Superoxide dismutase activity in human glomerulonephritis

ORIGINAL

INVESTIGATIONS

Superoxide Dismutase Activity in Human Glomerulonephritis Abul Kashem, MBBS, PhD, Masayuki Endoh, MD, Fumio Yamauchi, MD, Naohiro Yano, MD, Yasuo Nomoto, MD, Hideto Sakai, MD, Laszlo Pronai, MD, Masao Tanaka, PhD, and Hiroe Nakazawa, MD 0 Superoxide dismutase (SOD) in renal tissue biopsy specimens obtained from patients with immunoglobulin A nephropathy (13 cases) and non-immunoglobulin A mesangial proliferative glomerulonephritis (nine cases) was studied at the protein level by an enzyme-linked immunosorbent assay method and at the mRNA level by the reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Total SOD activity in the tissue supernatant was measured by applying an electron paramagnetic resonance/spin trapping method. Normal renal tissues obtained from kidneys removed for malignancies (six cases) were included as healthy controls. The copper and zinc form of SOD (Cu,Zn-SOD) activity at both the protein and mRNA levels was lower in the moderately or severely damaged tissues compared with that in the normal or mildly damaged tissues. On the other hand, manganese SOD (Mn-SOD) values at either the protein level or the mRNA level did not differ significantly between control and patient samples. In the histochemical study using a polyclonal rabbit anti-Cu,Zn-SOD antibody, the staining intensity for Cu,Zn-SOD antigen was lower in the areas with advanced histologic damage than in the intact tissues. A follow-up study showed that renal function deterioration was proportionately slower in patients whose SOD activity was within the range of healthy tissue levels at the time of biopsy. Our data suggest that a lower level of SOD activity, whether as a cause or a consequence of the disease process, might induce a decrease in the scavenger reaction of superoxide (O,-), thus causing the tissue to become more vulnerable to oxidative stress. 0 1996 by the National Kidney Foundation, Inc. INDEX

WORDS:

Superoxide

dismutase

activity;

human

renal

0

VER the last few years, it has been convincingly documented that reactive oxygen metabolites play a key intermediary role in the pathophysiologic process of clinical and experimental renal diseases. Superoxide dismutase (SOD) is one of the key antioxidant enzymes that participates in the cellular defense system against oxidative damage by catalyzing the dismutation of superoxide (02J to oxygen and HZ02.i9’ Previous studies have shown that experimental supplementation of SOD ameliorates the typical functional and morphologic abnormalities of glomeruli.3 Superoxide dismutase comprises a family of enzymes commonly characterized by the

From the Division of Nephrology and Metabolism, Department of Internal Medicine, and the Department of Physiology, Tokui University Isehara, Kanagawa, Japan; the Second Department of Medicine, Semmelweis University Medical School, Budapest, Hungary: and Nihon Kayaku, Japan. Received December 6, 1995; accepted in revised form March 12, 1996. Supported in part by grants from the Ministry of Health and Welfare and the Ministry of Education, Science and Culture of Japan. Address reprint requests to Abul Kashem, MBBS, PhD, Department of Internal Medicine, Tokai University, School of Medicine, Isehara City, Kanagawa 259-11, Japan. 0 1996 by the National Kidney Foundation, Inc. 0272-6386/96/2801-0002$3.00/O 14

American

Journal

tissues;

renal

tissue

injury.

metals they contain: the copper and zinc form of SOD (Cu,Zn-SOD), a soluble enzyme existing primarily in the cytoplasm of all mammalian cells, and manganese SOD (Mn-SOD), a mitochondrial enzyme.4*5 There is a dynamic balance between the defense system and free radical production. However, this balance can be broken naturally or experimentally either by increasing free radical production or by weakening the defense system. We recently demonstrated the involvement of 02- in the mechanism of glomerular damage in mesangial proliferative glomerulonephritis (PGN). 02- neutrophils and monocytes from both immunoglobulin A nephropathy (IgAN) and non-IgA mesangial PGN patients correlated with the renal dysfunction when stimulated with either a specific or nonspecific stimulus.6 The role of O,- in experimental renal diseases has been assessed by the use of SOD, the depletion of which was shown to aggravate the nephrosis.738 Therefore, renal tissue injury might not only be secondary to increased production of an offending mediator such as 02-, but impairment of the antioxidant defense system, such as a decrease in SOD activity, may be one of the liable factors as well. However, reports on the activities of SOD in the human renal tissue are very limited.’ Thus, detailed information regarding SOD activof Kidney

Diseases,

Vol 28, No 1 (July),

1996:

pp 14-22

SUPEROXIDE

DISMUTASE

AND

GLOMERULONEPHRITIS

ity in human renal tissues and their clinical implication in glomerulonephritis is not available. In this study, we assessed the SOD activity at both the protein and mRNA levels in renal tissue biopsy specimens from IgAN and PGN patients. MATERIALS

AND

15

water. The enzyme activity values were standardized by the tissue protein content measured by the method of Lowry et a1.13All proceedings were conducted in triplicate, and the results were expressed as mean values i SD. Since Cu,ZnSOD and Mn-SOD activities could not be differentiated by this method, the obtained activity was referred to as the total SOD activity.

