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Immunofluorescent demonstration of myoglobin in the kidney
lmmunofluorescent Demonstration Myoglobin in the Kidney
of
Case Report and Review of Forty-Three
Cases of Myoglobinemia
Myoglobinuria
Identified
and
Immunologically
LAWRENCE J. KAGEN, M.D. New York, New York
Myoglobin was identified in the kidney of a patient who died with renal failure six days after the onset of myoglobinuria, secondary to ischemia of the lower extremities. Its localization was made possible by the use of immunofluorescent staining with specific antimyoglobin serum. Myoglobin was present in distal tubular elements, as pigmented casts in the lumens and within the cytoplasm of tubule cells. Many such cells were degenerated. An outline of the principal causes of myoglobinemia and myoglobinuria in forty-three patients identified by immunoassay over a four year period is presented. In the absence of acidosis, and with otherwise normal renal hemodynamics, myoglobin itself may not be uniquely injurious to the kidney: however the frequent association of myoglobinuria with renal failure should alert the physician to the need for diagnosis and close observation of these patients. Myoglobin, the low molecular weight, heme-containing protein of striated muscle, has been found in the serum and urine of patients after various types of muscle injury. Its presence there is a consequence of release into the circulation from striated muscle and subsequent renal clearance. In
From the Department of Medicine, Columbia University College of Physicians and Surgeons, and the Edward Daniels Faulkner Arthritis Clinic of the Presbyterian Hospital, New York, New York. This study was supported in part by Public Health Service Grant 5 ROl AM 11659 NIAMD, National Institutes of Health, Bethesda, Mary land. Requests for reprints should be addressed to Dr. Lawrence J. Kagen. Department of Medicine, College of Physicians and Surgeons of Columbia University. 630 West 168th Street, New York, New York 10032. Manuscript received September 12, 1969.
Volume 48. May 1970
man as well as in the experimental animal [l-4], renal damage and death have at times been the sequel to myoglobinuric states. Antimyoglobin serum, which we have prepared [S], offers a greater possibility of specifically and sensitively ,detecting this potentially dangerous condition than previous chemical or physical methods. In addition, the use of specific antiserum coupled to fluorescein has permitted histologic localization of myoglobin in the muscle cell [6]. We report here immunofluorescent staining of myoglobin in the kidney of a patient who died with renal failure following myoglobinuria. The case report, discussion of the possible relationship of myoglobinuria to renal failure and a brief tabulation of the causes of this entity which we have encountered using the immune assay method are presented.
MATERIALS
AND METHODS
Antiserum to human myoglobin was prepared by immunizing rabbits with purified skeletal muscle myoglobin in complete Freund’s adjuvants 153. This antiserum formed precipitates with myoglobin but not with other components of human muscle. It did not react with liver or uterine extracts, hemoglobin, serum or urine. Immunologic measurement of myoglobin concentration by the
649
IMMUNOFLUORESCENT
DEMONSTRATION
OF
MYOGLOBIN
-
KAGEN
accident past history included a cerebrovascuiar carotid endarterectomy years earlier, bilateral years earlier (at another hospital) and an inguinal rhaphy sure and rales and
nine
months
earlier.
On admisslon,
eighteen fourteen hernior-
his blood
pres-
was 114/68 mm Hg, pulse rate 80 beats per minute temperature 99.4”F. Palmar erythema, fine crackling at the base of t,he right lung, normal cardiac findings a pulsatile mass in the lower abdominal quadrant were
noted. The right dorsalis pedis pulse was absent: other pulses in both lower extremities were preserved. The complete blood count was normal, as was the urine analysis. There was no proteinuria. Blood urea nitrogen was 18 mg per cent. Serum electrolytes and protein electrophoresis were normal. A chest roentgenogram demonstrated a dilated thoracic aorta, prominence of the left ventricle and evidence of pulmonary
emphysema.
At operation, a thin-walled fusiform aneurysm, 8 cm in diameter and 15 cm in length extending to t*he bifurcation of the abdominal aorta, was seen. A small aneurysm of the common iliac artery and arteriosclerotic changes of proximal and distal arteries were also noted. The aortic aneurysm was resected and replaced by a Dacron8 prosthesis extending from the proximal aorta to both common iliac arteries. During the time the aorta was clamped the lower extremities were flushed with diluted heparin-saline solution. The entire procedure took nine and a half hours and was performed
Fig. toxylin
1.
