Renal Pathology

Myoglobin-Induced Acute Tubular Injury (ATI)

Agnes B. Fogo
Vanderbilt University Medical Center
Nashville, Tennessee


Clinical History
This 64-year-old white retired man with a kidney transplant presented in August 2006 for evaluation of one-week history of weakness, muscle pain and diarrhea. Muscle aches and weakness had started in the lower legs and then involved the anterior thigh and calf. The pain rendered him unable to walk. He also had diarrhea for three days, and noticed red/brown urine, but maintained normal urine output. He had no other symptoms.

He had received a living non-related 0 antigen match kidney in June 2005 for end stage renal disease for diabetic nephropathy, and had been in stable health, with controlled glucose and moderately controlled blood pressure, but persistent hyperlipidemia. He had one episode of acute cellular rejection, biopsy proven, in January 2006. Serum creatinine stabilized after this at 2.6-2.7 mg/dl. His medications at that time included mycophenolate, cyclosporine (level 305 ng/ml), prednisone, furosemide, diltiazem, aspirin, simvastatin and insulin. His prescription for an angiotensin receptor-blocker (ARB) had not been filled at that time, as it was not on his hospital's formulary. An ARB was added to his medications and diuretic dose was increased in mid-July 2006, when he presented with edema and increased weight of 8-10lbs, but stable creatinine.

His past medical history was significant for 60-70 pack yrs smoking, but he had quit five years ago. He did not use alcohol. He had coronary artery disease with four-vessel bypass in 2000, implantable cardioverter defibrillator placement in March 2005 and elective cholecystectomy in 2005.

On presentation in August 2006, he appeared overweight and in mild distress. His blood pressure was 152/78 mmHg, temperature was 97.6F, pulse 88 beats/min and weight 259 lbs. He had tenderness of the posterior calves and anterior thighs and 1+ edema of the lower extremities, but otherwise normal exam, including neurological and remaining musculoskeletal exam. Admission laboratories (normal in brackets) showed sodium 130 mmol/L, potassium 7.0 mmol/L, chloride 95 mmol/L, CO2 19 mmol/L, serum creatinine 8.3 mg/dl, BUN 143 mg/dL, glucose 242 mg/dl, phosphorus 8.8 mg/dl, AST 503 U/L [15-37] and ALT, 204 U/L [30-65], alkaline phosphatase 80 U/L, total bilirubin 0.6 mg/dl, direct bilirubin 0.4 mg/dl, uric acid 7.8 mg/dL. His CBC showed a decreased hematocrit at 31.5%, with platelets 209,000/mm3, white blood cells 8,900/mm3, with lymphocytes 5.3%, PMNs 88.5%, monocytes 5.6%, eosinophils 0.5%, and basophils 0.1%. PT and PTT were normal. Creatine kinase (CK) was 16,393 U/L [normal 21-232], with CK-MB 123.9 ng/mL [normal 0-3.6]. Urinalysis showed positive dipstick for protein, large blood and urobilinogen, with negative glucose and bilirubin.

An ultrasound of the transplant kidney was unremarkable with no signs of obstruction. He was admitted and treated with fluids and cyclosporin, ARB and simvastatin were discontinued, and the patient was given Imuran. On day four, with creatinine remaining high, a renal biopsy was performed.


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Figure 1 - Low power, PAS stain shows widespread acute tubular injury with thinning of cytoplasm and loss of brush borders. Several mixed cellular and proteinaceous casts are present. The interstitium has a mild mononuclear infiltrate and edema. A small artery and two glomeruli are unremarkable.
Figure 2 - Similar features are present, but with even more casts are shown, sometimes with little PAS positivity.
Figure 3 - Similar features are present, but with even more casts are shown, sometimes with little PAS positivity.

Figure 4 - Higher power PAS stain shows granular pattern of casts and loss of brush borders from proximal tubular cells.
Figure 5 - Granular and weakly PAS positive pattern of casts is illustrated.

Figure 6 - Granular and dense redish brown ball-like cast material is present, typical of myoglobin.
Figure 7 - A normal glomerulus at high power (PAS).


