Altered renal kallikrein and renin gene expression in nephrotic rats and modulation by converting enzyme inhibition.

Urinary kallikrein excretion (UKE) is decreased in rats with passive Heymann nephritis (PHN), but increases after converting enzyme inhibition (CEI). Although CEI potentiates bradykinin activity, neither the effect of CEI on kallikrein secretion nor the abnormal renal kallikrein metabolism in PHN has been examined previously. To determine the mechanism by which CEI increases UKE, normal rats and PHN received enalapril, 40 mg/kg per d orally for 4 d. UKE was 85% lower in PHN than in normals and increased in both groups after CEI, although UKE in PHN remained significantly less than in normals. Kallikrein mRNA was significantly lower in PHN compared to normals but not in PHN treated with CEI and did not change in normal rats. Renin mRNA was significantly lower in PHN, and was stimulated by CEI only in normals. Renal kallikrein and renin content were not different and were not altered by CEI. Both kallikrein and renin genes appear to be transcriptionally suppressed in rats with PHN and the depressed kallikrein mRNA levels can be reversed by CEI. The modest increase in UKE despite normalization of kallikrein mRNA after CEI suggests that there is also a posttranscriptional defect in synthesis and/or secretion of kallikrein.

A low-protein diet restricts albumin synthesis in nephrotic rats.

High-protein diets increase albumin synthesis in rats with Heymann nephritis but albuminuria increases also, causing serum albumin concentration to be suppressed further than in nephrotic animals eating a low-protein diet. Experiments were designed to determine whether dietary protein augmentation directly stimulates albumin synthesis, or whether instead increased albumin synthesis is triggered by the decrease in serum albumin concentration. Evidence is presented that dietary protein augmentation directly stimulates albumin synthesis, accompanied by a proportional increase in steady-state hepatic albumin mRNA concentration (AlbmRNA) and by an increase in AlbmRNA transcription. When the increased albuminuria resulting from dietary protein augmentation is blunted with enalapril, serum albumin concentration is shown to increase in nephrotic rats. Both albumin synthesis and AlbmRNA increase in these animals despite the greater serum albumin concentration. Albumin synthesis correlates inversely with both serum albumin and serum oncotic pressure in nephrotic rats fed 40% protein, but does not correlate with serum albumin concentration in nephrotic rats fed 8.5% protein (LP), even when serum albumin concentration is reduced. Albumin masses are preserved in LP primarily because of reduced albuminuria. Reduced serum oncotic pressure and dietary protein augmentation combine to stimulate albumin synthesis in nephrotic rats at the level of gene transcription.

Proteinuria, not altered albumin metabolism, affects hyperlipidemia in the nephrotic rat.

It has been established previously that nephrotic hyperlipidemia is characterized by both an increase in lipid synthesis and a defect in removal of lipoproteins. The relationship between these defects and altered albumin metabolism is uncertain. One hypothesis is that hepatic lipogenesis increases in parallel with albumin synthesis. To test this hypothesis, albumin synthesis was increased in nephrotic rats fed an 8.5% protein diet (LPN) by increasing dietary protein to 40% (HPN). Proteinuria was modulated in half of the rats fed 40% protein by enalapril (HPE). Albumin synthesis was the same in both HPN and HPE, but proteinuria was reduced in HPE compared to HPN, and so were serum cholesterol and triglycerides (TG). To examine the effect of serum albumin on lipid clearance in the absence of proteinuria, plasma clearance of chylomicrons (CM) and VLDL was measured in Nagase analbuminemic rats (NAR) and found to be no different than in normal SD rats. When proteinuria was induced in NAR and in SD rats, a severe and identical defect in both CM and VLDL clearance was acquired in both groups and blood lipid levels were increased to a similar degree in both groups. Neither hyperlipidemia nor defective removal of lipoproteins from the circulation are linked to albumin synthesis or serum albumin concentration but result, at least in part, from proteinuria. Postheparin lipoprotein lipase (LPL) activity was reduced slightly in nephrotic animals compared to nonnephrotic controls, but the most striking finding was a highly significant decrease in postheraprin LPL activity in normal NAR compared to SD rats (P less than 0.001), suggesting that reduced LPL activity is not responsible for reduced clearance of CM and VLDL in nephrotic rats.