METHODS

Subjects and Sample Preparation for Super-oxide Dismutase Measurement The study protocol was approved by the Ethical Committee of Tokai University, and informed consent was obtained from all individuals after explaining the aim of the study. Open renal tissue biopsy specimens (approximately 10 mg) from 22 patients with IgAN (eight males and five females) and PGN (five males and four females) were included in the study after exclusion of systemic diseases, such as lupus erythematosus, liver cirrhosis, and diabetes mellitus. The parts of renal tissue remaining after routine histologic study for diagnosis (light microscopy, immunofluorescence, and electron microscopy) were used in this experiment. The advantages and detailed procedure of the open renal biopsy have been reported elsewhere.” None of the patients was receiving any drugs known to interfere with SOD activity at the time of biopsy. Severity of renal tissue damage was assessed by a semiquantitative analysis of light microscopic glomerular changes (mesangial proliferation, mesangial sclerosis, and crescent formation/adhesion to the capsule), tubulointerstitial changes (tubular atrophy, mononuclear/polymorphonuclear infiltration, and interstitial fibrosis), and vascular changes. All specimens were independently reviewed without knowledge of clinical and experimental information. The degree of the tissue lesions was graded into mild (changes affecting <20% of the samples), moderate (changes affecting <40%), and severe (changes affecting <40%). Normal renal tissue was obtained from six different kidneys nephrectomized due to renal cell carcinoma. All the renal tissue samples were frozen immediately and stored at -80°C until further use. Clinical progression of all patients was evaluated using proteinuria, hematuria, and serum creatinine levels that were determined at the time of biopsy and after an average interval of 2 years from the time of biopsy. All patients were receiving symptomatic treatment, such as antiplatelet and antihypertensive drugs, with the exception of five who had been treated with prednisolone.

Measurement of Total Superoxide Dismutase Activity Superoxide dismutase activity in the tissue supernatant was measured by an electron paramagnetic resonance/spin trapping method in which O,- generated in the hypoxanthine/ xanthine oxidase system is trapped by 5,5-dimethyl-l-pyrroline-N-oxide (DMPO), forming a relatively stable DMPOsuperoxide adduct. This adduct was monitored and used to determine the scavenger concentrations in samples, as described previously.“~” Sonicated tissue supematant, obtained after centrifugation at 20,OOOg for 20 minutes, was assayed for total SOD activity after appropriate dilution with distilled

Chemicals Xanthine oxidase was obtained from Boehringer-Mannheim GmbH (Mannheim, Germany); cytochrome c, xanthine, hypoxanthine, and desfenioxamine from Sigma Chemical Co (St Louis, MO); DMPO from Shonan Analytic Center (Tokyo, Japan); and recombinant human SOD from Nihon Kayaku (Tokyo, Japan).

Enzyme-Linked Immunosorbent

Assay

For the measurement of Cu,Zn-SOD and Mn-SOD proteins in the renal tissues, we used Cu,Zn-SOD and Mn-SOD enzyme-linked immunosorbent assay kits (Amersham Life Science, Buckinghamshire, England) according to the manual accompanying each kit. Sixteen sonicated tissue supematants from 12 IgAN and PGN patients and four healthy controls were used for SOD measurement after an appropriate dilution. In brief, 100 PL of Cu,Zn-SOD or Mn-SOD standard and unknown diluted tissue supematants were added to each well of the respective microtiter plates precoated with the respective antibody. The plates were incubated at room temperature (2 hours for Cu,Zn-SOD and 1 hour for Mn-SOD) and washed three times with a washing buffer. One hundred microliters of Cu,Zn-SOD or Mn-SOD monoclonal antibody coupled with peroxidase was added to each well, incubated at room temperature (2 hours for Cu,Zn-SOD and 1 hour for Mn-SOD), and re-treated with the washing buffer. Color was developed with a substrate solution (100 pL/well) containing 0.4 mg/mL o-phenylenediamine and 0.03% H,O, for 10 to 15 minutes at room temperature. The reaction was stopped by the addition of 50 /IL of 2 mol/L H,PO,, and the absorbance was measured at 492 nm. All standards and tissue supematants were set up in duplicate. The results were expressed in micrograms per milligram protein. Protein concentration was determined with a bicinchoninic acid (BCA) assay kit (Pierce, Rockford, IL).

