Elevated levels of 92-kd type IV collagenase (matrix metalloproteinase 9) in giant cell arteritis.

OBJECTIVE: To determine if circulating gelatinase activity and matrix metalloproteinase 9 (MMP-9) (gelatinase B, or 92-kd type IV collagenase) antigenic levels are elevated in sera of patients with giant cell arteritis (GCA), and to ascertain if MMP-9 messenger RNA (mRNA) is deposited in situ at sites of disease involvement. METHODS: Serum samples were collected from 12 patients with GCA and 12 healthy volunteers. Vascular tissue was obtained at the time of temporal artery biopsy. Type IV collagenase activity was determined by gelatin substrate zymography and the quantitative biotinylated gelatin substrate degradation assay. A double-sandwich immunoassay utilizing 2 different isotypes of monoclonal antibodies generated against MMP-9 was used for measuring serum MMP-9 antigenic levels. Finally, to localize sites of MMP-9 mRNA transcription in inflamed arteries, the method of reverse transcriptase in situ polymerase chain reaction (RTisPCR) was utilized. RESULTS: Serum gelatinase activity and MMP-9 titers were significantly increased in patients with GCA (mean +/- SEM 198.9 +/- 36.9 micrograms gelatin/hour/ml serum, versus 21.2 +/- 4.0 in controls; P = 0.0006). The differences in antigenic MMP-9 levels were even more prominent (3005.4 +/- 900.6 ng/ml and 31.6 +/- 9.8 ng/ml in GCA and control sera, respectively; P = 0.007). By RTisPCR, MMP-9 mRNA was mainly detected in cytoplasm of cells resembling smooth muscle cells and fibroblasts in regions of fragmented elastic tissue in the lamina media. CONCLUSION: Gelatinase activity, and specifically MMP-9 levels, are substantially elevated in sera of patients with GCA. Detection of MMP-9 mRNA in the lamina media of inflamed vasculature suggests that degradation of intercellular matrix, particularly elastic fibers, may play a key role in the pathogenesis of GCA. Further studies are needed to determine if the circulating MMP-9 level could be utilized as a clinical marker of disease activity.

Markedly elevated serum MMP-9 (gelatinase B) levels in rheumatoid arthritis: a potentially useful laboratory marker.

In an attempt to find a potentially useful serum marker in rheumatoid arthritis (RA) which reflects underlying pathogenic mechanisms, we measured the circulating levels of matrix-degrading metalloproteinase-9 (MMP-9), also termed gelatinase B, in sera and synovial fluid (SF) from patients with RA and also quantitated the deposition and local synthesis of MMP-9 in RA synovium. Clinical samples, subjected to gelatin substrate zymography, antigenic immunoassay, and a quantitative substrate degradation assay, revealed elevated 92- and 72-kDa proenzyme forms of MMP-9 and MMP-2 in RA sera and SF compared with healthy controls. Immunostaining on fresh RA synovial specimens revealed MMP-9 within vascular walls in fibroblast-like cells and macrophages; mRNA synthesis was detected using reverse transcriptase in situ PCR. In summary, MMP-9 levels are substantially elevated in the sera and SF from patients with RA. The RA synovium is a source of this MMP-9 production, with abundant mRNA and protein observed within several different type of rheumatoid synovial cells.

Recurrence of immunoglobulin A-kappa crystalline deposition disease after kidney transplantation.

Cases of immunoglobulin A heavy chain and kappa light chain deposition disease are rare and their clinical presentations vary. We report one patient with histopathologic and clinical findings of a microangiopathic glomerulonephritis due to immunoglobulin A-kappa deposition. Ultrastructural studies revealed highly ordered deposits in the capillary lumen, mesangium, and basement membrane. The disease recurred at 2.5 years after a cadaveric kidney transplantation. Pulse steroid therapy was repeatedly effective in retarding further progression of renal deterioration in this patient.

Morphine modulates 72-kDa matrix metalloproteinase.

