Association of decreased calcium-sensing receptor expression with proliferation of parathyroid cells in secondary hyperparathyroidism

BACKGROUND: The down-regulation of both calcium-sensing receptor (CaSR) and vitamin D receptor (VDR) in parathyroid (PT) glands of secondary hyperparathyroidism (HPT) caused by chronic renal failure has been associated with PT hormone hypersecretion as well as PT hypergrowth. To clarify the predominance of decreased expression of CaSR and VDR in the high proliferative activity of PT glands, we examined the relationship between the expression of both receptors and proliferative activity in human PT glands. METHODS: Serial sections of 56 PT glands, including 52 glands from secondary HPT and 4 normal PT glands resected together with thyroid carcinoma, were examined immunohistochemically with specific antibodies against CaSR, VDR, and Ki67. The Ki67-positive cell number was counted and expressed as the Ki67 score. The CaSR and VDR expressions were semiquantitatively analyzed. RESULTS: The expressions of both CaSR and VDR were markedly decreased in PT glands of secondary HPT, while the Ki67 score was significantly higher than it was in normal controls. When hyperplastic glands were classified into two subgroups, with [N(+)] or without [N(-)] nodular formation, CaSR expression was significantly decreased in N(+), while VDR expression was not different. Multiple regression analyses revealed that the decreased expression of CaSR could contribute significantly to the high proliferative activity, even if VDR expression was taken into account. CONCLUSION: The decrease in CaSR expression is associated with the high proliferative activity of PT glands in secondary HPT, independently of the decreased VDR expression. These findings provide a new insight into the pathogenesis of PT hyperplasia, which is refractory to vitamin D therapy in patients with severe secondary HPT.     

Persistent hyperparathyroidism in renal allograft recipients: vitamin D receptor, calcium-sensing receptor, and apoptosis

The phenotypic changes in parathyroid cells after successful renal transplantation remain to be elucidated. We compared 10 diffuse and 11 nodular hyperplastic parathyroid glands from five renal allograft recipients with persistent hyperparathyroidism, with five diffuse and 13 nodular hyperplasia from seven uremic patients on hemodialysis, and 13 normal glands. Comparisons included expressions of both vitamin D receptor (VDR) and calcium-sensing receptor (CaSR), proliferative activity (Ki67), and apoptosis (TUNEL). Immunoreactivity was assessed semiquantitatively and expressed as labeling index. The area/cell was also measured to assess cellular hypertrophy. The labeling indexes of VDR (587+/-71; mean+/-s.e.m.) and CaSR (45.0+/-2.8) in recipients' diffuse hyperplasia were significantly higher than those in uremic diffuse hyperplasia (224+/-44, 29.3+/-2.3, respectively) (P<0.01, each). However, these expressions remained low in recipients' nodular hyperplasia (42+/-8, 11.8+/-1.4, respectively). Ki67 labeling index in recipients' nodular hyperplasia (7+/-1) was significantly smaller than in uremic patients (24+/-6, P<0.01). TUNEL labeling index in recipients' diffuse hyperplasia (30+/-5) was the highest among the groups. The cell volume tended to be smaller in both patterns of hyperplasia in allograft recipients compared with uremic patients. Our results suggest that the phenotypic change in parathyroid cells after renal transplantation depends on the pattern of hyperplasia, where it is normalized only in diffuse hyperplastic glands in which the number of cells also regresses with significant induction of apoptosis.     

22-Oxacalcitriol upregulates p21(WAF1/Cip1) in human parathyroid glands. A preliminary report.

In the era of 22-oxacalcitriol (OCT), newly synthesized 1 alpha,25-dihydroxyvitamin D(3) analogue, against secondary hyperparathyroidism, the indications of parathyroidectomy (PTx) has been restricted. Recent investigations on animal models have revealed the inhibitory effects on PTH secretion after OCT treatment, whereas there has been no evidence about human parathyroid glands. A 38-year-old man with a 19-year history of hemodialysis was performed PTx after the failure of OCT treatment. Expressions of proliferative nuclear cell antigen (PCNA), calcium-sensing receptor (CaSR), vitamin D receptor (VDR), p53 and p21(WAF1/Cip1) were analyzed by Western blotting and immunohistochemistry on resected parathyroid glands. We confirmed up-regulations of CaSR and VDR, which contribute the reduction of serum PTH, by OCT treatment. Concomitant up-regulation of p21(WAF1/Cip1) but not p53, especially in nodular hyperplasia, can be considered to induce cell cycle arrest of the parathyroid cells, but not cytocidal effect of OCT.     

