Acute prostaglandin reduction with indomethacin and chronic prostaglandin reduction with an essential fatty acid deficient diet both decrease plasma flow to the renal papilla in the rat

Renal distribution of prostaglandin synthetase is mainly medullary, whereas the major degrading enzyme, prostaglandin dehydrogenase is primarily cortical. This suggests that prostaglandins (PG) released from the renal medulla could affect the medullary blood vessels. In two different experiments we studied the role of PG in the regulation of renal papillary plasma flow in the rat. First study: PG synthesis were stimulated in 34 adult Sprague-Dawley rats by bleeding from the femoral artery 1% of the body weight over a period of 10 minutes. Following this, indomethacin (a PG inhibitor, 10 mg/kg i.v.) was given slowly and then renal papillary plasma flow was measured 25 minutes after the end of infusion. In 17 indomethacin rats the renal papillary plasma flow averaged 18.8 ml/100 g/minute, whereas it averaged 23.0 in 17 non-indomethacin rats given diluent, an 18% reduction (p less than .025). Second study: Male Sprague-Dawley rats were made prostaglandin deficient by fasting rats for one week, followed by 10% dextrose fluid for one week and subsequent institution of an essential fatty acid (EFA) deficient diet for two weeks. With urinary PG excretion in prostaglandin deficient rats 28 ng/24 hours compared to 149 ng in control rats, they could be considered as prostaglandin deficient. When renal papillary plasma flow was measured, the 16 prostaglandin deficient rats had a 16% lower papillary plasma flow than 16 control rats, 21.6 vs 25.6 (p less than .005). These results clearly demonstrate that PG inhibition in rats decreases plasma flow to the papilla, strongly suggesting that PG are vasodilators for the vessels supplying the renal papilla.     

Changes in the incretory structures of the kidneys and the heterogeneity of the medulla under acute water and salt loading in rats

Juxtaglomerular complex (JGA) and kidney medullar interstitial cells (IC) in rats in acute water and salt loadings were studied. The animals were killed 1 and 2 hours later after water 0.9% and 2% natrium chloride per os administration in quantity of 5%-7% per total body weight. Plasma osmolality and plasma renin activity (PRA) was determined. In light microscopy the granulation of the JGA-cells was in accordance with PRA in all the experiment. IC were not altered in water loading. Marked diminishing of lipid granulation of the IC in all zones of papilla at 2% salt loading was observed. In electron microscopy changes in distribution and density of lipid granules of the IC in all zones of the renal papilla were noted. The lesions showed the dystrophic and necrobiotic changes in some IC, especially in central zone of papilla.     

Prostaglandin H synthase immunoreactivity localized by immunoperoxidase technique (PAP) in the small intestine and kidney of rabbit and guinea-pig.

Prostaglandins and inhibitors of prostaglandin synthesis have striking regulatory effects on intestinal muscularis externa. We suggested earlier that a population of macrophage-like cells, located between the external muscle layers might release prostaglandins with a local effect on enveloping interstitial cells of Cajal, postulated pacemaker cells of the gut. To determine cellular production site(s) of prostaglandin we applied monoclonal antibodies against prostaglandin H synthase combined with the PAP technique to sections of rabbit and guinea-pig small intestine and kidney. In rabbit small intestine muscle cells in the circular muscle layer and in the muscularis mucosae were positive, longitudinal muscle negative. Vascular endothelial cells and serosal mesothelial cells were stained. In guinea-pig all muscle layers were unstained but endothelial and mesothelial cells were stained together with unidentified cells in the outermost submucosa. In rabbit kidney, positive staining of collecting ducts, interstitial cells, the parietal layer of Bowman's capsule and arterial endothelial cells was present. Furthermore, we found prostaglandin synthase antigenicity in the epithelial cells lining the loop of Henle, not described before. In guinea-pig medullary collecting ducts were stained and the papilla was lined by stained epithelial cells. The results show a species variation in the distribution of recognizable levels of prostaglandin H synthase. The impressive reaction in the mesothelium must be considered, when enzyme distribution is examined biochemically with fractionated tissue. Our findings do not support our hypothesis that macrophage-like cells are more potent sources of prostaglandins than smooth muscle cells.     