Pigmented and
eosin
cast stain,
visible original
in
distal
magnification
tubular X
segment.
Hema-
63.
method of radial diffusion into agar premixed with specific antibody was performed as already described [5]. Fluorescent antibody staining of renal sections was carried out by the indirect technic, with antimyoglobin serum followed by fluorescent antigammaglobulin reagent as described [6]. The immune staining procedure was specific for myoglobin and failed to stain normal human kidney, lung, spleen, liver or
with
cyclopropane,
ether,
nitrous
oxide
and
oxygen anesthesia. Thiopental, 350 mg. d-tubocurarin?, 6 mg, and succinyl choline, 80 mg, were given intravenously during induction of anesthesia. D-tubocurarine, 12 mg, was given during surgery. A total of 6,250 ml of fluids plus 500 ml of whole blood were also administered, and the patient’s blood pressure remained normal throughout. Immediately after operation the patient’s left leg was pale and cold: no femoral pulse was detectable. A second procedure was then performed, the left femoral artery was opened, and a Fogarty cat,heter was inserted. However, it was impossible to clear the obstructed vessel. The abdominal wound was then re-entered, and the aneurysm of the left common iliac artery, which was found to contain thrombus, was removed and replaced with a Dacron prosthesis was joined to the distal end of the aortic prosthesis.
which Pulsa-
Fig.
of
uterus. Human renal tissue obtained at autopsy from our patient (J.O.M.) and from patients w,ho died of other diseases was quick frozen in isopentane cooled in a bath of liquid nitrogen. Sections were cut at a thickness of 6 p and fixed with acetone before staining [6]. No attempt was made to concentrate clinical samples of serum or urine before immunologic assay.
CASE REPORT The patient (J.O.M.), a sixty-five year old white man, was admitted for repair of a fusiform aneurysm of the abdominal aorta known to be present for nine months. During this time growth of the aneurysm had been noted on roentgenograms, and pain in t,he lower right abdominal quadrant, suggestively due to aneurysmal expansion, had appeared. In addition, the patient had a four to five year history of bilateral lower extremity claudication and was able to walk only about two city blocks at the time of admission. His
650
2.
mented original
Several casts
distal and
magnification
tubules
cell
(at
destruction.
right)
show
Hematoxylin
presence and
eosin
pigstain,
X 63.
The
American
Journel
of Medicine
IMMUNOFLUORESCENT
tion in the left lower color remained “poor.”
extremity
was
restored
but
the
DEMONSTRATION
OF MYOGLOBIN
-
KAGEN
leg
That evening the patient passed brownish urine. The “HematesW’ reaction was positive, and immunologic assay demonstrated 0.250 mg per ml of myoglobin. At this time 0.125 mg per ml of myoglobin was present in the serum. The left lower extremity was swollen by the next day, and loss of pulses in the right lower extremity occurred suddenly. An operation was then performed, and thrombus was removed from the right superficial femoral, popliteal and posterior tibia1 arteries with the use of Fogarty catheters. On this day the patient excreted 1,780 ml of reddish brown urine. Serum creatine phosphokinase was 1,107 units per ml, and blood urea nitrogen was 17 mg per cent. Muscle damage in the lower extremities evidently was marked. During the next four days the patient’s condition deteriorated rapidly. Oliguria and renal failure developed. Urinary output fell and by the third postoperative day was only 363 ml. The blood urea nitrogen rose to 111 mg per cent on the fourth postoperative day, and there was concomitant acidosis and hyperkalemia. Progressive muscle twitching and deepening lethargy developed. On the fifth postoperative day, electrolytes were sodium 132, potassium 5.6, chloride 90 and carbon dioxide 18 mEq per L; creatinini was 12.9 mg per cent, blood urea nitrogen 132 mg per cent, ‘calcium 4.0 and phosphorus 19.0 mg per cent. The hemoglobin was 11.1 gm per cent, blood patient was treated
pH 7.27 and urinary output 317 ml. The during this time with digoxin and with
stains for myoglobin. A, bright fluoresFig. 4. lmmunofluorescent cence seen in one oiemented cast. as well as in distal tubular cells if tubules not conta’ining casts. Medulla, original magnification X 100. 6. myoglobin seen as bright fluorescence in tubular cells and casts. Glomerulus (upper left) shows no fluorescence and contains no myoglobin. Cortex, original magnification X 100. C, myoglobin. present in cells of distal tubules which do not contain casts, revealed by fluorescence of tubules. Medulla, original magnification X 100. D, myoglobin containing cast demonstrates bright fluorescence. Medulla, original magnification X 400.
