Mark T. Houser

Assessment of proteinuria using random urine samples

To determine the feasibility of accurately assessing proteinuria using random urine samples, 24-hour urine samples were obtained from five healthy adult volunteers and 15 children (ages 5 to 17 years) with renal disease. Protein concentration was measured using a Coomassie blue binding technique, and total protein excretion was compared with the urinary protein/creatinine ratio (Up/Ucr). Total protein excretion varied from 28.3 mg to 12.3 gm, or 15.4 to 8500 mg/m2/day, and Up/Ucr ranged from 27.9 to 7650 micrograms/mg in the 24-hour samples; the correlation was highly significant (r = 0.99; P less than 0.001). Reference values for urinary protein concentration and Up/Ucr were established by examining random urine samples in 219 healthy individuals (ages 1 month to 61 years). Age-related differences in protein concentration were noted, and 95th percentiles were calculated as the upper limit of normal. The results of our study indicate that Up/Ucr is an accurate assessment of quantitative protein excretion and could largely replace the collection of timed urine samples for the assessment of proteinuria.

Assessment of urinary protein excretion in the adolescent: effect of body position and exercise.

We examined the effects of body position and exercise on the random urinary protein/creatinine ratio (Up/Ucr) in healthy adolescents, to provide reference data to be used in a simplified evaluation of proteinuria. Random urine samples were obtained during recumbency and after both ambulation and exercise in 116 subjects. The Up/Ucr was significantly (P less than 0.0001) higher with increasing levels of activity, and was widely variable, especially during the upright and postexercise periods. No sex-related differences in Up/Ucr were noted, except during recumbency, when values were significantly (P less than 0.001) higher in females. The urinary dipstick was found to be less sensitive as a tool to define abnormal degrees of recumbent proteinuria. We conclude that body position and exercise have significant effects on protein excretion, and suggest that the Up/Ucr in recumbent and upright urine samples will be useful in the evaluation of proteinuria.

The effect of unilateral nephrectomy on the nephrotoxicity of mercuric chloride in the rat.

Unilateral nephrectomy (UNX) induces a dramatic change in single-kidney structure and function. Therefore, the effects of nephrotoxins may be altered. To evaluate this possibility, mercuric chloride (2 mg/kg, sc) was given to male, Sprague-Dawley rats 2 days following either UNX or sham surgery. Nonoliguric acute renal failure developed and was qualitatively similar in both groups. Glomerular filtration rate (GFR) reached a nadir on Day 2 and was reduced to a greater extent in the UNX group. Furthermore, recovery of GFR was slower and occurred to a lesser extent by Day 10 in the animals subjected to UNX. Evidence of significant tubular dysfunction was present during the acute phase in both groups, as reflected by changes in the fractional excretion of sodium or lysozyme. Persistent tubular dysfunction was noted on Day 10 in both the sham and UNX groups, but the degree of dysfunction was greater in the UNX animals. The in vitro uptake of organic ions by renal cortical slices was reduced 24 hr following the injection of mercuric chloride although no difference was seen between the experimental groups. Mercury content within renal cortex was not increased in the UNX group at 1 or 3 hr but was higher 24 hr postinjection. Total urinary mercury excretion during the first day was not altered by UNX although single-kidney excretion was increased dramatically. These studies suggest that rats are more susceptible to mercuric chloride-induced nephrotoxicity 2 days following UNX. Although the mechanism(s) of this enhanced injury remains unclear, it does not appear to be completely related to an increase in renal cortical mercury content.

