Renal necrosis and the involvement of a single enzyme of the de novo pathway for the biosynthesis of platelet-activating factor in the rat kidney inner medulla

2-Bromoethylamine hydrobromide (BEA), when administered to rats, induces a highly specific papillary necrosis associated with the inner medulla. PAF levels in the blood were lowered by 50% and of the three enzymes that comprise the de novo route for PAF in the cortex/medulla, only the cholinephosphotransferase activity in the inner medulla microsomes was reduced (33%) by the BEA treatment. Moreover, BEA did not affect phosphatidylcholine synthesis in either the cortex or inner medulla. Our studies indicate that the de novo pathway for PAF synthesis in the renal inner medulla is responsible for the secretion of newly formed PAF into the blood stream and that a single enzyme in the de novo route accounts for the decreased rate of PAF synthesis during the development of renal necrosis.     

Urinary markers of nephrotoxicity following administration of 2 bromoethanamine hydrobromide a comparison with hexachlorobutadiene

2 bromoethanamine hydrobromide (BEA) has been widely considered to be a target selective nephrotoxin that causes necrosis of the medulla in 24-48 h, but recent reports suggest that early cortical injury is also associated with this lesion. In order to assess the cortical effects of BEA (100 mg kg^-1 bw single ip injection), several urinary markers of renal injury were evaluated over a 7 day period in male Wistar Albino rats. Hexachlorobutadiene (HCBD 150 mg kg^-1 bw in peanut oil ip), a renal toxin which targets selectively for the proximal tubule, was used as a comparison. After BEA treatment, urinary levels of alanine aminopeptidase, gamma-glutamyl-transpeptidase, alkaline phosphatase and glucose increased transiently. Each of the proximal tubule marker enzymes peaked earlier following HCBD treatment and elevation of alanine aminopeptidase and gamma glutamyl transpeptidase was sustained for longer periods than for BEA. Following BEA treatment, lactate dehydrogenase rose prominently on day 1 followed by a return to control values on day 2 and a further rise on day 3 and remained high until the end of the study. BEA also increased the urinary excretion of total protein and albumin. After HCBD treatment, lactate dehydrogenase showed a transient elevation and glucose levels were slightly increased. Based on the present observations the changes induced by BEA administration on urinary markers of renal injury are different from those observed following HCBD treatment. These findings suggest that BEA toxicity also involves other parts of the kidney besides the papilla.     

Urinary markers of nephrotoxicity following administration of 2 bromoethanamine hydrobromide a comparison with hexachlorobutadiene

2 bromoethanamine hydrobromide (BEA) has been widely considered to be a target selective nephrotoxin that causes necrosis of the medulla in 24-48 h, but recent reports suggest that early cortical injury is also associated with this lesion. In order to assess the cortical effects of BEA (100 mg kg^-1 bw single ip injection), several urinary markers of renal injury were evaluated over a 7 day period in male Wistar Albino rats. Hexachlorobutadiene (HCBD 150 mg kg^-1 bw in peanut oil ip), a renal toxin which targets selectively for the proximal tubule, was used as a comparison. After BEA treatment, urinary levels of alanine aminopeptidase, gamma-glutamyl-transpeptidase, alkaline phosphatase and glucose increased transiently. Each of the proximal tubule marker enzymes peaked earlier following HCBD treatment and elevation of alanine aminopeptidase and gamma glutamyl transpeptidase was sustained for longer periods than for BEA. Following BEA treatment, lactate dehydrogenase rose prominently on day 1 followed by a return to control values on day 2 and a further rise on day 3 and remained high until the end of the study. BEA also increased the urinary excretion of total protein and albumin. After HCBD treatment, lactate dehydrogenase showed a transient elevation and glucose levels were slightly increased. Based on the present observations the changes induced by BEA administration on urinary markers of renal injury are different from those observed following HCBD treatment. These findings suggest that BEA toxicity also involves other parts of the kidney besides the papilla.     