Renal Biopsy Findings
Light microscopic examination showed three pieces of cortex and one piece of cortex and medulla with eighteen glomeruli, none of which were globally or segmentally sclerosed. Glomeruli showed mild increase in mesangial matrix and cellularity, focal Bowman's capsule thickening and periglomerular fibrosis. The glomerular basement membranes were unremarkable. There was about 5% tubulointerstitial fibrosis, mostly in the subcapsular areas and associated with mild interstitial lymphocytic infiltrate with rare tubulitis in scarred areas and rare interstitial neutrophils and eosinophils. There was no lymphocytic infiltrate or tubulitis in the non-scarred cortex. Tubules were focally dilated with extensive epithelial cell flattenening and sloughing, surrounded by mild interstitial edema. There were frequent intratubular reddish-brown granular casts with occasional cell debris. None of these casts had a fractured appearance or surrounding syncytial reaction. There were no viral cytopathic changes. Arterioles showed mild hyalinosis. Interlobular arteries showed mild to moderate intimal fibrosis. There was no endothelialitis. Casts showed strong positivity with specific immunoperoxidase staining for myoglobin. Immunofluorescence staining for IgG and polyvalent antisera showed trace focal and segmental mesangial and capillary loop staining. There was no staining for IgA, IgM, C3 or C1q. Electron microscopic examination showed mild segmental expansion of lamina rara interna of the glomerular basement membrane and mild segmental increase in mesangial matrix, without any deposits. Podocytes and endothelial cells were unremarkable. Several tubular profiles contained globular casts with dark central core and slightly lighter periphery and no substructure, accompanied by tubular cell vacuolization and sloughing.

Pathologic Diagnosis
The presence of extensive tubular epithelial cell injury with granular reddish-brown casts, but minimal abnormalities in glomeruli and vessels by light microscopy and dark globular intratubular casts with slightly lighter periphery by electron microscopy is characteristic of myoglobin-induced acute tubular injury (ATI) [1]. This was confirmed by strong immunoperoxidase staining of intratubular casts with specific antibody against myoglobin. There was no evidence of acute cellular or vascular rejection or viral cytopathic changes.

Discussion
There are many causes of abrupt increase in creatinine in the transplant. Common etiologies include acute rejection, calcineurin inhibitor toxicity and ATI. In specific clinical circumstances, additional considerations, such as acute vasoconstriction or hypoperfusion (related to for instance volume depletion), drug hypersensitivity reaction, infection (particularly viral), obstruction, thrombotic microangiopathy (whether drug-related, idiopathic or recurrence of original disease), may be diagnostic considerations. Other rare conditions may also result in acute rise in creatinine, including recurrence of original disease, or de novo processes such as marked cholesterol embolization. The renal biopsy is essential in establishing the precise cause of acute onset of renal dysfunction in the graft. Most of the above conditions have distinct morphological lesions, which allow precise diagnosis of the underlying process, which may then direct appropriate treatment.

In our patient, the clinical differential diagnoses included acute ischemic injury due to volume depletion due to his preceding illness with diarrhea, calcineurin inhibitor toxicity or possibly rhabdomyolysis. Acute rejection due to inadequate immunosuppressive drug during his acute diarrheal illness was a lesser consideration. The renal morphological findings showed characteristic acute tubular injury with casts that by usual light microscopy were highly suspicious for myoglobin, confirmed by immunostaining.

The differential diagnosis of prominent casts includes ATI due to ischemia, with Tamm-Horsfall casts, which must be distinguished from the characteristic brownish-pigmented myoglobin casts of rhabdomyolysis. Pigmented intratubular casts may also be seen with hemoglobinuria. Hemoglobinuria may occur with acute hemolysis due to e.g. incompatible blood transfusion, malaria, quinine ingestion or paroxysmal hemoglobinuria. An additional rare consideration in the differential diagnosis of pigmented intratubular casts is bile casts. In patients with markedly elevated bilirubin levels, so-called bile or cholemic nephrosis may develop. Typically, tubular injury does not develop until bilirubin levels are greater than 20 mg/dl, in combination with hypoalbuminemia or endotoxemia. Rarely, renal biopsies have been performed in this clinical setting, revealing intratubular casts with bilirubin pigment [2]. These casts may have a more green hue than myoglobin casts. Specific immunostaining is helpful in differentiating these casts from myoglobin casts. Differentiation of myoglobinuric versus hemoglobinuric injury may also be done by examination of patient's blood and urine. The plasma is clear, usually with elevated CK in myoglobinuria, in contrast to light pink color plasma when hemolysis has caused hemoglobinuria. Both myoglobin and hemoglobin can be detected in the urine with a dipstick test, using the orthotolidine reaction, which reacts with the heme molecule.