Mesangial expansion is considered to be a precursor of glomerulosclerosis, a predominant glomerular lesion in heroin nephropathy. In addition to matrix synthesis, matrix degradation may also contribute to expansion of mesangium. In this study, we evaluated the effect of morphine on metalloproteinases (gelatinases) that degrade type IV collagen and are secreted by mesangial cells (MC). Gelatinolytic activity was significantly decreased in media of MC exposed to morphine for 1 wk compared with control [control, 2,411.6 +/- 198.7; morphine (10(-6) M), 954.4 +/- 112.2 ng.mg protein-1.3 h-1; P < 0.001]. A similar effect was seen at 2 wk [control, 17,010.6 +/- 1,789.5; morphine (10(-6) M), 8,925.2 +/- 1,623.5 ng.mg protein-1.3 h-1; P < 0.02]. Percent change in gelatinolytic activity was 39.58% (1 wk) and 47.53% (2 wk) compared with control. Morphine at concentrations of 10(-10) to 10(-6) M decreased gelatinolytic activity in MC. In in vivo studies, 24-h urines of morphine-treated rats showed a lower (P < 0.01) gelatinolytic activity when compared with controls. Isolated glomeruli from morphine-treated rats also showed decreased (P < 0.05) gelatinolytic activity compared with control. Naloxone, an opioid antagonist, did not inhibit the effect of morphine on gelatinolytic activity of MC. These results suggest that morphine may cause a decrease in degradation of type IV collagen in patients with heroin addiction. Accumulation of collagen because of lack of gelatinolytic activity in the mesangium may contribute to the expansion of mesangium.

Captopril inhibits the 72 kDa and 92 kDa matrix metalloproteinases.

Gelatinases are metalloproteinases in the kidney which can cleave type IV collagen as well as gelatin. We partially purified the 72 kDa and 92 kDa gelatinases. The gelatinolytic activity was measured by zymography and a quantitative biotin-avidin assay. By zymography, captopril in concentrations of 20 mM and 40 mM added to the incubation buffer reduced the gelatinolytic activity in a dose-dependent manner. The addition of zinc in a concentration of 50 to 100 microM reversed most of the inhibitory effect of captopril. By the biotin-avidin assay, captopril in a concentration of 30 to 50 nM reduced half of either the 72 kDa or 92 kDa gelatinolytic activity. Zinc in a concentration of 50 microM completely reversed the inhibitory effect of 1 microM captopril on both gelatinases. Lisinopril, a non-sulfhydryl ACE inhibitor, similarly inhibited the gelatinases, but a 100-fold higher concentration of the drug was needed. These findings suggest that captopril reversibly inhibits the 72 kDa and 92 kDa metalloproteinases by interacting with the zinc ion at their active sites. This inhibitory effect is observed with captopril levels comparable to the concentrations needed to inhibit the angiotensin converting enzyme in vivo and may at least partially explain some of the renoprotective effects seen with this drug.

Bradykinin generation by dialysis membranes: possible role in anaphylactic reaction.

Several recent reports have described a high incidence of anaphylactic reactions in patients being dialyzed with high-flux membranes while simultaneously using angiotensin-converting enzyme inhibitors. Many of these reports implicate polyacrylonitrile (PAN) as the membrane commonly involved in these reactions. To elucidate potential mechanisms of these anaphylactic reactions, whether dialysis membranes can activate the Hageman factor-dependent (contact) pathways as assessed by the in vitro generation of activated Hageman factor (Hfa), as well as the formation of kallikrein and subsequent bradykinin generation was examined. Both cuprophane (CUP) and PAN membranes were able to activate Hageman factor and convert prekallikrein to kallikrein as measured by an ELISA against kallikrein-C1-inactivator complexes. Subsequently, the active kallikrein was able to cleave bradykinin from its endogenous substrate, high-molecular-weight kininogen. However, it was found that the PAN membrane consistently led to an earlier and significantly higher formation of Hfa and kallikrein when compared with CUP. Importantly, there was also a pronounced but transient generation of bradykinin by the PAN membrane, in contrast to slower bradykinin formation by CUP, with both normal and uremic blood. It was proposed that the early and vigorous bradykinin generation induced by the contact of blood with PAN could explain, in part, the pathogenesis of the reported anaphylactoid reactions.