intravenous fluid and electrolytes, and exchange resins to lower serum potassium levels. Penicillin and streptomycin were also used. On the sixth postoperative day a fatal cardiac arrest occurred. At autopsy, the suture lines of the prostheses were closed and without signs of leakage. The renal arteries and veins were patent. A double renal artery was present on the right. The kidneys were swollen, each weighed 300 gm, and the capsule stripped off with difficulty. The cortex and medulla had a reddish brown coloration. There were several areas of infarction. Examination of muscle of t,he lower extremities was not performed. There was an acute focal interstitial pancreatitis.
RESULTS I. Immunologic and Histologic Staining of Kidney Sections. Figures 1 to 3 are sections of the kidney obtained at autopsy, The maior findines
stained with hematoxylin and eosin. can be classified as follows:
Glomeruli: Most glomeruli did not show gross abnormalities. Some were hyalinized, with focal lymphocytic infiltrates. There was congestion in cortical and parenchymal areas, with engorgement of smaller vessels. Capsular lining cells appeared degenerated, and the capsular space contained debris in places.
I
I Fig. 3. Distal tubules demonstrate pigmented casts with areas of cell degeneration. Hematoxylin and eosin stain, original magnification X 63.
Volume 4% May 1970
The tubules and pigmented casts: As shown, granular brownish pigmented casts occurred chiefly in the distal convoluted tubules, loops of Henle and collecting tubules, and were much more frequent nearer the medulla. Deterioration of the lining cells of the distal convoluted tubules and Henle’s loops which contained the pigmented casti also was frequent. Figure 4 shows results obtained when renal sections were stained by the immunofluorescent method using soecific antimvoelobin serum. The brownish oiemented -I~-~~ .,vm I casts demonstrated brilliant fluorescence. In addition, cells lining distal tubules which did not contain casts
651
IMMUNOFLUORESCENT
TABLE
I
DEMONSTRATION
OF
MYOGLOBIN
-
KAGEN
Conditions Associated with Myoglobinemia and Myoglobinuria, Detected by the Immune Precipitin Method,
1965-1969 Condition
No. of Patients
Postoperative Open heart surgery Spinal fusion Children after intravenous succinyl choline administration Dermatomyositis and polymyositis syndromes Idiopathic Femoral artery ischemia (thrombosis and embolus) Rigor or seizure disorders Tetanus Bacterial meningitis with seizures Alcoholic myopathy Severe burn Total
12 2 10 6 4 3 2 1 2 1 43
within their lumens had striking cytoplasmic fluorescence indicating the presence of myoglobin. Many of these cells appeared deteriorated. Myoglobin was not seen in glomeruli or in blood vessels. Control sections of three kidneys of patients with other diseases treated in the identical fashion failed to demonstrate this fluorescence. II. Experience with the Antiserum in the Detection of Myoglobinemia and Myoglobinuria. During the period 1965 to 1969, fourty-three patients were noted to have myoglobinemia and myoglobinuria (Table I). The occurrence of this condition after open heart surgery has already been reported [7]; almost 40 per cent of these patients were found to have this entity. Its occurrence after certain orthopedic procedures (i.e., spinal fusions) is now under study. Approximately one half of the children tested who received succinyl choline for anesthetic induction were found to have myoglobinemia [8]. Dermatomyositis and polymyositis syndromes frequently demonstrated myoglobinemia or myoglobinuria (six of fifteen patients tested during acute illness). Renal failure occurred in two of the six patients in this group. lschemia of one or both femoral arteries was another cause of severe myoglobinuria, complicated in each case by renal failure. Other disorders involving muscle (seizures and alcoholic myopathy) also were rarely associated with myoglobinuria. In one patient with an extensive and deep burn myoglobinuria also developed. Four patients have had recurrent bouts of myoglobinuria (associated with renal failure in two) without known cause. No patients with severe trauma were tested in the present series.