High protein diets stimulate albumin synthesis at the site of albumin mRNA transcription.

High dietary protein intake directly stimulates albumin synthesis ald albuminuria in rats with Heymann nephritis. Increased albumin synthetic rate might be due to the presence of increased amino acids available for protein synthesis causing more efficient translation of preformed albumin mRNA, or instead might be linked to increased steady state albumin mRNA levels in the liver. Albumin synthesis, hepatic albumin mRNA content, and albumin mRNA relative to beta actin mRNA (as an internal control) (Alb/beta Act), were measured in rats with Heymann nephritis fed either 8.5% protein (LP), or after protein intake was increased to 40% for 4 days (HP). enalapril (E) was used to modulate the proteinuric effect of HP, yielding four experimental groups, LPN (8.5% protein nephrotic, no enalapril), LPE (8.5% protein, enalapril treated), HPN (40% protein nephrotic, no enalapril), and HPE (40% protein, enalapril treated). Dietary protein augmentation increased the rate of albumin synthesis, steady-state albumin mRNA levels, and Alb/beta Act in both HPN and HPE, compared to either LPH or LPE, even though serum albumin concentration was greater in HPE than in either of the groups fed LP. Both albumin mRNA and Alb/beta Act correlated with the rate of albumin synthesis (r = 0.531, P less than 0.05; and 0.553, P less than 0.01 respectively). Nuclear run-on assays were performed using nuclei isolated from the livers of LPN or HPN to determine whether increased albumin mRNA resulted from an increase in the rate of albumin mRNA transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dietary protein intake and angiotensin converting enzyme inhibition in Heymann nephritis.

The effect of diets containing 8.5%, 21% or 40% protein on growth, urinary albumin excretion and serum albumin concentration was determined in rats with Heymann nephritis and in non-nephrotic control animals. Urinary albumin excretion was greater in nephrotic rats with each increment in dietary protein intake, and serum albumin concentration tended to be least in nephrotic rats fed 40% protein. Albuminuria decreased spontaneously and serum albumin concentration increased in nephrotic rats fed 8.5% protein for 25 days. Enalapril treatment caused a further reduction in urinary albumin excretion and an increase in serum albumin concentration in nephrotic rats fed 8.5% protein. Albuminuria did not decrease nor did serum albumin concentration increase in nephrotic rats fed 40% protein without enalapril treatment, but enalapril caused a significant reduction in urinary albumin excretion and an increase in serum albumin concentration in nephrotic rats fed either 8.5% or 40% protein. The rate of growth in normal rats was greatest when they were fed 21% protein, compared to either 8.5% or 40% protein. Growth rate was significantly reduced in nephrotic rats, regardless of dietary protein intake and regardless of treatment with enalapril, but the 21% protein diet still induced the most rapid rate of growth. Growth rate was not significantly different in nephrotic rats fed either 40% or 8.5% protein. The difference in weight between pair fed nephrotic and control animals fed 21% protein was due to a decrease in carcass and skin weight in nephrotic animals. Carcass protein was significantly reduced in the nephrotic animals, and carcass saponafiable fat tended to be reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Perinephric abscess: the missed diagnosis.

Perinephric abscess is a life-threatening but treatable process. Most infections of the perinephric space occur as a result of extension of an ascending urinary tract infection, commonly in association with nephrolithiasis or urinary tract obstruction. A large portion of the mortality is the result of failure to diagnose this entity in a timely fashion. This failure may be because of the frequently obscure or nonspecific nature of the clinical presentation. Blood cultures as well as urine cultures may fail to identify correctly the bacterial pathogens responsible for the abscess. Perinephric abscess should be considered in the differential diagnosis of any patient presenting with a urinary tract infection that fails to respond promptly to antibiotic therapy, particularly in those known to have anatomical abnormalities of the urinary tract or diabetes mellitus. Consideration of this diagnosis should enter into the differential diagnosis of fever with abdominal pain or flank pain. Early recognition of perinephric abscess and prompt drainage, either percutaneously or surgically, in combination with appropriate antibiotic coverage, should reduce dramatically the morbidity and mortality from this infection.

Proteinuria, hyperlipidemia, and the kidney.