Blocking the calcium cascade in experimental acute renal failure.

Calcium is believed to be responsible for initiating a deleterious cascade of events that leads to irreversible cell injury during prolonged ischemia. Theoretically, the calcium-dependent cascade of events can be interrupted at three distinct points: a) by reducing calcium inflow into the cytosol using a calcium channel blocker such as verapamil, b) by increasing the mitochondrial capacity to sequester calcium using ethane-1-hydroxy-1:1-diphosphonic acid (EHDP), and c) by inhibiting the activation of the calcium-calmodulin complex using trifluoperazine (TFP). To evaluate the protective role of these agents in prolonged ischemia, 190 unilaterally nephrectomized rats underwent total occlusion of the renal artery for 90 min. One hour before surgery, all the rats received an i.p. injection of either saline or one of the drugs. Of the 190 rats, 130 were used to determine survival and optimal drug doses; the remaining 60 rats were used to determine blood urea nitrogen and serum creatinine at 40 h and 5 days after surgery. Only 33% of the rats in the control group survived for 10 days. However, 87.5% (P less than 0.005), 90% (P less than 0.005), and 60% (P less than 0.01) of the rats pretreated with verapamil, TFP and EHDP respectively survived for 10 days. No differences, however, were seen in renal function tests among the control, TFP or EHDP groups. This suggests that calcium antagonists are successful in protecting the kidney from prolonged ischemic injury despite impaired renal function tests. It may also indicate that these agents delay or prevent the ischemic cells from undergoing irreversible damage.

Cyclosporine nephrotoxicity: blood volume, sodium conservation, and renal hemodynamics.

Glomerular filtration rate (GFR) and renal blood flow (RBF) are depressed by chronic cyclosporine treatment. We examined the hypothesis that depletion of extracellular or intravascular fluid volume contributes to the renal vasoconstriction of early cyclosporine nephrotoxicity (CCN). Control and CCN rats were given 10 mg/kg cyclosporine A or vehicle intramuscularly daily for 7 days. The effects of extracellular volume expansion, both acute (AVE, 10% body wt saline) and chronic (CVE, 10% body wt/day saline ip, 10 days including cyclosporine A treatment period), on renal hemodynamics were measured. In CCN, AVE completely normalized GFR and RBF, whereas CVE partially prevented the development of CCN. Renal autoregulatory ability was depressed in CCN but was largely restored by AVE. Intravascular volumes were measured with Evans blue and 51Cr-labeled red cells. Plasma and red cell volumes were reduced by 24% in CCN, indicating circulatory hypovolemia. Acute repletion of the deficit in blood volume by acute administration of an isoncotic solution (1.8 ml/100 g body wt of 5% albumin in isotonic saline) restored GFR and RBF to levels similar to those in control rats. Extracellular fluid volume, estimated as inulin space, was similar in both CCN and control groups. A metabolic study (7 day) showed stool Na loss in CCN to be twice that in controls but both groups remained in sodium balance. We conclude that the renal vasoconstriction produced in the rat by short-term cyclosporine treatment is, at least in part, prerenal in origin and related to the development of circulatory hypovolemia.

Cyclosporine nephrotoxicity: sodium excretion, autoregulation, and angiotensin II.