Prostaglandin H synthase immunoreactivity localized by immunoperoxidase technique (PAP) in the small intestine and kidney of rabbit and guinea-pig

Summary Prostaglandins and inhibitors of prostaglandin synthesis have striking regulatory effects on intestinal muscularis externa. We suggested earlier that a population of macrophage-like cells, located between the external muscle layers might release prostaglandins with a local effect on enveloping interstitial cells of Cajal, postulated pacemaker cells of the gut.To determine cellular production site(s) of prostaglandin we applied monoclonal antibodies against prostaglandin H synthase combined with the PAP technique to sections of rabbit and guinea-pig small intestine and kidney. In rabbit small intestine muscle cells in the circular muscle layer and in the muscularis mucosae were positive, longitudinal muscle negative. Vascular endothelial cells and serosal mesothelial cells were stained. In guinea-pig all muscle layers were unstained but endothelial and mesothelial cells were stained together with unidentified cells in the outermost submucosa. In rabbit kidney, positive staining of collecting ducts, interstitial cells, the parietal layer of Bowman's capsule and arterial endothelial cells was present. Furthermore, we found prostaglandin synthase antigenicity in the epithelial cells lining the loop of Henle, not described before. In guinea-pig medullary collecting ducts were stained and the papilla was lined by stained epithelial cells.The results show a species variation in the distribution of recognizable levels of prostaglandin H synthase. The impressive reaction in the mesothelium must be considered, when enzyme distribution is examined biochemically with fractionated tissue. Our findings do not support our hypothesis that macrophage-like cells are more potent sources of prostaglandins than smooth muscle cells.     

Demonstration of prostaglandin synthesis in collecting duct cells and other cell types of the rabbit renal medulla.

The renal medulla has a high capacity for prostaglandin production and the interstitial cells, which contain abundant lipid inclusions have been suggested to be the site of synthesis. However, histochemical studies have indicated that the collecting ducts are the main site of production. The object of the present study was to study the distribution of prostaglandin synthetase in the rabbit renal medulla by direct, quantitative determination of the enzyme activity in different cellular fractions. Slices were cut from rabbit renal papilla and immersed in a hypertonic saline solution. 92% of the collecting duct cells were then removed from the slices by suction through a micropipette. The remaining dissected slices thus contained mainly three cell types, cells of Henle's loop, endothelial cells, and interstitial cells. The isolated collecting duct fraction, the corresponding dissected slices, from which the colelcting duct cells were removed, as well as intact slices were assayed for prostaglandin synthetase activity using a quantitative assay with [14C] arachidonate as substrate. Of the prostaglandin in synthetase activity 39% was found in the collecting ducts, 53% in the dissected slices, and 7% in the dissection medium. It is thus concluded that significant prostaglandin synthetase activity is present in collecting duct cells as well as in at least one other cell type of the medulla.     

Salicylate nephropathy in the Gunn rat: potential role of prostaglandins

We examined the potential role of prostaglandins in the development of analgesic nephropathy in the Gunn strain of rat. The homozygous Gunn rats have unconjugated hyperbilirubinemia due to the absence of glucuronyl transferase, leading to marked bilirubin deposition in renal medulla and papilla. These rats are also highly susceptible to develop papillary necrosis with analgesic administration. We used homozygous (jj) and phenotypically normal heterozygous (jJ) animals. Four groups of rats (n = 7) were studied: jj and jJ rats treated either with aspirin 300 mg/kg every other day or sham-treated. After one week, slices of cortex, outer and inner medulla from one kidney were incubated in buffer and prostaglandin synthesis was determined by radioimmunoassay. The other kidney was examined histologically. A marked corticomedullary gradient of prostaglandin synthesis was observed in all groups. PGE2 synthesis was significantly higher in outer medulla, but not cortex or inner medulla, of jj (38 +/- 6 ng/mg prot) than jJ rats (15 +/- 3) (p less than 0.01). Aspirin treatment reduced PGE2 synthesis in all regions, but outer medullary PGE2 remained higher in jj (18 +/- 3) than jJ rats (9 +/- 2) (p less than 0.05). PGF2 alpha was also significantly higher in the outer medulla of jj rats with and without aspirin administration (p less than 0.05). The changes in renal prostaglandin synthesis were accompanied by evidence of renal damage in aspirin-treated jj but not jJ rats as evidenced by: increased incidence and severity of hematuria (p less than 0.01); increased serum creatinine (p less than 0.05); and increase in outer medullary histopathologic lesions (p less than 0.005 compared to either sham-treated jj or aspirin-treated jJ). These results suggest that enhanced prostaglandin synthesis contributes to maintenance of renal function and morphological integrity, and that inhibition of prostaglandin synthesis may lead to pathological renal medullary lesions and deterioration of renal function.     