Reverse Transcriptase-Polymerase Reaction Protocol

Chain

Total RNA was prepared from renal tissue homogenates using Isogen (Wake Chemical Inc, Osaka, Japan) based on a guanidinium-phenol-chloroform extraction method.14 Three micrograms of RNA was reverse transcribed with an Oligo (dT)18 primer (National Biosciences, Inc, Plymouth, MN) in 50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, 3 mmol/L MgC12, and 0.5 mmol/L dNTP mixture using 40 units of Moloney-Murine leukemia virus reverse transcriptase in the presence of 20 units of recombinant RNase inhibitor (Clontech Laboratories, Inc, Palo Alto, CA). We also obtained total RNA from glomeruli that were isolated from the renal tissue biopsy specimens using the microdissection method previously described with a minor modification.‘5 The microdis-

16 section solution (solution I) was a Hepes-buffer solution with the following concentrations: 135 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L Na,HP04 (7 HZO), 12 mmol/L MgS04 (7 H,O), 2 mmovL CaCl,, 1.2 mmol/L Na2S04, 55 mmol/L glucose, and 5 mmol& Hepes added after pH adjustment to 7.4. The collagenase solution (solution II) was of the same composition as the microdissection solution I, except that it contained 0.1% bovine serum albumin and 0.1% collagenase. Amplification of each polymerase chain reaction (PCR) was carried out in a final volume of 25 /IL (10 mmol/L Tris-HCl [pH 8.3],50 mmol/L KU, 1.5 mmol/L MgCl*, and 0.2 mmol/ L of all four dNTPs) containing 1 ,nL of each primer and 1 PL of templates with 2 units of AmpliTaq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT). Based on the genomic sequences previously reported, PCR primers for Cu,ZnSODI (5’-GCG ACG AAG GCC GTG TGC GTG-3’ for sense and 5’CGC TGC TTC CGG CAC ACG CAC-3’ for antisense), Mn-SOD’7 @-CGA CCT GCC CTA CGA CTA CGG-3’ for sense and 5’-CAA GCC AAC CCC AAC CTG AGC-3’ for antisense), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5’-GGC TGC TTT TAA CTC TGG TA-3’ for sense and 5’-TCT CCA TGG TGG TGA AGA CG-3’ for antisense) were designed using computer software, Oligo, and synthesized products obtained from TAKARA Biomedicals (Kyoto, Japan). The predicted sizes of the PCR products were 348 base pairs for Cu,Zn-SOD, 365 base pairs for Mn-SOD, and 250 base pairs for GAPDH. Thirty cycles of PCR (1 minute denaturing at 94°C 1 minute annealing at 62°C and 1.5 minute extension at 72°C) were performed. PCR products were analyzed by electrophoresis on 2% agarose gel (TAKARA Biomedicals) containing ethidium bromide (0.5 pL/mL), in Tris-acetate-EDTA buffer (pH 8.0). The gel was then photographed with Polaroid 667 film (Polaroid Co, Cambridge, MA), and the developed film was scanned on a flat bed scanner (ScanJet II cx; Hewlett Packard, Greeley, CO). The optical density of the gel image produced by the scanner was semiquantitated by a densitometric method using NIH Image 1.54, a computer program for image analysis.

Immunohistochemistry The avidin-biotin-peroxidase complex method was used to stain for Cu,Zn-SOD protein. I* Fresh-frozen sections were air dried and fixed in acetone for 10 minutes before staining; they were preincubated with normal rabbit serum before the primary antibody was applied. A polyclonal rabbit antiCu,Zn-SOD antibody (Nihon Kayaku)” was used to localize Cu,Zn-SOD protein. The sections were then incubated with biotinylated rabbit anti-mouse IgG and avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA). The final incubation was with 3,3’ diaminobenzidine tetrahydrochloride, giving a brown reaction product. The sections were counterstained in Meyer’s hematoxylin (Wako Chemical Inc).

Statistical Evaluation Data among different groups were compared using the ANOVA test and data between two groups were analyzed by the Spearman-Kendall and Fisher tests. All values were expressed as the mean t SD.