Glutathione monoethyl ester moderates mercuric chloride-induced acute renal failure

Glutathione (GSH)-dependent reactions are an important cellular defense against ischemic or oxidative injury, although their role in toxin-induced renal cellular injury is less clear. Because of the known sulfhydryl reactivity of mercury (M), we hypothesized that GSH could modify mercuric chloride (MC)-induced acute renal failure (ARF). Therefore, we evaluated the effects of glutathione monoethyl ester (GE), which produces high intrarenal levels of GSH, on the nephrotoxicity of MC. GE treatment in normal rats did not alter their creatinine clearance (CCr), fractional sodium (CNa/CCr) or lysozyme (CLy/CCr) excretion, but histologically resulted in prominent proximal tubular vacuolization. GE pretreatment in rats with MC-induced ARF resulted in partial preservation of their CCr, CNa/CCr and CLy/CCr. Renal histology also demonstrated a reduction in tubular necrosis. M content in the renal cortex 3 following MC was lower in the MC + GE group, but levels were higher in the liver and inner stripe/inner medulla as compared to animals receiving MC alone. No differences were seen in the outer stripe at 3 h or in any of the tissues 24 h following MC injection. Thus, GE moderated MC-induced ARF, likely by providing a large intracellular sulfhydryl pool and thereby reducing M reactivity with endogenous cellular proteins and enzymes.

Enhancement of renal and hepatic glutathione metabolism by dimethylthiourea

Dimethylthiourea (DMTU), a free radical scavenger, was administered to rats to study its effect on renal and hepatic glutathione metabolism, since it is a potential sulfhydryl donor. Six hours following DMTU, renal GSH content was significantly (P < 0.05) increased (10%), and was increased further after 24 h (28%) (P < 0.001). Hepatic GSH content was also significantly (P < 0.001) elevated at 6 and 24 h (5 and 33%, respectively). Seven days of daily DMTU therapy significantly (P < 0.001) increased renal and hepatic GSH content by 36 and 54%, respectively, which was associated with a significant (P < 0.001) increase in the renal activities of glutathione peroxidase (GP) by 38%, glutathione transferase (GT) by 92%, and glutathione reductase (GR) by 19% (P < 0.05). Significantly increased activities of hepatic GP by 84% (P < 0.01) and GT by 101% (P < 0.001) also occurred in DMTU-treated rats after 7 days of continuous therapy. From these data, we conclude that DMTU stimulates renal and hepatic GSH metabolism, which may be important in mediating DMTUs protective effect against free radical-induced tissue injury.

The Effects of Age and Urine Concentration on Lysozyme and N-acetyl-β-d-Glucosaminidase (NAG) Content in Urine

Random urine samples were obtained to evaluate potential age- or urine concentration-related differences in lysozyme or NAG content. The concentration and excretion of both enzymes was widely variable although no age-related differences were seen. Urine concentration, however, was an important variable as NAG concentration (per mL urine) and lysozyme excretion (per μmol creatinine) were significantly elevated and reduced, respectively, in samples with a higher specific gravity. The correlation coefficient between urine specific gravity and both parameters was significant. Lysozyme excretion is elevated in subjects undergoing a modest diuresis although NAG excretion is unaffected. These data may prove to be useful in the evaluation of renal dysfunction.

Glomerular injury in end-stage liver disease — role of circulating IgG and IgM immune complexes

The relationship of IgG- and IgM-bound circulating immune complexes and immune dysfunction to glomerular injury was evaluated in 15 children with end-stage liver disease (ESLD) awaiting liver transplantation. Compared with age-matched controls, children with ESLD had significantly (P<0.01) increased serum IgG, IgA, and IgM levels, as well as IgG- and IgM-bound circulating immune complexes. Furthermore, they showed a significant (P<0.05) depression of C3 and C4 levels compared with controls. Hematuria occurred in 66% of children with ESLD, and the urinary protein/creatinine ratio was also significantly (P<0.01) increased compared with controls (4.65±2.56 vs. 0.16±0.04mg/mg). Light microscopy of renal biopsy tissue obtained from 6 children with ESLD at the time of transplantation demonstrated mesangial proliferation and expansion with basement membrane splitting. This was associated with subendothelial deposits on electron-microscopic examination, compatible with a diagnosis of membranoproliferative glomerulonephritis. By immunofluorescence, deposition of IgG, IgA, and IgM occurred in various combinations with co-deposition of complement fragments. We conclude that membrano-proliferative glomerulonephritis is a common finding in children with ESLD, probably due to entrapment of circulating IgG- and IgM-bound immune complexes.

