Makoto Nodera

Increased apoptosis in a variety of tissues of zinc-deficient rats

Zinc deficient rats were prepared to investigate histopathological changes in thymus, testis, skin, esophagus, kidney and liver and the relationship between these changes and apoptosis. Seven-week-old male SD rats were given a Zn deficient diet (0% Zn diet) or a standard diet (0.02% Zn diet). The above-mentioned organs were excised 1, 2, 3, 4, 5, 10, 13, and 34 weeks after initiating diet administration. Then, these organs were examined morphologically, and apoptotic changes were analyzed by either the TdT- mediated dUTP - biotin nick end labeling (TUNEL) or electrophoresis. Significant morphological changes were seen only in rats on the 0% Zn diet. After 4 weeks, atrophy of the thymus was seen. After 5 weeks, oligospemia was observed, and after 10 weeks, testicular atrophy accompanied by the loss of sperm cells and spermatocytes was confirmed. In addition, after 10 weeks, thickening of epithelia was seen in the skin and esophagus of rats on the 0% diet. During the observation period, no marked morphological changes were observed in the liver or kidney. In the thymus and testis of rats on the 0% Zn diet, prior to detecting any morphological changes, increases in apoptosis were confirmed at 1 and 3 weeks after initiating diet administration, respectively. In the kidney and liver, TUNEL positive cells appeared after 13 and 34 weeks, respectively. These observations suggest that the functional and morphological changes in the thymus and testis of rats on the 0% Zn diet are caused by increased apoptosis, and that even when the supply of Zn is terminated for only a short period of time, immunocytes and germ cells can not survive or regenerate sufficiently. Again, the fact that even in the liver and kidney, apoptosis was observed when administration of the 0% Zn diet was prolonged suggests that the appearance of apoptosis is dependent on the amount of Zn in tissues. In addition, the fact that increases in apoptosis were confirmed in the skin of rats on the 0% Zn diet, but not in the esophagus of these rats suggests that apoptosis does not directly cause thickening of stratified squamous epithelium in Zn deficient rats.

Excessive zinc intake elevates systemic blood pressure levels in normotensive rats - potential role of superoxide-induced oxidative stress

Objectives The present study was designed to examine whether or not excessive Zn intake affects systemic blood pressure (BP) levels in a normotensive state. Methods Systolic BP (SBP) and mean arterial pressure (MAP) before and after administration of the nitric oxide synthase (NOS) inhibitor, Nω-nitro-L-arginine methyl ester (l-NAME) or the exogenous superoxide scavenger, tempol and the activity of the endogenous superoxide scavenger, Cu/Zn-superoxide dismutase (SOD) and levels of endothelial type (e)NOS mRNA and protein in the thoracic aorta were analyzed in male Sprague–Dawley rats fed a standard diet containing 0.005% Zn or a high Zn diet containing 0.5% Zn for 8 weeks. Results SBP and MAP levels observed at the end of dietary conditioning were significantly elevated in rats fed a high Zn diet relative to rats fed a standard diet. Administration of l-NAME caused an increase in MAP levels in rats fed a standard and a high Zn diet, demonstrating the involvement of the vasodilator, nitric oxide (NO) in the regulation of systemic BP in the two groups of rats. However, the expression of eNOS mRNA and protein in the thoracic aorta was not significantly different between rats fed a standard and a high Zn diet. On the other hand, administration of tempol led to a decrease in MAP levels in rats fed a standard and a high Zn diet, indicating the participation of the oxygen free radical, superoxide in the modification of systemic BP in the two groups of rats. As reported recently, the mechanism involved is due likely to a decrease in the action of the vasodilator, NO through the formation of peroxynitrite based on the non-enzymatic reaction of superoxide and NO. In addition, tempol treatment dramatically restored MAP levels in rats fed a high Zn diet to levels comparable with those observed in rats fed a standard diet, indicating that an elevation in systemic BP levels seen in rats fed a high Zn versus a standard diet is presumably brought by a reduction in the action of the vasodilator, NO resulting from an increase in the action of superoxide. The activity of Cu/Zn-SOD in the thoracic aorta was significantly reduced in rats fed a high Zn diet relative to rats fed a standard diet, appearing to at least in part, play a role in an increase in the action of superoxide in the vessel wall of rats fed a high Zn diet. Conclusions Excessive Zn intake may be a factor to elevate systemic BP levels in a normotensive state presumably through the oxidative stress caused by superoxide.

ZINC DEFICIENCY AGGRAVATES TUBULOINTERSTITIAL NEPHROPATHY CAUSED BY URETERAL OBSTRUCTION

Ureteral ligation causes tubulointerstitial nephropathy characterized by the tubular dilatation, the interstitial expansion, and a leukocyte infiltration into the tubulointerstitium. The present study was designed to explore whether zinc (Zn) deficiency affects the development of unilateral ureteral obstruction (UUO)-induced tubulointerstitial nephropathy. Compared to rats fed a standard diet, rats fed a Zn-deficient diet exhibited a greater influx of leukocytes into the tubulointerstitium of the cortex of the obstructed kidney 3 d following UUO. Furthermore, rats fed a Zn-deficient diet showed a slight invasion of leukocytes into the tubulointerstitium of the cortex of the contralateral nonobstructed kidney (CLK), although there was no infiltration of leukocytes into the CLK of rats fed a standard diet. These histological changes, however, were ameliorated by administration of enalapril, an angiotensin (ANG) I-converting enzyme inhibitor. Thus, it is suggested that Zn deficiency aggravates UUO-induced tubulointerstitial nephropathy via an increase in the action of ANG II.