COMMENTS Myoglobin could be identified in the kidney of who died with renal failure six days after the myoglobinuria, secondary to ischemia of the tremities. Its renal localization, demonstrated
652
a patient onset of lower exwith the
use of immunofluorescent staining, was restricted to the distal tubular elements in which it appeared within the lumen, as pigmented casts and within the cytoplasm of the tubule cells. Many of these tubular lining cells, which apparently had absorbed myoglobin from their lumens, appeared degenerated and broken. The reason for renal failure, which so often complicates myoglobinuria, is not well understood. Uremia has been produced in the rabbit after the injection of purified preparations of myoglobin [4]. In these experiments renal failure and death occurred in all animals with acid urine but not in those with alkaline urine. Death with renal failure has been seen frequently in patients with myoglobinuria, but in most such cases the mechanism has been unclear. Bywaters [l] described renal changes in twenty-two patients with myoglobinuria including renal swelling, congestion, glomerular capsule degeneration, pigmented casts in distal tubules and distal tubular degeneration similar to what was seen in the patient presented here. Hemoglobinuric renal failure has been experimentally induced in dehydrated rats [9]. Cast formation, tubular damage and hemoglobin in the lumen of vasa recta were major findings [lo]. In general, experimental evidence seems to support the notion that the heme proteins, myoglobin and hemoglobin, produce renal damage in certain physiologic settings marked by dehydration, acidosis or decrease in renal perfusion. The danger from myoglobinemic states would be expected to be greater since plasma protein binding is slight, and the molecular weight, 17,800, is relatively low. [11,12]. Bywaters [13] stated that plasma values might be less than 0.3 mg per 100 ml with pigment appearing in the urine. Hemoglobin, on the other hand, has a considerably higher renal threshold. Our patient represents the first instance in which myoglobin has been recognized in the kidney by means of immunologic staining. The findings have confirmed previous observations with conventional staining technics and added the fact that myoglobin is present in cytoplasm of many tubular lining cells even in the absence of pigmented luminal casts. Many such cells which contained myoglobin had degenerated. Immunologic assay was also used in this patient to identify myoglobinemia and myoglobinuria during life. This technique makes possible specific as well as sensitive quantitation in ranges below those approachable with other methods. With its use, as shown in Table I, forty-three patients with myoglobinemia and myoglobinuria have been identified over a four year period. The most common causative factors have been those connected with major surgery of long duration, the use of succinyl choline in children and dermatomyositis-polymyositis syndromes. There were several patients with renal failure after myoglobinuria in each of these categories. In all three patients with severe myoglobinuria after femoral artery occlusions renal failure developed. No patients with severe trauma were tested in this series. The antibody method has been useful in recognizing this potentially dangerous condition and in assessing the success of therapy.
The
American
Journal
of
ModkIn@
IMMUNOFLUORESCENT
DEMONSTRATION
OF MYOGLOBIN
-
KAGEN
REFERENCES 1. Bywaters EGL, Dible JH: The renal lesion in traumatic anemia. J Path Bact 54: 111. 1942. 2. Bywaters EGL, Stead JK: The production of renal failure following injection of solutions containing myohaemoglobin. Quart J Exp Physiol 33: 53, 1944. 3. Bywaters EGL: .lschaemic muscle necrosis (“Crush syndrome”). Brit Med Bull 3: 107, 1945. 4. Perri GC. Gorini P: Uraemia in the rabbit after injection of crystalline myoglobin. Brit J Exp Path 33: 440, 1952. 5. Kagen LJ, Christian CL: Immunologic measurement of myoglobin in human adult and fetal skeletal muscle. Amer J Physiol 211: 656, 1966. 6. Kagen LJ, Gurevich R: Localization of myoglobin in human skeletal muscle using fluorescent antibody technique. J Histochem Cytochem 15: 436, 1967.
Volume
48, M8y 1970
7. Kagen LJ: Immunologic detection of myoglobinuria after cardiac surgery. Ann Intern Med 67: 1183, 1967. 8. Hyman A, Kagen LJ, Ryan J: Unpublished observations. 9. Jaenike J: The renal lesion associated with hemoglobinemia. I. Its production and functional evolution in the rat. J Exp Med 123: 523, 1966. 10. Jaenike J: The renal lesion associated with hemoglobinemia. II. Its structural characteristics in the rat. J Exp Med 123: 537, 1966. 11. Lathem W: The binding of myoglobin by plasma proteins. J Exp Med 111: 65, 1960. 12. Whelsy MJ, Barrett 0, Jr, Crosby WH: Serum protein binding of myoglpbin, hemoglobin and hematin. Blood 16: 1579, 1960. 13. Bywaters EGL: Myoglobin and myoglobinuria. The Muscle (Pal. onovski M, ed) Paris: L’Expansion. 1952, p 213.
