Klaus Olgaard

Increased Expression of Adhesion Molecules in Uremic Atherosclerosis in Apolipoprotein-E–Deficient Mice

Chronic renal failure markedly accelerates atherosclerosis in apolipoprotein-E-deficient mice, but the mechanism is unknown. The recruitment of inflammatory cells in the arterial wall by vascular adhesion molecules plays a key role in the formation of classical atherosclerosis. This study examines whether the expression of vascular adhesion molecules is increased in uremic atherosclerosis. Uremia was induced by 5/6 nephrectomy; control mice were sham-operated. After 2 wk of uremia, no lesion formation could be demonstrated in uremic or control mice. After 12 wk, aortas from uremic mice had a 9.8-fold increase of the aortic plaque area fraction compared with control mice (P < 0.0001). The aortic expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in uremic mice was 215 +/- 31% (P < 0.05) and 243 +/- 55% (P < 0.05) of that in controls after 2 and 12 wk, respectively (n = 9 x 4). In contrast, aortic expression of vascular cell adhesion molecule-1 (VCAM-1) mRNA in uremic mice was unchanged after 2 wk but increased to 237 +/- 40% (P < 0.01) of that in control mice after 12 wk. On immunohistochemistry of aortas from uremic mice, ICAM-1 was predominantly present in endothelial cells both in nonlesioned and lesioned aortas, whereas VCAM-1 was predominantly present in the medial smooth muscle cell layer in lesioned aortas. The plasma concentration of soluble ICAM-1 (sICAM-1) (but not of sVCAM-1) was slightly elevated after 2 wk of uremia. In contrast, both sICAM-1 and sVCAM-1 plasma concentrations were markedly higher in uremic than control mice after 12 wk. These results suggest that uremic atherosclerosis is preceded by an upregulation of ICAM-1 expression in arterial endothelium and that formation of early uremic lesions is accompanied by upregulation of VCAM-1 expression in the medial smooth muscle cell layer.

Persistent Downregulation of Calcium-Sensing Receptor mRNA in Rat Parathyroids when Severe Secondary Hyperparathyroidism Is Reversed by an Isogenic Kidney Transplantation

Experimental severe secondary hyperparathyroidism (HPT) is reversed within 1 wk after reversal of uremia by an isogenic kidney transplantation (KT) in the uremic rats. Abnormal parathyroid hormone (PTH) secretion in uremia is related to downregulation of CaR and vitamin D receptor (VDR) in the parathyroid glands (PG). The aim of this investigation was to examine the expression of CaR and VDR genes after reversal of uremia and HPT in KT rats. 5/6 nephrectomized rats were kept on a normal or high-phosphorus (hP) diet for 8 wk to induce severe HPT (n = 8 in each group). In another group of seven uremic hP rats, uremia was reversed by an isogenic KT and PG were harvested within 1 wk posttransplant. Plasma urea, creatinine, total calcium, phosphorus, and PTH levels were measured. Parathyroid CaR and VDR mRNA were measured by quantitative PCR. Uremic hP rats had significantly elevated levels of creatinine, urea, and phosphorus (P < 0.001) and developed significant hypocalcemia (plasma calcium 1.83 +/- 0.2 mmol/L; P < 0.001) compared with normal control rats. After KT, the levels were normalized from day 3 to 7: creatinine from 0.117 +/- 0.016 to 0.050 +/- 0.002 mmol/L; urea from 23 +/- 4 to 7 +/- 0.3 mmol/L; phosphorus from 3.9 +/- 0.6 to 1.5 +/- 0.06 mmol/L; calcium from 1.8 +/- 0.2 to 2.5 +/- 0.02 mmol/L. Plasma PTH levels fell from 849 +/- 224 to a normal level of 38 +/- 9 pg/ml (P < 0.01). In uremic rats on a standard diet, CaR mRNA was similar to that of normal control rats, whereas VDR mRNA was significantly decreased. In uremic rats kept on hP diet, CaR mRNA was significantly decreased to 26 +/- 7% of control rats (P = 0.01) and VDR mRNA reduced to 36 +/- 11% (P < 0.01). In KT, previously hP uremic rats, both CaR mRNA and VDR mRNA remained severely reduced (CaR, 39 +/- 7%; VDR, 9 +/- 3%; P < 0.01) compared with normal rats. In conclusion, circulating plasma PTH levels normalized rapidly after KT, despite persisting downregulation of CaR and VDR gene expression. This indicates that upregulation of CaR mRNA and VDR mRNA is not necessary to induce the rapid normalization of PTH secretion from hyperplastic parathyroid glands.

