Judith B. Van Liew

Nitric oxide metabolism following unilateral renal ischemia/reperfusion injury in rats

Abstract. Renal ischemia/reperfusion (I/R) injury results in decreased glomerular filtration and renal blood flow (RBF) and increased urine output, characterized by natriuresis and impaired concentrating ability. We studied unilateral I/R in rats to assess renal handling of nitric oxide (NO). Prior to I/R, we measured urine flow rate (V), inulin clearance (CIN), para-aminohippuric acid clearance (CPAH), NO clearance (CNOx determined from metabolites NO2 and NO3), tubular transport of NOx (TNOx, filtered load ± urinary excretion), urine sodium and potassium excretion (UNaV, UKV), fractional excretion of sodium (FENa), and fractional excretion of NOx (FENOx) in each kidney. The left renal artery was then ligated for 30 min, followed by 30 min of reperfusion, and all measurements were repeated. CIN and CPAH were decreased in I/R kidneys compared with the contralateral kidney or pre-ischemia controls. V, FENa, and UKV were all significantly increased in I/R kidneys. Plasma NOx concentration was lower after injury in all animals (23.3±2.8 post injury vs. 30.4±7.7 μM pre injury, P <0.05). CNOx was significantly higher in I/R kidneys (0.14±0.05 ml/min per g kidney weight) than in pre-injury kidneys (0.03±0.02 right, 0.04±0.30 left) or the contralateral controls (0.04±0.02) (P <0.05 for all three controls). TNOx showed net tubular reabsorption of NOx in all kidneys (11±6 in post-ischemic left kidneys vs. 25±20 in left pre-ischemia, 33±13 in right pre-ischemia, and 21±4 right post-ischemia, nM/min per g kidney weight, P = NS). FENOx was higher in injured kidneys (28%±18) than in pre-injury (3%±0.6, 5%±3) or contralateral controls (6%±3) (P <0.05 for all three controls). Renal NOx excretion and clearance are increased despite decreased plasma levels of NO metabolites after I/R injury. This increased excretion is not dependent on RBF or glomerular filtration, but may be related to impaired tubular reabsorption of NOx combined with increased intra-renal NO production.

Factors influencing susceptibility of LEW rats to Heymann nephritis.

Although most LEW rats develop the proteinuria of Heymann nephritis (HN) within 2 months after immunization with Fx1A, protein excretion of some animals remains normal. We have compared nonproteinuric rats with those that developed HN in order to identify factors that influence susceptibility to immunologically medicated kidney disease. In the primary response to Fx1A, immunofluorescence tests showed that antibrush border titers in serum and immunoglobulin deposition in vivo were similar in all rats. However, complement was detected only in rats with proteinuria. Reimmunization with Fx1A at 30 weeks stimulated anamnestic antibody responses in all rats. Following reimmunization, 60% of nonproteinuric rats developed severe HN with an unusually rapid (1 week) onset. Once again, complement was present only in glomeruli of rats with proteinuria. It appears that titers of antibodies to brush border, measured by immunofluorescence tests, are not an index of the pathogenicity of the immune response to Fx1A. Immunological memory, leading to rapid expression of autoimmune disease upon reexposure to antigen, can be established by a primary immunization that does not produce clinical symptoms. Abnormal urine protein composition may provide a clue to subclinical immunopathology of the kidney.

Nitric Oxide-Inhibitory Effect of Aminoguanidine on Renal Function in Rats

Inhibition of nitric oxide (NO) synthesis by structural analogues of L-arginine reduces glomerular filtration, renal blood flow, sodium excretion, and urine output. N(G)-nitro-L-arginine methyl ester (L-NAME) inhibits constitutive and inducible isoforms of NO synthase, while aminoguanidine (AG) selectively inhibits inducible isoforms of NO synthase. We assessed the NO-inhibitory activity of AG on renal function. Rats were treated with aminoguanidine 50 mg/kg daily for 2 months, followed by L-NAME (25 mg/kg/day) for 1 week to inhibit all NO synthase isoforms. After treatment with L-NAME, we performed baseline renal function measurements, then infused L-arginine (2.5 mg/100 g BW x min) to reverse NO inhibition and assessed whether AG exerted NO-inhibitory activity independently of L-NAME. Prior to L-arginine infusion, AG-treated rats did not differ from controls with respect to body weight, kidney weight, systolic blood pressure, urine flow rate, urinary protein or albumin excretion, or urinary excretion of NO metabolites. After L-arginine infusion, all animals showed a 10-15% decrease in mean arterial blood pressure. L-Arginine-induced increases in urine flow, inulin clearance, PAH clearance, sodium excretion, and NO metabolite excretion were blunted in aminoguanidine-treated animals. To assess long-term effects of aminoguanidine, rats were treated for 12 months. Urinary excretion of NO metabolites was lower than controls. Inulin clearance was higher than controls. Aminoguanidine blunts the effect of L-ariginine on renal hemodynamics independently of the nitric oxide synthase inhibitor, L-NAME. However, the use of aminoguanidine for 12 months in rats did not adversely affect renal function.

