A Cohort Study Comes of Age

Over nearly two decades, a major national study of kidney disease led and coordinated at Penn has defined key risk factors in an all-too-common silent epidemic. And the way its scientists make discoveries has grown up along with the study itself.

By Steve Graff

Photos by Peggy Peterson

For Mark Paviglianiti, it started in 1962 when he was just six years old. While he lay in bed sick for weeks with a fever, doctors from his hometown of Lancaster, Pa., worked to figure out what was wrong with him. Eventually, they spotted high levels of protein in his urine—a surefire sign of trouble in the kidneys.

They sent him to the Children’s Hospital of Philadelphia, where the prominent pediatric kidney specialist Milton Rapoport, MD’31 put him on sulfa antibiotics to stabilize his kidney function, as well as prednisone. It worked. Three years later, he was off the medication and seemingly out of the woods, until his early 20s, when a kidney infection brought him back to a nephrologist. That infection, too, was fixed. Though Paviglianiti didn’t feel like he had a chronic disease, these incidents were the start of a years-long journey. He and his doctors would end up keeping a watchful eye on his kidneys and higher-than-normal protein levels over the next 35 years, battling other issues, like a mild heart condition and high blood pressure and cholesterol, along the way. 

Such is the life of a patient with chronic kidney disease (CKD).

 

While it’s rare for children to be diagnosed with the condition, the health problems Paviglianiti has faced are exceedingly common ones for adults with CKD. Patients’ kidneys—the nonstop workhorses that rid the body of waste—progressively fail over time. Today, this complicated and highly variable disease, further complicated by its comorbidities, afflicts nearly one in seven people in America, 90 percent of whom have no idea they have it because symptoms, like constant fatigue and vomiting, typically don’t appear until the last stage.

“Even as a young boy, my parents told me I had kidney disease, but nothing specific about it. Nor did they know how it happened,” said Paviglianiti, now 62. “And to this day, I don’t know what triggered it.”

Paviglianiti’s experience with CKD, in a way, mirrors one of the largest efforts to better understand it. As he progressed, so did a major epidemiological study that has been running for nearly two decades.

In what’s known as the Chronic Renal Insufficiency Cohort (CRIC, pronounced crick) study, more than 100 researchers from the Perelman School of Medicine and other institutions have discovered new insights into the drivers of CKD and how it impacts the body’s systems by closely following Paviglianiti and thousands of other patients. Almost 20 years ago, researchers couldn’t have predicted the paths CRIC would take, nor what questions they’d get to ask or even the methods they’d use to answer them. And today, in what would have probably seemed like science fiction back then, researchers are turning to a cutting-edge discipline and its smart technologies to keep it growing.

Birth of a Study

In the 1990s, before CRIC launched, many clinicians thought of kidney disease as “a binary thing, where it’s like you jump off a building and nothing is happening until you hit the ground,” said Harold I. Feldman, MD, MSCE, chair of Biostatistics, Epidemiology and Informatics in the Perelman School of Medicine, principal investigator of CRIC’s Scientific and Data Coordinating Center and the study’s national chair. “Like the rest of our community, I was awakened by the research that others were doing. I came to understand how important it was to think of CKD as a progressive erosion of health.”

The binary mindset had left a whole terrain of health consequences unexplored.

In the clinic, most patients fell into two categories: normal kidney function or at risk for failure. If physicians spotted a problem, typically with the urine test showing too much protein, known as proteinuria, it was diagnosed as “pre-end-stage renal disease” or, worse, “chronic renal failure,” a diagnosis that made it seem the kidneys had failed already. Clinicians would attempt to lower blood pressure and better manage patients’ diabetes, two well-known drivers of CKD, but that’s only if those problems existed. The cause of many cases was unknown. Eventually, sick patients on the edge of kidney failure—which wasn’t uncommon—prepared for dialysis or, if they were lucky, received a transplant.

During this time, CKD rates in the United States had jumped significantly. From 1990 to 2001, the number of patients more than doubled, with about 19 million Americans suffering from CKD in 2001, the year that CRIC began, according to the Centers for Disease Control and Prevention. A small percentage of cases were diagnosed in children, as Paviglianiti’s had been.

“Before CRIC, there was virtually no epidemiology data on the long-term consequences of kidney failure, nor was there much interest in understanding mechanisms by which kidney failure progresses, which could in turn lead to potential intervention,” said Raymond Townsend, MD, the lead CRIC clinical center investigator at Penn, and professor of Medicine in the Division of Renal-Electrolyte and Hypertension, who has now enrolled more than 700 patients in the study. “All physicians knew at CRIC’s start was that high blood pressure, the presence of diabetes, and protein excretion in the urine were risk indicators for CKD and its progression,” he said.