Parathyroid Hormone 7-84 Induces Hypocalcemia and Inhibits the Parathyroid Hormone 1-84 Secretory Response to Hypocalcemia in Rats with Intact Parathyroid Glands

Biologic effects of large C-terminal parathyroid hormone (PTH) fragments, opposite to those of N-terminal PTH, have been demonstrated. C-terminal PTH fragments are co-secreted with N-terminal PTH from the parathyroids. The aim of our study was to examine whether C-terminal PTH 7-84 regulates secretion of PTH 1-84 and affects the expression of genes of relevance for parathyroid function, PTH, calcium-sensing receptor (CaR), PTH type 1 receptor (PTHR1), and PTH-related peptide (PTHrP) genes in rat parathyroid glands. PTH 7-84 induced a significant decrease in plasma Ca2+ in rats with intact parathyroid glands. Despite the reduction of plasma Ca2+, no stimulation of PTH 1-84 secretion took place. Furthermore, the PTH 1-84 secretory response to EGTA-induced acute and severe hypocalcemia was significantly inhibited by PTH 7-84. During recovery from hypocalcemia, plasma Ca2+ levels were significantly lower in the PTH 7-84-treated group, as compared with the vehicle group, and at the same time plasma PTH 1-84 levels were significantly suppressed. The expression of PTH, CaR, PTHR1, and PTHrP genes in the rat parathyroid glands was not affected by PTH 7-84. The peripheral metabolism of PTH 1-84 was not affected by PTH 7-84. PTH 7-84 did not cross-react with the rat bioactive PTH 1-84 assay. In normal rats with intact parathyroid glands, PTH 7-84 inhibited the PTH 1-84 secretory response to hypocalcemia and induced a significant decrease in plasma Ca2+. These effects of PTH 7-84 on PTH 1-84 secretion and on plasma Ca2+ levels were not associated with significant changes in PTH, PTHR1, CaR, and PTHrP gene expressions in the rat parathyroid glands. It is hypothesized that PTH 7-84 regulates PTH secretion via an autocrine/paracrine regulatory mechanism.

The calcium/parathyroid hormone concept of the parathyroid glands.

Plasma ionized calcium is the major determinant of parathyroid hormone (PTH) secretion. The minute-to-minute secretory response of the parathyroids to changes in plasma ionized calcium is described by the calcium/PTH concept, but the detailed mechanism is not yet well understood. The recent cloning of a calcium-sensing receptor in the plasma membrane of the parathyroid cells will probably yield important information concerning the mechanisms by which calcium and other ions control the parathyroid function. It is likely that autocrine and paracrine factors also participate in the regulation of PTH secretion. PTH, chromogranin A, chromogranin A-related peptides and endothelin-1 have been suggested as autocrine factors. More documentation is needed on the impact of these factors in the physiology of the parathyroid gland. In-vivo investigations of the parathyroid function are difficult to interpret because of the complexity of the PTH secretory response to hypo- and hypercalcaemia. Rate dependency and the ability of the parathyroids to sense the direction of changes in calcium make the existing models for investigating the calcium/PTH relationship inappropriate. In vitro, the models are compromised by a rapid drop in the expression of the calcium-sensing receptor of the cultured parathyroid cells. We, therefore, recommend caution when using the calcium/PTH concept in clinical or experimental investigations.