Effect of acidosis, alkalosis and monofluoroacetate administration on citrate and ATP content of rat renal medulla and papilla

1. --Renal distribution of citrate showed that there is an increase in citrate content from cortex to medulla and a decrease from medulla to papilla. Alkalosis produced an increase in citrate content and acidosis a decrease in renal citrate content, in each of the studied renal area. Monofluoroacetate produced no significant change in citrate content of medulla or papilla; it did not interfere with the acido-basic related changes in cortex citrate content, but its effect was additive. 2. --Renal distribution of ATP significantly decreased from cortex to medulla and from medulla to papilla. Acid or basic diet had no influence on intratissular ATP content. Fluoroacetate decreased renal ATP content.     

Sulfur-containing amino acids that increase renal glutathione protect the kidney against papillary necrosis induced by 2-bromoethylamine

Papillary necrosis was observed in the kidneys of rats, 72 h after receiving a single injection of bromoethylamine (BEA). This effect was associated with renal glutathione (GSH) depletion 1 h after the administration of BEA. Stimulation of renal GSH synthesis by pretreatment of the animals either with glutamine + glycine + cystine or N-acetyl-L-cysteine was attempted. Low doses of these precursors administered previously to BEA, respectively, decreased or abolished the GSH depletion. Nevertheless, both pretreatments failed to modify the magnitude of renal papillary necrosis. High doses of these precursors did not modify the BEA-induced GSH depletion, but they significantly increased GSH levels 24 h after BEA administration. At this time, although a smaller intensity of renal papillary necrosis was observed with the amino acid mixture pretreatment, N-acetyl-L-cysteine pretreated rats showed no papillary necrosis. It is suggested that the observed protective effects against BEA-induced renal papillary injury may be ascribed in some measure, to a mechanism independent of GSH.     

N-Acetyl-l-cysteine abolishes the bromoethylamine-induced choline incorporation into renal papillary tissue

The role of regenerative processes in the protective effect of N-acetyl-L-cysteine (NAC) against bromoethylamine-induced renal papillary necrosis was assessed in rats given bromoethylamine (BEA) (1.2 mmol/kg), N-acetylcysteine (6 mmol/kg), or N-acetylcysteine plus BEA. Renal papillary slices were dissected after 15 hours of treatment, and ¹⁴C-choline incorporation into total phospholipid, lysophosphatidylcholine, sphingomyelin, and phosphatidylcholine was measured. Bromoethylamine elicited an increase in the incorporation of ¹⁴C-choline into choline-containing phospholipid, an effect that was abolished when N-acetylcysteine was administered prior to bromoethylamine. These studies indicate that the defensive mechanism of N-acetylcysteine against bromoethylamine-induced renal papillary necrosis is not related to regenerative processes and that N-acetylcysteine abolishes the bromoethylamine-induced choline incorporation into papillary phospholipid. © 1996 John Wiley & Sons, Inc.     

N-acetyl-L-cysteine improves renal medullary hypoperfusion in acute renal failure

This study evaluated the effects of N-acetyl-L-cysteine (NAC), a free radical scavenger, and N(omega)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthesis inhibitor, on the changes in renal function, intrarenal blood flow distribution (laser-Doppler flowmetry), and plasma peroxynitrite levels during the acute renal failure (ARF) produced by inferior vena cava occlusion (IVCO; 45 min) in anesthetized rats. Renal blood flow fell on reperfusion (whole kidney by -45.7%; cortex -58.7%, outer medulla -62.8%, and papilla -47.7%); glomerular filtration rate (GRF) also decreased (-68.6%), whereas fractional sodium excretion (FE(Na%)) and peroxynitrite and NO/NO plasma levels increased (189.5, 46.5, and 390%, respectively) after ischemia. Pretreatment with L-NAME (10 microg. kg(-1). min(-1)) aggravated the fall in renal blood flow seen during reperfusion (-60%). Pretreatment with NAC (150 mg/kg bolus + 715 microg. kg(-1). min(-1) iv) partially prevented those changes in renal function (GFR only fell by -29.2%, and FE(Na%) increased 119.4%) and laser-Doppler blood flow, especially in the outer medulla, where blood flow recovered to near control levels during reperfusion. These beneficial effects seen in rats given NAC seem to be dependent on the presence of NO, because they were abolished in rats pretreated with L-NAME. Also, the antioxidant effects of NAC prevented the increase in plasma peroxynitrite after ischemia. In conclusion, NAC ameliorates the renal failure and the outer medullary vasoconstriction induced by ICVO, effects that seem to be dependent on the presence of NO and the scavenging of peroxynitrite.     