Cyclosporine-induced nephrotoxicity (CIN) was studied in rats treated for 7 days with cyclosporine (10 mg x kg-1 x day-1 im) or vehicle (CON). CIN rats displayed characteristic reductions in glomerular filtration (GFR) and renal blood blood flow (RBF), and electron microscopy showed injury to proximal cells. Metabolic studies (7 day) showed significantly lower renal sodium excretion in conscious CIN rats compared with CON. In anesthetized rats at similar blood pressures, nephron GFR (SNGFR) was lower in CIN than CON, but fractional Na reabsorption was similar. In CIN, SNGFR, measured proximally to block flow to the sensing site of tubuloglomerular feedback (TGF) at the macula densa, was not significantly different than distal SNGFR. The rate of distal fluid delivery was significantly lower in CIN than in CON. Inhibition of the renin-angiotensin system (RAS) with captopril (CAP, 10 mg/kg iv), or saralasin (SAR, 0.3 mg x kg-1 x h-1 iv) caused marked arterial hypotension in CIN and a fall in renal vascular resistance (RVR). With arterial pressure controlled, CAP or SAR increased GFR and RBF, and reduced RVR in CIN, but did not reverse the renal deficits compared with similarly treated CON. RBF autoregulation in CIN was impaired between 90 and 140 mmHg but was partially restored by CAP. We conclude that both the filtered load and excretion rate of sodium in CIN are significantly reduced compared with controls, that SNGFR in CIN is not depressed by TGF in response to elevated distal fluid delivery, and that the RAS is not a primarily mediator of the renal vasoconstriction in CIN.

Reduced or absent serum anion gap as a marker of severe lithium carbonate intoxication.

Two patients with life-threatening lithium carbonate intoxication (serum levels, greater than 4 mEq/L [greater than 4 mmol/L]) presented with a reduced or absent serum anion gap. In both subjects, hemodialysis simultaneously removed the excess lithium ion and normalized the anion gap. Conversely, the anion gap was normal in subjects with therapeutic serum lithium ion levels. Severe lithium carbonate intoxication should be added to the category of illnesses (multiple myeloma, bromide intoxication) causing a marked reduction in the anion gap. In the comatose patient, a reduced anion gap may serve as an important clinical clue to the presence of this drug intoxication.

Decreased total and active urinary kallikrein in normotensive Dahl salt susceptible rats.

Abnormalities in the kallikrein-kinin system have been found in human and animal models of essential hypertension. The purpose of this study is to assess the kallikrein-kinin system in normotensive Dahl salt sensitive (S) and salt resistant (R) rats on a zero sodium diet. Urinary kallikrein was measured at 7 and 12 wk of age by different techniques. When kallikrein activity was assessed, by a kininogenase assay, S rats excreted 66% (P less than 0.001) and 75% (P less than 0.01) as much kallikrein as R rats at 7 and 12 wk of age. Using an artificial substrate method (Kabi S-2266), S rats excreted 30% (P less than 0.001) and 56% (P less than 0.05) as much kallikrein as R rats at 7 and 12 weeks, respectively. Using a technique to measure total kallikrein, S rats excreted 53% (P less than 0.001) and 65% (P less than 0.05) as much kallikrein as R rats at 7 and 12 wk of age. Normotensive S rats failed to increase maximally kallikrein activity or total kallikrein when the diet was switched from a .4% to a .0064% sodium chloride diet. There was no difference in inhibitors, as measured by the recovery of purified kallikrein added to S and R urine (56 +/- 21% vs. 53 +/- 13%). Km values for S and R urinary kallikrein were similar (3.1 +/- .5 X 10(-5) vs. 2.6 +/- .5 X 10(-5) M/liter). Trypsin-activatable kallikrein was equivalent in the S and R rats on the .0064% and .4% sodium chloride diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Independent action of prostaglandins and kinins on vasopressin-stimulated water flow.