Hyperlipidemia in the nephrotic syndrome is the result of abnormalities in both synthesis and catabolism of lipids and lipoproteins. The etiology of nephrotic hyperlipidemia has not been established, but both abnormal glomerular permeability to plasma proteins and reduced serum oncotic pressure may contribute. Although standard hypolipemic drugs are effective in nephrotic patients, therapies such as dietary protein restriction and angiotensin-converting enzyme inhibitors which reduce proteinuria and increase serum oncotic pressure ameliorate hyperlipidemia as well. Hyperlipidemia may also induce proteinuric renal disease in normal animals and worsen renal injury in a variety of animal models of kidney disease. Conversely, treatment of hyperlipidemia prevents renal injury and lessens proteinuria. Potential mechanisms by which hyperlipidemia may cause renal injury include inflammatory and immunologically mediated injury and alteration of glomerular paracrine function.

Hormonal modulation of proteinuria in the nephrotic syndrome.

Proteinuria is the primary manifestation of a variety of glomerular diseases which are characterized clinically by the nephrotic syndrome. In many cases there is little effective treatment for the primary disease process. However, reduction of proteinuria can frequently improve the hypoalbuminemia, hyperlipidemia and edema which are responsible for the morbidity of the nephrotic syndrome. Proteinuria can be reduced in nephrotic humans and experimental animal models by restriction of dietary protein intake, nonsteroidal anti-inflammatory drug, and by angiotensin-converting enzyme inhibitors. Each of these therapies modifies the activity of locally acting glomerular hormones, autocoids, suggesting that there is a component of proteinuria which is hormonally mediated. The effects of dietary protein, nonsteroidal anti-inflammatory drugs, and angiotensin-converting enzyme inhibitors on nephrotic proteinuria and their potential hormonal mechanisms of action is the subject of this review.

Albuminuria causes lysozymuria in rats with Heymann nephritis.

To determine if changes in dietary protein intake alter renal excretion of small molecular weight proteins in passive Heymann nephritis, 21 rats with passive Heymann nephritis were fed 8.5% protein for 12 days after injection with antiserum. Dietary protein intake was then increased to 40% in 10 rats (LP-HP) while 11 rats remained on 8.5% protein (LP-LP). Lysozymuria (UlysV) increased from 66.5 +/- 31.0 mcg/day to 457.5 +/- 98.0 mcg/day (P less than 0.001) after five days in LP-HP, but was unchanged in LP-LP. Albuminuria (UalbV) increased only in LP-HP, from 168 +/- 23 mg/day to 447 +/- 45 mg/day (P less than 0.001). Urinary lysozyme excretion correlated with UalbV (r = 0.737, P less than 0.001), and changes in UlysV correlated with changes in UalbV (r = 0.657, P less than 0.01). To determine whether the increase in UlysV was the direct effect of the change in diet, enalapril 40 mg/kg/day was administered to prevent the increase in UalbV that occurs when these rats are fed a high protein diet. Twelve rats were fed 8.5% (LP) and 10 were fed 40% protein (HP) from the time of injection with antiserum. Six LP (LPE) and five HP (HPE) received enalapril. UlysV was 873 +/- 391 mcg/day in HP and nearly undetectable in the other three groups. UalbV was significantly greater in HP (368 +/- 60 mg/day) compared to the other three groups (114 +/- 16 in LP, 136 +/- 44 in HPE, 95 +/- 21 in LPE.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of modulation of renal kallikrein-kinin system in the nephrotic syndrome.

Albuminuria (UAlbV) can be reduced by converting-enzyme inhibitors (CEI), but the hormonal mechanism responsible for this effect has not previously been defined. Since CEI increase kinin activity as well as reduce angiotensin II (ANG II) activity, experiments were performed to determine the effect of isolated alterations in kinin and ANG II metabolism on UAlbV in rats with passive Heymann pephritis. Phosphoramidon was used to potentiate kinin activity without altering ANG II synthesis. Aprotinin was utilized in combination with the CEI, enalapril, to prevent the increase in kinin activity caused by CEI. UAlbV and the fractional renal clearance of albumin (FCAlb) decreased significantly after either phosphoramidon or enalapril, although only enalapril reduced blood pressure. Glomerular filtration rate (GFR) was not affected by either drug. Phosphoramidon did not affect plasma renin activity (PRA) or the pressor response to angiotensin I (ANG I), indicating that ANG II synthesis was not altered. Aprotinin prevented the reduction in UAlbV and FCAlb produced by CEI but not the hypotension, elevated PRA, or ANG I pressor blockade produced by CEI. Aprotinin alone had no effect on UAlbV, GFR, PRA, or blood pressure. UAlbV can be reduced by increasing kinin activity by a mechanism that is not dependent on suppression of ANG II activity or reduction in GFR or blood pressure. CEI may reduce proteinuria as a result of their action on the kallikrein-kinin system rather than on the renin-angiotensin system.