of
Case Report and Review of Forty-Three
Cases of Myoglobinemia
Myoglobinuria
Identified
and
Immunologically
LAWRENCE J. KAGEN, M.D. New York, New York
Myoglobin was identified in the kidney of a patient who died with renal failure six days after the onset of myoglobinuria, secondary to ischemia of the lower extremities. Its localization was made possible by the use of immunofluorescent staining with specific antimyoglobin serum. Myoglobin was present in distal tubular elements, as pigmented casts in the lumens and within the cytoplasm of tubule cells. Many such cells were degenerated. An outline of the principal causes of myoglobinemia and myoglobinuria in forty-three patients identified by immunoassay over a four year period is presented. In the absence of acidosis, and with otherwise normal renal hemodynamics, myoglobin itself may not be uniquely injurious to the kidney: however the frequent association of myoglobinuria with renal failure should alert the physician to the need for diagnosis and close observation of these patients. Myoglobin, the low molecular weight, heme-containing protein of striated muscle, has been found in the serum and urine of patients after various types of muscle injury. Its presence there is a consequence of release into the circulation from striated muscle and subsequent renal clearance. In
From the Department of Medicine, Columbia University College of Physicians and Surgeons, and the Edward Daniels Faulkner Arthritis Clinic of the Presbyterian Hospital, New York, New York. This study was supported in part by Public Health Service Grant 5 ROl AM 11659 NIAMD, National Institutes of Health, Bethesda, Mary land. Requests for reprints should be addressed to Dr. Lawrence J. Kagen. Department of Medicine, College of Physicians and Surgeons of Columbia University. 630 West 168th Street, New York, New York 10032. Manuscript received September 12, 1969.
Volume 48. May 1970
man as well as in the experimental animal [l-4], renal damage and death have at times been the sequel to myoglobinuric states. Antimyoglobin serum, which we have prepared [S], offers a greater possibility of specifically and sensitively ,detecting this potentially dangerous condition than previous chemical or physical methods. In addition, the use of specific antiserum coupled to fluorescein has permitted histologic localization of myoglobin in the muscle cell [6]. We report here immunofluorescent staining of myoglobin in the kidney of a patient who died with renal failure following myoglobinuria. The case report, discussion of the possible relationship of myoglobinuria to renal failure and a brief tabulation of the causes of this entity which we have encountered using the immune assay method are presented.
MATERIALS
AND METHODS
Antiserum to human myoglobin was prepared by immunizing rabbits with purified skeletal muscle myoglobin in complete Freund’s adjuvants 153. This antiserum formed precipitates with myoglobin but not with other components of human muscle. It did not react with liver or uterine extracts, hemoglobin, serum or urine. Immunologic measurement of myoglobin concentration by the
649
IMMUNOFLUORESCENT
DEMONSTRATION
OF
MYOGLOBIN
-
KAGEN
accident past history included a cerebrovascuiar carotid endarterectomy years earlier, bilateral years earlier (at another hospital) and an inguinal rhaphy sure and rales and
nine
months
earlier.
On admisslon,
eighteen fourteen hernior-
his blood
pres-
was 114/68 mm Hg, pulse rate 80 beats per minute temperature 99.4”F. Palmar erythema, fine crackling at the base of t,he right lung, normal cardiac findings a pulsatile mass in the lower abdominal quadrant were
noted. The right dorsalis pedis pulse was absent: other pulses in both lower extremities were preserved. The complete blood count was normal, as was the urine analysis. There was no proteinuria. Blood urea nitrogen was 18 mg per cent. Serum electrolytes and protein electrophoresis were normal. A chest roentgenogram demonstrated a dilated thoracic aorta, prominence of the left ventricle and evidence of pulmonary
emphysema.
At operation, a thin-walled fusiform aneurysm, 8 cm in diameter and 15 cm in length extending to t*he bifurcation of the abdominal aorta, was seen. A small aneurysm of the common iliac artery and arteriosclerotic changes of proximal and distal arteries were also noted. The aortic aneurysm was resected and replaced by a Dacron8 prosthesis extending from the proximal aorta to both common iliac arteries. During the time the aorta was clamped the lower extremities were flushed with diluted heparin-saline solution. The entire procedure took nine and a half hours and was performed
Fig. toxylin
1.
Pigmented and
eosin
cast stain,
visible original
in
distal
magnification
tubular X
segment.