KASHEM ** 30.

ET AL

P
*

l * I

Iz.225.

w

J e 0

T

Control N-6

Mild Gr. N-6

Moderate

Gr.

Severe

N-7

Gr.

N-9

Fig 1. Superoxide dismutase activity in renal tissue specimens from healthy controls and patients. The vertical error bars represent the mean value plus 1 SD.

RESULTS

Superoxide Dismutase Activity in the Renal Tissue

The average values of SOD activity in the tissue supernatants from normal kidney and from mildly, moderately, and severely damaged tissues were 19.40 t 4.12, 18.40 ? 2.83, 10.93 t 3.95, and 9.24 +- 1.58 U/mg protein, respectively (Fig 1). The SOD activity in the moderately and severely damaged tissues was significantly lower than that in the normal and mildly damaged tissues. Superoxide dismutase activity in the mildly damaged tissue was not significantly lower compared with that in the controls. The SOD level in the healthy tissues showed an individual variation, but the level was always higher than 15 U/mg protein. Cu,Zn-SOD and &In-SOD Proteins in the Kidney

Cu,Zn-SOD and Mn-SOD proteins in the renal tissues were detected by an enzyme-linked immunosorbent assay (Fig 2). The mean level of Cu,Zn-SOD in normal healthy tissues was 4.18 f 1.73 pglmg protein, which was significantly higher than that in the patient tissues (2.88 t 1.08 pg/mg protein). The mean Mn-SOD level in the healthy tissues (1.42 t 0.43 pg/mg pro-

SUPEROXIDE

DISMUTASE

AND

GLOMERULONEPHRITIS

17

lated glomeruli, and we observed basically similar results as in the whole tissues except for lower intensity of expression for both target genes (data not shown).

* PcO.03

Localization

of Cu,Zn-SOD

The localization of Cu,Zn-SOD is of critical importance in evaluating many aspects of intracellular metabolism and in understanding how cells are protected against oxidants. Immunohistochemical staining showed that localization of Cu,Zn-SOD protein was predominantly in the

cu,zn-SOD

?&l-SOD

CONTROL (N=4)

Fig 2. Superoxide nal tissue specimens tients. The vertical value plus 1 SD.

12

Mn-SOD cu,zn-SOD PATIENT (N=12)

dismutase concentrations from healthy controls error bars represent

and the

in repamean

3

4

GAPDH -

tein) was lower than that in the patient tissues (1.86 + 0.41 pg/mg protein), but was not statistically significant. In the healthy renal tissues, the average level of Cu,Zn-SOD and Mn-SOD protein was 74.64% and 25.35%, respectively, of the total SOD. Cu,Zn-SOD and Mn-SOD mRiVA Expression Figure 3A shows representative PCR bands for GAPDH, Cu,Zn-SOD, and Mn-SOD; Fig 3B shows the results as arbitrary densitometry units for Cu,Zn-SOD. The intensity of the amplified GAPDH gene was almost uniform in all control and patient tissues, confirming that the efficiency of reverse transcription did not vary significantly between samples. The intensity of the Cu,ZnSOD gene showed a variation correlated with the severity of tissue damage, ie, specimens of moderately or severely damaged tissues disclosed comparatively weaker PCR bands than those of mildly damaged or healthy tissues. However, Mn-SOD expression did not differ significantly between control and patient tissues. The decrease of the Cu,Zn-SOD message appeared somewhat selective, as both GAPDH and MnSOD messages were unaffected irrespective of the tissue damage. Furthermore, we have also studied the Cu,Zn-SOD and Mn-SOD messages in the same amount of cDNA prepared from iso-

-r

1

I

CU,ZIl-SOD Fig 3. (A) A representative expression of GAPDH, CuZn-SOD. and Mn-SOD mRNA in renal tissues detected by .reverse transcriptase-PCR assay. PCR products of normal tissue (lane l), mildly damaged tissue (lane 2), moderately damaged tissue (lane 3), and severely damaged tissue (lane 4) were obtained from IgAN and PGN patients. (B) Representative data of the densitometric analysis for Cu,Zn-SOD expression.

