Progression of early phase diabetic nephropathy in streptozotocin-induced diabetic rats: evaluation of various kidney-related parameters

Diabetic nephropathy (DN) is one of the serious secondary complications of diabetes, which results in end-stage renal failure. Reports on the progressive nature of early phase DN especially with respect to kidney parameters such as kidney weight, type IV collagen excretion, total kidney and urinary glycosaminoglycans (GAGs) are few. This work was undertaken to determine systematically the progression of early phase DN in relation to various kidney-related parameters for a period of four months. Experimentally-induced diabetic rats were grouped based on fasting blood glucose levels. Various basic and kidney-related parameters such as kidney weight, microalbuminuria, urinary excretion of GAGs and type IV collagen, total kidney GAGs, histopathology, glomerular area and glomerular volume were examined in control and diabetic rats. There was a progressive increase in fasting blood sugar, urine sugar, kidney weight, microalbuminuria, urine glycosaminoglycans, urine type IV collagen, glomerular area and glomerular volume but there was a progressive decrease in kidney glycosaminoglycans. Glomerular sclerotic condition was aggravated with the increase in duration of diabetes from 1 to 4 months. Onset of DN in rats begins subtly after one month of diabetes but gets vitiated and more pronounced at the end of four months.     

Febuxostat (TMX-67), a novel, non-purine, selective inhibitor of xanthine oxidase, is safe and decreases serum urate in healthy volunteers

In order to evaluate the safety, pharmacological properties, and urate-lowering efficacy of febuxostat, a non-purine, selective inhibitor of xanthine oxidase, a Phase 1, 2-week, multiple-dose, placebo-controlled, dose-escalation study was conducted in 154 healthy adults of both sexes. Daily febuxostat doses in the range 10 mg to 120 mg resulted in proportional mean serum urate reductions ranging from 25% to 70% and in proportional increases in maximum febuxostat plasma concentrations and area under plasma concentration versus time curves. Accompanying the hypouricemic effect were increases in serum xanthine concentrations, decreases in urinary uric acid excretion, and increases in urinary xanthine and hypoxanthine excretion, confirming inhibition of xanthine oxidase activity by febuxostat. Hepatic conjugation and oxidative metabolism were the major pathways of elimination of febuxostat from the body, and renal elimination did not appear to play a significant role. Although not uncommon, adverse events were mild and self-limited, and no deaths or serious adverse events were observed. Febuxostat is a safe and potent hypouricemic agent in healthy humans.     

Comparison between perindopril and nifedipine in hypertensive and normotensive diabetic patients with microalbuminuria. Melbourne Diabetic Nephropathy Study Group.

OBJECTIVE--To compare the efficacy of angiotensin converting enzyme inhibition with calcium antagonism in diabetic patients with microalbuminuria. DESIGN--Randomised study of diabetic patients with microalbuminuria treated with perindopril or nifedipine for 12 months and monitored for one or three months after stopping treatment depending on whether they were hypertensive or normotensive. Patients were randomised separately according to whether they were hypertensive or normotensive. SETTING--Diabetic clinics in three university teaching hospitals. PATIENTS--50 diabetic patients with persistent microalbuminuria. In all, 43 completed the study: 30 were normotensive and 13 hypertensive; 19 had type I diabetes and 24 had type II diabetes. INTERVENTIONS--For 12 months 20 patients were given perindopril 2-8 mg daily and 23 were given nifedipine 20-80 mg daily. MAIN OUTCOME MEASURES--Albumin excretion rate, blood pressure, and glomerular filtration rate. RESULTS--Both perindopril and nifedipine significantly reduced mean blood pressure. During treatment there was no significant difference between those treated with perindopril and those treated with nifedipine with respect to albuminuria or mean blood pressure. Stopping treatment with both drugs was associated with a sustained increase in albuminuria and mean blood pressure. There was a significant correlation between mean blood pressure and albuminuria and also between the reduction in mean blood pressure and the decrease in albuminuria during treatment with both drugs. In hypertensive patients both drugs caused significant decreases in mean blood pressure and albuminuria. In normotensive patients there was no significant reduction in albuminuria with either regimen. CONCLUSIONS--In diabetic patients with microalbuminuria blood pressure seems to be an important determinant of urinary albumin excretion. Perindopril and nifedipine have similar effects on urinary albumin excretion, both preventing increases in albuminuria in normotensive patients and decreasing albuminuria in hypertensive patients.     