A Model of Reversible Uremia Employing Isogenic Kidney Transplantation in the Rat: Reversibility of Secondary Hyperparathyroidism

Kidney transplanted patients with normalized kidney function may still exhibit a variety of problems such as bone problems, vascularly problems, and hormonal dysfunctions. A part of the symptoms may be persisting uremic symptoms, secondary to the pretransplanted period of chronic uremia. An experimental rat model, designed to the study of the reversibility of the chronic uremic implications is therefore described. A stable, severe chronic uremia was induced by 5/6 nephrectomy to inbred Lewis rats. Ten weeks later uremia was reverted by a successful isogenic rat kidney transplantation. During the period of chronic uremia the p-urea was elevated to an average of 21.8 +/- 0.9 mmol/l and p-creatinine to 105.7 +/- 5.7 microM/l. The isogenic kidney transplantation resulted in reestablishment of normal kidney function with an average level of p-urea of 7.6 +/- 0.2 mmol/l and p-creatinine 42.5 +/- 1.9 microM/l perfectly corresponding to the sham-operated rats, i.e. one-kidney rats. Reversibility of the secondary hyperparathyroidism due to chronic uremia was investigated in the model. In rats with chronic renal failure PTH increased from 52 +/- 4.9 pg/ml to 152 +/- 12.2 pg/ml and was normalized after transplantation. It is therefore concluded that the present described technique of introducing long term uremia followed up by a successful kidney transplantation in the rat may be a useful model to study the reversibility of different uremic manifestations.

Effect of chronic uremia on the transcriptional profile of the calcified aorta analyzed by RNA sequencing

The development of vascular calcification (VC) in chronic uremia (CU) is a tightly regulated process controlled by factors promoting and inhibiting mineralization. Next-generation high-throughput RNA sequencing (RNA-seq) is a powerful and sensitive tool for quantitative gene expression profiling and the detection of differentially expressed genes. In the present study, we, for the first time, used RNA-seq to examine rat aorta transcriptomes from CU rats compared with control rats. Severe VC was induced in CU rats, which lead to extensive changes in the transcriptional profile. Among the 10,153 genes with an expression level of >1 reads/kilobase transcript/million mapped reads, 2,663 genes were differentially expressed with 47% upregulated genes and 53% downregulated genes in uremic rats. Significantly deregulated genes were enriched for ontologies related to the extracellular matrix, response to wounding, organic substance, and ossification. The individually affected genes were of relevance to osteogenic transformation, tissue calcification, and Wnt modulation. Downregulation of the Klotho gene in uremia is believed to be involved in the development of VC, but it is debated whether the effect is caused by circulating Klotho only or if Klotho is produced locally in the vasculature. We found that Klotho was neither expressed in the normal aorta nor calcified aorta by RNA-seq. In conclusion, we demonstrated extensive changes in the transcriptional profile of the uremic calcified aorta, which were consistent with a shift in phenotype from vascular tissue toward an osteochondrocytic transcriptome profile. Moreover, neither the normal vasculature nor calcified vasculature in CU expresses Klotho.

Cardiac structure and function in a mouse model of uraemia without hypertension

Kidney dysfunction is often associated with cardiac left ventricular hypertrophy and increased cardiovascular mortality. Objective. The aim of this study was to find out whether this reflects direct effects of uraemia on the heart or is dependent on accompanying hypertension. Material and methods. Apolipoprotein‐E (apoE)‐deficient C57BL/6 mice are resistant to development of hypertension after renal mass reduction. To evaluate the impact of uraemia without hypertension on the heart, apoE‐deficient mice underwent 5/6 nephrectomy (NX) or sham operation (Sh) and were randomized to treatment with the angiotensin converting enzyme inhibitor enalapril (12 mg kg−1 d−1) or no medication. Results. NX did not affect systolic blood pressure (BP), but reduced mean creatinine clearance, body weight and blood haemoglobin to 27 % (p<0.01), 82 % (p<0.0001) and 73 % (p<0.0001), respectively, of the values in Sh mice. Thirty‐six weeks after NX, heart wet weight, echocardiographic estimates of left ventricular mass and left ventricular diastolic and systolic functions were similar in NX and Sh mice. NX did not increase cardiac fibrosis or cardiac mRNA expression of biglycan, whereas it decreased the mRNA expression of procollagen (p<0.01). Enalapril reduced BP (p<0.001), heart wet weight and estimated left ventricular mass in both NX (p<0.01) and Sh mice (p<0.05), but did not affect cardiac diastolic or systolic function. Conclusions. The results suggest that uraemia does not impair cardiac structure or function in apoE‐deficient mice. Since NX has no effect on BP in apoE‐deficient mice, the results may indicate that hypertension is important for development of left ventricular disease in uraemia.

