HgCl2-induced changes in cytosolic Ca2+ of cultured rabbit renal tubular cells

Fura 2 was used to measure changes in cytosolic [Ca2+] ([Ca2+]i) in cultured rabbit kidney proximal tubule cells exposed to HgCl2. Treatment with 2.5-10 microM HgCl2 resulted in an extracellular [Ca2+] ([Ca2+]e)-independent 2- to 12-fold increase in [Ca2+]i above resting levels of about 100 nM. Treatment with 25-100 microM HgCl2 caused a rapid [Ca2+]e-independent 10- to 12-fold increase in [Ca2+]i within 1 min followed by a recovery to about 2-fold steady state by 3 min. With 25-100 microM HgCl2, both magnitude and rate of Ca2+ increase were similar, but recovery was greater with increasing doses. A slower, secondary increase in [Ca2+]i followed which varied with HgCl2 concentration and required [Ca2+]e. The first increase in [Ca2+]i represents release from intracellular pools. Calcium channel blockers, calmodulin inhibitors, and mitochondrial inhibitors do not alter the patterns of [Ca2+]i changes due to HgCl2. The recovery response with higher HgCl2 concentrations appears to be triggered by Hg2+ and not by the increased [Ca2+]i. Sulfhydryl modifiers N-ethylmaleimide, PCMB and PCMBS produced [Ca2+]e-independent [Ca2+]i increases similar to those induced by low HgCl2 concentrations. Cell killing with HgCl2 was about 50% greater with normal [Ca2+]e than with low [Ca2+]e, suggesting that [Ca2+]e influx is important in accelerating injury leading to cell death.     

Glycogen deposition in distal tubular cells during HgCl2 induced acute renal failure

An unusual cytoplasmic accumulation of glycogen within the distal tubular epithelium of the kidney was produced by subcutaneouse administration of a single dose of HgCl2 (4 mg/kg body weight), used to induce acute renal failure. Since the plasma immune-reactive insulin was increased while plasma and urine glucose levels remained normal, it was concluded that activation of glycogen synthase might have lead to this effect. Furthermore, the accumulated glycogen was considered to contribute to the protection of distal tubular cells against HgCl2-induced injury, since oxidative energy metabolism was severely depressed after HgCl2 administration.     

Urinary excretion of furosemide in rats with HgCl2-induced acute renal damage.

To examine the influence of mercuric chloride (HgCl2)-induced acute renal damage on urinary excretion of furosemide, HgCl2 (1 mg/kg) or its vehicle alone was given intraperitoneally to Wistar rats. The following two experiments were done. Study I: Three percent body weight (b.w.) of 1% NaCl solution or furosemide (30 mg/kg) in 3% b.w. of 1% NaCl solution was given orally before and after HgCl2 treatment, and an 8-hour urine was collected. Study II: Furosemide (30 mg/kg) was given orally, and blood samples were obtained at 1, 2, 3, 4, 6 and 8 hours after administration. Urinary excretion of N-acetyl-beta-D-glucosaminidase increased, and urine volume and urinary excretions of furosemide and sodium decreased in the HgCl2-treated rats. There were significant correlations between the urinary furosemide and its diuretic effects. Regression lines after HgCl2 were significantly different from those before treatment. The values of absorption as well as elimination rate constant were smaller, while the time to maximum concentration and the elimination half-life were longer in the HgCl2-treated rats compared to vehicle-treated animals. These results suggest that the urinary excretion of furosemide and the responsiveness of renal tubular cells to this agent are impaired in rats with HgCl2-induced acute renal damage.     

