Peter H. Bach

Chemically Induced Renal Papillary Necrosis and Upper Urothelial Carcinoma. Part 1

In the past, renal papillary necrosis (RPN) has been commonly associated with long-term abusive analgesic intake, but over recent years a wide variety of industrially and therapeutically used chemicals have been shown to induce this lesion experimentally or in man. Destruction of the renal papilla may result in: (1) secondary degenerative cortical changes which precede chronic renal failure or (2) a rapidly metastasizing upper urothelial carcinoma, which has a very poor prognosis. This article will briefly review the published data on the morphology, function, and biochemistry of the normal renal medulla and the pathology associated with RPN, together with the secondary changes which give rise to cortical degeneration or epithelial carcinoma. It will then examine in detail those chemicals which have been reported to cause RPN in an attempt to delineate structure-activity relationships. Finally, the many different theories that have been proposed to explain the pathophysiology of RPN will be examined and an hypothesis will be put forward to explain the primary pathogenesis of the lesion and its secondary consequences.

The role of metabolic activation of analgesics and non-steroidal anti-inflammatory drugs in the development of renal papillary necrosis and upper urothelial carcinoma

There has been no cogent hypothesis to explain the molecular basis of analgesic and non-steroidal anti-inflammatory drug (NSAID) associated renal papillary necrosis (RPN) and upper urothelial carcinoma (UUC). The microsomal cytochrome P-450 enzyme system may generate reactive intermediates which promote pathophysiological effects in the lung, liver and renal cortex, but the absence of P-450 activity in the medulla suggests that it is unlikely that similar events lead to RPN and UUC. Other enzymes (eg. peroxidases) convert substituted aromatics into benzoquinoneimines (an intermediate that has previously been defined in P-450-mediated toxicity). The medulla is rich in fatty acid peroxidases involved in the metabolism of arachidonic acid. NSAID and analgesics interact with key enzymes in this pathway, which could lead to the co-oxygenation of exogenous and endogenous compounds via the peroxidase, lipoxygenase, or prostaglandin hydroperoxidase enzymes. The generation of reactive molecules in the medulla could explain both RPN and UUC via the alkylation of macromolecules. The formation of free radicals would give rise to extensive lipid peroxidation, (there are large quantities of free polyunsaturated fatty acids in the medullary interstitial cells), an event of major potential importance to local cell destruction and genotoxic effects. At present this proposed mechanism of co-oxygenation offers the most attractive working hypothesis to explain the molecular pathogenesis of both RPN and UUC.

Changes in medullary glycosaminoglycan histochemistry and microvascular filling during the development of 2-bromoethanamine hydrobromide-induced renal papillary necrosis

A single dose of 2-bromoethanamine (BEA) hydrobromide induced an apex limited renal papillary necrosis (RPN) at low doses (50 mg/kg, ip and sc, or 100 mg/kg, po), and the total ablation of the medulla at high doses (greater than 150 mg/kg, ip and sc, or 1000 mg/kg, po) in both male and female Wistar rats. BEA (at levels of 50, 100, and 150 mg/kg ip to male rats) caused transient hydropic changes. By 12 to 24 hr the anatomical elements of the medulla were necrosed, and active regeneration and reepithelialization of the viable areas was apparent. The only cortical changes up to 120 hr were dilatation of the nephron and the Bowmans spaces. The medullary glycosaminoglycan matrix (assessed by histochemical staining with colloidal iron) gave an intense granular appearance 2 hr after BEA, which was more marked and diffuse in the apex of the medulla from 8 to 12 hr. The staining was lost from those areas where early necrotic changes had occurred by 12 to 24 hr, and there was no subsequent staining of matrix material up to 120 hr. Vascular filling with colloidal carbon particles suggested an early shift of blood flow to the outer medulla at 2 to 4 hr, followed by the sustained filling of the microvasculature of the medulla well into the period when frank necrotic change was apparent. These data suggest that changes in the medullary matrix might play a previously unrecognized role in the development of 2-bromoethanamine hydrobromide-induced renal papillary necrosis, and these changes may give rise to loss of the material that supports the medullary capillaries.

Effects of arsenite on hepatic mixed-function oxidase activity in rats

1. Injection of arsenite (As3+) to control rats results in losses of total hepatic cytochrome P-450 and significant decreases of ethoxycoumarin O-deethylase (ECOD) and ethoxyresorufin O-deethylase (EROD) activities. However, As3+ appears to decrease the activity of these enzymes differentially, with EROD showing greater sensitivity than ECOD. 2. Injection of As3+ to rats treated with phenobarbital and isosafrole significantly decreases the total content of hepatic cytochrome P-450 and various mixed function oxidase (MFO) activities, with the exception of ECOD which appears to be insensitive to As3+. 3. 3-Methylcholanthrene administration apparently protects against the effects of As3+ on the cytochrome P-450 system, since total content of the cytochrome P-450 and various MFO activities were all insensitive to this treatment.