Reduced p21, p27 and vitamin D receptor in the nodular hyperplasia in patients with advanced secondary hyperparathyroidism

BACKGROUND: In uremic patients with secondary hyperparathyroidism (2HPT), nodular hyperplasia of parathyroid gland shows a monoclonal pattern of cell proliferation, in which a decreased density of vitamin D receptor (VDR) also is demonstrated. The present study aimed at elucidating the mechanism of parathyroid cell proliferation in relation to cell cycle determinants in patients with advanced 2HPT. METHODS: The expression of cyclin-dependent kinase inhibitors, p21 and p27, and VDR were examined and compared among four groups of nodular (Nd; N = 23) or diffuse (Df; N = 6) hyperplastic parathyroid glands resected due to 2HPT, primary adenomas (Ad; N = 15), and histologically-normal parathyroid glands (C; N = 20) removed during thyroidectomy. Immunohistochemical analyses for VDR, p21, p27 and Ki67 antigen were performed in formalin-fixed paraffin-embedded tissues by using specific polyclonal antibody. The distribution and the intensity of immunoreactivity was quantified by using NIH imaging, and was expressed as the labeling index (LI) of positive nuclear staining in a random set of 1000 cells. RESULTS: p21 LI was significantly diminished in both Nd (85 +/- 110; mean +/- SD) and Ad (136 +/- 122) as compared to that in Df (360 +/- 191) or C (359 +/- 228; P < 0.01). p27 LI was also significantly diminished in both Nd (97 +/- 156) and Ad (187 +/- 196) as compared to that in Df (532 +/- 146) or C (631 +/- 170; P < 0.01). VDR LI in Nd (162 +/- 194) was also significantly lower than that in Df (495 +/- 337), Ad (383 +/- 262), or C (659 +/- 234), respectively (P < 0.01). Parathyroid sections with high nuclear VDR expression elicited high p21 and p27 expression. Both p21 and p27 LI in Nd correlated significantly with nuclear VDR LI (r = 0.92; P < 0.01, r = 0.76; P < 0.01), but not with p53 LI, and inversely correlated with the glandular weight (r = 0.44; P < 0.05, r = 0.41; P < 0.05). CONCLUSIONS: The reduced expression of p21 and p27, in a VDR-dependent manner, is a major pathogenic factor for a nodular parathyroid gland growth.     

p21WAF1 and TGF-alpha mediate parathyroid growth arrest by vitamin D and high calcium.

BACKGROUND: High dietary phosphorus (P) worsens uremia-induced parathyroid (PT) hyperplasia through increases in the growth promoter transforming growth factor-alpha (TGF-alpha). In contrast, P restriction prevents PT hyperplasia by inducing the cell cycle inhibitor p21. Since 1,25(OH)2D3-antiproliferative action in various cell types involve increases in p21, we studied whether induction of p21 by 1,25(OH)2D3 or the vitamin D analog, 19-Nor-1,25(OH)2D2, could counteract the PT hyperplasia induced by high dietary P in early uremia. METHODS: Normal (N) and uremic (U; 5/6 nephrectomized) female Sprague-Dawley rats were fed high P (HP), low P (LP) or high Ca (HCa) diets and administered intraperitoneally (IP) either vehicle or vitamin D metabolites for seven days, as follows: N-HP; U-HP + vehicle; U-HP + 1,25(OH)2D3 (4 ng/day); U-HP + 19-Nor-1,25(OH)2D2 (30 ng/day); U-LP; U-HCa. Serum PTH and PT gland weight assessed secondary hyperparathyroidism. Immunohistochemical quantitation of two markers of mitotic activity, Ki67 and PCNA measured PT hyperplasia. Immunohistochemical expression of PT p21 and TGF-alpha addressed potential mechanisms regulating PT cell growth. RESULTS: 1,25(OH)2D3 and 19-Nor-1,25(OH)2D2 were effective in suppressing both PTH secretion and PT hyperplasia induced by uremia and high dietary P independent of increases in ionized Ca. Both vitamin D compounds enhanced PT p21 expression and prevented high P-induced increases in PT TGF-alpha content. Induction of PT p21 and reduction of TGF-alpha content also occurred when uremia-induced PT hyperplasia was suppressed by high dietary Ca. CONCLUSIONS: In early uremia, vitamin D suppression of high P-induced PT hyperplasia and high dietary Ca arrest of PT growth involve induction of PT p21 and prevention of increases in TGF-alpha.     