Can Hyperparathyroid Bone Disease Be Arrested or Reversed

Parathyroid hyperplasia, oversecretion of parathyroid hormone (PTH), and hyperparathyroid bone disease are characteristic features of chronic uremia; they develop early in the course of uremia and often in a progressive way. This review focuses on the potential for arrest or regression of hyperparathyroid-induced bone disease. For this purpose, the review addresses investigations that have used bone histology and not investigations that indirectly attempted to demonstrate changes in the skeleton by measurements of bone mineral density or laboratory indices of bone turnover, other than PTH. A prerequisite for inducing regression of the hyperparathyroid bone disease is a significant suppression of PTH secretion or reversal of hyperparathyroidism and uremia. It is concluded, on the basis of paired bone biopsy studies in patients with established hyperparathyroid bone disease, that bone histology can be improved or normalized after treatment that diminishes PTH levels. Oversuppression of PTH levels, however, might lead to adynamic bone disease.

Epigenetic Methylation of Parathyroid CaR and VDR Promoters in Experimental Secondary Hyperparathyroidism

Secondary hyperparathyroidism (s-HPT) in uremia is characterized by decreased expression in the parathyroids of calcium sensing (CaR) and vitamin D receptors (VDR). Parathyroid hormone (PTH) is normalized despite low levels of CaR and VDR after experimental reversal of uremia. The expression of CaR in parathyroid cultures decreases rapidly. Methylation of promoter regions is often detected during epigenetic downregulation of gene expression. Therefore, using an experimental rat model, we examined changes in methylation levels of parathyroid CaR and VDR promoters in vivo and in vitro. Methods. Uremia was induced by 5/6 nephrectomy. Melting temperature profiling of CaR and VDR PCR products after bisulfite treatment of genomic DNA from rat parathyroids was performed. Real-time PCR measured expression of PTH, CaR, VDR, and klotho genes in vitro. Results. Parathyroids from uremic rats had similar low levels of methylation in vivo and in vitro. In culture, a significant downregulation of CaR, VDR, and klotho within two hours of incubation was observed, while housekeeping genes remained stable for 24 hours. Conclusion. In uremic s-HPT and in vitro, no overall changes in methylation levels in the promoter regions of parathyroid CaR and VDR genes were found. Thus, epigenetic methylation of these promoters does not explain decreased parathyroid expression of CaR and VDR genes in uremic s-HPT.

Basic Science and Dialysis: Parathyroid Growth and Suppression in Renal Failure

In advanced uremia, parathyroid hormone (PTH) levels should be controlled at a moderately elevated level in order to promote normal bone turnover. As such, a certain degree of parathyroid hyperplasia has to be accepted. Uremia is associated with parathyroid growth. In experimental studies, proliferation of the parathyroid cells is induced by uremia and further promoted by hypocalcemia, phosphorus retention, and vitamin D deficiency. On the other hand, parathyroid cell proliferation might be arrested by treatment with a low‐phosphate diet, vitamin D analogs, or calcimimetics. When established, parathyroid hyperplasia is poorly reversible. There exists no convincing evidence of programmed parathyroid cell death or apoptosis in hyperplastic parathyroid tissue or of involution of parathyroid hyperplasia. However, even considerable parathyroid hyperplasia can be controlled when the functional demand for increased PTH levels is removed by normalization of kidney function. Today, secondary hyperparathyroidism can be controlled in patients with long‐term uremia in whom considerable parathyroid hyperplasia is to be expected. PTH levels can be suppressed in most uremic patients and this suppression can be maintained by continuous treatment with phosphate binders, vitamin D analogs, or calcimimetics. Thus modern therapy permits controlled development of parathyroid growth. When nonsuppressible secondary hyperparathyroidism is present, nodular hyperplasia with suppressed expression of the calcium‐sensing receptor (CaR) and vitamin D receptor (VDR) has been found in most cases. An altered expression of some autocrine/paracrine factors has been demonstrated in the nodules. The altered quality of the parathyroid mass, and not only the increased parathyroid mass per se, might be responsible for uncontrollable hyperparathyroidism in uremia and after kidney transplantation.