Abnormalities in hepatic lipase in chronic renal failure: role of excess parathyroid hormone.

Post-heparin hepatic lipase activity is reduced in chronic renal failure (CRF). This could be due to reduced synthesis, decreased activity, and/or impaired secretion of the enzyme. Further, the factor(s) responsible for such derangements are not elucidated. We examined hepatic lipase metabolism in normal, 6-wk-old CRF rats, CRF-PTX (parathyroidectomized) rats, and CRF and normal rats treated with verapamil (CRF-V, normal-V) using liver homogenate, hepatic cell culture for 8 h, and in vitro liver perfusion. The Vmax of hepatic lipase in liver homogenate was significantly (P < 0.01) reduced and the Km was significantly (P < 0.01) increased in CRF rats, but the values were normal in CRF-PTX, CRF-V, and normal-V rats. Culture of hepatic cells for 8 h was associated with an increase in hepatic lipase activity but the increment in CRF rats was significantly (P < 0.01) lower than that of normal, CRF-PTX, CRF-V, and normal-V rats. Both parathyroid hormone (PTH)-(1-84) and 1-34 inhibited the production of hepatic lipase in cultured cells from normal, CRF-PTX, CRF-V, and normal-V rats. The expression of the mRNA of the hepatic lipase was significantly reduced in CRF animals with the ratio between it and that of house keeping gene G3DPH being 15 +/-3% compared to 40 +/- 1.3% in normal, 44+/-2.9% CRF-PTX, 44 +/- 5.4% in CRF-V, and 39 +/- 3.9% in normal-V rats. Infusion of heparin to the in vitro hepatic perfusion system increased the activity of hepatic lipase in the effluent in all groups of rat except in CRF animals. Infusion of PTH-(1-34) in dose of 10(-6) M into the liver perfusion system inhibited the increase in post-heparin hepatic lipase activity. The data show that in CRF (a) the mRNA of hepatic lipase is downregulated, and hepatic lipase production, activity and release are impaired, (b) that this is due to the state of secondary hyperparathyroidism of CRF since both acute and chronic excess of PTH were associated with these abnormalities, (c) and that prevention of excess PTH by PTX of CRF rats or blocking the effect of PTH by treatment with verapamil corrected the derangement in hepatic lipase metabolism.

Effects of parathyroid hormone on cytosolic calcium of rat adipocytes.

Available data indicate that adipocytes are targets for PTH action, and chronic excess of PTH increases calcium burden of fat tissue, suggesting that PTH increases entry of calcium into adipocytes. The present study examined the effects of PTH-(1-84) and its amino-terminal fragment, PTH-(1-34), on cytosolic calcium ([Ca2+]i) of adipocytes and evaluated the cellular pathways that mediate the potential effect of PTH on [Ca2+]i of these cells. PTH-(1-84) but not PTH-(1-34) produced a dose-dependent rise in [Ca2+]i of adipocytes. This effect occurred in the presence or absence of calcium in the media, but the magnitude of the rise in [Ca2+]i was significantly greater when calcium was present in the media. The PTH antagonist [Nle8,18Tyr34]bPTH(7-34)NH2, verapamil, and nifedipine blocked to variable degrees the PTH-induced rise in [Ca2+]i. The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate, and the GTP-binding protein (G protein) GTP gamma S also produced a dose-dependent rise in [Ca2+]i of adipocytes. These effects were inhibited by staurosporine and the G protein inhibitor guanosine 5'-O-1(2-thiodiphosphate), respectively. Similary, staurosporine, calphostin C, guanosine 5'-O-1(2-thiodiphosphate), and pertussis toxin inhibited the effect of PTH on [Ca2+]i of adipocytes. (Bu)2cAMP also increased [Ca2+]i of adipocytes, but PTH did not stimulate cAMP production by adipocytes, and N-[2(p-bromocin-namylamino)ethyl]5-isoquinoline-sulfonamide, an inhibitor of protein kinase A, did not affect the PTH-induced rise in [Ca2+]i of adipocytes. The data indicate that: 1) PTH-(1-84) increases [Ca2+]i of adipocytes; 2) this action of the hormone is receptor mediated; 3) the hormone uses a G protein activation of calcium channels and the phospholipase C pathway in mediating its action on [Ca2+]i; and 4) the rise in [Ca2+]i is due to both increased calcium influx into the adipocytes and mobilization of calcium from intracellular stores.

Central nervous dysfunction in uremia.