p21(WAF1) and transforming growth factor-alpha mediate dietary phosphate regulation of parathyroid cell growth

BACKGROUND: The parathyroid (PT) hyperplasia induced by renal failure can be further enhanced by high dietary phosphate (P) or completely abolished by P restriction. To identify potential mechanisms mediating these opposing effects of dietary P on PT growth, this study first focused on p21(WAF1) (p21) because high P reduces while low P enhances serum 1,25-dihydroxyvitamin D, whose potent antiproliferative properties result from the induction of p21. In addition to reducing p21, high P-induced PT growth could result from increased PT expression of the growth promoter transforming growth factor-alpha (TGF-alpha), known to be elevated in hyperplastic and adenomatous human PT glands. METHODS: The time course for dietary P regulation of PT expression of TGF-alpha and p21 was assessed for seven days after 5/6 nephrectomy in rats and correlated with the degree of PT hyperplasia and secondary hyperparathyroidism. RESULTS: In P-restricted 5/6 nephrectomized rats, PT-p21 mRNA and protein increased by day 2, independent of changes in serum 1,25-dihydroxyvitamin D, and remained higher than in the high P counterparts for up to seven days. The PT hyperplasia of the high P group could not be attributed to a reduction of PT-p21 expression from normal control values. Instead, PT-TGF-alpha protein was higher in uremic rats compared with normal controls and increased further with high dietary P intake. PT levels of proliferating cell nuclear antigen (PCNA), an index of cell mitoses, correlated inversely with p21 and directly with TGF-alpha. Consistent with these findings, PT gland size and serum PT hormone levels, similar in both dietary groups at day 2, were higher in the high P group by day 5. Induction of p21 by low P and of TGF-alpha by high P was specific for the PT glands. Dietary P had no effect either on intestinal growth or p21 or TGF-alpha protein content. CONCLUSIONS: These findings suggest that low P induction of p21 could prevent PT hyperplasia in early uremia, whereas high P enhancement of TGF-alpha may function as an autocrine signal to stimulate growth further.     

Direct maxacalcitol injection into hyperplastic parathyroids improves skeletal changes in secondary hyperparathyroidism

Direct maxacalcitol (OCT) injection into a parathyroid gland (PTG) ameliorates several important etiologic factors of resistance to medical treatments for secondary hyperparathyroidism (s-HPT): the upregulations of vitamin D receptor (VDR) and Ca-sensing receptor (CaSR) in PTGs and the regression of PTG hyperplasia by the induction of apoptosis. In this study, we evaluated the bone histomorphology on the basis of maintaining these effects in advanced s-HPT. Five/six nephrectomized Sprague-Dawley rats were fed a high-phosphorus and low-calcium diet for 8 weeks. These rats were divided into four treatment groups: (1) basic uremic (at the baseline), (2) direct OCT single injection into PTGs (DI-OCT) followed by OCT intravenous administration for 4 weeks (IV-OCT), (3) direct vehicle injection and IV-OCT, and (4) no treatment for an additional 4 weeks. The effects of these treatments on serum intact-parathyroid hormone (PTH) level, PTG weight, VDR and CaSR expression levels in PTGs, and bone histomorphometric parameters were investigated. In the DI-OCT+IV-OCT group, the significant decrease in serum intact-PTH level was maintained by the following IV-OCT. A significant decrease in PTG weight and the upregulations of VDR and CaSR expression levels in PTGs were also observed. Bone histomorphometric analysis showed significant improvements in osteitis fibrosa in both cancellous and cortical bones. However, these findings were not observed in the other groups. These results suggest that osteitis fibrosa caused by advanced s-HPT can be successfully reversed by a control of PTH at an appropriate level through the improvement of PTG hyperplasia as induced by DI-OCT+IV-OCT.     

