Application of Dolichos biflorus in immunoassay detection of kidney collecting duct biomarkers.

Currently there are no biomarkers for detecting collecting duct damage in man. Antibodies to several collecting duct-specific antigens exist but sandwich assays have been difficult to establish due to the need for two different antibodies to the same protein. We hypothesized that a collecting duct-specific lectin could be used in combination with a collecting duct-specific antibody to negate the need for two different antibodies. The collecting duct specificity of selected antibodies (NiCa II 13C2, Pap XI 3C7, HuPaP VII 2B11 and aquaporin 2), was verified by immunohistochemistry. Aquaporin 2 and Pap XI 3C7 were used successfully in setting up assays with the lectin Dolichos biflorus, using the Meso Scale Discovery (MSD) platform. Antigen expression was highest in the papillae of rat and human kidney (corresponding to the greatest density of collecting ducts) and was also present in normal urine. We propose that further qualification and validation would lead to an assay for detecting collecting duct damage in man.

[Diagnostic image (394). A man with a tissue fragment in the urine. Papillary necrosis]. / Diagnose in beeld (394). Een man met een weefselbrok in de urine. Necrose van een nierpapil.

A 54-year-old man with chronic renal failure due to diabetic nephropathy presented with macroscopic haematuria and excretion of necrotic tissue in the urine as a sign of papillary necrosis.

1H-Nuclear magnetic resonance pattern recognition studies with N-phenylanthranilic acid in the rat: time- and dose-related metabolic effects.

N-Phenylanthranilic acid (NPAA) causes renal papillary necrosis (RPN) in the rat following repeated oral dosing. Non-invasive early detection of RPN is difficult, but a number of potential biomarkers have been investigated, including phospholipid and uronic acid excretion. This study used 1H-nuclear magnetic resonance (NMR) spectroscopic analysis of urine to investigate urinary metabolic perturbations occurring in the rat following exposure to NPAA. Male Alderley Park rats received NPAA (300, 500 or 700 mg kg(-1) day(-1) orally) for 7 days, and urine was collected on days 7-8, 14-15, 21-22 and 28-29. In a separate study, urine was collected on days 1-2, 3-4, 5-6 and 7-8 from rats receiving 500 mg kg(-1) day(-1). Samples were analysed by 1H NMR spectroscopy combined with multivariate data analysis and clinical chemistry. Histopathology and clinical chemistry analysis of terminal blood samples was carried out following termination on days 4, 6, 8 and 29 (4 week time course) and days 2, 4, 6 and 8 (8 day study). Urine analysis revealed a marked, though variable, excretion of beta-hydroxybutyrate, acetoacetate and acetone (ketone bodies) seen on days 3-4, 5-6 and 7-8 of the study. It is postulated that the ketonuria might be secondary to an alteration in fatty acid metabolism due to inhibition of prostaglandin synthesis. In addition, an elevation in urinary ascorbate was observed during the first 8 days of the study. Ascorbate is considered to be a biomarker of hepatic response, probably reflecting an increased hepatic activity due to glucuronidation of NPAA.

Tratamiento acortado de pielonefritis aguda en mujeres / Shortened treatment in female acute pyelonephritis

Stamey, a fines de los setenta, demostró que la esterilización de la orina es un hecho muy precoz en el tratamiento de toda Infección Urinaria (IU), y mi experiencia personal de más de 20 años con tratamiento de tres días en IU en pacientes con cualquier condición clínica (febril o no), con buenos resultados, nos permite proponer tratamiento acortado en PNA. No hay acuerdo sobre la duración del tratamiento de PNA, pero en general, se propone más de 7 días. Se efectúo un protocolo prospectivo de tratamiento acortado de PNA, en pacientes sin patología obstructiva. Todas las pacientes fueron tratadas con 3 días de antimicrobiano parenteral (Gentamicina i.m., 160 mg aldía) por 3 días, y 20 a 30 días de profilaxis posterior con Nitrofurantoína oral 50 mg/noche. Se efectuó examen de orina de diagnóstico, de control al tercer día, y antes de suspender la Nitrofurantoína. Ingresaron al protocolo 40 pacientes consecutivas, con diagnóstico clínico de PNA, recibidos por el Serviciode Urgencia, con temperatura sobre 38 °C, vómitos, dolor lumbar, y examen de orina previo a tratamientocon leucocituria y bacteriuria (con cultivo positivo, confirmado posteriormente), y ecotomografía renal sin dilatación.

Increased urinary uronic acid excretion in experimentally-induced renal papillary necrosis in rats.