The kallikrein-kinin and the prostaglandin systems are both important modifiers of vasopressin action. This study examines whether the systems are dependent on one another for their action. Four groups of toad hemibladders were examined. In groups 1 and 2 animals the endogenous prostaglandin system was inhibited. Inhibition of kallikrein by aprotinin caused vasopressin-stimulated water flow to increase further (24.8 +/- 4.9 to 34.5 +/- 4.8 microliters/min) while potentiation of kinins by captropril caused vasopressin-stimulated water flow to decrease (45 +/- 6.3 to 30.5 +/- 5.4 microliters/min). In groups 3 and 4 endogenous kallikrein was inhibited by aprotinin. The addition of prostaglandin E2 caused vasopressin-stimulated water flow to decrease (17.5 +/- 2.7 to 5.71 +/- 1.0 microliter/min) while the inhibition of endogenous prostaglandins caused vasopressin-stimulated water flow to increase (26.7 +/- 3.4 to 39.2 +/- 3.5 microliters/min). Thus, the inhibitory effects of prostaglandins and kinins on vasopressin-stimulated water flow are independent of one another.

Renal dysfunction after arteriography.

Acute renal failure has been observed in patients undergoing angiography in which a hypertonic triiodinated contrast medium is used. To ascertain the incidence of renal dysfunction and the clinical risk factors, we did a prospective study in which creatinine clearance was measured before and immediately after 120 arteriographic procedures. Thirty-seven patients (31%) sustained a significant reduction in creatine clearance after arteriography. No specific risk factor could be determined. Our findings, however, indicate that patients with preexisting renal insufficiency or diabetes mellitus are not at a higher risk for sustaining a fall in creatinine clearance after angiography.

Reduced ratio of active-to-total urinary kallikrein in essential hypertension.

Urinary kallikrein excretion was studied in 34 patients with mild, normal-renin, essential hypertension without evidence of target organ damage and in 23 normotensive controls, using assays that measure both active (kininogenase activity) and total (active plus inactive) kallikrein. There was no significant difference in either active or total kallikrein excretion between the two groups. However, the ratio of active-to-total enzyme was decreased in the hypertensives (0.83 +/- 0.03 units/micrograms) compared to the normotensives (1.00 +/- 0.05 units/micrograms) (p less than 0.002). The active-to-total ratio was inversely related to sodium excretion in both groups, indicating that the proportion of active to inactive enzyme increased in response to reduced sodium intake. We conclude that, although absolute excretion of active and total kallikrein is not decreased, enzyme activity per microgram of total kallikrein excreted is reduced in mild, normal-renin essential hypertension. This abnormality may be due to a defective enzyme, or to a reduced excretion of active relative to inactive kallikrein. The latter could result from the presence of a urinary kallikrein inhibitor or to reduced activation of a proenzyme.

Effects of alterations in sodium and water metabolism on urinary excretion of active and inactive kallikrein in man.

Renal kallikrein is present in human urine in both an active and an inactive form. Several previous studies have examined the response of active kallikrein excretion to alterations in sodium and water metabolism, but the response of inactive kallikrein has not been evaluated systematically. We have developed a method for determining inactive kallikrein in urine using two assays. Active kallikrein is measured using a kininogenase assay. Total (active plus inactive) kallikrein is measured using a direct RIA. Inactive kallikrein is calculated from the difference between active and total kallikrein excretion. We have used this technique to study the effect of alterations in sodium and water metabolism on kallikrein excretion. Acute volume expansion with saline and moderate acute or chronic increases in water intake did not alter the excretion of either active or inactive kallikrein. Dietary sodium restriction increased the excretion of total kallikrein by 30%; active kallikrein increased 82%, while inactive kallikrein excretion was found to be unchanged. Spironolactone reduced total kallikrein excretion in subjects on a low salt diet. Again, the change in excretion was entirely attributable to the active form of the enzyme. Total kallikrein excretion increased during the first 3 days of fludrocortisone administration in subjects on a high salt intake and then plateaued. Active kallikrein increased progressively throughout the 7-day study period. Inactive kallikrein increased during the first 4 days, then fell to control levels. Thus, mineralocorticoid initially stimulates the formation and/or release into urine of both active and inactive kallikrein. Later, reciprocal changes in active and inactive enzymes occur.