Comparative study of the expression of proteins involved in the cell cycle in renal secondary hyperparathyroidism

BACKGROUND: In renal hyperparathyroidism, parathyroid cell proliferation seems to play a key role in the progression of the disease. Therefore, G1/S transition, a main cell cycle regulatory step, could be deregulated in these patients. METHODS: One hundred and one parathyroid glands, taken from parathyroidectomies performed on 41 patients on hemodialysis (HD), and 15 glands, taken from 7 patients with post-transplantation persistent hyperparathyroidism (HPT), were studied. Twelve normal parathyroid (PT) glands were used as the control. Biochemical data, immunohistochemical (IHC) profiles of G1/S transition regulators belonging to the two main pathways (cyclin D1/p16INK4A/pRb and p14ARF/p53/MDM2), and proliferation rate (Ki67) were correlated. RESULTS: All of the other proteins differed from normal IHC profiles in both groups that showed significant higher proliferating rates, decreases in p27KIP1, pRb, and cyclin D1, as well as increases in p16INK4A, p53, MDM2, and p21WAF1 levels, in comparison with normal PT glands, with the exception of cyclin D3. Contrary to patients with HPT who were on hemodialysis, in post-transplantation HPT, consistent correlations between biochemical data and IHC profiles were obtained. CONCLUSION: In both groups IHC profiles of proteins involved in G1/S transition regulation significantly differed from normal PT glands. The results support partial reversion to normal IHC profile in post-transplantation HPT.     

Decreased Parathyroid Klotho Expression Is Associated With Persistent Hyperparathyroidism After Kidney Transplantation

Although successful kidney transplantation usually corrects hyperparathyroidism, the condition persists in some patients. The present study was designed to determine whether Klotho or fibroblast growth factor 23, the key regulator of parathyroid hormone, is involved in persistent hyperparathyroidism in kidney transplant recipients (KTRs). Nineteen hyperplastic parathyroid glands were obtained from end-stage renal disease (ESRD) patients and KTRs; 6 normal parathyroid glands were used as controls. We compared the expression of Klotho, fibroblast growth factor receptor 1 (FGFR1) and calcium-sensing receptor (CaSR) in the KTRs and ESRD patients. Expressions of Klotho, FGFR1, CaSR and vitamin D receptor, as evaluated by immunohistochemistry, were quantified as the number of positive cells per unit area. The Klotho, FGFR1 and CaSR expressions in parathyroid glands of the post–kidney transplantation (PSKT) and the ESRD groups were significantly decreased compared with normal controls. In the ESRD group, Klotho expression and number of proliferating cell nuclear antigen–positive cells in the parathyroid gland were significantly decreased in parathyroid adenomas as compared with parathyroid hyperplasia. The expression of FGFR1 and CaSR in the parathyroid glands was significantly increased in the PSKT compared with the ESRD group. There was no significant difference in Klotho expression between the PSKT and ESRD groups. Incomplete recovery of Klotho levels in the parathyroid gland may play a role in the pathogenesis of tertiary hyperparathyroidism after kidney transplantation.     

The effects of calcitriol on ameliorating podocytes impairment and its possible mechanism in DN rats

Objective To investigate the effects and underlying mechanism of calcitriol on ameliorating podocytes impairment in DN rats. Methods SD rats were randomly divided into four groups: normal control (NC) group, calcitriol treatment (VD) group: calcitriol 0.1μg•kg-1•d-1, diabetic nephropathy (DN) group: streptozocin (STZ) 58 mg/kg, DN treated with calcitriol (DN+VD) group：calcitriol 0.1 μg•kg-1•d-1 + STZ 58 mg/kg. Rats were sacrificed at the end of 18 weeks. Results Compared with the DN group, the DN+VD group exhibited significantly lower proteinuria by 36%, improved renal histology at the end of the experiment (P＜0.05), and similar levels of blood glucose,serum urea nitrogen as well as body weight (P＞0.05). There were no significant differences in the serum concentrations of creatinine, calcium and phosphorus among the four groups (P＞0.05). In DN group, the expressions of nephrin, podocin, VDR, PI3K-p85 and p-Akt were significantly decreased and the expression of desmin was increased compared to NC group. Calcitriol treatment could attenuate the above changes. Additionally, a positive correlation was observed between the expressions of nephrin and VDR (r=0.776, P＜0.05). Likewise, the expression of nephrin was positively correlated with either PI3K-p85 or p-Akt (r=0.736, r=0.855, all P＜0.05). Conclusion Calcitriol can ameliorate podocytes injury in DN rats, which might be related with the further up-regulation of PI3K/p-Akt signaling pathway.     