We have evaluated the potential of urinary uronic acid measurement as an early indicator in the development of renal papillary necrosis (RPN). Urinary uronic acid was quantified with a range of other urinary biochemical parameters in rats given multiple doses of N-phenylanthranilic acid (NPAA) or mefenamic acid (MFA), each of which induces a dose-related papillary necrosis. In addition, histological examination was also carried out to confirm the development and presence of RPN. NPAA was administered to male wistar rats at p.o. doses of 100, 250, and 500 mg/kg and MFA at p.o. doses of 75, 150, and 300 mg/kg on days 1-4 and 8-11, and urine samples were collected for 16 hours each day. NPAA increased uronic acid excretion two-fold for both medium and high doses from day four. MFA increased uronic acid excretion to two and a half-fold by day 10 in the highest dose administered. Urinary creatinine was equally elevated in a dose-related manner following treatment with either NPAA or MFA. None of the other routine markers (urinary or serum) of nephrotoxicity showed any statistical changes. NPAA produced a dose- and time-related increase in excretion of uronic acid. Evidence of widespread papillary necrosis was seen histologically at the high doses of NPAA or MFA. The significant elevation of uronic acid in urine following treatment with either NPAA or MFA was well ahead of the development of RPN detectable by routine histology, suggesting that uronic acid measurement could serve as an early indicator of RPN. The assessment of urinary uronic acid may therefore provide a novel sensitive and selective marker of identifying the lesion earlier than is currently possible. An increase in urinary uronic acid following NPAA and MFA treatment supports the biochemical basis of these changes as a representative of acid mucopolysaccharides accumulation.

1H NMR spectroscopic and histopathological studies on propyleneimine-induced renal papillary necrosis in the rat and the multimammate desert mouse (Mastomys natalensis).

The renal papillary toxin, propyleneimine (PI), was administered at 20 or 30 microliters/kg i.p. to male Sprague Dawley (SD) rats (n = 5), Fischer 344 (F344) rats (n = 4), and to multimammate desert mice (Mastomys natalensis, n = 4). Urine was collected at time points up to 4 days p.d. and the toxicological response of the different animal models to PI compared using 1H NMR spectroscopy of urine, renal histopathology, and urinary assays for alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and gamma-glutamyl transpeptidase (gamma GT). The renal papillae of both F344 and SD rats showed extensive necrotic lesions 4 days post-dosing and in some cases sloughing of the papilla. However, only slight renal papillary necrosis (RPN) was observed in Mastomys treated with 20 microliters/kg PI and, although slight to moderate damage was observed at 30 microliters/kg, PI-treated Mastomys showed substantially less RPN than either group of PI-treated rats. 1H NMR urinalysis showed that PI treatment caused a decrease in the urinary concentrations of succinate (0-24 hr p.d.) and citrate (24-48 hr p.d.) and an increase in creatine (0-48 hr p.d.) in all animal models. Trimethylamine-N-oxide (24-48 hr) and 2-oxoglutarate concentrations decreased initially following the administration of PI and then rose above control levels. The 1H NMR-detected urinary biochemical effects of PI in all three models were similar. However, taurine concentrations were elevated in the urine of Mastomys following PI treatment, perhaps indicating a degree of liver damage, whereas taurinuria was not seen in either SD or F344 rats. These observations are discussed in relation to the potential mechanism of PI-toxicity.

Natriuretic effect of atriopeptin III in rats with papillary necrosis.

This study compared the effects of atriopeptin III (AP III) on sodium excretion and renal interstitial hydrostatic pressure (RIHP) in control rats and in rats pretreated with 2-bromoethylamine (BEA) to produce papillary necrosis. In control rats, infusion of AP III (100 ng.kg-1.min-1) increased sodium excretion from 2.2 +/- 0.7 to 6.4 +/- 0.9 microeq.min-1.g kidney wt-1 and RIHP from 6.8 +/- 0.7 to 8.7 +/- 0.9 mmHg, whereas glomerular filtration rate and renal blood flow were unaltered. Similar results were obtained in rats pretreated with BEA 48 h before the experiment. In rats studied 6 wk after BEA treatment, the papilla was absent and there was atrophy of juxtamedullary nephrons. AP III did not alter sodium excretion or RIHP in this group of rats. These results indicate that 1) an intact renal papilla and/or juxtamedullary nephron population may be required for the natriuretic effect of AP III; 2) the papillary injury 48 h after BEA is not sufficient to abolish the natriuretic response to AP III; and 3) elevations in RIHP may play a role in the natriuretic response to AP III.

Carbonic anhydrase independent bicarbonate reabsorption in rats with chronic papillary necrosis.