Demonstration of prostaglandin synthesis in collecting duct cells and other cell types of the rabbit renal medulla

The renal medulla has a high capacity for prostaglandin production and the interstitial cells, which contain abundant lipid inclusions, have been suggested to be the site of synthesis. However, histochemical studies have indicated that the collecting ducts are the main site of production. The object of the present study was to study the distribution of prostaglandin synthetase in the rabbit renal medulla by direct, quantitative determination of the enzyme activity in different cellular fractions.Slices were cut from rabbit renal papilla and immersed in a hypertonic saline solution. 92% of the collecting duct cells were then removed from the slices by suction through a micropipette. The remaining dissected slices thus contained mainly three cell types, cells of Henle's loop, endothelial cells, and interstitial cells. The isolated collecting duct fraction, the corresponding dissected slices, from whcih the collecting duct cells were removed, as well as intact slices were assayed for prostaglandin synthetase activity using a quantitative assay with [¹⁴C] arachidonate as substrate.Of the prostaglandin synthetase activity 39% was found in teh collecting ducts, 53% in the dissected slices, and 7% in the dissection medium. It is thus concluded that significant prostaglandin synthetase activity is present in collecting duct cells as well as in at least one other cell type of the medulla.     

Differences in lipid granulation as the basis for a morphologic differentiation between third-stage larvae of Uncinaria stenocephala and Ancylostoma caninum

Differences in the distribution of lipid granules between unstained third-stage larvae of Uncinaria stenocephala and Ancylostoma caninum cultured at 15 C was found to be an effective means for differentiating these 2 species of canine hookworms. In contrast, larvae cultured at 22 C were less easily differentiated based on the distribution of lipid granules. After culturing at 15 C, third-stage larvae of U. stenocephala were motile and exhibited 32 well-demarcated intestinal cells which contained intracellular lipid granules. Intestinal cells were easily visualized due to the absence of extraintestinal lipid granulation. Ancylostoma caninum third-stage larvae cultured under similar conditions were significantly less motile and contained extraintestinal accumulations of lipid granules which obscured intestinal cells. Both species exhibited an overall decrease in lipid granulation during a 14-day observation period following culture at 15 C. Morphologic differentiation of these 2 species after 14 days was based on the absence of intra- and extra-intestinal lipid in U. stenocephala and the presence of some lipid granules in both these locations in A. caninum. The first- and second-stage larvae of both species cultured at 15 C exhibited dense accumulations of extraintestinal lipid granules and were morphologically indistinguishable. This suggests that the observed difference in lipid granulation between the third-stage larvae of U. stenocephala and A. caninum cultured at 15 C is due to differences in lipid utilization during the third stage rather than differences in lipid synthesis by the first- and second-stage larvae and is related to the adaptation of these parasites to their respective climatic regions.     