Biochemical and cellular effects of direct maxacalcitol injection into parathyroid gland in uremic rats

The most important etiological factors of resistance to medical treatments for secondary hyperparathyroidism are the decreased contents of the vitamin D receptor (VDR) and Ca-sensing receptor (CaSR) in parathyroid cells and a severely swollen parathyroid gland (PTG) as a result of hyperplasia. The effects of direct maxacalcitol (OCT) injection into PTG in terms of these factors were investigated in this study. The PTG of Sprague-Dawley rats that were 5/6 nephrectomized and fed a high-phosphate diet were treated by a direct injection of OCT (DI-OCT) or vehicle (DI-vehicle). The changes in serum intact parathyroid hormone (PTH), Ca(2+), and phosphorus levels, in VDR and CaSR expression levels in parathyroid cells, and in Ca(2+)-PTH curves were examined. Apoptosis was analyzed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method and DNA electrophoresis for PTG. DI-OCT markedly decreased serum intact PTH level, and a significant difference in this level between DI-OCT and DI-vehicle was observed. However, serum Ca(2+) and phosphorus levels did not changed markedly in both groups. The upregulations of both VDR and CaSR, the clear shift to the left downward in the Ca(2+)-PTH curve, and the induction of apoptosis after DI-OCT were observed. These findings were not observed in the DI-vehicle-treated rats. Moreover, these effects of DI-OCT were confirmed by the DI-OCT into one PTG and DI-vehicle alone into another PTG in the same rat. DI-OCT may introduce simultaneous VDR and CaSR upregulations and the regression of hyperplastic PTG, and these effects may provide a strategy for strongly suppressing PTH levels in very severe secondary hyperparathyroidism.     

乳腺分叶状肿瘤EGFR、Ki67的表达和意义

目的：探讨乳腺分叶状肿瘤（PT）中EGFR、Ki67的表达和意义。方法：采用免疫组织化学EnVi-sion两步法，检测33例乳腺分叶状肿瘤间质细胞中EGFR和Ki67的表达，比较良性、交界性和恶性PT中EGFR、Ki67表达的差异。结果：EGFR在良性PT中的阳性表达率为33.3％（5/15），交界性PT为80.0％（8/10），恶性PT为100％（8／8），恶性PT的强阳性表达率为62.5％（5／8），EGFR在良恶性PT病变中的表达有统计学意义（P〈0.05）。Ki67蛋白的阳性表达率在良性PT组为13.3％（2／15），均为弱阳性表达，交界性PT为60.0％（6／10），恶性PT为100％（8/8），其中50.0％（4／8）为强阳性表达，Ki67蛋白表达在良恶性PT病变中有统计学意义（P〈0.05）。EGFR和Ki67抗原表达与PT组织学分级相关。在良性PT中EGFR和Ki67表达率低，交界性、恶性PT表达率高，随着PT级别的增高，其表达率和表达强度增加，两指标呈正相关（r=0.6073，P〈0.001）。结论：EGFR和Ki67有可能作为判断PT恶性度的指标。EGFR在交界性和恶性PT中的高表达，提示针对EGFR的靶向治疗在恶性难治性PT术后辅助治疗中有一定的临床应用前景。     

Vitamin D–Independent Therapeutic Effects of Extracellular Calcium in a Mouse Model of Adult‐Onset Secondary Hyperparathyroidism