Intratubular casts are of course also an essential component of light chain cast nephropathy (LCCN). These casts typically have a surrounding syncytial cell reaction with a fractured appearance of the casts, and the casts frequently, but not always, show light chain restriction staining by immunofluorescence. However, LCCN is quite uncommon in the transplant, although it may occur, even in patients who were not diagnosed with multiple myeloma or related disease as their primary disease [3]. Recently, a novel tubular injury associated with rapamycin has been recognized. Rapamycin caused ATI accompanied by intratubular casts with very similar appearance to those seen in LCCN. However, the casts were composed of keratin, representing remnants of degenerated epithelial cells rather than monoclonal light chains [4].

Rhabdomyolysis causes release of myocyte contents, including myoglobin and CK into blood. A rise in serum myoglobin level precedes an increase in serum CK. The half-life of myoglobin is only 1 to 3 hours and its concentration usually returns to normal levels 1 to 6 hours after the injury due to its rapid clearance through kidneys or its metabolism to bilirubin [5]. CK-MM is the predominant CK isoenzyme in muscles, with a half-life of 1.5 days. Serum CK, the most sensitive biochemical indicator of rhabdomyolysis, increases 2 to 12 hours after the onset of muscle injury and peaks at 3 to 5 days [6]. Thus, in the initial phases of rhabdomyolysis, serum CK may be normal and myoglobinuria may precede and resolve prior to an increase in CK [6]. Muscle damage also leads to electrolyte abnormalities such as hyperkalemia, early hypocalcemia and later hypercalcemia.

Rhabdomyolysis is estimated to underlie approximately 5-25% of all cases of acute renal failure (ARF) [7]. ARF is the most serious complication of rhabdomyolysis and occurs in up to 16.5% of patients with myoglobinuria [8]. Drugs and alcohol are the most common (up to 81%) etiologies of rhabdomyolysis [9]. Other etiologies include toxins (including illicit drugs), trauma, excessive exercise, long-term immobility, hereditary muscle enzyme defects, infections, metabolic/endocrine disorders, and hypo/hyperthermia [7]. The classic presenting features of rhabdomyolysis include muscle injury and/or pain, pigmented urine and renal dysfunction. However, in drug-induced rhabdomyolysis, other symptoms may predominate the clinical findings and thus a subclinical presentation of rhabdomyolysis without these common features may be overlooked [10].

Rhabdomyolysis is an important but rare adverse effect of statins. The incidence of all myotoxic effects of statins in the general population is between 1 and 7%, most of which are myalgias that are reversible within 2 to 3 weeks after statin withdrawal [11]. Fatal rhabdomyolysis following statins is very rare and its estimated incidence among all patients is 0% for fluvastatin, 0.04% for pravastatin, 0.12% for simvastatin and 0.19% for lovastatin [12]. Importantly, statins have interactions with other drugs, and their metabolism is affected by the major hepatic cytochrome P450 isoenzyme CYP3A4 [13]. Cyclosporin and clarithromycin are CYP3A4 inhibitors and thereby increase statin levels, such that patients receiving either of these drugs in addition to statins that are metabolized by CYP3A4 enzymes (including simvastatin, lovastatin, atorvastatin and cerivastatin), have increased risk of muscle toxicity. Cerivastatin had a remarkably increased risk of rhabdomyolysis when combined with cyclosporine, and therefore was withdrawn in 2001 [14]. Rhabdomyolysis occurring in transplant patients has mostly been related to a combination of cyclosporin with either lovastatin or simvastatin. In contrast, pravastatin and fluvastatin, because of their different metabolism are least likely to cause muscle toxicity in transplant patients. Other important statin interactions are those of fluvastatin, thought to be a substrate of the CYP2C9 enzyme, which may have an interaction with diclofenac, which is metabolized by the same enzyme. These considerations of risk are important in selecting optimal therapy for high-risk patients such as renal transplant patients [15].