GFR increases before renal mass or ODC activity increase in rats fed high protein diets.

Consumption of a high protein diet causes renal hypertrophy and increased glomerular filtration rate (GFR). To determine the relationship between increases in GFR, renal ornithine decarboxylase activity (ODC), arginase activity, and renal growth, dietary protein intake was increased from 8.5% to 40% in 50 male Sprague-Dawley rats (HP). Forty-one rats remained on 8.5% protein as time controls (LP). Eight to 17 animals were killed daily for measurement of kidney weight (kidney wt), ODC and arginase activities, total kidney protein and DNA content. GFR increased within the first 24 hours after the increase in dietary protein and reached a maximum within 48 hours, ODC increased from 9.7 +/- 0.8 U/g to a peak of 170 +/- 35 U/g at 48 hours, decreasing to a stable value of 28.6 +/- 8.0 U/g at 72 hours and 25.4 +/- 5.1 U/g at 168 hours, a value significantly greater than that at time zero. Arginase activity did not change. Kidney wt as percent body weight (body wt) increased after the initial increase in both GFR and in ODC activity. The peak in ODC activity corresponded with the maximum increase in GFR and preceded the increase in renal mass. After GFR stabilized, ODC activity decreased to a plateau and renal growth relative to body wt ceased. The increase in kidney weight was accompanied by a parallel increase in total kidney protein. Kidney protein/kidney DNA ratio increased significantly by 96 hours, indicating that renal hypertrophy had occurred. The sequence of these events suggests that increasing GFR may trigger the rise in ODC activity.

The antiproteinuric action of angiotensin-converting enzyme is dependent on kinin.

Converting enzyme inhibitors (CEI) reduce proteinuria in nephrotic humans and animals, but the mediator(s) of this effect has not been identified definitively. To determine whether enhanced kinin activity contributes to the antiproteinuric action of CEI, rats with passive Heymann nephritis were treated with the B2 kinin receptor antagonist HOE 140, 300 micrograms/kg per day, for 3 days and then the CEI enalapril (ENAL), 35 mg/kg per day, was given for another 4 days while HOE 140 was continued (HOE/ENAL). Additional groups of nephrotic rats were untreated (CON), received HOE 140 only (HOE), or received ENAL only. ENAL alone produced a > 60% decrease in albuminuria after 4 days, whereas HOE 140 alone had no effect on albuminuria. In HOE/ENAL, pretreatment with HOE 140 prevented the decrease in albuminuria observed in ENAL. GFR increased significantly over time in all groups but was not different among the groups on any day. The clearance of albumin decreased significantly in ENAL (P < 0.001) and was significantly lower than in CON, HOE, or HOE/ENAL on Day 10. The fractional clearance of albumin decreased in all groups as a result of the increase in GFR but was significantly lower in ENAL compared with the other three groups at Day 10 and was not different between CON, HOE, and HOE/ENAL. Plasma renin activity and concentration were increased significantly in both ENAL and HOE/ENAL, indicating that converting enzyme was effectively inhibited in both groups. It was concluded that enhanced kinin activity contributes to the antiproteinuric action of CEI in this model of nephrotic syndrome.

Branched-chain amino acids augment neither albuminuria nor albumin synthesis in nephrotic rats.