The mechanisms of central nervous system dysfunction in uremia are multifactorial and only partially characterized. Studies using sealed presynaptic nerve terminals (synaptosomes) for in vitro ion transport and metabolism of neurotransmitter in chronic renal failure (CRF) neuronal cell culture and in vivo brain structure microdialysis generated significant new information. An increase in total calcium content of the cerebral cortex accompanied by increased levels of cytosolic calcium ([Ca(2+)]i) in synaptosomes are common findings in rats with CRF. Mechanisms leading to the increase in [Ca(2+)]i include increased calcium uptake mediated by parathyroid hormone and decreased activity of Na(+),K(+)-adenosine triphosphatase (ATPase) and Ca(2+)-ATPase of synaptosomes in CRF rats. Moreover, these synaptosomes respond inappropriately to depolarization, which can impair neurotransmitter metabolism. Brain gamma-aminobutyric acid content, norepinephrine, and acetylcholine release uptake and degradation are affected by uremia. These may lead to certain somatic, behavioral, and motor dysfunctions in uremia. Many derangements of the central nervous system in uremia appear to be mediated by secondary hyperparathyroidism of CRF because parathyroidectomy of animals with CRF prevented the increase in basal levels of [Ca(2+)]i and derangements in neurotransmitter metabolism. The role of other neurotoxins, such as guanidinosuccinic acid, are also reviewed.

Dysfunction of polymorphonuclear leukocytes in uremia: role of parathyroid hormone.

Polymorphonuclear leukocytes (PMNLs) from uremic patients have elevated basal levels of cytosolic calcium ([Ca2+]i), reduced calcium signal after activation of Fc(gamma) RIII receptor, and impaired phagocytosis. Chronic excess of parathyroid hormone (PTH) in uremia mediates its effect on PMNL's metabolism and function through the sustained elevation of their [Ca2+]i. Because calcium channel blockers interfere with this effect of PTH on PMNLs, treatment of patients on hemodialysis with verapamil, nifedipine, or amlodipine was associated with an improvement in metabolism and phagocytosis of PMNLs in humans. The therapy with calcium channel blockers should be continued in order to maintain its beneficial effects.

Defects in B-cell function and metabolism in uremia: role of parathyroid hormone.

Patients with chronic renal failure have impaired humoral immunity, inadequate B-cell proliferation and antibody production, and elevated basal levels of cytosolic calcium ([Ca2+]i) in their B cells. Multiple mechanisms can be involved in generation of these derangements. This article reviews data suggesting that high levels of parathyroid hormone (PTH) of uremia affect the metabolism and function of B cells. We also review studies on the role of normalization of [Ca2+]i in these abnormalities. Small but well-documented studies suggest that treatment of dialysis patients with calcium channels blockers can reverse the elevation of [Ca2+]i in B cells, which was followed by improvement of B-cell function. Thus, therapy with calcium channel blockers has the potential to decrease the infectious complication of uremia.

Niridazole as an adjunct to "conventional" immunosuppression in kidney allograft transplantation. Long-term follow up.

To assess the value of niridazole as adjuvant immunosuppressant to conventional steroid and azathioprine therapy, a prospective randomized clinical study in 26 cadaver kidney recipients had been performed. No beneficial effect was observed on the kidney graft survival with the addition of niridazole. Neither was there any additional immunosuppressive action demonstrated in the serum of the patients in this group. On the basis of our limited clinical experience niridazole can not be recommended as an adjunct agent for kidney graft recipients.

Immunogenicity of allogeneic pancreatic islet grafts: the effect of in vitro culture and the site of transplantation.

Freshly isolated or cultured for 14 days rat allogeneic pancreatic islets were transplanted either intramuscularly or intratesticularly. After 14 days, the specific recipients' antigraft humoral and cellular immunity evaluated by means of leukoagglutination assay and indirect splenocyte migration inhibition test, respectively. Islet preculture resulted in significant decrease of humoral and in absence of cellular antigraft immunity irrespectively of site of transplantation. Intratesticular grafting did not evoke cellular response after transplantation of both fresh and cultured islets. However, antibody production after intratesticular transplantation of fresh islets remained unaffected. It may be concluded that preculture of rat pancreatic islets affects their immunogenicity. Moreover, testis appears to be a suitable immunoprivileged site for islet grafting.

The influence of the H-2 receptor antagonist ranitidine on cadaver kidney allograft function.

To assess the influence of ranitidine on kidney allograft function a double-blind, placebo-controlled clinical study in 42 cadaver kidney recipients has been performed. Patients received ranitidine or placebo for 1 month. Four years after transplantation 81% of the patients in the control group were alive; 67% in the control and 71% in the ranitidine group with functioning grafts. These differences were statistically not significant. However, a slightly more intensive steroid therapy was given to keep the ranitidine patients adequately immunosuppressed. Ranitidine thus can be safely used in kidney graft recipients. Because of a lack of gastrointestinal complications in both groups, no conclusions can be drawn regarding the prophylactic value of ranitidine.