Ureteral Obstruction Enhances Eicosanoid Production in Cortical and Medullary Tubules of Rat Kidneys

We examined prostaglandin (PG) E2, 6-keto PGF1alpha, and thromboxane B2 (TxB2) production in cortical and medullary tubules from sham-operated control (SOC) rats and rats with bilateral ureteral obstruction (BUO) of 24 h duration. In SOC rats medullary tubules produced significantly greater amounts of the three eicosanoids than cortical tubules. Again, the production of PGE2, 6-keto PGF1alpha, and TxB2 by cortical and medullary tubules was significantly greater in BUO rats than in SOC rats. To elucidate the mechanisms involved, we examined the activity of phospholipase A2 (PLA2) reactive against phosphatidylcholine or phosphatidylethanolamine (PE), the activity of phospholipase C (PLC), and the levels of cyclooxygenase (COX) in cortical and medullary tubules from SOC and BUO rats. In SOC rats the activity of phosphatidylcholine-PLA2 and PE-PLA2, the activity of PLC, and the mass of COX were significantly greater in medullary tubules than in cortical tubules. On the other hand, the activity of PLC in membranes of cortical tubules and the activity of PE-PLA2 and PLC in membranes of medullary tubules, which were in active location, were significantly greater in BUO rats than in SOC rats. COX levels were also significantly greater in cortical and medullary tubules of BUO rats than in those of SOC rats. Thus, we indicate that medullary tubules from SOC rats have greater production of eicosanoids through increased activity of the PLA2 and PLC-COX pathway than cortical tubules from the same group of rats. Again, in rats with BUO, the tubular eicosanoid production may be enhanced via activation of the PLC-COX pathway in cortical tubules or through activation of the PE-PLA2 and PLC-COX pathway in medullary tubules. The enhanced production of tubular eicosanoids observed in rats with BUO may affect tubular function, particularly sodium and water reabsorption.

Detection of prepro-ET-1 mRNA in normal rat kidney by in situ RT-PCR.

Background: Available evidence has shown that endothelin-1 (ET-1) acts in an autocrine/paracrine fashion rather than as a hormone or cytokine. Therefore, the analysis of local ET-1 production is a crucial step toward understanding its physiological and pathophysiological importance. In this study, in situ RT-PCR was utilized to detect tubular expression of prepro-ET-1 mRNA in normal rat kidney. Methods: Kidneys were taken from normal Sprague-Dawley rats weighing approximately 200 g. In situ RT-PCR was carried out using the preparations embedded in paraffin and cut at a thickness of 8 µm. Furthermore, we tried semiquantitation of the prepro-ET-1 mRNA expression along different nephron segments by densitometric analysis. Results: Prepro-ET-1 mRNA expression was detected in all tubular segments of the kidney from normal rats. Densitometric analysis demonstrated its highest expression in cortical collecting duct (CCD) and outer medullary collecting duct (OMCD). The expression was the lowest in thin descending limb of Henle’s loop (TDL). Conclusion: This study showed that all tubular segments have the ability to synthesize ET-1 in rat kidney. It would be worth evaluating the levels prepro-ET-1 mRNA expression in various diseases by in situ RT-PCR to understand its pathophysiological role in such settings.

Altered expression of endothelin-1 and endothelial nitric oxide synthase in the juxtaglomerular apparatus of rats with HgCl2-induced acute renal failure

A progressive fall in glomerular capillary plasma flow (QA) is observed in mercury chloride (HgCl2)-induced acute renal failure (ARF) although the site of the main lesion of this ARF is the proximal tubule. To elucidate this mechanism, we examined the expression of endothelin (ET)-1 and endothelial nitric oxide synthase (eNOS) protein at the glomerulus level in the kidneys of control rats and rats with HgCl2-induced ARF. Both ET-1 and eNOS protein were detected in the juxtaglomerular cells of afferent arterioles. The expression of ET-1 was significantly increased in ARF rats when compared to control rats. Inversely, the expression of eNOS protein was markedly reduced in ARF rats as opposed to control rats. These observations suggest the participation of the vasoconstrictor, ET-1 and the vasodilator, NO in a reduction in QA observed in HgCl2-induced ARF.

Zinc-Excess Intake Causes the Deterioration of Renal Function Accompanied by an Elevation in Systemic Blood Pressure Primarily Through Superoxide Radical-Induced Oxidative Stress

Using rats fed 22 g/d of a control diet containing 0.005% zinc (Zn) or 2 Zn-excess diets containing 0.05% or 0.2% Zn for 4 weeks, we examined the mechanisms involved in the deterioration of renal function induced by Zn-excess intake. An increase in Zn intake elevated mean blood pressure (BP) and reduced renal blood flow (RBF) and inulin clearance in a dose-dependent manner. This decline in inulin clearance may be derived from a fall in RBF. Administration of the nitric oxide (NO) synthase inhibitor, Nω-nitro-l-arginine methyl ester, markedly increased mean BP and significantly decreased RBF in the 3 groups of rats. Administration of the exogenous superoxide radical (OO−) scavenger, tempol, significantly decreased mean BP and substantially increased RBF in all groups of rats. These observations suggest that both an elevation in systemic BP and a reduction in RBF seen in the 2 Zn-excess diet groups result from a decrease in the action of the vasodilator, NO, through the formation of peroxynitrite based on the nonenzymatic reaction of NO and increased OO−. Indeed, the activity of the endogenous OO− scavenger, copper/Zn-superoxide dismutase, was significantly reduced in the vessel wall of rats fed 2 Zn-excess diets versus a control diet. 8-Hydroxy-2′-deoxyguanosine formation caused by OO− generation was notably elevated in the kidneys of rats fed 2 Zn-excess diets relatively to rats fed a control diet. Thus, Zn-excess intake leads to the aggravation of renal function concomitantly with an increase in systemic BP predominantly through the oxidative stress caused by OO−.