, Mascarell S et al. A question well put. N Engl J Med 2009;360:1446-51.

Magen D, Berger L, Coady MJ et al. A loss of function mutation in NaPiIIa and renal Fanconi’s syndrome. N Engl J Med 2010;362:1102-9.

Thorleifsson G, Holm H, Edvardsson V et al. Sequence variants in the CLDN14 gene associate with kidney stones and bone mineral density. Nat Genet 2009;41:926-30.

NPT2 cotransporter

Tenenhouse HS, Beck L. Renal Na+-phosphate cotrasporter gene expression in X linked Hyp and Gyro mice. Kidney Int 1996;49:1027-32.

Beck L, Karaplis AC, Amizuka N et al. Targeted inactivation of Npt2 in mice leads to severe renal phosphate wasting, hypercalciuria and skeltal abnormalilites. Proc Natl Acad Sci USA 1998;95:5372-7.

Tenenhouse HS, Martel J, Gautier C et al. Renal expression of the sodium/phosphate cotransporter gene, Npt2, is not required for regulation of renal 1 alpha-hydroxylase by phosphate. Endocrinology 2001;142:1124-9.

White KE, Jonsson KB, Carn G et al. The autosomal dominant hypophosphatemic rickets (ADHR) gene is a secreted polypeptide overexpressed by tumors that cause phosphate wasting. J Clin Endocrinol Metab 2001;86:497-500.

Segawa H, Yamanaka S, Ohno Y et al. Correlation between hyperphosphatemia and type II Na-Pi cotransporter activity in KLOTHO mice. Am J Physiol Renal Physiol 2007;292:F769-F779.

NHERF

Mahon MJ, Donwitz M, Yun CC et al. Na/H exchanger regulatory factor 2 directs parathyroid hormone 1 receptor signalling. Nature 2002;417:858-61.

Shenolikar S, Voltz JW, Minkoff CM et al. Targeted disruption of the mouse NHERF1 gene promotes internalization of proximal tubule sodium-phosphate cotransporter type IIa and renal phosphate wasting. Proc Natl Acad Sci USA 2002;99:11470-5.

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PHEX

The HYP Consortium. A gene (PEX) with homologies to endopeptidases is mutated in patients with X-linked hypophosphatemic rickets. Nat Genet 1995;11:130-6.

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FGF23

The ADHR Consortium. Autosomal dominant hypophosphatemic rickets is associated with mutations in FGF23. Nat Genet 2000;26:345-8.

Shimada T, Mizutani S, Muto T et al. Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia. Proc Natl Acad Sci USA 2001;98:6500-5.

Strewler GJ. FGF23, hypophosphatemia, and rickets:has phosphatonin been found? Proc Natl Acad Sci USA 2001;98:5945-6.

White KE, Carn G, Lorenz-Depiereux B et al. Autosomal-dominant hypophosphatemic rickets (ADHR) mutations stabilizes FGF-23. Kidney Int 2001;60:2079-86.

Shimada T, Muto T, Urakawa I et al. Mutant FGF23 responsible for autosomal dominant hypophosphatemic rickets is resistant to proteolytic cleavage and causes hypophosphatemia in vivo. Endorinology 2002;143:3179-82.

Shimada T, Hasegawa H, Yamazzaki Y et al. FGF23 is a potent regulator of vitamin D metabolism and phosphate homeostasis. J Bone Miner Res 2004;19:429-35.

Shimada T, Urakawa I, Yamazaki Y et al. FGF23 transgenic mice demonstrated hypophosphatemic richets with reduced expression of sodium phosphate cotransporter type Iia. Biochem Biophys Res Commun 2004;314:409-14.

Oncogenic Rickets

Brendt T, Craig TA, Bowe AE et al. Secreted frizzled-related protein 4 is a potent tumor derived phosphaturic agent. J Clin Invest 2003;112:785-94.

Kurose K, Sakaguchi N, Nasu Y et al. Decreased expression of REIC/Dkk-3 in human renal clear cells carcinoma. J urol 2004;171:1314-8.