Febuxostat (TMX‐67), a Novel,Non‐Purine,Selective Inhibitor of Xanthine Oxidase,Is Safe and Decreases Serum Urate in Healthy Volunteers

In order to evaluate the safety, pharmacological properties, and urate‐lowering efficacy of febuxostat, a non‐purine, selective inhibitor of xanthine oxidase, a Phase 1, 2‐week, multiple‐dose, placebo‐controlled, dose‐escalation study was conducted in 154 healthy adults of both sexes. Daily febuxostat doses in the range 10 mg to 120 mg resulted in proportional mean serum urate reductions ranging from 25% to 70% and in proportional increases in maximum febuxostat plasma concentrations and area under plasma concentration versus time curves. Accompanying the hypouricemic effect were increases in serum xanthine concentrations, decreases in urinary uric acid excretion, and increases in urinary xanthine and hypoxanthine excretion, confirming inhibition of xanthine oxidase activity by febuxostat. Hepatic conjugation and oxidative metabolism were the major pathways of elimination of febuxostat from the body, and renal elimination did not appear to play a significant role. Although not uncommon, adverse events were mild and self‐limited, and no deaths or serious adverse events were observed. Febuxostat is a safe and potent hypouricemic agent in healthy humans.     

Stop-codon read-through for patients affected by a lysosomal storage disorder

Lysosomal storage disorders are a group of inherited diseases that can result in severe and progressive pathology due to a specific lysosomal dysfunction. Current treatment strategies include bone-marrow transplantation, substrate reduction, chemical-chaperone and enzyme-replacement therapy. However, each of these treatments has its limitations. Enhanced stop-codon read-through is a potential alternative or adjunct therapeutic strategy for treating lysosomal-storage-disorder patients. Premature stop-codon mutations have been identified in a large cohort of patients with a lysosomal storage disorder, making stop-codon read-through a possible treatment for this disease. In lysosomal-storage-disorder cells (mucopolysaccharidosis type I, alpha-L-iduronidase deficient), preclinical studies have shown that gentamicin induced the read-through of premature stop codons, resulting in enzyme activity that reduced substrate storage.     

Alterations in Oxidative Markers in the Cerebellum and Peripheral Organs in MPS I Mice

Mucopolysaccharidosis type I is a lysosomal storage disease with alterations in several organs. Little is known about the pathways that lead to the pathology. Evidences point oxidative stress on lysosomal storage diseases and mucopolysaccharidosis type I. The aim of the present study was to evaluate oxidative biomarkers on mucopolysaccharidosis type I mice model. We evaluated antioxidant enzymatic activity, protein damage and lipid peroxidation in the forebrain, cerebellum, heart, lung, diaphragm, liver, kidney and spleen. Superoxide dismutase activity was increased on cerebellum, lung, diaphragm, liver and kidney of mucopolysaccharidosis type I mice. Catalase activity was increased on cerebellum, spleen and lung. There was no alteration on glutathione peroxidase activity on any of the analyzed organs. Mucopolysaccharidosis type I mice showed increased carbonyl groups on cerebellum, heart and spleen. There was a decrease of thiobarbituric acid-reactive substances on the cerebellum of mucopolysaccharidosis type I mice. The results indicate a oxidative imbalance in this model. As lysosomes are very susceptible to oxidative damage, leading inclusive to cellular death, and lysosomal storage diseases present several alterations on this organelles, this finding can help to elucidate the cellular damage pathways on mucopolysaccharidosis type I.     

Characterization of plasma glycosaminoglycans in hemodialysis patients]

