A team of Mayo Clinic pathologists have discovered a new tissue biomarker, DNAJB9, for fibrillary glomerulonephritis (FGN), a rare kidney disease of unknown pathogenesis and poor outlook—nearly half of all patients end up on dialysis within four years of diagnosis. DNAJB9 is the first glomerulonephritis biomarker discovered by laser microdissection/mass spectrometry, as well as the first biomarker for FGN. This pathogenic protein had remained elusive since the disease was first described in 1977. “The diagnosis of FGN has always relied on using histological, histochemical, and clinical features that overlap with other glomerular diseases,” says Surendra Dasari, Ph.D., a bioinformatician in Mayo’s Department of Health Sciences Research, who co-authored the study paper (published in Kidney International Reports).

“The discovery of DNAJB9 opens doors for more specific diagnostics, which could lead to more targeted therapies. It also allows for the simplification of FGN diagnostic algorithms,” says Dr. Dasari.

According to Mariam Priya Alexander, M.D., a renal pathologist in Mayo’s Department of Laboratory Medicine and Pathology (DLMP), who also co-authored the paper, the DNAJB9 breakthrough was a direct result of the team’s interest in evaluating a series of cases best described as Congophilic fibrillary glomerulonephritis. “On studying the proteomic profile of this cohort by mass spectrometry and comparing it to the proteomic profile of cases of amyloidosis (a similar disease to FGN in which abnormal protein fibrils build up in tissue), and several other glomerular diseases, we found that DNAJB9 was unique to fibrillary glomerulonephritis, regardless of affinity to Congo red,” says Dr. Alexander. “We were quick to then evaluate the sensitivity and specificity of immunohistochemistry (IHC) of DNAJB9 in the diagnosis of this disease.” The resulting new DNAJB9 IHC test—which went live this past September—is highly sensitive (98%) and specific (99.7%) to FGN.

Difficulties of Diagnosis FGN forms nanometer-sized fibrils in glomeruli, which are the main filtration system in the kidney. Over time, these fibrils accumulate in the glomeruli and cause damage to kidney function as a blood filtration system. “Patients present with proteinuria, meaning the kidneys are leaking protein,” says Samih Nasr, M.D., a renal pathologist in Mayo’s DLMP and co-author of the study papers. “And these patients develop hypertension, and hematuria, where the kidneys are leaking blood. They also sometimes develop swelling from excess fluid in the body.” After a series of tests (including urinalysis, which detects the blood and protein) and serology testing, a nephrologist usually finds that the patient has renal insufficiency, or renal failure—when the kidney is no longer getting rid of waste in the body.

“This particular disease has no effective therapy, and most patients will progress to kidney failure and need dialysis,” says Dr. Nasr.

“Some of them might be lucky enough to receive a kidney transplant. But in one-third of those cases, the disease returns and can cause the loss of the new kidney.” Since renal insufficiency or failure are nonspecific symptoms, accurate diagnosis of FGN has, hitherto, required a rather complicated testing process, which demands a specialized, cumbersome technology. Simplicity of Immunohistochemistry versus Electron Microscopy “When a renal biopsy is obtained for kidney dysfunction, FGN fibrils can’t be seen using a traditional light microscope because the fibrils are too small,” says Dr. Dasari. “Until now, electron microscopy (EM) was needed to diagnose FGN because it can probe ultra-small structures like these fibrils.” However, even with EM technology, FGN can be difficult to distinguish from other glomerular diseases that exhibit fibrils, including amyloidosis. “The problem is that electron microscopy isn’t available in most pathology laboratories, especially in developing countries, because it’s labor-intensive and very costly for hospitals to maintain,” says Dr. Nasr. “It’s also costly to the patient and insurance companies. And in most laboratories, it takes several days to obtain the electron microscopy results, versus one day for our new IHC test.” Compared to EM, the IHC test is much more user-friendly and inexpensive. It can even be performed by hand, if necessary. And since glomerular deposition of DNAJB9 is specific to FGN, the IHC test can effectively replace the EM requirement for diagnosing the disease. “Any small laboratory in the world can develop this simple immunohistochemical stain,” says Dr. Nasr. “Or they can just send one paraffin section slide to Mayo Medical Laboratories (the reference laboratory of Mayo Clinic), for example, and we can make the diagnosis for this disease.” The Mass Spectrometry Edge