Cell proliferation and PTH secretion in the parathyroid gland are known to be regulated by vitamin D and extracellular calcium. Here, we examined the vitamin D–independent effects of correction of extracellular calcium in an adult‐onset secondary hyperparathyroidism (sHPT) model, using mice with a nonfunctioning vitamin D receptor (VDR). Wildtype and homozygous VDR mutant mice were kept on a rescue diet (RD) containing 2% calcium (Ca), 1.25% phosphorus (P), and 20% lactose until they were 4 mo or 1 yr of age. Subsequently, 4‐mo‐old mice were switched to a challenge diet (CD) containing the following: 0.5% Ca, 0.4% P, and 0% lactose. After 2 mo on the CD, groups of VDR mutant mice were either fed CD, a normal mouse chow with 0.9% Ca, 0.7% P, and 0% lactose, or the RD for another 3 mo. Feeding the RD protected VDR mutants against sHPT over 1 yr, showing that vitamin D is not essential for long‐term control of the function and proliferation of parathyroid cells. When 4‐mo‐old VDR mutants were switched from the RD to the CD for 2 mo, they developed severe sHPT associated with hypertrophy and hyperplasia of parathyroid glands and profound bone loss. Subsequent feeding of the RD during a 3‐mo therapy phase fully corrected sHPT, reduced chief cell proliferation, and reduced maximum parathyroid gland area by 25% by cell atrophy. There was no evidence of RD‐induced chief cell apoptosis. We conclude that signaling by the calcium‐sensing receptor regulates chief cell function and size in the absence of signaling through the VDR.     

Development and progression of secondary hyperparathyroidism in chronic kidney disease: lessons from molecular genetics

The identification of the calcium-sensing receptor (CaSR) and the clarification of its role as the major regulator of parathyroid gland function have important implications for understanding the pathogenesis and evolution of secondary hyperthyroidism in chronic kidney disease (CKD). Signaling through the CaSR has direct effects on three discrete components of parathyroid gland function, which include parathyroid hormone (PTH) secretion, PTH synthesis, and parathyroid gland hyperplasia. Disturbances in calcium and vitamin D metabolism that arise owing to CKD diminish the level of activation of the CaSR, leading to increases in PTH secretion, PTH synthesis, and parathyroid gland hyperplasia. Each represents a physiological adaptive response by the parathyroid glands to maintain plasma calcium homeostasis. Studies of genetically modified mice indicate that signal transduction via the CaSR is a key determinant of parathyroid cell proliferation and parathyroid gland hyperplasia. Because enlargement of the parathyroid glands has important implications for disease progression and disease severity, it is possible that clinical management strategies that maintain adequate calcium-dependent signaling through the CaSR will ultimately prove useful in diminishing parathyroid gland hyperplasia and in modifying disease progression.     

Effects of 12-lipoxygenase and angiotensinⅡ on p21, p27 and p57 in rat diabetic glomeruli

Objective To investigate the effects of 12-lipoxygenase (12-LO) and angiotensin Ⅱ(AngⅡ) on the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs) p21, p27 and p57 related to cell hypertrophy. Methods Mesangial cells were treated with high glucose for 24 hours and 48 hours respectively. 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and AngⅡ were infused to rats by osmotic mini-pump for 1 week and 2 weeks respectively. Rats fed high fat diet were received low dose streptozotocin (STZ) to make type 2 diabetes (DN). The rats were divided into normal control group, DN group, DN+AngⅡ type 1 receptor blocker (ARB) group or 12-LO inhibitor (CDC) group. DN+ARB rats were treated by losartan for 6 weeks, and DN+CDC rats were treated for 8 weeks. Urine albumin and protein expressions of p21, p27 and p57 were detected by ELISA and Western blotting respectively. Glomeruli injury and expressions of p21 and p27 were detected by PAS staining and immunohistochemistry respectively. Results High glucose increased p21 and p27 protein expression in mesangial cells significantly compared with the relative control (all P＜0.05), but had no effect on p57. AngⅡ increased p27 protein expression in glomeruli significantly (P＜0.05), but had no effect on p21 and p57 protein expression. 12(S)-HETE increased both p21 and p27 protein expression in glomeruli significantly (all P＜0.05), but had no effect on p57 protein expression. Blood glucose, kidney/body weight, urinary protein, and glomerular p21 and p27 protein expressions were increased in DN group (all P＜0.05) compared with those in control group, with little change of p57 protein expression (P＜0.05). Moreover, glomerular hypertrophy and extra cellular matrix accumulation were observed in DN group. However, urine protein,kidney/body weight, renal injury, but not blood glucose, were decreased in DN+ARB group and DN+CDC group compared with DN group respectively (P＜0.05). Further DN+CDC rats had decreased both p21 and p27 protein expressions in glomeruli, but DN+ARB rats only had decreased p27 protein expression (all P＜0.05). Conclusions 12-LO may induce both p21 and p27 protein expression in DN glomeruli,but AngⅡ may induce only p27 expression.     