Glycosaminoglycans are heteropolysaccharides composed of disaccharide repeating subunits, each one containing a uronic acid component (glucuronic or iduronic acid) and a hexosamine (N-acetyl-glucosamine or N-acetyl-galactosamine, which may be differently sulphated). The presence of GAGs in human plasma has been demonstrated in several studies; they are bound to plasma proteins through non-covalent linkages. However, very little is known about either their origin or their physiological role. Due to their anionic charge, they may influence some metabolic processes, such as blood coagulation, and they could also have a role in urolithiasis and atherogenesis. Moreover, they may be important in modulating the metabolism of some lipoproteins by affecting the rate of their catabolism. Modifications of GAG pattern have been described in a few pathological conditions such as mucopolysaccharidosis, connective tissue diseases and kidney diseases. A high frequency of accelerated atherosclerosis has been observed in haemodialysis patients (HD), probably associated with the altered lipoprotein profile, which is often described in these subjects. Since GAGs may play a role in lipoprotein metabolism, we isolated and characterized plasma GAGs from a group of HD patients and a group of normal matched subjects. Quantitative analysis of plasma GAGs showed a significant increase of these polysaccharides in the HD group. Circulating levels of GAGs were 8.21 +/- 1.89 micrograms/ml in control subjects, and 15.08 +/- 3.13 micrograms/ml in the HD group (p < 0.0001). The isolation of plasma GAGs by ion-exchange chromatography produced two uronic acid containing families: a low-charge (peak I) and a high-charge (peak II) species. Both of these contained GAGs associated with plasma proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selected indicators of calcium-phosphate metabolism in patients with diabetes mellitus

The changes in blood serum concentrations of calcium, magnesium, inorganic phosphate, total activity of alkaline phosphatase and the activity of its bone fraction, as well as urinary excretion of calcium, phosphate, hydroxyproline and oxalate have been measured in 31 patients with insulin-dependent (type I) diabetes, in 31 patients with non-insulin-dependent (type II) diabetes and in 29 healthy subjects in the condition of low-calcium diet. The elevated urinary excretion of calcium, phosphate, hydroxyproline and oxalate, lowered blood serum level of magnesium, and increased total and bone fraction activities of alkaline phosphatase were found in diabetic patients. The urinary excretion of calcium and hydroxyproline, and the activity of bone fraction alkaline phosphatase were significantly higher in patients with type II diabetes than in those with type I diabetes. It was concluded that there is a significant relation between the state of metabolic normalization of diabetes and the degree of biochemical aberrations concerning calcium-phosphate metabolism.     

The suramin-treated rat as a model of mucopolysaccharidosis: reversibility of biochemical and morphological changes in the liver

Rats treated with the trypanocidal drug suramin, a potent inhibitor of several lysosomal enzymes, develop a storage disorder which mimics the features of mucopolysaccharidosis (Constantopoulos et al. 1983). In this paper we have examined the reversibility of the biochemical and pathological changes induced in the liver of the suramin-treated rat. Rats were injected with a single intravenous dose of suramin (250 mg/kg) and allowed to survive for periods of up to 6 months. The liver was examined for suramin content, pathological changes, biochemical storage of glycosaminoglycans (GAGs) and for the blockade of the relevant hydrolytic enzymes. GAG storage in the liver peaked at approximately 14 days after administration of suramin when there was a five-fold increase in the GAG content. Thereafter GAGs decreased in parallel with the fall of suramin concentrations so that within 6 months the content had returned to control levels. The activity of most of the enzymes tested had also returned to control levels within 6 months. The pathological changes which are evident in the liver 1-2 weeks after administration of the drug had diminished considerably within 6 months. These results indicate that significant reversibility of both the biochemical and pathological changes induced by suramin occurs and they support the suitability of the suramin treated rat as a model to assess the value of therapeutic treatments of mucopolysaccharidosis.     

Hertiable Disorders of Mucopolysaccharide Metablism

The heritable diseases of mucopolysaccharide metabolism have undergone reclassification during the past several years as a result of advances in knowledge. Recognition of clinical, genetic and biochemical differences has made it possible to delineate six types of mucopolysaccharidosis. All types are characterized by excessive excretion of one or more acid mucopolysaccharides in the urine.The underlying defect is not known at present, but recent investigations have suggested possible defects in protein binding and increased biosynthesis and storage of the various mucopolysaccharides.Treatment of these disorders has been unrewarding, although administration of corticosteroids may be of some benefit.Several diagnostic tests are now available for the determination of excessive urinary acid mucopolysaccharide excretion.     