Salicylate nephropathy in the Gunn rat: Potential role of prostaglandins

We examined the potential role of prostaglandins in the development of analgesic nephropathy in the Gunn strain of rat. The homozygous Gunn rats have unconjugated hyperbilirubinemia due to the abscence of glucuronyl transferase, leading to marked bilirubin deposition in renal medulla and papilla. These rats are also highly susceptible to develop papillary necrosis with analgesic administration.We used homozygous (jj) and phenotypically normal heterozygous )jJ) animals. Four groups of rats (n = 7) were studied: jj and jJ rats treated either with aspirin 300 mg/kg every other day or sham-treated. After one week, slices of cortex, outer and inner medulla from one kidney wre incubated in buffer and prostaglandin synthesis was determined by radioimmunoassay. The other kidney was examined histologically.A marked corticomedullary gradient of prostaglandin synthesis was observed in all groups, PGE2 synthesis was significantly higher in outer medulla, but not cortex or inner medulla, of jj (38 ± 6 mg/mg prot) than jJ rats (15 ± 3) (p<0.01). Aspirin treatment reduced PGE2 synthesis in all regions, but outer medullary PGE2 remained higher in jj (18 ± 3) than jJ rats (9 ± 2) (p<0.05). PGE2α was also significantly higher in the outer medulla of jj rats with and without aspirin administration (p<0.05). The changes in renal prostaglandin synthesis were accompanied by evidence of renal damage in aspirin-treated jj but not jJ rats as evidenced by: increased incidence and severity of hematuria (p<0.01); increased serum creatinine (p<0.05); and increase in outer medullary histopathologic lesions (p<0.005 compared to either sham-treated jj or aspirin-treated jJ).These results suggest that enhanced protaglandin synthesis contributes to maintenance of renal function and morphological integrity, and that inhibition of protaglandin synthesis may lead to pathological renal medullary lesions and deterioration of renal function.     

Increased renal tubular synthesis of prostaglandins in the rabbit kidney in response to ureteral obstruction

The hydronephrotic rabbit kidney exhibits elevated basal prostaglandin synthesis and supersensitivity to peptide stimulation of vascular prostaglandin and thromboxane formation. In this study the distribution of the prostaglandin-forming cyclooxygenase in hydronephrotic and contralateral rabbit kidneys following one and four day ureteral obstructions was compared using immunohistofluorescence. No alterations were detected in the distribution or intensity of cyclooxygenase-positive fluorescence in the renal vasculature in response to ureteral obstructions. However, two significant differences were noted between hydronephrotic and contralateral kidneys in the staining of renal tubules: (a) the intensity of fluorescent staining in cortical and medullary collecting tubules of the hydronephrotic kidney was increased and (b) cyclooxygenase antigenicity appeared in the thin limbs of Henle's loop in the hydronephrotic organ. Although alterations in prostaglandin formation by the renal vasculature have been documented previously, our results indicate that ureteral obstruction also causes increased prostaglandin synthesis by renal tubules.     

Increased renal tubular synthesis of prostaglandins in the rabbit kidney in response to ureteral obstruction.

The hydronephrotic rabbit kidney exhibits elevated basal prostaglandin synthesis and supersensitivity to peptide stimulation of vascular prostaglandin and thromboxane formation. In this study the distribution of the prostaglandin-forming cyclooxygenase in hydronephrotic and contralateral rabbit kidneys following one and four day ureteral obstructions was compared using immunohistofluorescence. No alterasions were detected in the distribution or intensity of cyclooxygenase-positive fluorescence in the renal vasculature in response to ureteral obstructions. However, two significant differences were noted between hydronephrotic and contralateral kidneys in the staining of renal tubules: (a) the intensity of fluorescent staining in cortical and medullary collecting tubules of the hydronephrotic kidney was increased and (b) cyclooxygenase antigenicity appeared in the thin limbs of Henle's loop in the hydronephrotic organ. Although alterations in prostaglandin formation by the renal vasculature have been documented previously, our results indicate that ureteral obstruction also causes increased prostaglandin synthesis by renal tubules.     

Lipid granularity and topographic characteristics of renomedullary interstitial cells in genetically spontaneous hypertension in rats

Light microscopy was used to study lipid granularity of the renomedullary interstitial cells (IC) in spontaneously hypertensive rats (SHR) aged 4 months and in Wistar rats (control) of the same age. Altogether 2000 IC were examined per animal on an average. The concentric distribution of the granular IC in the renal papilla was demonstrated. Lipid granularity of the IC was discovered to be lowered and the zone of granular IC to be enlarged in the whole papilla in SHR rats as compared with control. Also, the differences were found in lipid granularity of the IC in individual zones of the papilla in both SHR and normotensive animals, which seems likely to indicate different functional significance of the individual parts of the renal papilla.     