Haematopoietic lineage-committed bone marrow cells, but not cloned cultured mesenchymal stem cells, contribute to regeneration of renal tubular epithelium after HgCl 2 -induced acute tubular injury

Abstract.   Objective : Our previous studies have demonstrated that endogenous bone marrow cells (BMCs) contribute to renal tubular regeneration after acute tubular injury. The aim of this study was to examine which fraction of BMCs, haematopoietic lineage marrow cells (HLMCs) or mesenchymal stem cells (MSCs), are effective. Materials and methods : Six-week-old female mice were lethally irradiated and were transplanted with female enhanced green fluorescent protein-positive (GFP⁺), plastic non-adherent marrow cells (as a source of HLMCs) plus cloned cultured male GFP⁻ MSCs. Four weeks later, they were assigned into two groups: control mice with vehicle treatment and mice treated with HgCl₂. Tritiated thymidine was given 1 h before animal killing which occurred at intervals over 2 weeks. Kidney sections were stained for a tubular epithelial marker, cell origin indicated by GFP immunohistochemistry or Y chromosome in situ hybridization; periodic acid-Schiff staining was performed, and samples were subjected to autoradiography. One thousand consecutive renal tubular epithelial cells per mouse, in S phase, were scored as either female (indigenous) GFP⁺ (HLMC-derived) or male (MSC-derived). Results : Haematopoietic lineage marrow cells and MSCs stably engrafted into bone marrow and spleen, but only HLMC-derived cells, not MSCs, were found in the renal tubules and were able to undergo DNA synthesis after acute renal injury. A few MSCs were detected in the renal interstitium, but their importance needs to be further explored. Conclusion : Haematopoietic lineage marrow cells, but not cloned cultured MSCs, can play a role not only in normal wear-and-tear turnover of renal tubular cells, but also in repair after tubular injury.     

Blunted renal dopaminergic system activity in HgCl2-induced membranous nephropathy

The present study evaluated the possible role of the renal dopaminergic system in the sodium retention of HgCl2-induced nephrotic syndrome. The time courses of urinary excretion of sodium, protein, dopamine and the precursor l-3,4-dihydroxyphenylalanine (L-Dopa) were evaluated in HgCl2-treated and control rats up to day 21. The renal aromatic l-amino acid decarboxylase (AADC) activity, the enzyme responsible for the synthesis of renal dopamine, was evaluated during negligible proteinuria accompanied with enhanced sodium retention (day 7), increased proteinuria accompanied with greatest sodium retention (day 14) as well as during increased proteinuria accompanied with negative sodium balance (day 21). Also, the influence of volume expansion (VE, 5% bw) and the effects of the D1-like agonist fenoldopam (10 microg kg bw(-1) min(-1)) on natriuresis and on proximal tubular Na+,K+-ATPase activity were examined on day 14. The daily urinary dopamine output and urinary dopamine/L-Dopa ratios were reduced in HgCl2-treated rats from day 2 and beyond. This was accompanied by a marked decrease in renal AADC throughout the study. During VE, the fenoldopam-induced inhibition of proximal tubular Na+,K+-ATPase activity was similar between HgCl2-treated and control rats. However, the urinary sodium excretion during fenoldopam infusion was markedly increased by 60% to 120% in control rats but was not altered in HgCl2-treated rats. It is concluded that HgCl2 nephrosis is associated with a blunted renal dopaminergic system activity. However, the lack of renal dopamine in HgCl2 nephrosis does not appear to be related with the overall renal sodium retention in a state of proteinuria.     

D-penicillamine-induced autoimmunity in Brown-Norway rats. Similarities with HgCl2-induced autoimmunity

D-penicillamine (DP) has been previously shown to induce an autoimmune disease in Brown-Norway (BN) rats, characterized by a dermatitis, by the production of antinuclear antibodies, by the formation fo circulating immune complexes, and by linear IgG deposits along the glomerular basement membrane. These manifestations are quite similar to those observed in mercuric chloride (HgCl2)-induced autoimmunity. The mechanism of the latter disease has been recently partly elucidated. The aim of this study was to compare DP and HgCl2-induced autoimmunity in BN rats and to compare the mechanisms involved in both situations. A transient increase in the number of spleen cells, affecting B cells and CD4+ T cells, and an increase in serum IgE concentration, previously reported in HgCl2-induced autoimmunity, were observed during DP treatment. Autoreactive anti-class II T cells able to proliferate not only in the presence of autologous B cells but also in the presence of syngeneic normal B cells were found in DP-treated BN rats. Spontaneous regulation occurred, associated with the disappearance of autoreactive T cells. Suppressor CD8+ T cells were not involved in this phenomenon. Mechanisms involved in both the induction and the regulation of DP-induced autoimmunity seem to be quite similar to those reported in HgCl2-induced autoimmunity.     