It was only as recent as the late 1990s when “kidney disease” came to be recognized as “chronic kidney disease,” a condition whose progressive nature was poorly understood.

In 1999, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) convened a series of workshops on the issues, and Feldman attended. A decision was ultimately made at the behest Josephine Briggs, MD, then the director of NIDDK’s Division of Kidney, Urologic, and Hematologic Diseases, to put out a request for grant applications and start a kidney disease counterpart to the Framingham Heart Study, which famously tracked cardiovascular health in thousands of volunteers from an old factory town outside Boston for decades, beginning in 1949. The study, which is still ongoing, is credited with saving millions of lives and introducing the words “risk factor” into the lexicon, in addition to implicating cigarette smoking and high cholesterol in heart disease, among other findings that emerged over time.

Penn was chosen to lead this new venture, along with six other clinical sites, with initial funding of $40 million for five years. Feldman would serve as principal investigator of the Scientific and Data Coordinating Center (see sidebar), along with J. Richard Landis, PhD, a professor of Biostatistics at Penn. CRIC’s charge: to better understand traditional risk factors, like hypertension, and search for previously unknown factors for CKD progression and cardiovascular diseases, with an eye toward interventional trials to slow or stop it. Like the Framingham study, CRIC would recruit thousands of adult patients at different stages of CKD and follow them over time to find patterns: What was the relationship between cardiovascular diseases and CKD? What else may be driving progression? Were there any genetic mutations or biomarkers tied to CKD progression?

New questions—and answers—branched out from there, as meaningful associations and newer research techniques surfaced.

First Steps

In the study’s first phase, CRIC researchers recruited a diverse group of 3,600 people from around the country, roughly 45 percent white, 46 percent black, and the rest Hispanic. Half of the patients had diabetes. Fifty-four percent were men.

Paviglianiti was among the first wave to enter into the study at Penn in 2003—the 43^rd patient, to be precise. When he was in his late 30s, a routine check-up revealed high blood pressure and high levels of protein, so he took himself back to a Penn nephrologist, Robert Grossman, MD. A few years later, Grossman, then a CRIC investigator, suggested he join the study, given his overall good health and the state of his condition. CKD is categorized into five stages, and at the time Paviglianiti enrolled, he was stage 2.

During that first eight-hour study visit, the clinical research team took his blood pressure on all four limbs, along with his height, weight, and waist size. They performed an electrocardiogram, or EKG, to gauge his heart health. They took his medical history and did mental tests. They extracted blood. Asked him to pee in a cup. Clipped his finger and toe nails. This happened, give or take a few tests, every December, along with a phone call every six months.

“Any way you look at, I felt like it was a great opportunity to not only learn more about myself but also for the scientific community to learn more about the disease through me,” Paviglianiti said. “Maybe the findings would help progress the science and help some other people down the road.”

It wasn’t long before analyses of these patients and their medical histories, using traditional statistical approaches, began revealing meaningful characteristics, or phenotypes. Meanwhile, just as CRIC began, the National Kidney Foundation published new guidelines to help physicians recognize and better define the stages of CKD, as well as other outcomes of interest beyond its progression.

On one front, CRIC confirmed what had been known: CKD disproportionately affected patients with a lower socioeconomic status and of color, and CKD patients were more likely to develop or die of CVD. But its prospective nature also helped see those events more clearly, even after only five or six years of follow-up data.

“Going into the study, the expectation was that the cardiovascular outcomes participants would most likely experience would be atherosclerotic ones, like myocardial infarctions and strokes,” said Amanda Anderson, PhD, an associate professor of Epidemiology at Tulane University and long-time CRIC investigator, who formerly served on the faculty at Penn. “It was really striking to all of us when we started adjudicating all the hospitalizations and found that it was heart failure occurring at a much higher rate.”

Knowing who is at high risk and for which diseases can help determine which patients may need preventive therapy.

Patients, regardless of their diabetic status, were also at a higher risk of peripheral arterial disease and stroke, CRIC researchers reported in studies in the American Journal of Nephrology and American Journal of Kidney Diseases.

For Paviglianiti, the realities of CVD risks began to crystalize four years into the study. An EKG test from one of his clinical visits revealed a mild condition called Mobitz 1, where the heart regularly skips a beat, which led him to Frank E. Silvestry, MD’90, an associate professor of Medicine in the Division of Cardiovascular Medicine at Penn. Silvestry discovered that Paviglianiti had high cholesterol, too, a known risk factor for heart problems, so he put him on a statin, which stabilized his levels.