High Resolution Light Microscopic Morphological and Microvascular Changes in an Acutely Induced Renal Papillary Necrosis

Morphological changes were followed in semi-thin glycolmethacrylate sections, after treating male Wistar rats with a single ip dose of 2-bromoethanamine (BEA) hydrobromide (100 mg/kg) to induce renal papillary necrosis. Medullary interstitial cells had irregular nuclei at 4 hr and focal necrosis by 8 hr which spread from the papilla tip to the cortico-medullary junction from 12 hr. Increased mucopolysaccharide staining was observed in the papilla tip at 4 hr, and was lost from those regions where necrosis had developed by 48 hr. Endothelial platelet adhesion, first seen at 8 hr, was very marked at 18 hr, but affected capillaries in necrotic regions only, up to 144 hr. The absence of extravasated Monastral Blue B demonstrated the integrity of the medullary microvascular endothelia. The distal nephron showed degenerative changes at 12 hr and cell exfoliation at 18 hr. Cortical changes were confined to PAS-positive casts in the collecting duct and loop of Henle from 8 hr and dilatation of distal and proximal tubules at 8 and 72 hr, respectively. There was active repair at the junction between viable and necrotic tissue in the papilla from 24 hr with mitoses in the collecting ducts and loops of Henle. Normally the urothelium is <3–4 cells thick, but upper urothelial proliferation followed BEA administration. Hyperplasia was especially marked at the mouth of the ureter and in the pelvis opposite the region of necrosis (7–8 cells thick at 18 hr) and had only partially resolved by 144 hr. These data show that the interstitial cells are the primary target in BEA-induced renal papillary necrosis and that other morphological changes, including a localized hyperplasia, are secondary.

Experimentally Induced Renal Papillary Necrosis and Upper Urothelial Carcinoma

Publisher Summary This chapter discusses experimentally induced renal papillary necrosis and upper urothelial carcinoma. It presents several morphological, histochemical, and functional data to support several distinct series of pathological changes following the administration of 2-bromoethanamine (BEA). The earliest histochemical changes take place in the medullary matrix that appears to undergo depolymerization. The renal medullary interstitial cells are the first cell type to undergo degenerative change that is rapidly followed by damage to the delicate elements of the medulla. The collecting ducts and endothelial changes are late and generally follow the necrosis of other anatomical regions of the medulla. At present, the lipid changes in the medulla are not well understood, but they are similar to those already reported in human analgesic abusers. The early subtle degenerative changes in the proximal tubule do not appear to be central to the development of the papillary lesion, but the subsequent exfoliation of the brush border and proximal tubular cells are important components of the protein casts that begin to form in the distal nephron. These subsequently appear to play at least some role in the development of functional changes that cause marked proximal tubular dilatation. The chapter illustrates the time course of the major pathophysiological changes associated with the development of RPN, and its secondary consequences of cortical degeneration and upper urothelial hyperplasia.

The effect of N-phenylanthranilic acid-induced renal papillary necrosis on urinary acidification and renal electrolyte handling

The oral administration of a suspension of N-phenylanthranilic acid (N-PAA), over the range of 0.5 to 2 mmol/kg for 14 consecutive days, caused a dose-related renal papillary necrosis (RPN), which involved no more than 30% of the medullary apex. This area of necrosis was no greater following daily doses of 3 and 5 mmol/kg of N-PAA for 14 days, but cortical degenerative changes were induced. The area of the necrotic lesion was greater in the left kidneys of individual rats than in the right kidneys. The apex-limited histopathological changes associated with the administration of low doses of N-PAA were not reflected by altered electrolyte or water homeostasis and only high doses of N-PAA caused significant changes. Urinary volume was significantly increased (in animals treated with 5 mmol/kg), whereas urinary osmolality (greater than 2 mmol/kg N-PAA), and Na+ (5 mmol/kg), K+ (5 mmol/kg), and Cl- (5 mmol/kg) excretion was decreased compared to controls. Blood urea nitrogen was increased at doses greater than 3 mmol/kg in association with cortical degenerative changes. When untreated rats were dosed orally with NH4Cl (400 mg/kg) there was a lag period between 0 and 2 hr (when no changes in H+ excretion occurred), but the urinary pH was depressed in the 2- to 4-hr collection period. Only those rats treated with the highest dose of N-PAA (5 mmol/kg) showed a significantly impaired urinary acidification after NH4Cl loading. There was, however, a statistically significant dose-related decrease in the excretion of Cl- following NH4Cl dosing, provided urine was sampled between 0 and 2 hr. These data highlight the failure of the commonly used renal function tests (such as urinary volume, osmolality, and electrolyte excretion) to reflect apex-limited RPN, unless cortical degenerative changes were also present. The dose-related depression of Cl- excretion in the 0- to 2-hr period following oral NH4Cl loading, suggests that appropriately timed sampling of this urinary anion could offer an improved criterion for the diagnosis of RPN.