Immunoreactive atrial natriuretic polypeptides in the adrenal medulla and sympathetic ganglia

Atrial natriuretic polypeptide (ANP)-like immunoreactivity was found in the rat adrenal gland by using indirect immunofluorescence and peroxidase-antiperoxidase techniques. ANP-like immunostaining was present in most of chromaffin cells with varying degrees of immunoreactivity. The majority of medullary cells displayed very intense immunostaining, and several clusters revealed weaker immunostaining. No staining was found in the adrenal cortex or in the nerve fibers in this organ. In the consecutive sections treated for dopamine-beta-hydroxylase (DBH), apparently all medullary cells had intense immunofluorescence for DBH and its distribution pattern was very similar to that for ANP-like immunoreactivity. While phenylethanolamine N-methyltransferase (PNMT) immunoreactive cells largely corresponded to the intensely stained ANP-like immunoreactive cells, suggesting that adrenaline cells contained a large amount of ANP-like substance, noradrenaline cells contained a smaller amount of this substance than adrenaline cells. Ultrastructural study showed that end-products due to the immunoreaction with the ANP antiserum were primarily associating with chromaffin granules. In addition, the presence of ANP-like immunoreactivity was investigated in several sympathetic ganglia of the rat. No principal ganglion cells were ANP-positive, whereas a few small intensely fluorescent (SIF) cells were ANP-immunoreactive. The present findings suggest that catecholamines coexist with ANP which has a natriuretic and vasodilating effect, in adrenal medullary cells and SIF cells in several rat sympathetic ganglia, but not in principal ganglion cells.     

Work in progress correlation of renal medullary prostaglandin content and renal interstitial cell lipid droplets

Indomethacin administration and hydronephrosis in rabbits has been found to produce increases in the number and changes in the composition of the lipid droplets in the renal medullary interstitial cells. The response to indomethacin, a prostaglandin synthetase inhibitor, was dose dependent.Work is in progress to assess the effects of other non-steroidal antiinflammatory drugs on the renal inner medulla and the interstitial cell lipid droplets.     

Dissociation between renal medullary PGE2-synthesis and urine PGE2-excretion. Antagonism by bumetanide of chlorazanil induced urine PGE2-excretion in rats

Normal conscious female Sprague-Dawley rats were treated with chlorazanil (3 mg/kg i.p.), and urine was collected for 3 hours. Urine prostaglandin E₂-excretion increased from 25±3 to 271±32 ng/kg/3 h. The enhancement of urine PGE₂-excretion was inhibited by pretreatment with bumetanide (75 mg/kg p.o.). In separate experiments the papillary quantity of PGE₂ was determined in freshly homogenized tissue. The basal level (14±2 ng PGE₂/papilla) was increased by chlorazanil to 51±11 ng PGE₂/papilla and 24±7 ng PGE₂/papilla at one and two hours respectively after drug administration. The capacity of chlorazanil to increase medullary PGE₂ accumulation was unaffected by bumetanide dissociated the medullary PGE₂ level from the excretion of PGE₂ in urine, when the former was elevated by chlorazanil.     

Prostaglandin E2 production by renal inner medullary tissue slices: effect of metabolic inhibitors

Increasing oxygen from 5 to 95% has previously been shown to increase prostaglandin (PG) production in renal inner medullary slices. The possible role of oxidative phosphorylation in this process was investigated. The oxidative phosphorylation inhibitors, dinitrophenol (DNP), oligomycin, and cyanide were evaluted for their effects on PGE2 production and ATP levels. None of the inhibitors affected PGE2 synthesis, although they lowered ATP levels at the concentrations tested. In contrast, incubation of inner medullary tissue slices with 0% oxygen resulted in decreases both in PGE2 and ATP levels. This suggests that the effect of oxygen on prostaglandin synthesis may be due to substrate limiting effects rather than an effect on oxidative phosphorylation. When 22 mM 2-deoxyglucose was added to the incubation medium or when glucose was omitted, PGE2 levels increased. Sodium fluoride, presumably acting as a glycolytic inhibitor, increased PGE2 levels, with a maximal effect at 10 mM. ATP levels were 37% of control values with 20 mM NaF. This indicates that glucose may inhibit prostaglandin synthesis. These results indicate that oxygen (substrate) availability can limit inner medullary PGE2 production. In view of the low pO2 in the inner medulla, especially during antidiuresis, oxygen can potentially regulate prostaglandin production in this tissue.     