Renal Injury and Proteinuria in Female Spontaneously Hypertensive Rats

This investigation describes the evolution of functional and morphological changes in the kidney of female spontaneously hypertensive rats (SHR; 5-90 weeks) and control animals of the same genetic strain, Wistar-Kyoto (WKY). Systolic blood pressure in SHR was 162.0 +/- (SD) 14.1 mm Hg up to 45 weeks, then increased to 189.4 +/- (SD) 16.6 mm Hg (50-90 weeks). Blood pressure in WKY controls remained constant (117.8 +/- 13.6 mm Hg, 5-100 weeks). Protein excretion in the SHR was higher than controls (1.5 +/- 0.5 mg/24 h x 100 g BW, 5-100 weeks) from week 30 on. At 90 weeks, excretion rose to 26.1 +/- 9.8 mg/24 h x 100 g BW and is predominately an albuminuria. After 70 weeks superficial tubular fluid albumin concentration in the SHR is significantly increased (p less than 0.001) over controls. Microscopic changes were evident in glomeruli, tubules, interstitium and arterial vessels at 72-90 weeks and demonstrated an increasing gradient of severity from outer to inner cortex. Glomeruli show sclerosis, fibrinoid necrosis and pericapsular fibrosis. Glomeruli studied with a polyanionic stain revealed a marked decrease in staining affinity of deep, compared to superficial glomeruli. The arterial lesions consisted of thickening of the intima and hyperplasia of the media. WKY kidneys were unremarkable. The selectivity of renal injury and proteinuria in the female SHR is similar to that in the male SHR. However, the evolution of these changes is delayed and may be related to a lower blood pressure until 50 weeks of age.

Endogenous creatinine clearance in the rat: strain variation.

The clearance of endogenous creatinine was examined in five strains of rats (Wistar, Wistar Kyoto, Spontaneously Hypertensive Rats, Biobreeding/Worcester diabetic prone and diabetic resistant rats). Creatinine clearance was compared with inulin clearance as the standard. Conditions for clearance measurements were also varied (anesthesia with constant infusion, overnight collection of urine, fed vs. unfed state, single-injection technique). The clearance of creatinine adequately reflects the glomerular filtration rate in three strains (Wistar, Wistar-Kyoto and the Spontaneously Hypertensive Rat). In the two strains of the Biobreeding/Worcester rat creatinine clearance is consistently lower than the inulin clearance. When creatinine clearance is measured from an overnight collection of urine with food withheld it is always lower than when food is present. This clearance should always be validated by comparison with inulin clearance measured simultaneously or under comparable conditions. The ease with which endogenous creatinine clearance can be measured makes it a reasonable method when large numbers of repeated determinations of glomerular filtration rate are required.

Absence of Sodium and Water Retention in Rats with Severe Proteinuria

Studies with two models of immunologically mediated glomerular disease in the rat, chronic serum sickness and Heymann nephritis, show that fluid retention can be dissociated from other signs of the nephrotic syndrome (excessive proteinuria, hypoalbuminemia, hypercholesterolemia). Clinical evidence of fluid retention (increased body weight, decreased hematocrit, ascites) was only detected in severe chronic serum sickness and coincided with an abrupt drop in urinary sodium concentration and sodium excretion. Severe proteinuria was not associated with sodium and water retention in moderate chronic serum sickness and in Heymann nephritis. These observations support the hypothesis that, in conditions of severe proteinuria, an intrarenal defect in sodium excretion rather than a systemic factor, leads to fluid retention.

Single Nephron Hemodynamics in Spontaneously Hypertensive Rats

Abstract This study was designed to determine whether glomerular hypertension develops as a function of age in the spontaneously hypertensive rat (SHR). Male SHR and age-matched Wistar-Kyoto (WKY) normotensive controls were divided into three groups for measurements of whole kidney and single nephron hemodynamics at 5, 10, and 15 months of age. As reported previously, SHR developed significant proteinuria which was predominantly an albuminuria, after 5 months of age. There were no differences in whole kidney or single nephron glomerular filtration rates between SHR and WKY. Afferent glomerular capillary hydraulic pressure (P GC) was slightly increased in SHR compared with WKY at 10 months of age. At 15 months of age, P GC in SHR was significantly lower than WKY. Our studies indicate that increased capillary pressure is not a major factor in the development and progression of renal injury in the spontaneously hypertensive rat.