18

Fig 4. Localization of Cu,Zn-SOD protein in the renal tissues detected predominantly glomerular capsular areas. The staining intensity is stronger in tissue from the healthy tubulointerstitial damaged tissues (B and C). Negative control shows no specific positive normal IgG (D). (Magnifications x200.)

peritubular and glomerular capsular areas. The intensity of staining was lower in the damaged interstitium than in the histologically intact one. No remarkable staining was observed in the glomerular cells except for some weak focal staining in some glomeruli. Blood vessel endothelium and smooth muscle were consistently negative (Fig 4). Superoxide Dismutase Activity and Tissue Damage Superoxide dismutase activity in the tissues was compared with the severity of renal tissue damage to assess the involvement of SOD in the mechanism of tissue injury. Superoxide dismutase level disclosed an inverse correlation with the severity of tubulointerstitial lesions (Fig 5); however, no such correlation was observed with the glomerular changes (data not shown).

in the controls staining

KASHEM

ET AL

peritubular (A) than with the

and in the rabbit

Superoxide Dismutase Activity and Clinical Course To evaluate the functional significance of SOD levels in chronic glomerulonephritis patients, 19 patients (11 with IgAN and eight with PGN) whose renal function was normal (serum creatinine < 1 mg/dL) at the time of renal biopsy were followed for an average of 24.5 + 2.4 months after the renal biopsy by re-examining proteinuria, hematuria, and serum creatinine levels (Table 1). The patients were divided into two groups on the basis of SOD levels: group 1 patients had activity 2 15 units (ie, patients whose SOD levels were either equal or above the lowest value of normal tissue) and group 2 patients had activity <15 units (ie, patients with lower than normal tissue values). Changes in proteinuria, hematuria, and serum creatinine were minor in group I than in group II patients, although renal function in a

SUPEROXIDE

DISMUTASE

AND

GLOMERULONEPHRITIS

Tubulointerstitial +

SOD level 4 5 units

lesion +I

000 000

+H

000 v:

Fig 5. A comparison between renal SOD level and tubulointerstitial damage in both IgAN and PGN patients. Tubulointerstitial damage appeared to be increased in parallel to the decreased SOD activity. -, No lesion; +, mild lesion: ++, moderate lesion; and +++, severe lesion; 0, control; 0, IgAN Pt; 0, PGN pt; P < 0.005.

few patients was shown to be unstable even with a higher SOD level. Figure 6 shows that the change of serum creatinine ratio/year (the second value was divided by the first study value and expressed in years) was inversely correlated with the SOD levels (P < 0.01) in a group of patients who were not receiving any drugs known to interfere with SOD activity. However, prednisolonetreated patients (indicated by asterisks) were shown to preserve a comparatively better renal function during the course of follow-up (Table 1). DISCUSSION

Among various antioxidant systems equipped within the aerobic cells, SOD works as one of the key enzymes in reducing local levels of reactive oxygen metabolites.” The enzyme distributed in cytosol and/or mitochondria can abase superoxide before it can interact to form more cytotoxic metabolites and thus prevent reactive oxygendependent tissue damage. Our present study at the protein and mRNA levels provided strong evidence that the total SOD activity was lower in patients with histologically advanced tissue damage than in patients with less tissue damage or in healthy controls. Until now, SOD activities in the renal tissue were inferred mainly from experimental studies, except for very limited reports on human nephritis.’ Although the mechanism(s) by which SOD, especially Cu,Zn-SOD,

19

decreases in the nephritic tissue could not be elucidated in this study, the cause(s) is possibly the inactivation of SOD. Superoxide dismutase inactivation by HzOz, a dismutation product of O,through destruction of a histidine residue, has been reported by Bray and Cockle.21 Self-inactivation of SOD in activated neutrophils through their own O,- generation also has been documented previously22; a decrease in SOD activity of human neutrophils was observed when neutrophils were incubated with an O,--producing system. Our study and other previous studies have demonstrated that 02- generation from neutrophils and monocytes was increased in IgAN and PGN patients, and the increase was more pronounced in patients with advanced renal dysfunction.6,23 On the whole, it is conceivable that SOD may be inactivated by O,- in a dose-dependent manner, and that the imbalance between the production of 02- and scavenging agents such as SOD might contribute significantly to the disease process of glomerulonephritis. In our study, the parallel decrease of Cu,ZnSOD activity at the protein level as well as at the mRNA level in patients with moderate/severe tissue damage suggests another possible mechanism whereby the decrease in Cu,Zn-SOD activity is due to the suppression of SOD at the message level. Previous reports have shown that control of SOD activity in the mammalian cells is largely dependent on the regulation of SOD at the mRNA leve1.24A very recent study has shown that transforming growth factor-pl, whose glomerular expression was observed to be increased in IgAN,25 can directly suppress the expression of antioxidant enzymes, including Cu,Zn-SOD and Mn-SOD genes in cultured rat hepatocytes.26 Although SOD activity in the present study includes both the Cu,Zn-SOD and Mn-SOD activities, our results disclosed that the tissue SOD activity appears to be predominantly dependent on Cu,Zn-SOD activity, and we found that Cu,Zn-SOD constitutes a relatively large (70% to 80%) amount of the total SOD activity in normal renal tissue. We also noticed a trend of increasing Mn-SOD expression in some patients, but not up to the significance level seen in previous experimental in vitro studies, in which a selective Mn-SOD induction in a number of cell types in response to inflammatory mediators27 or oxidant stress2’