Effects of indomethacin on renal inner medullary plasma flow

The effects of the prostaglandin system on renal hemodynamics were studied by treating rats with a single intraperitoneal dose of indomethacin, an inhibitor of prostaglandin synthesis. Medullary plasma flow was significantly reduced 30–45 minutes after indomethacin, but was elevated 3–6 hours after indomethacin. These changes in medullary plasma flow correlated well with circulating levels of prostaglandins A and E. Total renal blood flow decreased following indomethacin treatment, but returned to normal levels within an hour. These results indicate that the inhibition of prostaglandin synthesis following a single intraperitoneal dose of indomethacin is short-lived and is followed by a significant elevation in prostaglandin synthesis. It is likely that prostaglandin levels play an important role in the control of renal medullary plasma flow.     

Histology and histochemistry of the seal ovaries and the age-related changes in the ovaries of the Greenland seal]

Histological and histochemical methods were used to study the ovaries of Greenland seal (Pagophoca groenlandica) from birth-time up to 30 years of age and mature females of Phoca vitulina and Erignathus barbatus. The ovary of the new-born Greenland seal has fetal medullary substance which is a provisory endocrinous gland producing not only sex hormones but also corticosteron. In other species of seals the intestinal cells of the medullary substance are the equivalent of this gland. Within 3-4 weeks after birth the reduction of the fetal medullary substance is completed, it is substituted by the connective tissue and the ovary acquires its typical structure. The rest of the fetal medullary substance is in the depth of the cortex and near the infundibulum of the ovary as lipofuscincontaining cells. When the maturation period approaches, the process of the follicle atresia regularly changes: the epithelium dies quicker, and the multiplication of intestinal cells increases. The ovaries of seals are rich in interstitial cells. Their amount cyclically changes. The cells producing steroid hormones always well hydrolize AS naphthyl-phosphates, the reaction with glycerophosphate is more variable. The connective tissue is poor in acid mucopolysaccharides, its amorphous substance in the ovary cortex is rich in protein. Senile changes of the ovary are noticed in the seal beginning from 20 years of age.     

Stimulation of prostaglandin synthesis by ascorbic acid via hydrogen peroxide formation

Ascorbic acid causes an increase in prostaglandin (PG) synthesis in human lung fibroblasts in culture. This is accompanied by an increase in fatty acid release from cellular lipid stores. The effects of ascorbic acid on prostaglandin synthesis and fatty acid release are erased if the cultures are treated simultaneously with catalase, but not if treated with superoxide dismutase. The action of catalase points to a role of hydroperoxides in the synthesis of prostaglandins. The addition of hydrogen peroxide itself increases prostaglandin synthesis by these cells.     

The participation of hydroperoxides and oxygen radicals in the control of prostaglandin synthesis

Our results demonstrate that the organic hydroperoxide t-butyl-hydroperoxide (TBHP) influences the synthesis of prostaglandins in the human embryo lung fibroblast. TBHP inhibits or stimulates prostaglandin synthesis as a function of its concentration. Regardless of the concentration employed in these experiments however, TBHP stimulated the release of arachidonate from lipid stores. When the arachidonate release step in prostaglandin (PG) synthesis was bypassed by the addition of free arachidonate to the cell cultures, t-butyl hydroperoxide further stimulated PG synthesis, indicating that the hydroperoxide activates arachidonate conversion to prostaglandins. 4-Hydroxy-2,2,6,6-tetramethylpiperidinoxy radical, a scavenger of oxygen radicals, when added to cell cultures alone had no measurable effect on either arachidonate release or prostaglandin synthesis. When 4-hydroxy-2,2,6,6-tetramethylpiperidinoxy radical was administered to cell cultures in combination with t-butyl hydroperoxide, it increased prostaglandin synthesis while inhibiting arachidonate release by the hydroperoxide. The participation of hydroperoxides and trappers of oxygen radicals in the regulation of PG synthesis is not unique to lung fibroblasts. Endothelial cells from the vasculature as well as fibroblasts from the cornea also appear to be affected by these compounds with respect to prostaglandin synthesis.