Hema-
63.
method of radial diffusion into agar premixed with specific antibody was performed as already described [5]. Fluorescent antibody staining of renal sections was carried out by the indirect technic, with antimyoglobin serum followed by fluorescent antigammaglobulin reagent as described [6]. The immune staining procedure was specific for myoglobin and failed to stain normal human kidney, lung, spleen, liver or
with
cyclopropane,
ether,
nitrous
oxide
and
oxygen anesthesia. Thiopental, 350 mg. d-tubocurarin?, 6 mg, and succinyl choline, 80 mg, were given intravenously during induction of anesthesia. D-tubocurarine, 12 mg, was given during surgery. A total of 6,250 ml of fluids plus 500 ml of whole blood were also administered, and the patient’s blood pressure remained normal throughout. Immediately after operation the patient’s left leg was pale and cold: no femoral pulse was detectable. A second procedure was then performed, the left femoral artery was opened, and a Fogarty cat,heter was inserted. However, it was impossible to clear the obstructed vessel. The abdominal wound was then re-entered, and the aneurysm of the left common iliac artery, which was found to contain thrombus, was removed and replaced with a Dacron prosthesis was joined to the distal end of the aortic prosthesis.
which Pulsa-
Fig.
of
uterus. Human renal tissue obtained at autopsy from our patient (J.O.M.) and from patients w,ho died of other diseases was quick frozen in isopentane cooled in a bath of liquid nitrogen. Sections were cut at a thickness of 6 p and fixed with acetone before staining [6]. No attempt was made to concentrate clinical samples of serum or urine before immunologic assay.
CASE REPORT The patient (J.O.M.), a sixty-five year old white man, was admitted for repair of a fusiform aneurysm of the abdominal aorta known to be present for nine months. During this time growth of the aneurysm had been noted on roentgenograms, and pain in t,he lower right abdominal quadrant, suggestively due to aneurysmal expansion, had appeared. In addition, the patient had a four to five year history of bilateral lower extremity claudication and was able to walk only about two city blocks at the time of admission. His
650
2.
mented original
Several casts
distal and
magnification
tubules
cell
(at
destruction.
right)
show
Hematoxylin
presence and
eosin
pigstain,
X 63.
The
American
Journel
of Medicine
IMMUNOFLUORESCENT
tion in the left lower color remained “poor.”
extremity
was
restored
but
the
DEMONSTRATION
OF MYOGLOBIN
-
KAGEN
leg
That evening the patient passed brownish urine. The “HematesW’ reaction was positive, and immunologic assay demonstrated 0.250 mg per ml of myoglobin. At this time 0.125 mg per ml of myoglobin was present in the serum. The left lower extremity was swollen by the next day, and loss of pulses in the right lower extremity occurred suddenly. An operation was then performed, and thrombus was removed from the right superficial femoral, popliteal and posterior tibia1 arteries with the use of Fogarty catheters. On this day the patient excreted 1,780 ml of reddish brown urine. Serum creatine phosphokinase was 1,107 units per ml, and blood urea nitrogen was 17 mg per cent. Muscle damage in the lower extremities evidently was marked. During the next four days the patient’s condition deteriorated rapidly. Oliguria and renal failure developed. Urinary output fell and by the third postoperative day was only 363 ml. The blood urea nitrogen rose to 111 mg per cent on the fourth postoperative day, and there was concomitant acidosis and hyperkalemia. Progressive muscle twitching and deepening lethargy developed. On the fifth postoperative day, electrolytes were sodium 132, potassium 5.6, chloride 90 and carbon dioxide 18 mEq per L; creatinini was 12.9 mg per cent, blood urea nitrogen 132 mg per cent, ‘calcium 4.0 and phosphorus 19.0 mg per cent. The hemoglobin was 11.1 gm per cent, blood patient was treated
pH 7.27 and urinary output 317 ml. The during this time with digoxin and with
stains for myoglobin. A, bright fluoresFig. 4. lmmunofluorescent cence seen in one oiemented cast. as well as in distal tubular cells if tubules not conta’ining casts. Medulla, original magnification X 100. 6. myoglobin seen as bright fluorescence in tubular cells and casts. Glomerulus (upper left) shows no fluorescence and contains no myoglobin. Cortex, original magnification X 100. C, myoglobin. present in cells of distal tubules which do not contain casts, revealed by fluorescence of tubules. Medulla, original magnification X 100. D, myoglobin containing cast demonstrates bright fluorescence. Medulla, original magnification X 400.