20

KASHEM

Table 1. A Follow-up an Interval Following

Study the

Showing the Measurement

Changes in Proteinuria, of Superoxide Dismutase

Proteinuria

Hematuria (RBCIHPF)

wa Patient No. Group

and Serum Creatinine in Renal Tissue Biopsy

SCr (mg/dL)

Levels After Specimens

2nd

Change of SCr Ratiofyr

Time Between First and Second Study

0.7 0.7 0.8 0.8 0.7 0.8 0.8

0.7 0.7 0.8 1.0 0.8 0.8 1.0

0.40 0.50 0.54 0.60 0.57 0.60 0.68

30 24 22 25 24 20 22

0.7 0.8 0.8 0.9 0.8 0.9 0.9 0.9 0.9 0.8 0.8 0.7

0.9 0.8 1.0 1.0 1.4 1.4 1.2 0.9 1.3 1.0 1.2 1.3

0.64 0.60 0.68 0.56 1.05 0.62 0.69 0.50 0.60 0.58 0.90 1 .Ol

24 20 22 24 20 30 23 24 30 26 20 22

Age/Sex

SSA (Units)

1st

2nd

1st

2nd

1st

23/F 21/M 20/F* 38/M 36/M 30/F 39/F

15.7 15.3 22.0 20.2 19.8 17.0 17.9

0.1 1.3 1.4 0.1 0.8 0.3 1.2

0.1 1.0 0.5 0.4 0.4 0.2 0.8

3+ 4+ 5+ 4+ + 3+ 5+

2+ 2+ 2+ 3+ + + 3+

20/M 40/F 50/M 51/M* 32/M 39/F

12.6 13.2 10.2 10.5 10.0 11.2

29/F

10.0

0.2 1.1 1.3 0.6 1.4 0.9 0.7 0.9 1.1

4+ 3+ 3+ 4+ 3+ 4+ 2+ 4+ 3+

2+ 3+ 4+ 3+ 4+ 5+ 3-k 2+ 2+

1

1

2 3 4 5 6 7 Group 8 9 10 11 12 13 14 15 16 17 18 19

Hematuria, Activity

ET AL

2

35/M* 58/M*

8.2 6.5

0.5 1.3 0.8 1.4 0.7 0.7 0.8 1.1 2.2

39/M*

6.1

1.2

0.2

4+

+

52/M 53/M

8.3 8.5

1.3 3.0

2.1 1.9

4+ 3+

5+ 2+

NOTE. Patients with higher SOD levels showed a comparatively satisfactory with lower SOD levels. Abbreviations: SSA, superoxide scavenging activity; RBC, red blood cells; biopsy; 2nd, 2 years after biopsy. Symbols: +, ~1; 2+, 1 to 4; 3+, 5 to 9; 4+, 10 to 29; 5+, >30. * Receiving prednisolone.

was observed. This apparent difference may be due to either heterogeneity of experimental models, experimental conditions, or cell types used for the different sets of study. Moreover, an increase of Mn-SOD expression in remaining viable cells, which is possibly because of its induction by inflammatory mediators likely to prevail in the diseased tissues, is masked by a decrease in the number of cells able to produce adequate levels of this enzyme. However, the contribution of Mn-SOD to the total SOD activity may not be sufficient to protect tissues from oxidant injury, since it has been shown previously that induction of Mn-SOD without corresponding changes in other antioxidant enzymes in the cells does not increase resistance against oxidants.29 The localization of SOD in the human renal tissue is of some interest. Our mRNA assay revealed that both glomerular and nonglomerular

clinical HPF,

course

high-power

compared field;

lst,

with

patients

at the time

of

cells have the ability to produce both Cu,Zn-SOD and Mn-SOD, although the intensity of expression was less in the glomerular samples. In our histochemical study, Cu,Zn-SOD staining was localized predominantly in the peritubular and glomerular capsular areas, and the intensity of staining was comparatively less in the tissues with advanced tubulointerstitial damage, possibly because of suppression of the expression by disease-related inflammatory agents. Therefore, the decrease in tissue SOD levels to the extent of tissue damage further exposes tissue more vulnerable to oxidant injury; thus, the low SOD might be involved in the pathogenesis of the disease. A follow-up study also showed that renal function in patients with SOD activities in the control range tended to deteriorate rather more slowly than in patients with lower SOD levels. This is

SUPEROXIDE

26.