Hypovolemic models of acute tubular necrosis in the rat kidney.

Studies were undertaken to determine whether a hypotensive episode under variable conditions is capable of inducing experimental acute renal failure in rats. Animals were subjected to hypovolemic shock by withdrawing volumes of blood necessary to maintain a systolic pressure of 30-40 mm Hg for 105-110 min. The blood was then reinfused and the animal was allowed to recover for 48 h prior to sacrifice. In an attempt to increase the injury, a second group of animals was salt-depleted prior to injury, a third group was volume-depleted by being deprived of H2O for 72 h prior of injury, a fourth group received 7.5 mg/kg indomethacin 30 min prior to injury, and a fifth group had 30% of the blood which was removed to produce shock hemolyzed and returned following the injury. In all groups examined, light microscopy revealed a moderate to severe acute tubular necrosis localized mainly in the outer stripe of the outer zone as defined by Peter (1909). Tubular damage was confined to the medullary pars recta of the proximal tubule and only in the most severe cases did injury involve the cortical pars recta and pars convoluta. Casts were present in the distal tubules and collecting ducts. Despite these significant histologic alterations, BUN values from all experimental groups remained within control levels. These studies clearly show that extensive necrosis of the medullary pars recta can be dissociated from the development of acute renal failure.     

The specificity of disease-associated anti-fibrillarin autoantibodies compared with that of HgCl2-induced autoantibodies

Autoantibodies against nucleolar components are a common serological feature of patients suffering from scleroderma, a collagen vascular autoimmune disease. An important target of these autoantibodies is a protein with an apparent molecular weight of 36 kDa and a pI value of 8.5, located in the dense fibrillar component of the nucleolus and therefore termed fibrillarin. Animal models in which abundant anti-nucleolar antibodies appear spontaneously have not yet been described; however, high levels of anti-fibrillarin antibodies can be induced by treating susceptible strains of mice with sub-toxic amounts of mercuric chloride. In this study, we have analysed the specificity of anti-fibrillarin autoantibodies of human and murine origin. Our results suggest that both species have similar, if not identical conformational epitopes that are the target of anti-fibrillarin autoantibodies; these epitopes require the presence of a 30-kDa fragment of the fibrillarin molecule. Post-translational modifications such as the dimethylation of arginines in the N terminus of the protein are not essential for antibody recognition.Abbreviations ANA Antinuclear autoantibodies - ANolA Antinucleolar autoantibodies - snoRNA small nucleolar RNA - snoRNP small nucleolar ribonucleoprotein     

Amelioration with vessel dilator of acute tubular necrosis and renal failure established for 2 days

Seventeen Sprague-Dawley rats had ischemic nonoliguric acute renal failure (ARF) induced by vascular clamping resulting in their preischemic blood urea nitrogen (BUN) and creatinine levels of 16 +/- 1 and 0.56 +/- 0.05 mg/dl to increase to 162 +/- 4 and 8.17 +/- 0.5 mg/dl, P < 0.001, respectively, at day 4 of postischemia. Vessel dilator, a 37-amino-acid cardiac peptide hormone (0.3 microg x kg(-1) x min(-1) ip), decreased the BUN and creatinine levels to 53 +/- 17 mg/dl and 0.98 +/- 0.12 mg/dl (P < 0.001) in another seven animals where ARF had been established for 2 days. Water excretion doubled with ARF and was further augmented by vessel dilator. Transthoracic echocardiography revealed left ventricular dilation as a probable cause of the increase in vessel dilator in the circulation with ARF, and vessel dilator infusion reversed this dilation. At day 6 of ARF, mortality decreased to 14% with vessel dilator from 88% without vessel dilator. Acute tubular necrosis was <5% in the vessel dilator-treated rats compared with 25% to >75% in the placebo-treated ARF animals. We conclude that vessel dilator improves acute tubular necrosis and renal function in established ARF.     