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Tumoral calcinosis

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Topaz O, Shurman DL, Bergman R et al. Mutations in GALNT3, encoding a protein involved in O-linked glycosylation, cause familial tumoral calcinosis. Nat Genet 2004;36:579-81.

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Larsson T, Davis SI, Garringer HJ et al. FGF23 mutants causing familail tumor calcinosis are differentially processed. Endocrinology 2005;146:3883-91.

FGF References

Saunders JW Jr. The proximo-distal sequence of the origin of the parts of the chick wing and the role of ectoderm. J Exp Zool 1948;108:363-403.

Naski MC, Colvin JS, Coffin JD et al. Repression of hedgehog signaling and BMP4 expression in growth plate cartilage by fibroblast growth factor receptor 3. Development 1998;125:4977-88.

Hu MC, Qiu WR, Wang YP et al. FGF-18, a novel member of fibroblast growth factor family, stimulates hepatic and intestinal proliferation. Mol Cell Biol 1998;18:6063-6074

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Sun X et al. Conditional inactivation of FGF4 reveals complexity of signalling during limb bud development. Nature Genet 2000;25:83-6.

FGF-Receptor linked diseases

Keegan K, Johnson DE, Williams LT et al. Isolation of an additional member of the fibroblast growth factor receptor family, FGFR-3. Proc Natl Acad Sci USA 1991;88:1095-9.

Rousseau F, Bonaventure J, Legeai-Mallet L et al. Mutations in the gene encoding fibroblast growth factor receptor-3 in achondroplasia. Nature 1994;371:252-4

Delezoide AL, Benoist-lasselin C, Legeai-Mallet L et al. Spatio-temporal expression of FGFR 1,2 and 3 genes during human embryo-fetal ossification. Mech Dev 1998; 77:19-30.

Tavormina PL, Shiang R, Thompson LM et al. Thanatophoric dysplasia /(types I and II) caused by distict mutations in fibroblast growth factor receptor 3. Nat Genet 1995;9:321-8

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Friday, April 9, 2010

Localized osteomyelitis as cause of prosthetic joint long term failure.

 

PIDAY

 

Known since antiquity, and found as causes of death of Pharahons in Egyptian mummy, osteomyelitis is a difficult-to-treat infection characterized by the progressive inflammatory destruction of bone, with unuseful activation of osteoblasts. The pathogenesis of osteomyelitis has been explored in various animal models; these studies have found that normal bone is highly resistant to infections. However infections can occur as a result of very large inocula, traumatic events, or by the presence of foreign bodies, such as prosthetic joint devices.

Quite recent studies demonstrated that infections associated with prosthetic joints is typically caused by microorganisms that growth in “biofilms”. The definition of biofilm structure has been explained by Greenberg EP on Sience article on 1999 as a common cause of persistent infections. It is formed by microorganisms enclosed in a polymeric matrix into an high density microbial structure, where the volume is so limited to increase the density of cell-to-cell signaling molecules in a rate sufficient to activate bacterial genes, a phenomenon called “quorum sensing”. In the biofilm bacteria are protected from antimicrobial agents and from host immune responses. Biofilm organisms have much greater resistance to antimicrobial killing than do planktonic bacteria. This resistance is related to reduced growth rate of biofilm organisms, whic enter a stationary phase of growth. Probably because of a incomplete penetration of metabolic substances such as glucose and oxygen.

 

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Biomechanics of foof ulcers : in panel A biomechanics of gait, in panel B friction, compression, pressure and shear forces acting during dynamic walking; in panel C consequences of callus formation with abnormal movements, excessive stress and breakdown of connective tissue and muscles. ( adapted from Habershaw and Chzran in NEJM issue n. 11 September 14, 2000)-

The most common microorganism is the coagulase-negative staphylococcus aureus ( both methycillin sensitive or methycillin resistant ) and the molecular mechanisms involved into bacterial adhesion to skin and after to prosthetic joint structures or any foreign bodies is directly related to the presence on two group of factors:

- Non specific factors: such as surface tension, hydrophobicity, and electrostatic interactions

- Specific factors: synthesis of bacterial molecules called adhesins coded by a well known gene tightly regulated by a intercellular adhesin operon (ica).