Enzyme Histochemical Changes in an Acutely Induced Renal Papillary Necrosis

Enzyme histochemistry was assessed in semi-thin glycolmethacrylate sections after 100 mg/kg 2-bromoethanamine (BEA) hydrobromide had been given ip to male Wistar rats to induce renal papillary necrosis. Changes in the proximal tubular marker enzymes alkaline phosphatase (Alk Phos), gamma-glutamyltranspeptidase (GGT) and adenosine triphosphatase (ATPase) were not apparent before 8 hr, but there was a progressive loss up to 144 hr. The proteinaceous PAS-positive casts in the loops of Henle and the collecting ducts stained for Alk Phos and GGT (from 12 hr) and for ATPase (from 18 hr). Acid phosphatase (Acid Phos) staining was increased in the proximal tubule lysosomes from 18 hr. There was a marked increase in Alk Phos in all hyperplastic upper urothelial cells from 8 to 24 hr, and a mosaic of staining remained in the pelvis adjacent to the necrosed papilla at 144 hr. At 12 hr, there was an increase in the staining of the pelvic, ureter and bladder vascular endothelial ATPase, the intensity and area of which increased progressively from 18 hr and almost occluded the capillary lumens in the worst affected areas by 144 hr. These data show several distinct series of pathological changes after the administration of BEA. The subtle degenerative changes in the proximal tubule followed the papillary lesion, but exfoliated brush border and proximal tubular cells were important components of the protein casts in the distal nephron. Similarly, the intense Alk Phos staining in the hyperplastic regions of the upper urothelium and the increased pelvic, ureteric and bladder endothelial ATPase staining suggested they develop as a consequence of the papillary lesion.

Assessment of heavy metal nephrotoxicity in vitro using isolated rat glomeruli and proximal tubular fragments.

Nephrotoxic metals are thought to affect mainly the proximal tubule, but the pathophysiology of acute renal failure (ARF) caused by some of these compounds cannot be explained by damage to this part of the nephron alone. To compare toxic effects on different parts of the nephron, metabolic studies (de novo protein synthesis as assessed by amino acid incorporation and fatty acid oxidation) were performed in freshly isolated rat glomeruli and proximal tubular fragments (PTF) in the presence of increasing concentrations of mercury (Hg), chromium (Cr), and cadmium (Cd) salts. Glomerular protein synthesis was very sensitive to Hg (concentration to reduce protein synthesis by 50%: 3.4 microM) and Cr (15 microM), while in PTF amino acid incorporation was similarly affected by Cd and Hg (32 and 34 microM). Glomerular fatty acid synthesis was also more sensitive to Hg than that in PTF (3.2 vs 55 microM, p less than 0.005). In experiments to study the effects of reduced glutathione (0.5 and 1 mM) on the metal toxicity, preincubation of the fragments with reduced glutathione failed to protect glomeruli against subsequent exposure to the metals, but partially protected PTF (greater than 100 microM for Hg and Cd). These data show that isolated glomeruli are more susceptible to those metals with the potential to cause ARF in vivo, with Hg being the most potent toxin. The results suggest that the glomerular sensitivity to Hg may indicate an important target region of the nephron in the development of ARF which has previously not been recognized.

Relevance of a rat model of papillary necrosis and upper urothelial carcinoma in understanding the role of ochratoxin A in Balkan endemic nephropathy and its associated carcinoma.

Ochratoxin A is nephrotoxic and has been implicated in the genesis of Balkan endemic nephropathy (BEN), a condition that leads to end-stage renal disease and upper urothelial tumours. This compound induces renal parenchymal carcinoma in male mice only, and is not considered to be a potent carcinogen nor is there experimental evidence of its propensity to cause upper urothelial carcinoma. There is, however, evidence that exposure to more than one mycotoxin may be an important factor in the clinical spectrum of BEN. Analgesic nephropathy is clinically different, but is also associated with an upper urothelial carcinoma. The combination of urothelial initiation and an acute papillary necrosis in rats produces upper urothelial carcinoma. This two-stage experimental model offers the potential to assess the role of ochratoxin A in BEN-associated upper urothelial carcinoma under experimental conditions.