HgCl2 increases the methemoglobin prooxidant activity. Possible mechanism of Hg2+-induced lipid peroxidation in erythrocytes

In an attempt to elucidate the mechanism of initiation of peroxidation in HgCl2-treated erythrocytes, the effect of HgCl2 on methemoglobin-catalyzed lipid peroxidation was studied. It was found that HgCl2 reinforces the prooxidant action of methemoglobin. This effect seems not to be due to dissociation or degradation of the hemoglobin molecule to heme-containing fragments or iron-containing products of low molecular weight. The results obtained indicate that Hg2+ increases the binding of oxy- and methemoglobin to liposomes. A suggestion is made that the acceleration of methemoglobin-catalyzed peroxidation by HgCl2 is mainly due to increased binding of methemoglobin to liposomes. On the basis of these results and the results obtained previously the possible mechanism of initiation of peroxidation in Hg2+-treated erythrocytes is discussed.     

Opposite effects of diabetes on nephrotoxic and ischemic acute tubular necrosis

The effect of streptozotocin-induced diabetes mellitus on two different models of acute tubular necrosis (ATN), was studied: (i) the nephrotoxic model of HgCl2-induced ATN and (ii) the ischemic model of renal artery clamping for 60 min. Induction of ATN with HgCl2 in normal rats decreased CrCl from 0.67 +/- 0.05 to 0.1 +/- 0.019 ml/min (P less than 0.001) after 24 hr, and it deteriorated further to 0.03 +/- 0.013 ml/min after 48 hr; whereas, in the diabetic rats, HgCl2 decreased CrCl from 0.98 +/- 0.11 only to 0.31 +/- 0.037 ml/min (P less than 0.0001), but CrCl recovered to 0.50 +/- 0.08 ml/min after 48 hr. Bilateral clamping of renal arteries for 60 min in control and diabetic rats extremely decreased CrCl in both groups. Twenty-four hours after clamping, two of nine rats from the diabetic group died, whereas none from the control group died. Forty-eight hours after clamping, all nine rats from the diabetic group died, whereas only two rats from the control group died, and in the four surviving rats CrCl recovered slightly. Our study shows that streptozotocin-induced diabetes could not confer a general protection against ATN. It was protective against a nephrotoxic insult but aggravated the ischemic insult. An attempt to reconcile these discrepant effects is made in the Discussion.     

人脐带间充质干细胞在急性肾小管坏死模型犬的体内分布及趋化性

目的 观察人脐带间充质干细胞在家犬急性肾小管坏死模型的体内分布及归巢.方法 健康家犬18 只随机分为3 组.模型1 组:肌注新鲜配制的0.2﹪二氯化汞溶液7 ml/kg建立急性肾小管坏死模型,采用经外周静脉注射法输注体外分离培养并用4',6- 二脒基-2- 苯基吲哚(DAPI)标记的人脐带间充质干细胞.模型2 组:造模...     

Intrarenal distribution of calcium and magnesium in the dog. Effect of an osmotic diuresis (author's transl)]

1 The determination of Na, Ca, Mg, and K concentrations was performed in four different regions of the dog kidney (cortex, outer medulla, inner medulla, and papilla) during antidiuresis and during an osmotic diuresis. 2 The results show a medullary concentration gradient for calcium. This gradient is much higher than that found for sodium. 3 An inverse concentration gradient from cortex to inner medulla for Mg and K is found. 4 An osmotic diuresis (hypertonic mannitol) decreases the corticomedullary gradient of Na, but does not alter significantly the intrarenal distribution of Ca, Mg and K. 5 These results consistent with an intracellular localization of Mg and K in the renal tissue. It is suggested that the medullary concentration gradient for Ca may be due either to a countercurrent multiplier system similar to that for Na, or to a higher tissular fixation of Ca in the inner medulla and papilla than in the outer medulla and cortex.