Glomerular tubular balance of renald-glucose transport during hyperglycemia

Abstractd-Glucose transport in the kidney of glucose loaded rats was investigated using clearance and micropuncture techniques. The range of plasma glucose concentration in clearance experiments was 20–140 mmol·l−1 and in micropuncture experiments 17–94 mmol·l−1.1.During hyperglycemia, the glucose concentration in endproximal tubular fluid was elevated above that in arterial plasma. At plasma concentrations above 60 mmol l−1 intratubular glucose concentration was found to be about 1.2 times higher than in plasma.2.At endproximal puncture sites TF/Posmol was unity throughout the investigated range of hyperglycemia.3.Proximal tubular glucose reabsorption during hyperglycemia is close to saturation which is compatible to aKm=10.8 mmol l−1 as determined previously.4.Passive glucose permeability does not change during hyperglycemia. The permeability constant of 2.03·10−5 cm·s−1 does not differ significantly from that during normoglycemia, 1.9·10−5 cm·s−1.5.Single nephron glomerular filtration rate (SNGFR) and fluid reabsorption in the proximal convolution (C) were significantly correlated during hyperglycemia (r=0.78,P<0.001). Fractional volume reabsorption during hyperglycemia was decreased to 0.36 as compared to control, but during hyperglycemia it was not affected by the magnitude of the glucose plasma concentration.6.During hyperglycemia, proximal tubular glucose reabsorption (TG) was correlated to SNGFR (r=0.64,P<0.001). This relation became insignificant when the influence of volume reabsorption (C) is controlled for (r=0.17,P>0.5). However, the significance of the correlation between TG and C persists when the influence of SNGFR is held constant.7.Calculations indicate that when glucose reabsorption was doubled, et sodium transport was increased about fourfold.8.In hyperglycemia, renal transport rate (TG), when factored by renal glomerular filtration rate (GFR) seems to be linearly related to glucose plasma concentration. Up to endproximal puncture site 25.5% and by the entire kidney 68.2% of the tubular glucose load were reabsorbed. The difference may be attributed either to glucose transport systems which are localized distal to the proximal convoluted tubules and/or to an inhomogenity of the glucose transport in the different types of nephrons.d-Glucose transport in the kidney of glucose loaded rats was investigated using clearance and micropuncture techniques. The range of plasma glucose concentration in clearance experiments was 20–140 mmol·l−1 and in micropuncture experiments 17–94 mmol·l−1.

Enalapril and Pressure-Diuresis in Hypertensive Rats Transgenic for Mouse Renin Gene

The recent development of a transgenic rat strain bearing the mouse ren-2 renin gene [TGR(mRen2)27] has provided a new monogenetic model of hypertension. Other hypertensive rat strains are characterized by a blunted pressure-diuresis-natriuresis response such that higher renal perfusion pressures are required to excrete normal amounts of water and sodium. Dysfunction of the renin-angiotensin and nitric oxide systems may cause in this abnormality. This study examined the effect of enalapril on the pressure-natriuresis response and urinary nitric oxide metabolite excretion in 6-month-old TGR(mRen2)27 rats. The slope of the line relating renal perfusion pressure and urine flow rate in TGR (0.08+/-0.01 microl x min(-1) x g kidney weight(-1) mm Hg[-1]) was significantly lower than that in control rats (0.15+/-0.01 microl x min(-1) x g kidney weight(-1) mm Hg[-1]). Pressure-natriuresis responses were also shifted to higher pressure levels in TGR. Treatment with enalapril for 3 months lowered the mean arterial pressure from 94+/-2 to 84+/-4 mm Hg in control rats and from 146+/-3 to 89+/-3 mm Hg in TGR. The slopes of lines relating renal perfusion pressure and urine flow rate as well as sodium excretion were significantly increased by enalapril in control and transgenic animals. Urinary nitric oxide metabolite excretion rose similarly with increasing renal perfusion pressure in both control and TGR rats and was not affected by enalapril. These results confirm that older TGR rats have a blunted pressure-diuresis-natriuresis response that can be corrected by inhibition of the renin-angiotensin system and suggest that their production of nitric oxide is normal.

The Renal Handling of IgG in the Aging Rat and in Experimental Kidney Disease

Abstract The urinary excretion of total protein, low-MW proteins, albumin, high-MW proteins, and intact IgG was measured in male Wistar rats between the ages of 5-52 weeks, and in rats with experimentally induced glomerular or tubular proteinuria. About 25% of aging rats spontaneously developed focal glomerulosclerosis and a mild glomerular proteinuria. By age 52 weeks, total protein excretion in rats with glomerulosclerosis exceeded that of unaffected rats by a factor of seven (39.5 vs 5.4 mg/24 hr × 100 g body wt), and albumin excretion was seven times higher than IgG excretion in affected rats (21.2 vs 2.9 mg/24 hr × 100 g body wt). Rats with chromate toxicity exhibited a reversible tubular proteinuria, with low-molecular weight protein excretion reaching 16.8 mg/24 hr × 100 g body wt (75% of total protein excretion) at the time of peak toxicity. IgG excretion remained less than 0.6 mg/24 hr × 100 g body wt. Aminonucleoside induced a massive but reversible glomerular proteinuria (204 mg/24 hr × 100 g body wt), with IgG excretion reaching 11.4 mg/24 hr × 100 g body wt (6% of total protein excretion) at the time of peak toxicity. Biochemical and immunochemical studies showed that, while some intact IgG is present in normal rat urine, most IgG immunoreactivity is derived from low-molecular weight catabolic fragments of IgG which interfere with the immunoassay of intact urinary IgG. One of these fragments, probably Fc fragment, may be involved in the pathogenesis of focal segmental glomerulosclerosis.