Foxp3 staining in BK virus allograft nephropathy and comparison with acute cellular rejection.

BACKGROUND: Foxp3(+)CD4(+)CD25(+) regulatory T cells are involved in maintaining immunologic self-tolerance. These cells have been investigated in acute cellular rejection (ACR) of renal allografts. In this retrospective pathological study, we evaluated Foxp3(+) immunostaining in BK virus nephropathy (BKVN). In some circumstances, BKVN may be difficult to distinguish histologically from ACR. METHODS: Sequential sections were made of 30 allograft core biopsies and stained for hematorylin and eosin (H&E), C4d, cytomegalovirus (all negative), SV40, CD3, CD20, and Foxp3. Twelve biopsies were from diagnosed BKVN cases, 12 were from diagnosed ACR cases, and six showed neither BKVN nor ACR (controls). The 100x field of maximum cellular inflammation was located and marked on the H&E stain. The same area on the CD3, CD20, and Foxp3 slides was marked. Staining lymphocytes were counted under 400x magnification. Degree of BKVN was assessed according to the Drachenberg scale; degree of ACR was assessed by the Banff criteria. RESULTS: The range of Foxp3(+) staining (cells/mm(2)) was much larger in BKVN (0-270) compared to ACR (0-35). The mean difference did not reach statistical significance owing to a large degree of overlap between the two groups. In BKVN, the Foxp3(+) infiltrate correlated with the degree of CD3(+) infiltrate (P = .012), and median Foxp3(+) infiltrate increased with Drachenberg grade of BKVN. CD3(+) cell levels were not significantly different in BKVN versus ACR. CONCLUSIONS: BKVN cases with high levels of Foxp3(+) graft infiltrates may be manifesting an immune response different from that of ACR. Positive Foxp3 correlation with Drachenberg grade suggests a down-regulatory response.

PTH, chronic renal failure and myocardium.

The heart is a target organ for parathyroid hormone (PTH), and the action of this hormone on the myocardium may be mediated through the ability of PTH to increase cytosolic calcium ([Ca2+]i) in the myocardial cells. Such a property of PTH may be responsible for rise in [Ca2+]i in chronic renal failure (CRF). Our study examined these issues. The data from this study showed: (1) PTH increases [Ca2+]i of cardiac myocytes, (2) this action is receptor-mediated and is produced by activation of the L-type calcium channels following stimulation of G protein(s), (3) the rise in [Ca2+]i is due to both augmented entry of calcium into the myocytes and mobilization of calcium from sarcoplasmic reticulum by a calcium-induced calcium release mechanism, (4) CRF is associated with a significant rise in basal levels of [Ca2+]i of cardiac myocytes, (5) this effect is mediated by the state of secondary hyperparathyroidism of CRF, and (6) the pathways through which excess PTH in CRF generates this effect include both increased entry of calcium into cardiac myocytes and decreased exit of this ion out of these cells.

Parathyroid hormone stimulates the generation of inositol 1,4,5-triphosphate in brain synaptosomes.

Parathyroid hormone (PTH) increases the levels of the second messenger, inositol 1,4,5 triphosphate (I1,4,5P3) in kidney and bone cells. It has been reported the I1,4,5P3 increases calcium uptake by brain synaptosomes. Because PTH also augments calcium entry in brain synaptosomes, it is possible that PTH induces the generation of I1,4,5P3 in these structures as well. The current study examined the effect of PTH-(1-84) on myoinositol turnover in vitro in rat brain synaptosomes. PTH-(1-84) in concentration of 10(-6)mol/L significantly (P < 0.01) increased the IP3 production (35 +/- 52%). The results indicate that PTH activates the phosphoinositol turnover in brain synaptosomes and that this pathway may be involved in the PTH-induced increase in [Ca2+]i in brain synaptosomes.

Role of elevated cytosolic calcium in the pathogenesis of complications in diabetes mellitus.

Both type I and type II diabetes mellitus are associated with derangements in the regulation of intracellular calcium. Hyperglycemia causes an acute rise in cytosolic calcium ([Ca2+]i) due to increased calcium influx and in certain cells to mobilization of intracellular calcium stores as well. The increase in calcium entry is secondary to the activation of calcium channels inhibitable by verapamil, nifedipine, or amlodipine. The stimulation of these calcium channels is mediated by the activation of G protein(s), leading to stimulation of various cellular pathways. Chronic hyperglycemia is also associated with decreased calcium exit from cells. The combination of increased calcium influx and decreased calcium efflux leads to sustained elevation in basal levels of [Ca2+]i. The latter abnormality may adversely affect cell function. Treatment of diabetic animals with calcium channel blockers normalizes cell [Ca2+]i and prevents and/or reverses the derangements in cellular function.