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.     

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.     

The volume and ionic composition of cells in incubated slices of rat renal cortex, medulla and papilla

The apparent extracellular space in incubated slices of rat renal cortex, medulla and papilla has been measured using three differently sized marker molecules, mannitol, sucrose and inulin. Cellular volumes have been estimated by following the efflux of 3-O-methyl-D-glucose from equilibrated slices. Sucrose appears to be the most accurate extracellular marker in each of the regions examined, in that the sum of its volume of distribution plus cellular volume approximates most closely to the total slice fluid volume. Inulin has the same volume of distribution as sucrose in cortical slices, but under-penetrates medullary and papillary tissue. Mannitol overestimates the extracellular space in all three regions, although its larger volume of distribution, relative to that of sucrose, was not statistically significant in papillary slices. When cell volume and composition are estimated (a) using sucrose as extracellular marker and (b) making appropriate allowance for the presence of bound tissue electrolytes, it is found that cells in each region have low Na+ and high K+ concentrations and contents. When papillary slices are incubated in medium of very high osmolality (NaCl plus urea, 2000 mosmol/kg H2O) there is a moderate (approx. 23%) decrease in cell volume and an increase in cell fluid Na+ and Cl- concentrations equal to approx. 50% of the increase in the extracellular concentrations. Cell K+ concentrations remain unchanged. The results show that cells in renal slices are able to maintain high K+-to-Na+ ratios when incubated in isosmotic (cortex) or moderately hyperosmotic media (medulla and papilla), and suggest that regulation of papillary cell volume following hyperosmotic shock can only partly be ascribed to uptake of extracellular electrolytes.     

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.     

Expression of multiple water channel activities in Xenopus oocytes injected with mRNA from rat kidney

To test the hypothesis that renal tissue contains multiple distinct water channels, mRNA prepared from either cortex, medulla, or papilla of rat kidney was injected into Xenopus oocytes. The osmotic water permeability (Pf) of oocytes injected with either 50 nl of water or 50 nl of renal mRNA (1 microgram/microliter) was measured 4 d after the injection. Pf was calculated from the rate of volume increase on exposure to hyposmotic medium. Injection of each renal mRNA preparation increased the oocyte Pf. This expressed water permeability was inhibited by p-chloromercuriphenylsulfonate and had a low energy of activation, consistent with the expression of water channels. The coinjection of an antisense oligonucleotide for CHIP28 protein, at an assumed > 100-fold molar excess, with either cortex, medulla, or papilla mRNA reduced the expression of the water permeability by approximately 70, 100, and 30%, respectively. Exposure of the oocyte to cAMP for 1 h resulted in a further increase in Pf only in oocytes injected with medulla mRNA. This cAMP activation was not altered by the CHIP28 antisense oligonucleotide. These results suggest that multiple distinct water channels were expressed in oocytes injected with mRNA obtained from sections of rat kidney: (a) CHIP28 water channels in cortex and medulla, (b) cAMP-activated water channels in medulla, and (c) cAMP-insensitive water channels in papilla.     

