total intracellular PTH was the non-PTH (1-84), most likely A novel mechanism for skeletal resistance in uremia.PTH 7-84. Background. In treating secondary hyperparathyroidism, Conclusion. In patients with chronic renal failure, the presthe target level of serum intact parathyroid hormone (I-PTH) ence of high circulating levels of non-1-84 PTH fragments should be three to five times normal to prevent adynamic bone (most likely 7-84 PTH) detected by the "intact" assay and the disease. In circulation, there is a non-(1-84) PTH-truncated antagonistic effects of 7-84 PTH on the biological activity of fragment, likely 7-84, which, in addition to PTH 1-84, is mea-1-84 PTH explain the need of higher levels of "intact" PTH sured by most I-PTH immunoradiometric (IRMA) assays, givto prevent adynamic bone disease. ing erroneously high I-PTH values. We have developed a new IRMA assay in which the labeled antibody recognizes only the first six amino acids of the PTH molecule. Thus, this new IRMA assay (Whole PTH) measures only the biologically active 1-84 Parathyroid hormone (PTH), a single-chain polypep-PTH molecule. tide of 84 amino acids [1], plays a critical role in the Methods. Using this new IRMA assay (Whole PTH) and the Nichols "intact" PTH assay, we compared the ability of regulation of mineral metabolism. Ionized calcium, caleach assay to recognize human PTH (hPTH) 1-84 and hPTH citriol, and phosphorus are the three major regulators 7-84 and examined the percentage of non-1-84 PTH in circulaof PTH homeostasis in humans. tion and in parathyroid glands. Possible antagonistic effects of The human PTH gene is located on the short arm of the 7-84 PTH fragment on the biological activity of 1-84 PTH chromosome 11. The coding region spans more than 4 in rats were also tested. Results. In 28 uremic patients, PTH values measured with kb and consists of three exons. The first exon contains the Nichols assay, representing a combined measurement of the 5Ј untranslated region of the PTH transcript. Theboth hPTH 1-84 and hPTH 7-84, were 34% higher than with coding sequence spans exons 2 and 3. The spliced cytothe Whole assay (hPTH 1-84 only); the median PTH was 523 plasmic transcript is 772 bases long. The primary translaversus 318 pg/mL (P Ͻ 0.001). Similar results were found in tion product, pre-pro-PTH (115 amino acids), is formed 14 renal transplant patients. In osteoblast-like cells, ROS 17.2, 1-84 PTH (10 Ϫ8 mol/L) increased cAMP from 18.1 Ϯ 1.25 to in the rough endoplasmic reticulum of parathyroid chief 738 Ϯ 4.13 mmol/well. Conversely, the same concentration of cells [2] and is converted within seconds to pro-PTH (90 7-84 PTH had no effect. In parathyroidectomized rats fed a amino acids) [3]. In the Golgi apparatus, pro-PTH is calcium-deficient diet, 7-84 PTH was not only biologically inacconverted to intact PTH (I-PTH; 84 amino acids) approxtive, but had antagonistic effects on 1-84 PTH in bone. Plasma calcium was increased (0.65 mg/dL) two hours after 1-84 PTH imately 15 minutes after the biosynthesis of the original tre...

Unilateral ureteral obstruction (UUO) is a model of renal injury characterized by progressive tubulointerstitial fibrosis and renal damage, while relatively sparing the glomerulus and not producing hypertension or abnormalities in lipid metabolism. Tubulointerstitial fibrosis is a major component of several kidney diseases associated with the progression to end-stage renal failure. Here we report that when a critical renal developmental morphogen, osteogenic protein-1 (OP-1; 100 or 300 microg/kg body wt), is administered at the time of UUO and every other day thereafter, interstitial inflammation and fibrogenesis are prevented, leading to preservation of renal function during the first 5 days after obstruction. Compared with angiotensin-converting enzyme inhibition with enalapril treatment, OP-1 was more effective in preventing tubulointerstitial fibrosis and in preserving renal function. The mechanism of OP-1- induced renal protection was associated with prevention of tubular atrophy, an effect not shared with enalapril, and was related to preservation of tubular epithelial integrity. OP-1 blocked the stimulation of epithelial cell apoptosis produced by UUO, which promoted maintenance of tubular epithelial integrity. OP-1 preserved renal blood flow (RBF) during UUO, but enalapril also stimulated RBF. Thus OP-1 treatment inhibited tubular epithelial disruption stimulated by the renal injury of UUO, preventing tubular atrophy and diminishing the activation of tubulointerstitial inflammation and fibrosis and preserving renal function.