RENAL TUBULAR PROTEIN HANDLING IN EXPERIMENTAL RENAL DISEASE

Competitive inhibition of renal tubular transport occurs between low- and high-molecular-weight proteins following intravenous infusion, but this relationship is less clear following de novo glomerular or renal tubular injury. The present study evaluated renal lysozyme and albumin handling following renal tubular injury induced by both low- and high-dose mercuric chloride (0.5 and 2.0 mg/kg) and maleic acid (50 and 400 mg/kg), and following glomerular injury induced by puromycin aminonucleoside (5 mg/100 g) or Adriamycin (5 mg/kg). Subtle renal tubular injury induced only mild isolated albuminuria, while severe tubular injury caused dramatic lysozymuria and moderate albuminuria. However, increased filtration of albumin in these models of glomerular injury did not inhibit lysozyme transport.

Role of glutathione metabolism in the reduction of proteinuria by dimethylthiourea in adriamycin nephrosis.

Glutathione (GSH) metabolism, a tissue detoxification pathway, was evaluated in rats with adriamycin nephrosis (AN) treated with dimethylthiourea (DMTU), a free radical scavenger. After 7 days of DMTU, a significant reduction in proteinuria occurred as compared to AN controls (62.4 +/- 13.3 vs. 155.0 +/- 24.0 mg/24 h). A significant increase in renal cortical GSH content as well as glutathione peroxidase (GP) and transferase (GT) activities occurred in DMTU-treated rats as compared to controls. Glutathione monoethyl ester (GME) administration alone reduced proteinuria by 21% in AN, which was not significant despite a large increase in the renal GSH content, however, GP and GT activities were not increased by GME. We conclude that DMTU ameliorates glomerular injury in AN by stimulating GSH metabolism.

Synergistic Effects of Fish Oil Diet and Dimethylthiourea in Acute Adriamycin Nephrosis

The synergistic effects of combining fish oil (FO) diet, which reduces thromboxane A production, with the free radical scavenger, dimethylthiourea (DMTU), were evaluated in acute adriamycin nephrosis, because proteinuria in adriamycin nephrosis is mediated by increased renal thromboxane A and free radical production. The effects of combined evening primrose oil (EPO) and DMTU were compared with the DMTU + FO combination because EPO increases prostaglandin E but not thromboxane A. After 7, 14, and 21 days, proteinuria was significantly (p < 0.05) reduced in rats receiving either DMTU + corn oil (CO) or DMTU + FO compared with untreated control rats. However, after 21 days, rats receiving DMTU + FO had significantly reduced urine protein excretion compared with those receiving DMTU + CO (103.9 ± 20 mg daily vs 351.8 ± 29.8 mg daily; P < 0.05). In contrast to FO, rats receiving EPO + DMTU had similar urine protein excretion to rats receiving DMTU + CO after 21 days (170.2 ± 20.34 mg daily vs 179.45 ± 26.38 mg daily). The mean serum cholesterol concentration was significantly (P < 0.01) reduced in rats receiving DMTU + FO (195.2 ± 23.8 mg/dL) compared with DMTU + CO (377.9 ± 28.5 mg/dL). Serum triglyceride levels also were significantly (P < 0.01) reduced in rats receiving DMTU + FO (52.5 ± 26.4 mg/dL) compared with DMTU + CO (100.5 ± 36.9 mg/dL). No significant differences in serum cholesterol concentrations or triglycerides occurred between rats receiving DMTU + CO and DMTU + EPO. Renal glutathione content was significantly (P < 0.05) increased by 23% in normal rats receiving FO diet and by 34% in rats receiving combined DMTU + FO compared with CO alone. Evening primrose oil did not enhance renal glutathione content. The authors conclude that FO diet, but not EPO, is synergistic with DMTU in reducing proteinuria and hyperlipidemia in adriamycin nephrosis, possibly by enhancing the free radical scavenging ability of DMTU.