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Yale researchers dig for new kidney biomarkers

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Kevin B. O’Reilly

October 2016—An automated immunoassay has been created for symmetric dimethylarginine, or SDMA, a biomarker that can detect chronic kidney disease between 10 to 17 months earlier than creatinine, with 100 percent sensitivity and 91 percent specificity. And, unlike with creatinine, a patient’s muscle mass does not influence the biomarker’s reliability. SDMA has already been incorporated into the kidney-function testing advice that guides clinician ordering worldwide. Since the automated SDMA test was launched in July 2015, 5 million samples have been analyzed and 80 percent of clinicians are aware of the test.

There is a hitch in SDMA’s forward march to a place of prominence in chronic kidney disease testing: It has gone to the dogs—and cats.

The automated SDMA assay is available only from Idexx Laboratories, a Westbrook, Me., company with a 40 percent share of the veterinary lab testing market. In veterinary medicine, the weaknesses of serum creatinine as a CKD biomarker are pronounced because there are no estimated glomerular filtration calculations for laboratories to use and report.

So veterinary clinicians have been faced with the task of how to accurately interpret the meaning of a creatinine level in a serum sample from a Chihuahua or a Saint Bernard. Creatinine levels are normalized by breed, but that solution is short of ideal given the scope of the problem: One-third of cats and 10 percent of dogs develop kidney disease during their lives. Idexx now includes SDMA in all of its routine chemistry panels.

SDMA’s value should go beyond diagnosing CKD in dogs and cats, says Murthy Yerramilli, PhD, vice president of research and development at Idexx Laboratories.

“We want to take this test to human medicine,” Dr. Yerramilli says. “We want to collaborate and show that similar clinical findings happen in human medicine. We do think that this is going to be extremely useful for human health as well.”

That is more than just talk. The company has handed off its automated SDMA assay, which runs on Beckman and Roche instruments, to Yale University School of Medicine for validation in human samples.

Joe El-Khoury, PhD, is handling the validation task. He is an assistant professor of laboratory medicine at Yale and co-directs the clinical chemistry laboratory at Yale New Haven Health. His PhD research focused on developing a high-throughput, liquid chromatography-mass spectrometry assay to measure SDMA, along with asymmetric dimethylarginine and arginine (El-Khoury JM, et al. Anal Bioanal Chem. 2012;402[2]:771–779), so he has had a long-running interest in SDMA’s potential utility. (Dr. El-Khoury has received honoraria and travel reimbursement for two speaking engagements at Idexx, but the company is not paying him for the validation studies on its SDMA assay.)

The need for an alternative CKD biomarker is great, Dr. El-Khoury argued in a presentation at this year’s annual meeting of the American Association for Clinical Chemistry. The endogenous biomarkers used to estimate GFR based on creatinine and cystatin C have disadvantages. Creatinine, he said, has poor sensitivity and specificity and is affected by extra-renal factors such as age, gender, ethnicity, race, diet, muscle mass, and medication. While eGFRcystatinC is more accurate than creatinine-based eGFR, it is influenced by thyroid functioning and may be affected by obesity, inflammation, and smoking.

Using the more sophisticated Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPI), there is a 30-point spread in eGFRcreatinine, Dr. El-Khoury said during the session, “Emerging Biomarkers of Acute Kidney Injury and Chronic Kidney Disease.”

“In somebody whose GFR value comes back as 60, your range could really be between all the way down to 30 or all the way up to 90,” he said. “So you could be normal, or you could be at stage three or stage four chronic kidney disease. Even though the equations that have been developed have improved, there is still a lot of room for improvement, and there are still a lot of extra-renal effects and variability.”

Cystatin C represents an improvement in accuracy, but in high-stakes clinical situations when diagnostic accuracy is critical—for example, whether to accept a donor kidney for transplantation—clinicians often double-check the organ’s functioning using an exogenous marker such as iothalamate. But that silver standard for measuring GFR is time-consuming and involves radioactive exposure for patients and thus is not a good choice for routine CKD screening, Dr. El-Khoury said.

Dr. El-Khoury

Dr. El-Khoury

The ideal biomarker for CKD “must have a constant production rate so that as you’re rendering it into urine it is not affected by changes in production,” he added. “And it must be freely filtered. Obviously, you want a concentrated marker and it can’t be something that cannot get through the glomerulus. And it should neither be reabsorbed nor secreted by the renal tubules, so it’s not affected by other factors related to the filtration process. Also, it should not be metabolized or eliminated in extra-renal pathways.”

SDMA appears to fit this bill of particulars.

“It is a methylated product of arginine residues. It is produced by post-translational modification of arginine residues in proteins that are in histones,” Dr. El-Khoury said. “So this is a very highly preserved process, and it is produced at a fairly stable rate. And you have constant turnover and production of the marker. Over 90 percent is renally cleared and filtered through the glomerulus.”