Vascular calcification is associated with cardiovascular disease, the most common cause of death in chronic kidney disease (CKD). Patients with CKD are treated with vitamin D receptor activators (VDRAs); therefore, we determined if this treatment affects vascular calcification. Uremic rats were given vehicle, calcitriol, paricalcitol, or doxercalciferol three times a week for 1 month. Calcitriol significantly increased the serum calcium-phosphate product and aortic calcium content. Paricalcitol had no effect but the same dose of doxercalciferol significantly increased the calcium-phosphate product and the aortic calcium content, the latter being confirmed by von Kossa staining. To see if the increased aortic calcium was due to an increased serum calcium-phosphate product or to a differential effect of the two VDRAs, we lowered the dose of doxercalciferol and increased the dose of paricalcitol. A lower doxercalciferol did not increase the calcium-phosphate product but increased the aortic calcium content. A higher dose of paricalcitol still had no effect. Doxercalciferol treatment increased the mRNA and protein expression of the bone-related markers Runx2 and osteocalcin in the aorta, whereas paricalcitol did not. Hence, different VDRAs have different effects on vascular calcification in uremic rats. The effects are independent of the serum calcium-phosphate product suggesting independent mechanisms.

Monotherapy with angiotensin-converting enzyme inhibitors has been shown to be beneficial in suppressing the progression of experimentally induced kidney diseases. Whether such therapy provides additional benefits when combined with vitamin D or an analog of vitamin D has not been established. Rats were made uremic by 5/6 nephrectomy and treated as follows: Uremic ؉ vehicle (UC), uremic ؉ enalapril (30 mg/L in drinking water; E), uremic ؉ paricalcitol (19-nor; 0.8 g/kg, three times a week), and uremic ؉ enalapril ؉ paricalcitol (E ؉ 19-nor). A group of normal rats served as control (NC). BP was significantly elevated in the UC and 19-nor groups compared with the NC group but was indistinguishable from normal in the E and E ؉ 19-nor groups. The decrease in creatinine clearance and the increase in the excretion of urinary protein that were observed in the UC group were ameliorated by the use of E alone or by E ؉ 19-nor (P < 0.05 versus UC). The glomerulosclerotic index was significantly decreased in both the 19-nor (P < 0.01) and E ؉ 19-nor groups (P < 0.01) compared with the UC group. Tubulointerstitial volume was significantly decreased in both the E (P < 0.05) and E ؉ 19-nor groups (P < 0.01) compared with the UC group. Both macrophage infiltration (ED-1-positive cells) and production of the chemokine monocyte chemoattractant protein-1 were significantly blunted in E ؉ 19-nor compared with E group. TGF-␤1 mRNA and protein expression were increased in the UC group (mRNA: 23.7-fold; protein: 29.1-fold versus NC). These increases were significantly blunted in the 19-nor group (mRNA: 7.1-fold; protein: 8.0-fold versus NC) and virtually normalized in the E ؉ 19-nor group (protein: 0.8-fold versus NC). Phosphorylation of Smad2 was also elevated in the UC group (7.6-fold versus NC) but less so in the 19-nor-treated rats (5.5-fold versus NC). When rats were treated with E ؉ 19-nor, the phosphorylation of Smad2 was normal (1.1-fold versus NC). Thus, 19-nor can suppress the progression of renal insufficiency via mediation of the TGF-␤ signaling pathway, and this effect is amplified when BP is controlled via renin-angiotensin system blockade.

Mutant forms of TRPC6 can activate NFAT-dependent transcription in vitro via calcium influx and activation of calcineurin. The same TRPC6 mutants can cause FSGS, but whether this involves an NFAT-dependent mechanism is unknown. Here, we generated mice that allow conditional induction of NFATc1. Mice with NFAT activation in nascent podocytes in utero developed proteinuria and glomerulosclerosis postnatally, resembling FSGS. NFAT activation in adult mice also caused progressive proteinuria and FSGS. Ultrastructural studies revealed podocyte foot process effacement and deposition of extracellular matrix. NFAT activation did not initially affect expression of podocin, synaptopodin, and nephrin but reduced their expression as glomerular injury progressed. In contrast, we observed upregulation of Wnt6 and Fzd9 in the mutant glomeruli before the onset of significant proteinuria, suggesting a potential role for Wnt signaling in the pathogenesis of NFAT-induced podocyte injury and FSGS. These results provide in vivo evidence for the involvement of NFAT signaling in podocytes, proteinuria, and glomerulosclerosis. Furthermore, this study suggests that NFAT activation may be a key intermediate step in the pathogenesis of mutant TRPC6-mediated FSGS and that suppression of NFAT activity may contribute to the antiproteinuric effects of calcineurin inhibitors.