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Staphylococcus Epidermidis biofilm in foreign body (Robin Patel Mayo Clinic College of Medicine with courtesy)

Probably depending on site of penetration, time required, and foreign bodies molecular structure ( titanium, tantalum, carbonic, ferrous presence ) microorganisms adhere with host proteins such as collagen fibrils, fibrinogen, vonWillebran factor, fibronectin covering foreign bodies immediately after penetration into our body. Adhesins are commonly referred such as MSCRAMM ( microbial surface components recognizing adhesive matrix molecules ).Foreign bodies remain devoid of a microcirculation, which is crucial to host defences and also from delivery of antibiotic drugs.

In particular in an old work Burke JF on 1961 demonstrated that using guinea pig model the subcutaneous injection of Staphylococcus aureus infection was prevented by administration of antibiotics before or shortly after the inoculation of skin with S. aureus, reducing the size of reducing the size of the ensuing skin lesions markedly and that with each delay of an hour in antibiotic administration, the resulting lesion became larger until the third hour. By the fourth hour, the lesion was the same size as in untreated control animals.

The clinical validity of this experimental observation was established in 1969 by Polk and Lopez-Mayor in a study of perioperative and postoperative administration of cephaloridine in which the patients receiving drugs perioperatively presented significantly lower rates of infections compared to patients receiving same drugs postoperatively.

However the presence of subcutaneous foreign body reduces the minimal inoculum needs for S. aureus that is required to cause infection due to the formation of “biofilm” and “quorum sensing effect” by a factor of more than 100.000. This defect is partially related, according to Zimmerli, to a locally acquired granulocyte defect, activation of neutrophils on foreign surfaces results in the release of human neutrophi peptide that deactivated the granulocytes. In fact despite the use of perioperative antimicrobial prophylaxis, since less than 100 colony-forming units of microorganisms can cause an infection.

This experimetal finding has been confirmed by the clinical demonstration that prosthetic joint bone infection can occur not only during or shortly after surgical implants but also during the entire lifetime of the implants and are responsible of most reimplantation needs.

Normal bone remodeling requires the coordination of BMU forming cells (oseoclasts and osteoblasts ). This process is done through the release of a complex network of growth factors from medullary and haematogenous lineages in particular of M-CSF, RANK/OPG/RANKL, IL-1 alfa, IL-7, forming a very complex paracrine pathway able to regulate any single BMU activity.

These bone growth factors deriving mainly by lymphocytes and machrophagic lineages are the same involved into inflammatory answers in any site of our body. It has been postulated that several bacterial components act directly or indirectly as bone modulating factors acting on BMU regulatory pathway. The continuous release of these cytokine signals is increased in states of increased bone remodeling such as during bone fracture repair processes, both sugically or traumatically produced. So that an increase in granulocytes concentration, due to the presence of bacterial biofilm account for the production of granulocytes degeneration with lipid membrane peroxidation, free oxigen radical release, cell necrosis, forming finally a structure like a true ascessual formation.

Pus spreads into vascular channels, raising the intraosseous pressure and impairing blood flow. The ischemic necrosis of bone results in the separatio of devascularized fragments, which are visible also on plain radiographic films, as radiotrasparent areas of demineralized bone called “sequestra”.

In acute forms of osteomyelitis the main features are the presence of microorganisms, infiltration of neutrophils, congested and thrombosed blood vessels, whereas the chronic forms are characterized by necrotic bone whithout living osteocytes.

Concerning the pharmachologic prophylaxis with antibiotics it is clearly evident from these experimental biologic data that they are useful only two hours before the possible subcutaneous access of external bodies, if any advantage is present at all as prophylactic use.

We have to distinguish between antibiotic that can be used prophylactically and those that can be used for therapy; the first ones should have a short half life and higher bioavailability, the second ones should have long half life and should have large spectrum activity, they should have a bactericidal activity against the S. aureus or other bacteria species.