Other reported risk factors for statin-induced myopathy include advanced age, small body size, multisystem disease including chronic renal failure, alcohol abuse, perioperative period, large quantities of grape fruit juice, and concomitant consumption of fibrates, azole anti-fungals, nicotinic acid (rarely), macrolide antibiotics, HIV protease inhibitors, nefazodone, verapamil or amiodarone [16]. Of note, fatal rhabdomyolysis has been reported in two patients taking concomitant diltiazem, a weak CYP3A4 inhibitor, and simvastatin, both drugs that our patient was taking [17] .

Despite the risks of adverse drug interactions outlined above, statins are an essential component of intervention for renal transplant patients to decrease their remarkably increased cardiovascular morbidity and mortality. Post-transplant hyperlipidemia, a known risk factor for cardiovascular disease, is seen in 16% to 78% of recipients [18] contributed to by immunosuppressive therapy, especially corticosteroids and cyclosporine. Statins effectively reduce hyperlipidemia and, subsequently, its related cardiovascular risk [19]. Early initiation of statins following renal transplantation significantly reduces major cardiovascular events [20]. Moreover, experimental studies suggest that statins may be beneficiary through lipid-independent mechanisms, such as improving endothelial function, reducing proinflammatory cytokines and adhesion molecules, upregulation of eNOS, antioxidant effects and some immunomodulatory properties [21]. The postulated beneficial effects of statins on acute cellular or vascular rejection remain controversial at the moment [21].

The mechanisms of statin-induced muscle damage are unknown, but hypotheses include defects in cell membrane structure, mitochondrial dysfunction, and impaired myocyte duplication [22]. Experimental studies suggest that myoglobinuria alone is insufficient to induce ARF, and that additional hypovolemia, renal vasoconstriction or urine acidification is required for nephrotoxicity [7]. The proposed mechanisms of myoglobinuria-associated ARF include obstruction by casts, tubulotoxic effect of ferrihemate, a breakdown product of myoglobin at pH <5.6 and renal vasoconstriction secondary to inhibition of nitric oxide synthesis (74). To our knowledge, direct nephrotoxicity of statins has not been systematically studied. Nevertheless, findings suggestive of direct renal tubular toxicity on high-dose statin therapy without rhabdomyolysis was reported in a patient with familial hypercholesterolemia [23].

Current therapeutic modalities include withdrawal of myotoxic agents and predisposing conditions, intravenous volume expansion, urinary alkalinization and forced diuresis [24]. Other treatments include supportive treatment with correction of electrolyte abnormalities including hyperkalemia, hypocalcemia and hypophosphatemia. Prognosis of rhabdomyolysis depends on severity and comorbid conditions, with mortality of 52% in patients with ARF, compared to 14% in those without ARF, in a series of critically ill intensive care unit patients with rhabdomyolysis [25].

Clinical Follow-up
Following admission, the patient was given i.v. fluids, and cyclosporine, simvastatin, mycophenolate mofetil, furosemide and angiotensin receptor blockers were stopped, and azathioprine was started. A week later, he had no significant myalgia but still had weakness, CK was less than 1000 U/L, transaminases were markedly improved and urine became clear. Serum creatinine decreased to 3.2 mg/dl in two weeks and to his baseline level of 2.5 mg/dl at three months follow-up.

Summary
In summary, this patient had rhabdomyolysis and myoglobin-induced ATI, most likely secondary to simvastatin. He had additional risk factors for rhabdomyolysis, including taking cyclosporine and diltiazem and reduced GFR, possibly with reduced renal blood flow secondary to recent diarrhea, increased furosemide and angiotensin receptors blockers. Prompt recognition and diagnosis based on characteristic clinical and pathologic findings with appropriate therapy restored his renal function to baseline. Rhabdomyolysis and myoglobinuric ARF, although rare, are serious complications of statin therapy. Renal transplant patients are at greater risk of these complications due to concomitant use of cyclosporine and possible renal dysfunction.

Acknowledgment
I thank Dr. Behzad Najafian for his contributions to this syllabus, which also is the basis for our in press Renal Biopsy Report (Kidney Int, Najafian, Franklin, Fogo, in press)

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