Both albuminuria (UalbV) and albumin synthesis (AlbSyn) are modulated by dietary protein in nephrotic rats, but the agent(s) linking diet to altered UalbV and AlbSyn is unknown. Others have reported that branched-chain amino acids (BCAA) cause neither increased renal blood flow nor glomerular filtration rate (GFR) normally induced by dietary protein nor increased blood glucagon thought to be necessary for protein-mediated effects on renal hemodynamics. The effect of BCAA on UalbV is unknown. Because BCAA increase AlbSyn in tissue culture and after a fast, it is possible that feeding BCAA may increase AlbSyn but not UalbV in nephrosis. Nephrotic rats were fed either 8.5% casein (LP); 21% casein (NP); 8.5% casein supplemented with valine, leucine, and isoleucine to the total amount provided by a 21% casein diet (2.37%) (LBC); or 8.5% casein plus 12.5% BCAA providing a diet isonitrogenous to 21% casein (HBC). UalbV and AlbSyn were significantly greater in NP compared with LP, LBC, or HBC and were the same in the latter three groups. Glucagon was infused into nephrotic rats fed 8.5% casein either subcutaneously or intraperitoneally in quantities sufficient to increase plasma levels to over 10 times control but had no effect on UalbV. The ability of dietary protein to increase AlbSyn or UalbV is not a result of total alpha-amino nitrogen intake but is a result of the specific amino acid composition of the diet and must result entirely from the effect of one or more non-BCAA. Increased blood glucagon alone has no effect on UalbV.

The effect of angiotensin-converting enzyme inhibition and dietary protein restriction in the treatment of proteinuria.

Both angiotensin-converting enzyme inhibitors and dietary protein restriction have been reported to reduce urinary protein losses in patients with chronic glomerular diseases. We evaluated these two therapies in 12 such patients ingesting a constant metabolic diet containing 1.6 g protein/kg body weight per day. After a steady-state was achieved during a 3-week baseline period, patients were randomly assigned to either enalapril, titrated to reduce mean arterial pressure by 10 mm Hg, or an isocaloric 0.8 g/kg protein diet. Five patients in each group completed 3 additional weeks of observation during the treatment period. Enalapril resulted in an average reduction in urinary protein and albumin losses of 26% and 33%, respectively, without reducing creatinine clearance. Albumin synthesis was unchanged and nitrogen balance increased slightly (+142.8 +/- 85.7 mmol/d [+2.0 +/- 1.2 g/d], P = 0.075). Dietary protein restriction had no consistent effect on proteinuria or albuminuria, whereas albumin synthesis (25.9 +/- 3.4 v 21.5 +/- 2.9 g/d/1.73 m2, P less than 0.05) and nitrogen balance (-135.6 +/- 92.8 mmol/d [-1.9 +/- 1.3 g/d], P = 0.10) decreased. Both therapies resulted in a modest increase in plasma potassium concentration. Whether the maintenance of albumin synthesis in the presence of a reduction in urinary protein losses will convey a long-term advantage to treatment of proteinuric patients with angiotensin-converting enzyme inhibitors remains to be determined.

Differential effects of diabetes and glomerulonephritis on glomerular basement membrane composition.

The hallmark of renal diseases involving the glomerulus is the presence of proteinuria. While the routes of pathogenesis of proteinuria have not been established, alterations in the barrier function of the glomerular basement membrane (GBM) have been implicated. We evaluated the effect of streptozotocin diabetes and passive Heymann nephritis (PHN) over time on the macromolecular composition of rat GBM to determine if changes in composition correlate with proteinuria. Six to twelve rats from each group (control, diabetic, and PHN) were sacrificed 1, 5, 28, 56, or 84 days after induction of disease. Identical amounts of GBM were subjected to a sequential extraction procedure, and type IV collagen, entactin, laminin, fibronectin, and anionic charge content were quantitated in the extracts. Type IV collagen and entactin content did not change with time or disease. Both laminin and fibronectin contents increased with time in GBM in all groups, but this increase was significantly greater in diabetic GBM. A significant decrease in anionic charge content of GBM coincided with the onset of albuminuria at Day 28 in diabetes, but no change was seen in PHN. In diabetic rats, the increase in laminin content over control preceded the onset of albuminuria, while the increase in fibronectin was not apparent until after albuminuria was present. In PHN, no differences in type IV collagen, entactin, laminin, fibronectin, or anionic charge content of GBM were found compared with control, even though profound albuminuria was evident from Day 5 through 84. Thus, while alterations in laminin and fibronectin content may contribute to the loss of glomerular permselectivity in streptozotocin diabetes, such changes apparently are not involved in PHN.

Differing actions of dietary protein and enalapril on renal function and proteinuria.