Autocatalytic cleavage of ADAMTS-4 (Aggrecanase-1) reveals multiple glycosaminoglycan-binding sites

ADAMTS-4, also referred to as aggrecanase-1, is a glutamyl endopeptidase capable of generating catabolic fragments of aggrecan analogous to those released from articular cartilage during degenerative joint diseases such as osteoarthritis. Efficient aggrecanase activity requires the presence of sulfated glycosaminoglycans (GAGs) attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In the present study, we demonstrate that full-length ADAMTS-4 (M(r) approximately 68,000) undergoes autocatalytic C-terminal truncation to generate two discrete isoforms (M(r) approximately 53,000 and M(r) approximately 40,000), which exhibit a marked reduction in affinity of binding to sulfated GAGs. C-terminal sequencing and mass analyses revealed that the GAG-binding thrombospondin type I motif was retained following autocatalysis, indicating that sites present in the C-terminal cysteine (cys)-rich and/or spacer domains also effect binding of full-length ADAMTS-4 to sulfated GAGs. Binding-competition experiments conducted using native and deglycosylated aggrecan provided direct evidence for interaction of the ADAMTS-4 cysteine-rich/spacer domains with aggrecan GAGs. Furthermore, synthetic peptides mimicking putative (consensus) GAG-binding sequences located within the ADAMTS-4 cysteine-rich and spacer domains competitively blocked binding of sulfated GAGs to full-length ADAMTS-4, thereby identifying multiple GAG-binding sites, which may contribute to the regulation of ADAMTS-4 function.     

Immobilization on the day 14th does not disrupts the basic diurnal rhythm of bone resorption

Weight bearing and physical activity are important mechanical stimuli to bone growth and metabolism, and microgravity, such a space flight and/or bed rest, induces bone resorption and bone loss. An increased excretion of urinary Ca, an increased bone resorption and a decreased bone mineral density (BMD) have been observed in bed rest experiment of healthy subjects. Bone resorption markers show the specific circadian rhythms in human. Cross-linked carboxyl-terminal telopeptide of type I collagen (ICTP) and the urinary excretion of deoxypyridinoline (Dpy) are the highest in the early morning and the lowest late at night. Bed rest immobilization might influence these rhythms, due to no mechanical loading with loss of daily life activity. Bone resorption markers in healthy subjects had been compared between before and during bed rest to determine disruption of diurnal rhythms of bone resorption.     

Experimental mucopolysaccharidosis: preservation and ultrastructural visualization of intralysosomal glycosaminoglycans by use of the cationic dyes cuprolinic blue and toluidine blue

The cationic dyes Cuprolinic Blue (CB) and Toluidine Blue (TB) were used to preserve the intralysosomal storage material accumulating in tilorone-induced mucopolysaccharidosis. As shown in previous studies, the stored glycosaminoglycans (GAGs) are leached during the conventional fixation procedure, with the result that the lysosomes appear empty. In the present study, the liver, spleen, and cornea-conjunctiva of tilorone-treated rats were examined. The application of CB in the presence of 0.1 M or 0.3 M MgCl2 simultaneously with, or subsequently to the primary fixative yielded electron-dense precipitates within the storage lysosomes. When TB (0.1%) was added to the primary fixative, the storage lysosomes contained filamentous structures arranged in reticular patterns. With increasing TB concentrations (up to 1%) the lysosomes increasingly often showed apparently amorphous storage material which was continuous with the reticular filamentous structures. Similar ultrastructural patterns were obtained with GAG-TB complexes prepared in vitro. The intralysosomal storage material preserved by TB is interpreted as GAG-TB precipitates. In conclusion, the use of CB provides a method which allows direct cytochemical demonstration of the subcellular sites of GAG-storage. The use of TB represents an easy method to obtain electron micrographs pathognomonic of the mucopolysaccharidosis induced by tilorone and congeners. Either method may be helpful to detect this adverse drug effect at the subcellular level.     

Effects of barefoot habituation in winter on thermal and hormonal responses in young children--a preliminary study

This study investigated how socks-wearing habit or habitual barefoot in the cold winter affected skin temperatures of distal lower extremities, the urinary excretion of adrenaline, noradrenaline and cortsisol in young children. In Experiment I for preschool children, the measurements of foot and leg skin temperatures were conducted for 30 min in the classroom controlled at 23+/-2 degres C and 50+/-5%RH, and the excretion of urinary catecholamines and cortisol during nocturnal sleep were analyzed. In Experiment II for elementary school children, nocturnal secretion of urinary catecholamines and cortisol was analyzed. While leg skin temperature tended to be lower in barefoot group than in socks group during Experiment I, foot skin temperature was not significantly different between the two groups. Fall of leg skin temperature during 30 min measurement tended to be smaller in barefoot group than in socks group. Urine volume and urinary excretion of cortisol tended to be greater in barefoot group than in socks group for preschool children. Urinary noradrenaline was significantly greater and cortisol tended to be greater in barefoot group than in socks group for elementary school children. Considering that most of the findings shown above were in the proximity of the established level of statistical significance, it was provisionally concluded that young children with barefoot habituation might show more effective cold adaptation of metabolic type than those without the habituation do, by keeping their skin temperatures higher even in the cold and enhancing the metabolic rate.     