The present study was designed to indirectly localize the tubular sites of carbonic anhydrase independent bicarbonate reabsorption in the rat. Papillary necrosis was induced in rats by intravenous administration of bromoethyleneamine hydrobromide (BEA) 6 weeks prior to the study, in order to assess the role of deep nephrons in this process. Acetazolamide alone, acetazolamide plus amiloride, and acetazolamide, amiloride plus furosemide were infused into rats with intact papillae (groups I, III, V) and rats with BEA-induced papillary necrosis (groups II, IV, VI). Our results show that chronic papillary necrosis does not alter carbonic anhydrase independent bicarbonate reabsorption, since the fractional excretion of bicarbonate (FEHCO3) was not significantly higher when acetazolamide was infused into animals with BEA-induced papillary necrosis as compared to those rats with intact papillae (FEHCO3 group I vs. group II: NS). The addition of amiloride hydrochloride, a blocker of distal acidification at the administered doses, increased FEHCO3 significantly in both, animals with intact papillae and those with papillary necrosis, to a similar degree. The addition of furosemide to acetazolamide and amiloride further induced a significant increase in FEHCO3 only in the group of animals with papillary necrosis (FEHCO3 group V 43.0 +/- 2.9% vs. group VI 52.1 +/- 0.9%; p less than 0.05). It appears from our study that deeper nephrons and papillary structures are not indispensable for carbonic anhydrase independent bicarbonate reabsorption in the rat on a chronic basis. The cortical collecting duct appears to have a significant capacity to reabsorb bicarbonate independent of carbonic anhydrase which can be blocked by amiloride.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta 2-microglobulin excretion and urinary cytology in analgesic nephropathy.

Patients with analgesic nephropathy are at risk from uro-epithelial malignancy. Enhanced secretion of beta 2-microglobulin occurs from epithelial cancer cells. In order to find a screening test for malignancy in analgesic nephropathy, urinary levels of this protein were measured in patients with analgesic nephropathy with urine cytological abnormalities and were compared to a control group with glomerulonephritis. Mean fractional excretion of beta 2-microglobulin was higher (8.61 +/- 1.76 SEM) in patients with analgesic nephropathy than in those with glomerulonephritis (1.13 +/- 0.76) (P less than 0.025). Those patients with analgesic nephropathy who had malignant cells in the urine had higher mean fractional excretion (18.56 +/- 5.77) than those with only atypical cells (8.5 +/- 2.0) (P less than 0.05) who in turn had higher mean values than those with normal cytology (2.12 +/- 0.62) (P less than 0.0025). It is suggested that the increased beta 2-microglobulin excretion in analgesic nephropathy is due to secretion from abnormal urothelial cells as well as reduced tubular catabolism. Beta 2-microglobulin may be of use as a screening test for malignancy in analgesic nephropathy.

The effects of chronic papillary necrosis on acid excretion.

Complete papillary necrosis in rats can be induced within 1 month following a single injection of 2-bromoethylamine hydrobromide (BEA) (50 mg, i.v.). Utilizing a combination of clearance and balance techniques the effects of complete absence of the papilla was examined as regards urinary acidification, whole kidney glomerular filtration rate (GFR), single nephron GFR, and morphology. Whole kidney GFR was not different from control, however, the percent filtering juxtamedullary nephrons was markedly diminished (87.2 +/- 2.1 vs. 31.5 +/- 3.6% filtering, control vs. BEA, respectively, P less than 0.001) and significantly reduced in the superficial nephrons (80.6 +/- 3.6 vs. 62.2 +/- 6.1% filtering, control vs. BEA, respectively, P less than 0.05). There was a significant decrease in juxtamedullary single nephron GFR and an increase in the superficial single nephron GFR as assessed by the quantitative Hanssen's technique in the animals with chronic papillary necrosis. Complete papillary necrosis was associated with normal arterial bicarbonate concentration, pH, and plasma electrolyte concentrations. At the same degree of acidemia (induced by NH4Cl administration) minimal urinary pH, ammonium excretion, and titratable acid excretion were not different than seen in age matched controls. The response to Na2SO4 infusion and phosphate infusion was the same in both groups of animals. The urine-blood (U-B)pCO2, and index of urinary acidification, was identical in BEA and control animals. Scanning electron microscopy showed scarring of the juxtamedullary glomeruli one month after BEA. The papilla was sloughed and lying free in the renal pelvis in every experimental animal. These data demonstrate that complete papillary necrosis is not associated with acidosis nor a defect in urinary acidification.

Functional characterization of drug-induced experimental papillary necrosis.