Renal medullary electrolytes: effects of furosemide assessed by studies in vivo of electrical admittance

In anaesthetized rabbits electrical admittance (a reciprocal of impedance) of the kidney in situ was recorded using electrodes located in the cortex, outer medulla, inner medulla and papilla. Renal haemodynamics, clearances and Na+ concentration in tissue slices were also determined. Admittance changes in response to i.v. furosemide, 1.5 or 3 mg/kg body weight, and to 15% mannitol infusion, reflected changing interstitial electrolyte concentration and, indirectly, changes in tubular reabsorption of NaCl. The large dose of furosemide and mannitol infusion decreased admittance in all renal zones whereas the small dose affected only the inner medulla and papilla. The rapid onset of the fall in admittance of the inner medulla, even in absence of changes within the outer medulla, suggests that the drug's action is not confined to the thick ascending limb but includes the thin ascending segment.     

The volume and ionic composition of cells in incubated slices of rat renal cortex, medulla and papilla

The apparent extracellular space in incubated slices of rat renal cortex, medulla and papilla has been measured using three differently sized marker molecules, mannitol, sucrose and inulin. Cellular volumes have been estimated by following the efflux of from equilibrated slices. Sucrose appears to be the most accurate extracellular marker in each of the regions examined, in that the sum of its volume of distribution plus cellular volume approximates most closely to the total slice fluid volume. Inulin has the same volume of distribution as sucrose in cortical slices, but under-penetrates medullary and papillary tissue. Mannitol overestimates the extracellular space in all three regions, although its larger volume of distribution, relative to that of sucrose, was not statistically significant in papillary slices. When cell volume and composition are estimated (a) using sucrose as extracellular marker and (b) making appropriate allowance for the presence of bound tissue electrolytes, it is found that cells in each region have low Na⁺ and high K⁺ concentrations and contents. When papillary slices are incubated in medium of very high osmolality (NaCl plus urea, 2000 mosmol/kg H₂O) there is a moderate (approx. 23%) decrease in cell volume and an increase in cell fluid Na⁺ and Cl⁻ concentrations equal to approx. 50% of the increase in the extracellular concentrations. Cell K⁺ concentrations remain unchanged. The results show that cells in renal slices are able to maintain high K⁺-to-Na⁺ ratios when incubated in isosmotic (cortex) or moderately hyperosmotic media (medulla and papilla), and suggest that regulation of papillary cell volume following hyperosmotic shock can only partly be ascribed to uptake of extracellular electrolytes.     

Angiotensin converting enzyme predominates in the inner cortex and medulla of the rat kidney

Regional distribution of angiotensin converting enzyme(ACE) in the rat kidney was studied. The ACE activities in the inner cortex and outer medulla were about 10 and 5 times those in the outer cortex, respectively. The activity in the inner medulla or papilla was much the same as that in the outer cortex. Immunofluorescence was greatest in the proximal tubules in the inner cortex, while the outer medulla and the inner medulla or papilla showed a weak fluorescence. The brush border membranes isolated from the inner cortex also possessed about 10 times the ACE activity seen in the outer cortex. The results indicate that the major source of renal ACE is not the proximal convoluted tubules in the outer cortex, but rather the brush border membranes of proximal tubules in the inner cortex. The contribution of ACE in the inner cortex would therefore be predominant.     

Studies of tissue electrical admittance: relation to tissue sodium for different zones of rabbit kidney

Tissue electrical admittance (reciprocal impedance) and Na+ concentration were determined in slices of rabbit renal cortex, outer medulla, inner medulla and the papilla. In each zone admittance was highly and significantly correlated to tissue Na+ (r = 0.71 to 0.91, p less than 0.001). The cortex admittance proved a relatively insensitive index of tissue electrolyte concentration. The highest sensitivity was observed for the outer medulla: values for the inner medulla and papilla were slightly lower. The data confirm the usefulness of admittance measurement for dynamic assessment of the cortico-papillary electrolyte gradient but show that the values measured in the outer medulla cannot be directly compared with those for the inner medulla and the papilla.     

Effect of salt loading in the rat on adenylate cyclase and phosphodiesterase activity in kidney cortex, medulla and papilla.