Effect of potassium chloride on cytosolic calcium of brain synaptosomes of rats with chronic renal failure.

Norepinephrine (NE) release from brain synaptosomes is dependent in major part on an adequate rise in cytosolic calcium ([Ca2+]i). A smaller or a greater calcium signal (delta [Ca2+]i) or delta[Ca2+]i/basal [Ca2+]i ratio in response to stimuli may interfere with NE release from brain synaptosomes. In order to further evaluate the mechanism of reduced NE release from brain synaptosomes in chronic renal failure (CRF), we examined the changes in synaptosomal [Ca2+]i in response to KCl in normal, CRF, and normocalcemic, parathyroidectomized (PTX) CRF rats (CRF-PTX) and in CRF rats treated with verapamil (CRF-V). CRF rats displayed significantly (p less than 0.01) higher basal levels of [Ca2+]i and a higher delta[Ca2+]i and delta [Ca2+]i/basal [Ca2+]i ratio than in the other three groups of rats. These parameters were not different among normal, CRF-PTX and CRF-V rats. The data are consistent with the notion that the higher calcium signal and the higher ratio between signal and the basal [Ca2+]i in response to a stimulus in CRF are deleterious to synaptosomal function and are responsible, in part, for the reduced NE release by these structures in CRF.

Verapamil reverses PTH- or CRF-induced abnormal fatty acid oxidation in muscle.

Chronic renal failure (CRF) is associated with impaired long chain fatty acids (LCFA) oxidation by skeletal muscle mitochondria. This is due to reduced activity of carnitine palmitoyl transferase (CPT). These derangements were attributed to the secondary hyperparathyroidism of CRF, since prior parathyroidectomy in CRF rats reversed these abnormalities and PTH administration to normal rats reproduced them. It was proposed that these effects of PTH are mediated by its ionophoric property leading to increased entry of calcium into skeletal muscle. A calcium channel blocker may, therefore, correct these derangements. The present study examined the effects of verapamil on LCFA oxidation, CPT activity by skeletal muscle mitochondria, and 45Ca uptake by skeletal muscle obtained from CRF rats and normal animals treated with PTH with and without verapamil. Both four days of PTH administration and 21 days of CRF produced significant (P less than 0.01) reduction in LCFA oxidation and CPT activity of skeletal muscle mitochondria, and significant (P less than 0.01) increment in 45Ca uptake by skeletal muscle. Simultaneous treatment with verapamil corrected all these derangements. Administration of verapamil alone to normal rats did not cause a significant change in any of these parameters. The data are consistent with the proposition that the alterations in LCFA in CRF or after PTH treatment are related to the ionophoric action of the hormone and could be reversed by a calcium channel blocker.

Derangements in acetylcholine metabolism in brain synaptosomes in chronic renal failure.

Chronic renal failure causes abnormalities in the central nervous system function and in norepinephrine metabolism of brain synaptosomes. The present study examined the effect of renal failure on the metabolism of another neurotransmitter, acetylcholine, which is involved in the modulation of behavioral and motor function. We measured acetylcholine content and release, choline content, uptake and release and activity of choline kinase in synaptosomes from rats with renal failure with various duration, renal failure-parathyroid-ectomized rats maintained normocalcemic, renal failure and normal rats treated with verapamil. Acetylcholine content increased while choline content decreased proportionally and significantly (P < 0.01) with the duration of renal failure; choline kinase activity was reduced (P < 0.01). These derangements were prevented by parathyroidectomy of renal failure rats or by their treatment with verapamil. Choline uptake and release were elevated in renal failure and these abnormalities were not corrected by parathyroidectomy or verapamil therapy. Acetylcholine release was elevated in renal failure and parathyroidectomy prevented this derangement. Verapamil reduced acetylcholine release in both normal and renal failure rats. The data show that: (a) renal failure causes significant derangements in acetylcholine metabolism leading to its accumulation in and an increase in its release from brain synaptosomes; (b) this is mainly due to reduced activity of choline kinase, most likely, mediated by the state of secondary hyperparathyroidism of renal failure; (c) blocking the parathyroid hormone-induced calcium influx into synaptosomes by verapamil prevented the abnormalities in acetylcholine metabolism; and (d) the derangement in choline uptake and release in CRF is not related to excess parathyroid hormone since parathyroidectomy or verapamil treatment did not correct them.