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Patient affected by diabetes, with four days history of fever, left side shoulder and neck pain with numbness and paresthesias in the fingers of hand of the same side. Underwent Positron Emission Tomogrtaphy and  CT scan, because it was possible to perform an MRI scan. It has been demonstrated an abnormal hypermetabolic area on the fourth cervical vertebral body that was consistent with the presence of an infection. After gentamicin and penicillin given endovenously the patient recovered promptly. (From NEJM issue n.23 June 19, 2003)

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An intraoperative photograph taken at first dèbridement procedure (B), in a patient with necrotic mandibular bone (A), 6 weeks postoperatively picture shows healing of a recent extraction site but persistently exposed bone at the site of recent dèbridement. This finding may be seen in patients taking bisphosphonates. (from NEJM issue n.12 March 20, 2008)

The basis of surgical behaviour have been developed after the Results of the Harvard Medical Practice Study published on the Journal in 1991 in two parts. The first part talks about incidence of adverse events and negligence in hospitalized patients, whereas the second art talks abot the nature of these aderse events. From this large longitudinal study it has been identified as source of adverse envents in hospitalized patients Thoracic and Cardiac surgery (10.8 %), Vascular surgery (16.1%), Neurosurgery (9.9%), General surgery (7.0 %), Urologic surgery (4.9%), Orthopedic surgery (4.1 %), General Medicine (3.0 %) according to Diagnosis Related Groups (DRGs) classification. Interestingly rates of adverse events increase with age so that people 65 years old or more have an increased risk (more than doubled) compared to people from 16 to 44 years of age. In particular 16-44 y.o. group shows a rate of adverse event of 2.6%, 45-64 y.o. group shows a rate of adverse eventof 4.4 %, above 65 y.o. the patietns shows a rate of adverse event of 5.7%.

Four classes of DRGs formerly based om Fetter RB classification of 24 subspecialities based on diagnosis related groups, using ICD-9 classification codes, were identified to obtain a DRGs risk groups on a scale from 1 (low) to 6 (high).

1. DRG 1.82 % adverse events acceptable risk event

2. DRG 3.34 % adverse events

3. DRG 4.26 % adverse events

4. DRG 7.13 % adverse events non acceptable risk event

According to causes types of adverse events are reported in this percentage:

Operative risks

Wound infections 13.6 %

Technical complications 12.9 %

Late complications 10.6 %

Nontechnical complications 7.0 %

Surgical failure 3.6 %

Nonoperative risks

Drugs related 19.4 %

Diagnostic mishap 8.1 %

Therapeutic mishap 7.5 %

Procedure related 7.0 %

Fall 2.7 %

Fracture 1.2 %

Postpartum 1.1 %

Anesthesia related 1.1 %

Neonatal 0.9 %

Between drugs related events antibiotic events account for great proportion (16.2 %), then antitumor drugs ( 15.5%), anticoagulant drugs (11.2 %), cardiovascular drugs ( 8.5 %), antiseizure ( 8.1 %), antidiabetic drugs (5.5 %), antihypertensive drugs (5.0 %), analgesic drugs ( 3.5%), antiasthmatic drugs (2.8%), sedative and hypnotics (2.3 %), antidepressant drugs (0.9 %), antipsychotic (0.7 %), peptic ulcer drugs (0.5 %).

Concerning use of antibiotic therapy , it’s quite clear from these statistical notes and the previously mentioned experimental evidences that the emergence of multi-drugs resistant bacteria or the emergence of Staphyloccus aureus new classification based on coagulase negative or positive groups and methycillin resitance or sensitivity, as well’s the resistance to chinolonic drugs can be attributed mainly to the widespread use of these antibiotic therapy and in particular of penicillin classes of drugs in presurgical wrong times (i.e. before two hours from intervention until 24-48 hours before intervention).

The use on two hours and not more before is a quite recent approach of general surgical preparations and notions: only after the pubblication of the study of JP Burke on The Journal issue of 30 January 1992.

If we consider also the different weight in percentage relevance of surgery specialities such as Vascular surgery (16%) compared to General Medicine (3.0%) we can understand the great role of Hospital derived wrong use of antibiotic treatment compared to use in General Practice in the emergence of antibiotic resistence in bacteria.

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