Albuminuria (UalbV) increases in proportion to dietary protein in rats with passive Heymann nephritis. To determine whether a similar relationship existed in normal animals, 14 normal rats were switched from an 8.5% protein diet (LP) to a 40% protein diet (HP). Initially UalbV and glomerular filtration rate (GFR) increased in parallel, but GFR ceased to increase after 48 h while UalbV continued to increase, causing a significant increase in fractional renal clearance of albumin (FCalb). In contrast, HP for 4 days caused only a transient increase in GFR in nephrotic rats but effected a threefold sustained increase in UalbV. Pretreatment of nephrotic rats with enalapril blunted but did not entirely prevent the increase in UalbV after switching to HP and did not affect the increase or subsequent decline in GFR. Treatment with enalapril for 10 days reduced UalbV and FCalb in nephrotic rats fed either LP or HP. The similar pattern of changes in urinary albumin excretion in normal and nephrotic rats after dietary protein augmentation suggests that dietary protein may modify UalbV by the same process in both normal and nephrotic animals. The increases in UalbV and GFR resulting from dietary protein augmentation represent parallel but independent processes, since only the proteinuric response is modified by converting enzyme inhibition. Dietary protein restriction and converting enzyme inhibition exert an additive effect to reduce UalbV.

Dietary protein intake modulates glomerular eicosanoid production in the rat.

The quantity of protein in the diet modulates glomerular function. To study the effect of dietary protein intake on glomerular eicosanoid production, rats were randomized to either a high- (40%) or low- (8.5%) protein isocaloric diet. Ten to fourteen days later glomeruli were isolated and incubated in the absence (basal) and presence (stimulated conditions) of arachidonic acid, and production rates of prostaglandin (PG) E2, PGF2 alpha, and thromboxane B2 (TxB2) were determined by direct radioimmunoassay. Under basal conditions, glomerular production of all three eicosanoids was significantly greater in rats ingesting the high-protein diet. Glomerular production of PGE2 and TxB2 was also greater in animals fed the high-protein diet in the presence of arachidonic acid, suggesting that glomerular cyclooxygenase activity was augmented. In contrast, ingestion of a high-protein diet was not associated with a significant increase in eicosanoid production by renal papillae or in TxB2 release by clotting blood. To investigate the potential role of the renin-angiotensin system in the dietary protein-induced modulation of glomerular eicosanoid production, rats ingesting a high- or low-protein diet were randomized to treatment with an angiotensin-converting enzyme inhibitor or no therapy. Enalapril attenuated the dietary protein-induced augmentation in glomerular eicosanoid production. This effect occurred only when administered in vivo, since the active metabolite enalapril did not alter PGE2 production by isolated glomeruli when added in vitro. Dietary protein intake also modulated glomerular eicosanoid production in three models of experimental renal disease in the rat (streptozotocin-induced diabetes mellitus, Heymann nephritis, and partial renal ablation).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dietary protein intake on albumin homeostasis in nephrotic patients.

Animals with experimental renal disease maintained on diets restricted in protein develop less severe renal lesions and less proteinuria than do animals maintained on a normal or high protein diet. To determine whether restriction of dietary protein will reduce urinary albumin excretion in patients with established nephrosis and whether such dietary restriction will result in decreased albumin pools, we performed paired studies on nine nephrotic patients. They were fed sequential diets with a protein content of 1.6 and then 0.8 g/kg body wt, each for 2 weeks. Caloric intake remained constant at 35 Kcal/kg. In six patients the high protein diet was fed first; in three the order of dietary administration was reversed. Urinary albumin excretion was reduced on the low protein diet in all patients regardless of dietary order. Both the renal clearance of albumin and the fractional renal albumin clearance were reduced significantly on the low protein diet. The rate of albumin synthesis was greater on the high protein diet, but so was the rate of albuminuria. Despite the higher rate of albumin synthesis during the period of high protein intake, serum albumin concentration and plasma albumin mass were both less than during the period of low protein intake. Thus, dietary protein restriction in patients with established nephrosis results in decreased urinary albumin excretion in excess of any reduction in creatinine clearance. Total albumin mass is preserved and plasma albumin mass is actually increased during the period of dietary protein restriction. Protein restriction may be feasible in nephrotic patients.(ABSTRACT TRUNCATED AT 250 WORDS)