Low sulfated glycosaminoglycans are excreted in patients with the Lowe syndrome

Glycosaminoglycans (GAGs) were prepared from the urine of three patients and from normal individuals by cetylpyridinium chloride precipitation and Pronase digestion. The GAGs were analyzed by electrophoresis, anion-exchange chromatography, and enzymatic and chemical degradation. Each of the three patients showed a four- to fivefold increase in urinary GAG excretion compared to normal controls and in one patient a tenfold increase was measured during a period of behavioral agitation which included joint swelling. Urinary GAGs from affected individuals were characterized by a high proportion of low sulfated molecules. The predominant low sulfated component was chondroitin-4-sulfate (C4S); however, small amounts of chondroitin-6-sulfate (C6S) were also present. Heparan sulfate (HS) was present in normal proportion (5-10%) and most of it was not low sulfated. Abnormal excretion of chondroitin (Ch), hyaluronic acid (HA), and dermatan sulfate (DS) was not detected. These findings suggest that the clinical manifestations of Lowe syndrome may be caused by a defect in GAG metabolism.     

On the alcianophilia of the drug suramin used as a tool for inducing experimental mucopolysaccharidosis

The trypanocidal drug suramin was previously reported to induce mucopolysaccharidosis in rats; apart from the biochemical demonstration of increased tissue concentrations of sulfated glycosaminoglycans (GAGs), a strongly positive staining reaction with the cationic dye Alcian Blue was taken as indicating GAG-storage (Constantopoulos et al. 1983). The purpose of the present report is to point out a methodical pitfall. In model experiments it was found that suramin itself, being a polysulfated compound, gives a strongly positive reaction with Alcian Blue at pH 1. It is known that suramin is accumulated in the lysosomes and that high drug concentrations are retained in the tissues for weeks. Therefore a positive staining reaction with Alcian Blue observed in a given cell cannot be conclusively attributed to the storage of sulfated GAGs as has been done in the past. The present report may be a warning that, in the case of the suramin-induced animal model of mucopolysaccharidosis, the usual histochemical strategy, i.e. staining with cationic dyes, is not suitable for analysing the cellular distribution pattern of GAG-storage, since the inducing drug by itself reacts with the indicator dye.     

On the alcianophilia of the drug suramin used as a tool for inducing experimental mucopolysaccharidosis

Summary The trypanocidal drug suramin was previously reported to induce mucopolysaccharidosis in rats; apart from the biochemical demonstration of increased tissue concentrations of sulfated glycosaminoglycans (GAGs), a strongly positive staining reaction with the cationic dye Alcian Blue was taken as indicating GAG-storage (Constantopoulos et al. 1983). The purpose of the present report is to point out a methodical pitfall. In model experiments it was found that suramin itself, being a polysulfated compound, gives a strongly positive reaction with Alcian Blue pH 1. It is known that suramin is accumulated in the lysosomes and that high drug concentrations are retained in the tissues for weeks. Therefore a positive staining reaction with Alcian Blue observed in a given cell cannot be conclusively attributed to the storage of sulfated GAGs as has been done in the past. The present report may be a warning that, in the case of the suramin-induced animal model of mucopolysaccharidosis, the usual histochemical strategy, i.e. staining with cationic dyes, is not suitable for analysing the cellular distribution pattern of GAG-storage, since the inducing drug by itself reacts with the indicator dye.     