Adenylate cyclase (AC) and phosphodiesterase (PDE) activities were studied in the cortex, medulla and papilla of the rat kidney. Sodium loading in vivo for 14 days resulted in a decrease of AC activity in the cortex, a small increase in the medulla and a substantial increase of AC activity in the papilla. Sodium loading caused reciprocal effects on PDE activity: an increase in kidney cortex and a decrease in kidney papilla. Loading of glucose in vivo or chronic administration of antidiuretic hormone in vivo did not cause the changes in AC or PDE observed after sodium loading. The possible significance of these findings is discussed.     

Expression of adrenomedullin in hypoxic and ischemic rat kidneys and human kidneys with arterial stenosis

To investigate regional aspects of hypoxic regulation of adrenomedullin (AM) in kidneys, we mapped the distribution of AM in the rat kidney after hypoxia (normobaric hypoxic hypoxia, carbon monoxide, and CoCl(2) for 6 h), anemia (hematocrit lowered by bleeding) and after global transient ischemia for 1 h (unilateral renal artery occlusion and reperfusion for 6 and 24 h) and segmental infarct (6 and 24 h). AM expression and localization was determined in normal human kidneys and in kidneys with arterial stenosis. Hypoxia stimulated AM mRNA expression significantly in rat inner medulla (CO 13 times, 8% O(2) 6 times, and CoCl(2) 8 times), followed by the outer medulla and cortex. AM mRNA level was significantly elevated in response to anemia and occlusion-reperfusion. Immunoreactive AM was associated with the thin limbs of Henle's loop, distal convoluted tubule, collecting ducts, papilla surface epithelium, and urothelium. AM labeling was prominent in the inner medulla after CO and in the outer medulla after occlusion-reperfusion. The infarct border zone was strongly labeled for AM. In cultured inner medullary collecting duct cells, AM mRNA was significantly increased by hypoxia. AM mRNA was equally distributed in human kidney and AM was localized as in the rat kidney. In human kidneys with artery stenosis, AM mRNA was not significantly enhanced compared with controls, but AM immunoreactivity was observed in tubules, vessels, and glomerular cells. In summary, AM expression was increased in the rat kidney in response to hypoxic and ischemic hypoxia in keeping with oxygen gradients. AM was widely distributed in the human kidney with arterial stenosis. AM may play a significant role to counteract hypoxia in the kidney.     

Adult human CD133/1(+) kidney cells isolated from papilla integrate into developing kidney tubules

Approximately 60,000 patients in the United States are waiting for a kidney transplant due to genetic, immunologic and environmentally caused kidney failure. Adult human renal stem cells could offer opportunities for autologous transplant and repair of damaged organs. Current data suggest that there are multiple progenitor types in the kidney with distinct localizations. In the present study, we characterize cells derived from human kidney papilla and show their capacity for tubulogenesis. In situ, nestin(+) and CD133/1(+) cells were found extensively intercalated between tubular epithelia in the loops of Henle of renal papilla, but not of the cortex. Populations of primary cells from the renal cortex and renal papilla were isolated by enzymatic digestion from human kidneys unsuited for transplant and immuno-enriched for CD133/1(+) cells. Isolated CD133/1(+) papillary cells were positive for nestin, as well as several human embryonic stem cell markers (SSEA4, Nanog, SOX2, and OCT4/POU5F1) and could be triggered to adopt tubular epithelial and neuronal-like phenotypes. Isolated papillary cells exhibited morphologic plasticity upon modulation of culture conditions and inhibition of asymmetric cell division. Labeled papillary cells readily associated with cortical tubular epithelia in co-culture and 3-dimensional collagen gel cultures. Heterologous organ culture demonstrated that CD133/1(+) progenitors from the papilla and cortex became integrated into developing kidney tubules. Tubular epithelia did not participate in tubulogenesis. Human renal papilla harbor cells with the hallmarks of adult kidney stem/progenitor cells that can be amplified and phenotypically modulated in culture while retaining the capacity to form new kidney tubules. This article is part of a Special Issue entitled: Polycystic Kidney Disease.