Effects of hepatocyte growth factor (HGF) administration were examined in a model of acute ischemic renal injury induced by bilateral renal artery occlusion in rats. Compared with rats administered vehicle, rats administered 20 micrograms HGF subcutaneously 30 min postischemia had significantly lower serum creatinine and blood urea nitrogen levels over the course of 7 days postocclusion, enhanced insulin clearances measured on day 2 postocclusion, reduced mortality, and much less injury evident by examination of kidney histologies 7 days postinjury. The tubular regeneration that occurred postischemic injury was reflected by increased incorporation of 5-bromo-2'-deoxyuridine (BrdU) in cortical tubular epithelium compared with incorporation in kidneys from noninjured rats. HGF enhanced BrdU incorporation compared with vehicle, indicating enhanced mitogenesis. The weight loss that occurs postischemic injury was not ameliorated by the dose of HGF we employed. We conclude that administration of HGF postischemic injury to rats stimulates the recovery of normal kidney function and the regeneration of proximal tubular epithelium.

Wrinkle-free (wrfr) is a previously uncharacterized, spontaneous, autosomal recessive mouse mutation resulting in very tight, thick skin. wrfr mutant mice exhibit severe breathing difficulties secondary to their tight skin and die shortly after birth. This phenotype is strikingly similar to a very rare human genetic disorder, restrictive dermopathy. wrfr mutant mice display a defective skin barrier, which is normally imparted by the cornified envelope, a composite of protein and lipid that prevents loss of water from within and entry of potentially harmful substances from without. In addition, hair growth from grafted wrfr skin is impaired. Positional cloning of the wrfr mutation revealed a retrotransposon insertion into a coding exon of Slc27a4, the gene encoding fatty acid transport protein (FATP)4. FATP4 is the primary intestinal FATP and is thought to play a major role in dietary fatty acid uptake; it therefore is viewed as a target to prevent or reverse obesity. However, its function in vivo had not been determined. Our results demonstrate an unexpected yet critical role for FATP4 in skin and hair development and suggest Slc27a4 to be a candidate gene for restrictive dermopathy.T he skin of mammals is composed of a dermis and an epidermis separated by a basement membrane. The epidermis is stratified, consisting of basal keratinocyte, spinous͞ prickle, granular, and squamous͞cornified layers. Keratinocytes move upward from the basement membrane and progress through a scheduled program of differentiation, with cell death marking their final differentiation step. During this differentiation program, keratinocytes flatten and accumulate lipids that are discharged into the intercellular space (1). In the stratum corneum the lipids are crosslinked with a number of proteins including loricrin and involucrin to form the cornified envelope (2). The insoluble cornified envelope is a composite of protein and lipid that serves as a barrier to loss of water from within and to entry of potentially harmful substances from without. Without such a barrier, life on land could not exist (3).We discovered a spontaneous, autosomal recessive mutation in our mouse colony causing extremely tight, thick skin. We named the mutation wrinkle-free (wrfr). The wrfr phenotype is similar to a rare human disease called restrictive dermopathy (4, 5). Newborn wrfr Ϫ͞Ϫ mice have difficulty breathing because of the tight skin, do not suckle, exhibit a defective skin barrier, and die several hours after birth. There are also defects in hair follicle morphogenesis and hair growth. Because of the presumed fundamental importance of the mutated gene to skin development and its potential relevance to human disease, we sought to identify the affected gene. Here we present a characterization of the wrfr phenotype and the positional cloning of the wrfr mutation. Materials and MethodsMicrosatellite Marker Analysis. Genomic DNA was prepared from tissues by standard proteinase K digestion, phenol͞chloroform extraction, and ethanol precipitation (6) or by ...

The effects of administering insulin-like growth factor I (IGF-I) were examined in a model of lschemic acute tubular necrosis in rats. Injury was induced by 75 min of bilateral renal artery occlusion. Compared to rats administered vehicle, rats administered IGF-I (100 pg/day via continuous subcutaneous infuio) had significantly ower serum creatinine and blood urea nitrogen levels over the course of 7 days postocclusion. Glomerular filtration rate as determined by inulin clearance was examined on day 2 postocclusion and was siicantly increased in IGF-I-treated animals (0.16 ± 0.02 ml per min per 100 g of body weight) compared to vehicle-treated controls (0.08 ± 0.02 ml per min per 100 g of body weight). The weight io that occurred during the course of acute tubular necrosis was ameliorated by IGF-I. Mortality was reduced from 36.7% in vehicle-treated rats to 7.1% in rats administered IGF-I. Histologically, there was much ls renal injury evident at day 7 postocclusion in the IGF-I-treated rats compared to vehicle-treated controls. In contrast, growth hormone (200 pzg adminitered subcutaneously for 4 days) did not affect recovery of renal function or reduce mortality postreperfusion. This report demonstrates a beneficial effect of IGF-I a staon in the setting of acute tubular necrosis. Several properties of IGF-I render it a pharmacological agent with excellent potential for treatment of this condition in humans.