Receptor type I and type II binding regions and the peptidyl-prolyl isomerase site of cyclophilin B are required for enhancement of T-lymphocyte adhesion to fibronectin

Cyclophilin B (CyPB), a cyclosporin A (CsA) binding protein, interacts with two types of binding sites at the surface of T-lymphocytes. The type I sites correspond to functional receptors involved in endocytosis and the type II sites to sulfated glycosaminoglycans (GAGs). Mutational analysis of CyPB has revealed that W128, which is part of the CsA-binding pocket, is implicated in the binding to the functional type I receptors and that two amino acid clusters located in the N-terminus ensure the binding to GAGs. The peptidyl-prolyl isomerase activity of CyPB is not required for receptor binding. We have recently demonstrated that CyPB enhances adhesion of peripheral blood T-lymphocytes to fibronectin, a component of the extracellular matrix. We intended to identify additional amino acids involved in the binding of CyPB to its functional type I receptor and to determine regions responsible for the stimulation of peripheral blood T-lymphocyte adhesion. We determined that residues R76, G77, K132, D155, and D158 of the calcineurin (CN) interacting region were implicated in the recognition of type I receptor but not of GAGs. We also found that two different changes in the N-terminal extension that abated binding to GAGs prevented adhesion of peripheral blood T-lymphocytes to coated CyPB, whereas abbrogation of the PPIase activity had no effect. On the other hand, the adhesion of peripheral blood T-lymphocytes to coated fibronectin was not stimulated by CyPB mutants devoid of either type I receptor or GAGs binding activity or by mutants of the PPIase site. Altogether, the results demonstrate that different regions of CyPB are involved in peripheral blood T-lymphocyte activation and imply a novel important physiological function for peptidyl-prolyl isomerase activity.     

Urinary chiro-inositol and myo-inositol excretion is elevated in the diabetic db/db mouse and streptozotocin diabetic rat

Inositol phosphoglycan molecules containing either D-chiro-inositol or myo-inositol have been isolated from various mammalian tissues and are putative mediators of insulin action. Urinary excretion of inositols appears to be altered in diabetes mellitus; however, the relationships with different types of diabetes are unclear. The objective of this study was to determine the urinary excretion of chiro- and myo-inositol in diabetic animal models, including streptozotocin (STZ) rats, db/db mice, and fa/fa Zucker rats. In STZ rats (type 1 diabetes), 12-hr urinary excretion of chiro-inositol was elevated 336-fold and myo-inositol excretion was elevated 47-fold compared with their nondiabetic counterparts. When corrected for creatinine, chiro-inositol excretion was 259-fold higher and myo-inositol excretion was 36-fold higher in STZ rats than in normal rats. The same pattern was observed in db/db mice (type 2 diabetes), where 12-hr urinary chiro-inositol excretion was elevated 247-fold compared with normal mice. When corrected for creatinine, chiro-inositol excretion was 2455-fold higher and urinary myo-inositol excretion was elevated 8.5-fold in db/db mice compared with normal mice. The fa/fa Zucker rats (impaired glucose tolerance) had a pattern of urinary inositol excretion that was similar to the nondiabetic animals (lean Zucker rats, C57BL/6 mice, and Sprague-Dawley rats). In summary, urinary chiro-inositol and myo-inositol excretion was elevated in animal models of type 1 and type 2 diabetes mellitus, concomitant with hyperglycemia and glucosuria.     

Effect of calcium administration on renal responsiveness to parathyroid hormone in pseudohypoparathyroidism type I and II -- in comparison with normals, idiopathic and surgical hypoparathyroidism.

A 31-year-old man and a 12-year-old girl were diagnosed as pseudohypoparathyroidism (PHP) Type I because of a failure to respond to the administration of parathyroid hormone (PTH) with increased urinary excretion of phosphate and cyclic adenosine-3', 5'-monophosphate (cAMP). A 22-year-old woman was diagnosed as PHP Type II because there was no increase in the urinary excretion of phosphate despite of a marked increase in urinary cAMP excretion. With the combined calcium-PTH infusion or PTH infusion after vitamin D therapy, renal response was improved in these patients. Also dibutyryl adenosine-3'-5'-cyclic monophosphate (dbcAMP) infusion evoked an increased urinary phosphate excretion in all of the patients. The metabolic defect of our patients with PHP Type I may be caused not by a lack or defective form of PTH-sensitive receptor adenylate cyclase complex but rather by an abnormal conformation in the plasma membrane-associated receptor adenylate cyclase enzyme complex in kidney. In the patient with PHP Type II, as cAMP generation is intact, the metabolic defect might be related to a defect of calcium mobilization in renal tubular cells in response to PTH.