Thanks to their discovery of DNAJB9, the members of this Mayo team also developed a new mass spectrometry (MS) assay, which can distinguish FGN from amyloidosis and other glomerular diseases. “We did mass spectrometry on a large number of cases of FGN and on many different glomerular diseases that are in the differential diagnosis of FGN,” says Paul Kurtin, M.D., a hematopathologist who directs the Clinical Tissue Proteomics Laboratory (CTPL), a premiere mass spectrometry facility at Mayo. “DNAJB9 was detected in all the FGN cases—but in none of the samples from the other glomerular diseases. So, we not only discovered, but also confirmed, using mass spectrometry, that the presence of DNAJB9 in glomeruli has high diagnostic sensitivity and specificity for FGN.” This formed the basis for a new MS test for FGN. Because the method for detecting DNAJB9 by MS is virtually identical to that for typing amyloid in paraffin-embedded tissue, this test is particularly useful for cases where FGN is difficult to distinguish from amyloidosis. “The advantage of mass spectrometry over the DNAJB9 immunohistochemical stain is that, if the case turned out to be amyloidosis and not FGN, then MS can determine the type of amyloidosis with high degree of certainty,” says Dr. Nasr. “This is more applicable to the subset of cases of FGN that are Congo red-positive.” The entire diagnostic process can be accomplished in a single mass spectrometry assay. But then, using MS to characterize proteins in this way is routine at Mayo—for example, CTPL has analyzed more than 15,000 cases of amyloidosis involving many anatomic sites, including the kidney. An Under-Diagnosed Disease The team’s work showed that the DNAJB9 IHC and MS tests are superior to existing methods for diagnosing FGN. Hence, incorporation of this biomarker into clinical practice will now allow more rapid and accurate diagnosis of this disease—especially in developing countries that don’t have electronic microscopy. This is profound when one considers that FGN has probably gone under-diagnosed—many patients have the disease, but they and their treating physicians don’t know it. Or, patients are misdiagnosed with some other renal disease. “FGN is, for sure, an under-diagnosed disease in developing countries, where electron microscopy is not routinely done in the practice of renal pathology,” says Dr. Nasr.

Dr. Alexander adds, “We believe DNAJB9 immunohistochemistry will be a valuable and cost-effective test, which means we can now expect more cases of fibrillary glomerulonephritis to be diagnosed from all corners of our globe.”

Hope on the Horizon for More Targeted Treatments Currently, most patients with this disease are nonspecifically treated with immunosuppressive therapy with very little success. This is because immunosuppressive therapies don’t target DNAJB9, but they instead target the immune reaction to DNAJB9 fibrils. Such therapy can also mean harsh side effects for patients. The discovery of DNAJB9 in FGN implies that, in the next few years, more targeted therapies might be developed that could directly eliminate or reduce the deposition of DNAJB9 in FGN glomeruli. This could lead to better outcomes for patients. “We know that there are tons of this protein present in the glomeruli of these patients,” says Dr. Nasr. “And if someone could develop a therapy that dissolved this protein from the glomeruli, that would be a magic therapy for this disease. But at this point, this is speculation. What we know now is that DNAJB9 is the magic biomarker for this disease.” Both the mass spectrometry and IHC tests for DNAJB9 are now available from Mayo Medical Laboratories as stand-alone tests for the diagnosis of FGN. This breakthrough would not have been possible without the expertise of Mayo’s tissue mass spectrometry laboratory and immunohistochemistry laboratory team (that includes Drs. Dasari and Kurtin; Julie Vrana, Ph.D.; Karen Rech, M.D.; and Jason Theis), working closely with the renal pathology expertise of Drs. Nasr and Alexander. “Mayo’s renal pathology group will be adding both these assays to their diagnostic algorithms in their consultation practice—and of course, they are available as stand-alone tests for institutions that already have strong nephropathology expertise,” says Dr. Kurtin. “These novel findings have the potential to revolutionize the diagnosis of FGN.”