Expression of cyclin-dependent kinase inhibitors, p21cip1 and p27kip1, during wound healing in rats

Wound healing is a physiological process in which growth of cells is stringently regulated. Cell growth is controlled by cell cycle-related proteins in which the cyclin kinase inhibitors cause cell cycle arrest and inhibit proliferation. However, little is known about the expression and the role of cyclin kinase inhibitors during wound healing in vivo. This study was mainly designed to examine the expression of p21cip1 and p27kip1 in excisional wounds of full-thickness skin in rats. Concomitant expression of proliferation marker Ki67 was also examined. Proliferation predominantly occurred in the first week after injury, peaking at postwounding day 5. Expression of both p21cip1 and p27kip1 at the gene and protein levels did occur during wound healing and showed an inverse gradient to that of Ki67. Constitutive p27kip1 was expressed throughout wound healing with low levels during the proliferating period of days 3 and 5 and increased levels during post-mitotic and remodeling stages. In contrast, p21cip1 was expressed transiently with detectable levels only between days 7 and 14 by Western blot analysis. Immunohistochemically, epithelial cells, endothelial cells and fibroblasts all could express both p21cip1 and p27kip1. In conclusion, the overall results suggested that p21cip1 and p27kip1 may play a key role in supervising the growth resulting from cell proliferation in tissue repair.     

A role for enhanced integrin and FAK expression in uremia-induced parathyroid hyperplasia

Parathyroid (PT) hyperplasia is a major feature of secondary hyperparathyroidism (SH) in uremia. The transforming growth factor-alpha (TGFalpha) / epidermal growth factor receptor (EGFR)Ethgrowth loop is the main contributor to uremia-induced PT hyperplasia. Since integrin beta1 and focal adhesion kinase (FAK) are known to directly activate cell growth and enhance EGFR-driven growth, these studies examined their contribution to PT hyperplasia in uremia. Western blot analysis was used to measure the expression of EGFR, integrin beta1, and the non-receptor integrin-sensitive FAK, in PT glands from 8 hemodialysis patients with various degrees of SH at the time of the surgery, and in a normal human PT gland. In all patients, PT EGFR expression was higher than in the normal control. Integrin beta1, a direct activator of EGFR-driven growth, was increased in 5 of the 8 hyperplastic glands, whereas 7 out of 8 PT glands showed a marked enhancement in FAK expression, an elevation unrelated to increases in integrin beta1, but directly associated to time in hemodialysis. Similar increases in PT FAK content were observed after 1 month after the onset of uremia by 5/6 nephrectomy in rats. These findings suggest that in kidney disease, the increased PT cell growth driven by enhanced EGFR could be further aggravated through elevations in integrin beta1 and FAK expression.     

Regulation of parathyroid vitamin D receptor expression by extracellular calcium

Low extracellular calcium (Ca) stimulates parathyroid hormone (PTH) secretion and also increases the renal synthesis of calcitriol (CTR), which is known to decrease PTH production. This study began with the hypothesis that the parathyroid cell response to CTR may be modulated by extracellular Ca concentration through an effect on parathyroid cell vitamin D receptor (VDR). In the present study, rat parathyroid glands were incubated in low (0.6 mM) and high (1.5 mM) Ca concentration. The parathyroid VDRmRNA was higher in 1.5 than 0.6 mM Ca. Furthermore, this effect was not observed in incubated slices of kidney cortex and medulla, tissues which also possess both Ca and vitamin D receptors. Experiments were also performed to evaluate the effect of Ca on VDR expression in vivo. Male Wistar rats received intraperitoneal injections of CaCl(2) or a single intramuscular injection of EDTA to obtain 6 h of hypercalcemic (ionized Ca, 1.4 to 1.6 mM) or hypocalcemic (ionized Ca, 0.85 to 0.95 mM) clamp; a third group of rats was used as control. A small dose of CTR was administered to hypercalcemic rats to match the serum CTR levels of hypocalcemic rats. Parathyroid gland VDRmRNA and VDR protein were increased in hypercalcemic rats as compared with hypocalcemic rats. Increasing doses of CTR upregulated VDRmRNA and VDR only in hypercalcemic rats. Additional experiments showed that the decrease in VDR in hypocalcemic rats prevented the inhibitory effect of CTR on PTHmRNA. In conclusion, our study shows that extracellular Ca regulates VDR expression by parathyroid cells independently of CTR and that by this mechanism hypocalcemia may prevent the feedback of CTR on the parathyroids.     