DISMUTASE

y--14.66162x

24.

AND

+ 23.4923;r-

GLOMERULONEPHRITIS

21

0.66

P
p 22. 3 2 a 20. g ‘;

18.

jj

14.

8

12.

.= 5 - 16. g m

10. .3

.4

.5

.6

Change

.7 of Skr.

.8

.9

1

1.1

ratio/year

Fig 6. Changes in serum creatinine ratio/year (ie, ratio between the second and first study values) showing an inverse correlation with the degree of SOD activity (P < 0.01).

possibly due to the increased scavenging ability of higher SOD levels, preventing oxidant injury. The preservation of better renal function in prednisolone-treated patients is possibly due to corticosteroids limiting 0, production, and could also be related to a positive regulation of SOD at the mKNA level. Previous reports have shown reduced production of O,- in neutrophils with prednisolone treatment, which also had an upregulatory affect on SOD gene expression.30 Kawamura et a131reported the protection of glomeruli from oxidative injuries by activating glomerular antioxidant enzymes with glucocorticoid. However, our data could not indicate whether the preservation of better renal function is due to up-regulating SOD expression or to limiting 02- production, which remains to be determined by further study. In conclusion, it is suggested that the SOD, mainly Cu,Zn-SOD, among other defense mechanisms, might play an important protective role in determining the renal tissue injury caused by oxidants, but it remains to be determined whether the altered SOD activity in the kidney is a cause or a consequence of the disease process. REFERENCES 1, Getzoff ED, Tainer JA, Weiner PK, Kollman ardson JS, Richardson DC: Electrostatic recognition

PA, Richbetween

superoxide and copper, zinc superoxide dismutase. Nature 306:287-290, 1983 2. Fridovich I: Superoxide dismutases, in Meister A (ed): Advances in Enzymology. New York, NY, Wiley, 1986, pp 61-97 3. Diamond JR, Bonventre JV, Karnovsky MJ: A role for superoxide free radicals in aminoacid nucloside nephrosis. Kidney Int 29:478-483, 1986 4. Slot JW: Geuze HJ, Freeman BA, Crapo JD: Intracellular localization of the copper and manganese superoxide dismutases in the liver parenchymal cells. Lab Invest 55:363371, 1986 5. Kurobe N, Suzuki F, Okajima K, Kato K: Sensitive enzyme immunoassay for human Cu,Zn-superoxide dismutase. Clin Chim Acta 187:11-20, 1990 6. Kashem A, Endoh M, Nomoto Y, Sakai H, Nakazawa H: FcaR expression on polymorphonuclear leukocyte and superoxide generation in IgA nephropathy. Kidney Int 45868-875, 1994 7. Adachi T, Fukuta M, Ito Y, Hirano K, Sugiura M, Sugiura K: Effect of superoxide dismutase on glomerular nephritis. Biochem Pharmacol 35:341-345, 1986 8. Hara T, Miyai H, Iida T, Futenma A, Nakamura S, Kato K: Aggravation of puromycin amminonucleoside nephrosis by the inhibition of endogenous SOD. Tokyo, Japan, Springer-Verlag, Proceedings of the XIth International Congress on Nephrology, 1990, p 442 9. Futenma A, Yamada H, Miyai H, Yamada K, Iida T, Kato K: Cu,Zn-superoxide dismutase localization in the glomeruli of IgA nephropathy. J Clin Biochem Nutr 11:59-67, 1991 10. Nomoto Y, Tomino Y, Endoh M; Suga T, Miura M, Nomoto H, Sakai H: Modified open renal biopsy: Results in 934 patients. Nephron 45:224-228, 1987 11. Pronai L, Blazovics A, Horvath M, Lang I, Feher I: Superoxide scavenging activity of dihydroquinoline type derivative. Free Radic Res Commun 19:287-296, 1993 12. Pronai L, Nakazawa H, Ichimori K, Saigusa Y, Ohkubo T, Hiramatsu K, Arimori S, Feher J: Time course of superoxide generation by leukocytes-The MCLA chemiluminescence system. Inflammation 16:437-450, 1992 13. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 198:265-275, 1951 14. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987 15. Owada A, Tomita K, Terada Y, Sakamoto H, Nonoguchi H, Marumo F: Endothelin (ET)-3 stimulates cyclic guanosine 3’5.monophosphate production via ETB receptor by producing nitric oxide in isolated rat glomerulus and in cultured mesangial cells. J Clin Invest 93:556-563, 1994 16. Hallewell RA, Masiarz FR, Najarian RC, Puma JP, Quiroga MR, Randolp A, Sanchez-Pescador R, Scandella CJ, Smith B, Sreimer KS, Mullenbach GT: Human Cu/Zn superoxide dismutase cDNA: Isolation of clones synthesizing high levels of active or inactive enzyme from an expression library. Nucleic Acids Res 13:217-234, 1985 17. Ho YS, Crapo JD: Isolation and characterization of complementary DNAs encoding human manganese-contaming superoxide dismutase. FEBS Lett 229:256-260, 1988