Previous research has evaluated SDMA’s performance in comparison with creatinine and cystatin C, where it frequently showed strong correlation in the 0.80 range. However, Dr. El-Khoury explained, it is not sufficient to compare SDMA with existing biomarkers that may be less accurate. What appears as a false-positive in comparison with creatinine, for example, could be a false-negative given by creatinine. Only a handful of studies have compared SDMA with more accurate silver-standard mGFR markers—finding correlations ranging from 0.78 to 0.90—and all of these were done only using samples from patients with CKD or diabetes (Schwedhelm E, et al. Nat Rev Nephrol. 2011;7[5]:275–285).

To prove its mettle as a biomarker useful in screening, Dr. El-Khoury said, SDMA must be evaluated in healthy patients too. And so that is what he and his colleagues did.

“The reason why you want to look at healthy and CKD patients is to try to separate normals from abnormals,” Dr. El-Khoury tells CAP TODAY. “The marker has to perform equally well in both categories. It might only correlate in chronic kidney disease but have no correlation in healthy patients. Then we might not catch someone with declining function into the CKD stages.”

Dr. El-Khoury and his colleagues tested a series of 40 consecutive patients who had some reason to get tested for mGFR using iothalamate.

“These are very tough patient populations to get,” said Dr. El-Khoury, co-director of Yale’s clinical chemistry fellowship program. “We don’t frequently get patients who need silver-standard GFRs or measured GFR. In this case, the reason these patients showed up is they were either being evaluated as kidney donors, so this was our healthy population, or they were CKD patients or drug-dosing patients who needed to have their drug concentrations adjusted and the clinicians wanted to make sure their kidney-function result was still accurate.”

In addition to the mGFR reference method, the patients were tested for creatinine and cystatin C using the Roche Cobas 8000 and SDMA using the LC-MS/MS method Dr. El-Khoury developed. Compared with the silver-standard reference method, creatinine had just a -0.70 correlation, while cystatin C showed a higher correlation of -0.86. SDMA, meanwhile, had a correlation of -0.84 (El-Khoury JM, et al. Clin Biochem. Epub ahead of print July 21, 2016. doi:10.1016/j.clinbiochem.2016.07.009).

“That is a similar performance to cystatin C and much better than creatinine,” Dr. El-Khoury said. He said further research on SDMA should be done among a larger pool of healthy patients, one that is more diverse as 88 percent of the participants in this study were white and all were adults. He added that SDMA is influenced by age and, to some degree, gender. Those factors must be better understood and equations developed to account for them in generating an SDMA-based eGFR.

However, that is unlikely to happen if the test available for SDMA in humans is done using an LC-MS or ELISA method. While the sample-to-result run time for the LC-MS SDMA method he used in the lab is about 90 minutes, it must be run in batches, Dr. El-Khoury says.
“Today, the biggest reason why SDMA hasn’t been picked up—other than a lack of data, though there is a lot of convincing data—is the lack of an automated assay,” he says. “What is available now is highly specialized techniques like LC-MS or very manual techniques like ELISAs. [Idexx] is the first one to offer an automated platform that could run just like creatinine or cystatin C. That’s really a game changer. Once you can do this in high-volume laboratories, a lot more research can be done with it [SDMA].”

An automated SDMA immunoassay such as the Idexx test already used in millions of cats and dogs is essential “to bring this test to general clinical practice,” Dr. El-Khoury adds. “When you’re looking at screening for kidney function, you want something that can be done relatively quickly. And you’re not going to switch from a test like creatinine or cystatin C to a test that would require hours and very slow throughput to give you a result. Typically, these results are needed right away. People order these things when patients are waiting for radioactive dye or drug-dosing information. You can’t do that for a test that’s super slow.”

Dr. El-Khoury believes that a combination of cystatin C and SDMA may someday overtake creatinine in testing for chronic kidney disease.

“Hopefully, in the future SDMA will overtake creatinine, because creatinine is good but it does have known limitations,” he told the AACC crowd. “Let’s use the best biomarkers available and not have them impacted by one that has poorer performance.”

“Creatinine,” Dr. El-Khoury said, “is so 1950s.”

The defects of creatinine also was a recurring theme of the other portion of the AACC session, which focused on emerging biomarkers in acute kidney injury.

Nephrologist Chirag Parikh, MD, PhD, compared the slow progress made in understanding and treating acute kidney injury since the 1950s and the great strides made in treating acute myocardial infarction. In the ’50s, the principal way to diagnose a heart attack was white blood cell count. That moved to LDH in the ’60s, CPK in the ’70s, CK-MB in the ’80s, troponin T in the ’90s, and troponin I in the new millennium.

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