intravenous fluid and electrolytes, and exchange resins to lower serum potassium levels. Penicillin and streptomycin were also used. On the sixth postoperative day a fatal cardiac arrest occurred. At autopsy, the suture lines of the prostheses were closed and without signs of leakage. The renal arteries and veins were patent. A double renal artery was present on the right. The kidneys were swollen, each weighed 300 gm, and the capsule stripped off with difficulty. The cortex and medulla had a reddish brown coloration. There were several areas of infarction. Examination of muscle of t,he lower extremities was not performed. There was an acute focal interstitial pancreatitis.
RESULTS I. Immunologic and Histologic Staining of Kidney Sections. Figures 1 to 3 are sections of the kidney obtained at autopsy, The maior findines
stained with hematoxylin and eosin. can be classified as follows:
Glomeruli: Most glomeruli did not show gross abnormalities. Some were hyalinized, with focal lymphocytic infiltrates. There was congestion in cortical and parenchymal areas, with engorgement of smaller vessels. Capsular lining cells appeared degenerated, and the capsular space contained debris in places.
I
I Fig. 3. Distal tubules demonstrate pigmented casts with areas of cell degeneration. Hematoxylin and eosin stain, original magnification X 63.
Volume 4% May 1970
The tubules and pigmented casts: As shown, granular brownish pigmented casts occurred chiefly in the distal convoluted tubules, loops of Henle and collecting tubules, and were much more frequent nearer the medulla. Deterioration of the lining cells of the distal convoluted tubules and Henle’s loops which contained the pigmented casti also was frequent. Figure 4 shows results obtained when renal sections were stained by the immunofluorescent method using soecific antimvoelobin serum. The brownish oiemented -I~-~~ .,vm I casts demonstrated brilliant fluorescence. In addition, cells lining distal tubules which did not contain casts
651
IMMUNOFLUORESCENT
TABLE
I
DEMONSTRATION
OF
MYOGLOBIN
-
KAGEN
Conditions Associated with Myoglobinemia and Myoglobinuria, Detected by the Immune Precipitin Method,
1965-1969 Condition
No. of Patients
Postoperative Open heart surgery Spinal fusion Children after intravenous succinyl choline administration Dermatomyositis and polymyositis syndromes Idiopathic Femoral artery ischemia (thrombosis and embolus) Rigor or seizure disorders Tetanus Bacterial meningitis with seizures Alcoholic myopathy Severe burn Total
12 2 10 6 4 3 2 1 2 1 43
within their lumens had striking cytoplasmic fluorescence indicating the presence of myoglobin. Many of these cells appeared deteriorated. Myoglobin was not seen in glomeruli or in blood vessels. Control sections of three kidneys of patients with other diseases treated in the identical fashion failed to demonstrate this fluorescence. II. Experience with the Antiserum in the Detection of Myoglobinemia and Myoglobinuria. During the period 1965 to 1969, fourty-three patients were noted to have myoglobinemia and myoglobinuria (Table I). The occurrence of this condition after open heart surgery has already been reported [7]; almost 40 per cent of these patients were found to have this entity. Its occurrence after certain orthopedic procedures (i.e., spinal fusions) is now under study. Approximately one half of the children tested who received succinyl choline for anesthetic induction were found to have myoglobinemia [8]. Dermatomyositis and polymyositis syndromes frequently demonstrated myoglobinemia or myoglobinuria (six of fifteen patients tested during acute illness). Renal failure occurred in two of the six patients in this group. lschemia of one or both femoral arteries was another cause of severe myoglobinuria, complicated in each case by renal failure. Other disorders involving muscle (seizures and alcoholic myopathy) also were rarely associated with myoglobinuria. In one patient with an extensive and deep burn myoglobinuria also developed. Four patients have had recurrent bouts of myoglobinuria (associated with renal failure in two) without known cause. No patients with severe trauma were tested in the present series.