The functional expression of papillary necrosis was investigated with a model of drug-induced papillary necrosis. Bromoethylamine hydrobromide (BEA) administration to rats uniformly resulted in the development of papillary necrosis. All studies were performed 24 hours after BEA administration with the exception of the electrolyte balance studies, which were performed during the 72 hours after the induction of papillary necrosis. GFR was not different between BEA-treated and sham rats. BEA-treated rats had a significantly lower maximal urine osmolality and free water reabsorption than did sham rats. Renal tissue concentrations of sodium, potassium, and water were not different between BEA-treated and sham rats. During water diuresis, free water clearance was not significantly different between the two groups. During sodium bicarbonate administration, maximal bicarbonate reabsorption and urine-blood Pco2 gradient (at comparable urine bicarbonate concentrations) were not significantly different between the two groups. During sodium sulfate infusion, there was no difference in minimum urine pH, ammonium excretion, and net acid excretion between chronically acidotic BEA-injected and sham rats. In rats on "zero" sodium intake, BEA administration resulted in a significant increase in urine flow and sodium excretion, whereas sham rats remained in sodium balance. In rats with restriction of both sodium and chloride, BEA administration resulted in a significant wastage of sodium, chloride, and calcium. There was no difference in potassium excretion between BEA-treated and sham rats during hydropenia, bicarbonate administration, sodium sulfate infusion, or ingestion of a normal potassium diet. When potassium intake was restricted to "zero," BEA-treated rats developed potassium wastage; when potassium intake was increased to 21 mEq/day, BEA-treated rats had a significantly lower potassium excretion than did sham rats. These findings may result from alterations in collecting duct transport, but damage to deep medullary structures may also contribute.

Urine cytology findings in analgesic nephropathy.

Urine cytology screening for neoplasm led to the detection of 3 urothelial carcinomas and 1 severe urothelial dysplasia in 98 patients with analgesic-induced papillary necrosis. A further 18 patients had changes suggesting that they were at risk of incurring malignancy in the near future. Routine urine cytology is recommended in all cases of analgesic nephropathy.

In vitro lysis of target cells by rat polymorphonuclear leukocytes isolated from acute pyelonephritic exudates.

Polymorphonuclear (PMN) leukocytes from acute pyelonephritic exudates of rats were examined for cytoxicity against various target cells. Pyelonephritic PMN leukocytes caused lysis in vitro of syngeneic renal epithelial cells in 24 to 48 hr. They also caused lysis of allogeneic and xenogeneic target cells. The susceptibility to PMN-induced cytolysis was different among various target cell lines. Cell lysis proceeded at low ratios of effector to target cells and was not dependent on the addition of antibody or lectins. PMN isolated from peritoneal exudates (starch or endotoxin induced) were also cytoxic for the target cells. Cell-free supernatants from pyelonephritic PMN-target cell mixtures, or pyelonephritic PMN leukocytes cultured alone contained cytolytic substances which were nondialyzable and heat stable Finally, the urine of pyelonephritic rats was cytotoxic. These results suggest that PMN leukocytes and their cytotoxic mediators are directly involved in the pathogenesis of renal injury in pyelonephritis.

Nephrosis and papillary necrosis after pyelonephritis.

We present a case of nephrotic syndrome complicating acute pyelonephritis in a 45-year-old man. His first attack of acute bacterial pyelonephritis had two unusual features: transient nephrotic syndrome and chronic recurrent episodes of papillary necrosis. The former, which lasted for two weeks, was characterized by edema, excretion of 7.7 g of urinary protein per 24 hours and hypoproteinemia (1.8 g per 100 ml). A percutaneous renal biopsy two weeks after the height of the nephrotic state showed normal glomeruli by light and electron microscopy and immunohistologic studies. Interstitial changes were noted. Over two years the patient has passed approximately 50 fragments, characterized as necrotic tissue containing tubular structures. He has no evidence of diabetes mellitus, urinary-tract obstruction or ureteral reflux, analgesic abuse or atypical vasculitis. He is afebrile but has recurrent bacteriuria despite antibiotics. This case demonstrates that acute pyelonephritis must be added to the list of diseases causing the nephrotic state.

Experimental papillary necrosis of the kidney. IV. Medullary plasma flow.

To test the thesis that vasoconstriction plays a significant role in the pathogenesis of papillary necrosis caused by bromoethylamine hydrobromide (BEA), medullary plasma flow was determined in rats treated with BEA. Medullary blood flow was normal (1/2) to 1 hour after BEA treatment, and was actually elevated 6 hours after BEA. There was no increase in plasma levels of prostaglandins A and E, which would have been expected if there had been medullary ischemia. Pretreatment with reserpine, which inhibited the development of papillary necrosis, had little effect on medullary plasma flow. These observations do not support the notion that vasoconstriction is the mechanism by which BEA causes papillary necrosis.

Experimental papillary necrosis of the kidney. 3. Effects of reserpine and other pharmacologic agents on the lesion.

Reserpine is able to exert a pronounced inhibitory effect on the development of papillary necrosis following the administration of bromoethylamine hydrobromide to the rat. This inhibitory effect has been observed using light microscopy, histochemistry, indigo carmine excretion and urine output. These observations suggest that vasoconstriction may play a significant role in the pathogenesis of papillary necrosis, but the evidence for this is incomplete.