22 18. Hasui K, Sate E, Sakae K, Goto M, Tokunaga M: Immunohistological quantitative analysis of SlOO proteinpositive cells in T-cell malignant lymphomas, especially in adult T-cell leukemia/lymphomas. Path01 Res Pratt 188:4-5, 1992 19. Tanaka M, Hashimoto Y, Minezaki K, Shinozaki Y, Nakazawa H, Tsukamoto H, Komatsu M, Watanabe K: The fate of exogenously administration of superoxide dismutase in myocardium, in Kelvin JA, Davies KJA (eds): Oxidative Damage and Repair. Tokyo, Japan, Pergamon Press, 1990, pp 706-710 20. Fantone JC, Ward PA: Role of oxygen-derived free radicals and metabolites in leukocyte-dependent inflammatory reactions. Am J Path01 107:397-418, 1982 21. Bray RC, Cockle SA: Reduction and inactivation of superoxide dismutase by hydrogen peroxide. Biochem J 139:43-48, 1974 22. Pronai L, Hiramatsu K, Saigusa Y, Nakazawa H: Low superoxide scavenging activity associated with enhanced superoxide generation by monocytes from male hypertriglyceridemia with and without diabetes. Atherosclerosis 90:39-47, 1991 23. Chen HC, Tomino Y, Yaguchi Y, Fukui M, Yokoyama K, Watanabe A, Koide H: Oxidative metabolism of polymorphonuclear leukocyte (PMN) in patients with IgA nephropathy. J Clin Lab Anal 6:35-39, 1992 24. Hass MA, Iqbal J, Clerch LB, Frank B, Massaro D: Rat lung copper,zinc-superoxide dismutases isolation and sequence of full-length cDNA and studies of enzyme induction. J Clin Invest 83:1241-1246, 1989

KASHEM

ET AL

25. Sakai H, Naka R, Suzuki D, Nomoto Y, Miyazaki M, Nikolic-Paterson DJ, Atkins RC: In situ hybridization analysis of TGF-p in glomeruli from patients with IgA nephropathy. Contrib Nephrol 111:107-115, 1995 26. Kayanoki Y, Fujii J, Suzuki K, Kawata S, Matsuzawa Y, Taniguchi N: Suppression of antioxidative enzyme expression by transforming growth factor-b1 in rat hepatocytes. J Biol Chem 269:15488-15492, 1994 27. Visner GA, Dongall WC, Wilson JM, Burr IA, Nick HS: Regulation of manganese superoxide dismutase by lipopolysaccharide, interleukin-1, and tumor necrosis factor. J Biol Chem 265:2856-2864, 1990 28. Yoshioka T, Homma T, Meyrick B, Takeda M, MooreJarrett T, Kon V, Ichikawa I: Oxidants induce transcriptional activation of manganese superoxide dismutase in glomerular cells. Kidney Int 46:405-413, 1994 29. Kinnula VL, Pietarinen P, Aacto K, Virtanen I, Raivio KO: Mitochondrial superoxide dismutase induction does not protect epithelial cells during oxidant exposure in vitro. Am J Physiol 268:L71-L77, 1995 30. Macconi D, Zanoli AF, Orisio S, Longaretti L, Magrini L, Rota S, Radice A, Pozzi C, Remuzzi G: Methyl prednisolone normalizes superoxide anion production by polymorphs from patients with ANCA positive vasculitides. Kidney Int 44:215-220, 1993 31. Kawamura T, Yoshioka T, Bills T, Fogo A, Ichikawa I: Glucocorticoid activates glomerular antioxygen enzymes and protects glomeruli from oxidative injuries. Kidney Int 40:291-301, 1991