Parathyroid hyperplasia, adenomas, and carcinomas: differential expression of p27Kip1 protein

The histologic spectrum of proliferative parathyroid lesions (hyperplasia, adenoma, and carcinoma) often overlap, and differentiation between these lesions may at times be difficult. p27kip1 (p27) is a cyclin-dependent kinase inhibitor that helps regulate the transition from the G1 to the S phase of the cell cycle. Significantly higher levels of p27 expression have been detected in some normal tissues than in their neoplastic counterparts. The authors analyzed a series of parathyroid lesions to determine if expression of this cell cycle protein may be useful in distinguishing between parathyroid hyperplasia, adenomas, and carcinomas. Formalin-fixed paraffin-embedded tissues from randomly selected patients (22 histologically normal parathyroid glands, 33 cases of hyperplasia, 43 adenomas, and 17 carcinomas) were analyzed for expression of p27 by immunostaining. All cases were also immunostained for Ki67 with antibody MIB-1. The distribution of immunoreactivity was analyzed by quantifying the percentage of positive nuclei that was expressed as the labeling index (LI). In situ hybridization (ISH) for p27 mRNA was done using a cRNA probe with 30 of these cases. Normal parathyroid glands had the highest p27 LI (89.6 +/- 1.4), followed by hyperplasia (69.6 +/- 7.5), adenomas (56.8 +/- 3.4), and carcinomas (13.9 +/- 2.6). ISH showed no differences in p27 mRNA, indicating that the expression of the p27 gene was controlled at a posttranslational level in parathyroid tissues. Ki67 expression was significantly higher in carcinomas (LI = 8.4 +/- 1.9) than in adenomas (LI = 2.7 +/- 0.2) and hyperplasia (LI = 3.3 +/- 0.4). These results suggest that both p27 and Ki67 may be helpful in the diagnosis of histologically difficult parathyroid lesions.     

Sevelamer hydrochloride reverses parathyroid gland enlargement via regression of cell hypertrophy but not apoptosis in rats with chronic renal insufficiency.

BACKGROUND: Dietary phosphate restriction suppresses parathyroid hormone (PTH) secretion, synthesis, and parathyroid cell proliferation in experimental animals with chronic renal insufficiency (CRI), independently of serum calcium and 1,25(OH)2D3 levels. This study was conducted to examine whether sevelamer hydrochloride (sevelamer), a metal-free phosphate binder, could regress an advanced parathyroid gland (PTG) hyperplasia and enlargement in rats with CRI. METHODS: Male Sprague-Dawley rats were fed a diet containing adenine for 6 weeks to establish CRI. Normal rats and adenine-treated rats were sacrificed to obtain the PTG (baseline group). The adenine diet was changed to a normal diet or diet containing 1 or 3% sevelamer for another 4 weeks. Time course changes of serum levels of calcium, phosphorus, and PTH were measured. At the end of the study, the PTG was weighed and examined histologically. RESULTS: Adenine-treated rats developed severe CRI with marked elevation of serum phosphorus and PTH. The PTG weight markedly increased with enlarged cell volume (i.e. cell hypertrophy) at baseline. Sevelamer treatment rapidly lowered serum phosphorus and PTH levels within 6 days, and after 4 weeks, reduced the PTG weight by 38% compared to adenine-treated rats at baseline. The reduction in PTG weight was due to regression of cell hypertrophy, but not to decreased cell number by apoptosis. Decreased expression of calcium receptor in the PTG at baseline was partially recovered by the sevelamer treatment. CONCLUSIONS: The sevelamer treatment can reduce the PTG weight with a reduction in serum PTH levels via regression of cell hypertrophy but not apoptosis in rats with CRI. Reduced PTG function might contribute to the regression of cell hypertrophy.