COMMENTS Myoglobin could be identified in the kidney of who died with renal failure six days after the myoglobinuria, secondary to ischemia of the tremities. Its renal localization, demonstrated
652
a patient onset of lower exwith the
use of immunofluorescent staining, was restricted to the distal tubular elements in which it appeared within the lumen, as pigmented casts and within the cytoplasm of the tubule cells. Many of these tubular lining cells, which apparently had absorbed myoglobin from their lumens, appeared degenerated and broken. The reason for renal failure, which so often complicates myoglobinuria, is not well understood. Uremia has been produced in the rabbit after the injection of purified preparations of myoglobin [4]. In these experiments renal failure and death occurred in all animals with acid urine but not in those with alkaline urine. Death with renal failure has been seen frequently in patients with myoglobinuria, but in most such cases the mechanism has been unclear. Bywaters [l] described renal changes in twenty-two patients with myoglobinuria including renal swelling, congestion, glomerular capsule degeneration, pigmented casts in distal tubules and distal tubular degeneration similar to what was seen in the patient presented here. Hemoglobinuric renal failure has been experimentally induced in dehydrated rats [9]. Cast formation, tubular damage and hemoglobin in the lumen of vasa recta were major findings [lo]. In general, experimental evidence seems to support the notion that the heme proteins, myoglobin and hemoglobin, produce renal damage in certain physiologic settings marked by dehydration, acidosis or decrease in renal perfusion. The danger from myoglobinemic states would be expected to be greater since plasma protein binding is slight, and the molecular weight, 17,800, is relatively low. [11,12]. Bywaters [13] stated that plasma values might be less than 0.3 mg per 100 ml with pigment appearing in the urine. Hemoglobin, on the other hand, has a considerably higher renal threshold. Our patient represents the first instance in which myoglobin has been recognized in the kidney by means of immunologic staining. The findings have confirmed previous observations with conventional staining technics and added the fact that myoglobin is present in cytoplasm of many tubular lining cells even in the absence of pigmented luminal casts. Many such cells which contained myoglobin had degenerated. Immunologic assay was also used in this patient to identify myoglobinemia and myoglobinuria during life. This technique makes possible specific as well as sensitive quantitation in ranges below those approachable with other methods. With its use, as shown in Table I, forty-three patients with myoglobinemia and myoglobinuria have been identified over a four year period. The most common causative factors have been those connected with major surgery of long duration, the use of succinyl choline in children and dermatomyositis-polymyositis syndromes. There were several patients with renal failure after myoglobinuria in each of these categories. In all three patients with severe myoglobinuria after femoral artery occlusions renal failure developed. No patients with severe trauma were tested in this series. The antibody method has been useful in recognizing this potentially dangerous condition and in assessing the success of therapy.
The
American
Journal
of
ModkIn@
IMMUNOFLUORESCENT
DEMONSTRATION
OF MYOGLOBIN
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KAGEN
REFERENCES 1. Bywaters EGL, Dible JH: The renal lesion in traumatic anemia. J Path Bact 54: 111. 1942. 2. Bywaters EGL, Stead JK: The production of renal failure following injection of solutions containing myohaemoglobin. Quart J Exp Physiol 33: 53, 1944. 3. Bywaters EGL: .lschaemic muscle necrosis (“Crush syndrome”). Brit Med Bull 3: 107, 1945. 4. Perri GC. Gorini P: Uraemia in the rabbit after injection of crystalline myoglobin. Brit J Exp Path 33: 440, 1952. 5. Kagen LJ, Christian CL: Immunologic measurement of myoglobin in human adult and fetal skeletal muscle. Amer J Physiol 211: 656, 1966. 6. Kagen LJ, Gurevich R: Localization of myoglobin in human skeletal muscle using fluorescent antibody technique. J Histochem Cytochem 15: 436, 1967.
Volume
48, M8y 1970
7. Kagen LJ: Immunologic detection of myoglobinuria after cardiac surgery. Ann Intern Med 67: 1183, 1967. 8. Hyman A, Kagen LJ, Ryan J: Unpublished observations. 9. Jaenike J: The renal lesion associated with hemoglobinemia. I. Its production and functional evolution in the rat. J Exp Med 123: 523, 1966. 10. Jaenike J: The renal lesion associated with hemoglobinemia. II. Its structural characteristics in the rat. J Exp Med 123: 537, 1966. 11. Lathem W: The binding of myoglobin by plasma proteins. J Exp Med 111: 65, 1960. 12. Whelsy MJ, Barrett 0, Jr, Crosby WH: Serum protein binding of myoglpbin, hemoglobin and hematin. Blood 16: 1579, 1960. 13. Bywaters EGL: Myoglobin and myoglobinuria. The Muscle (Pal. onovski M, ed) Paris: L’Expansion. 1952, p 213.