“I became more aware of the possible connection to heart-related illnesses and failure because of the CRIC study,” said Paviglianiti, who was also on angiotensin-converting enzyme, or ACE, inhibitors, to control his high blood pressure. “And from that, I have maintained a patient relationship with Dr. Silvestry, to closely monitor my heart health and any potential disease progression.”

Follow-up data from the cohort also told researchers that patients with a lower estimated glomerular filtration rate, or eGFR, were more likely to suffer CVD events, and that the eGFR could predict their risk of having one. The equation used to calculate eGFR, as opposed to just considering the levels of the waste product creatinine, is now commonly used in clinics to screen and stage patients, thanks in part to data from CRIC and other studies.

Longer-term data also offered up more precise information about blood pressure’s effect on patients. CRIC Study researchers, reporting in the Annals of Internal Medicine, found that blood pressure above 130/80 was associated with progression of the disease, a lower figure than what was previously believed to drive it. This new target is appreciated more today in clinical settings, too.

“This is a space where observational outcomes, like from CRIC, in conjunction with other data, sometimes push practitioners across some treatment decision threshold,” Feldman said.

ACE inhibitors helped get Paviglianiti’s blood pressure down, which lowered his risk for future heart problems. Still, it wouldn’t stop the progression of his CKD.

Growth Spurts

During CRIC’s initial phase, 300 more Hispanic participants were brought in through a supplementary grant, bringing the total to 3,900. CRIC was subsequently renewed in 2008 and 2013 for its next two phases during which another 1,850 participants would eventually be added, including more than 200 additional participants at Penn and 339 more Hispanic participants recruited in Chicago.

And so, as CRIC continued to grow, every December, year after year, Paviglianiti returned to Penn, providing samples of blood and urine during a three-hour visit. Thousands of others at sites across the country did the same. Clinical teams would pack up their samples daily, and ship them to Penn, where they were carefully checked in, barcoded, and filed away in racks within biobank “freezer farms.” Researchers stopped collection of finger and toe nails after the first phase of the study.

At the same time, Penn shipped thousands of samples to NIDDK’s National Repository for researchers working on the parent study, as well as ancillary studies, to access. These side studies, led by CRIC and non-CRIC investigators, were funded by both the NIH and other sources.

“I think it is fair to say that the CRIC study is the best-funded study, with respect to ancillary studies, at the NIDDK by far, and that is a tribute to Harv [Feldman], the principal investigators, and many other people who were not involved as investigators but came on board through our outreach,” said John Kusek, PhD, a former NIDDK project scientist for CRIC who retired in 2017. “That’s a very unique and robust success story for the CRIC team.”

The wide array of ancillary studies conducted across the country are among CRIC’s biggest strengths, adding tens of millions of dollars to the parent project, which has received roughly $8 million a year from the NIH. The ancillary studies are where CRIC really expanded its footprint and contributions to the field.

Work led by a Johns Hopkins University researcher showed that older patients were on a cognitive decline as their CKD progressed or went on dialysis. Another study out of Penn published in the American Journal of Nephrology found that CKD patients were more likely to have poor physical performance and became frailer as their disease progressed. In another series of studies, Juan E. Grunwald, MD, a professor of Ophthalmology at Penn, using non-invasive, photographic tests, found that patients with damage to the retinal vasculature, known as retinopathy, had a greater chance of developing end-stage renal disease and CVD.

Cognitive decline, frailty, and retinopathy—each of these co-morbidities not only highlights the burden of disease patients face, but each also can also be used to gauge the severity of CKD in patients, if clinicians know to screen for them early on. The outcomes, for the most part, were unrecognized by the nephrology community before.

Another approach came from Townsend early on in CRIC. Townsend believed stiffness in large arteries was involved in the connections between high blood pressure and diabetes and some cases of CKD and related CVD. He began gathering preliminary data on arterial stiffness by measuring with technology called pulse wave velocity; that eventually led to a 10-year NIH grant. The data, ultimately published in 2018 articles in the American Journal of Kidney Diseases and in Hypertension, showed he was right. He found that stiffness in the large artery is a potent predictor of kidney disease progression, death, and cardiovascular complications. However, the work never made it past that initial phase nor into any interventional trials, much to Townsend’s chagrin. “Meanwhile, the Europeans remain ahead of us in that area,” he said. “And Asian countries incorporate arterial stiffness into clinical practice. The rest of the world has gotten on board; we’re just a little behind here in the states.”

Next Sequence

At the end of CRIC’s second phase and throughout its third phase, now with 10 years’ worth of data, investigators began in earnest to utilize more of the stored specimens. That’s blood and urine, and other samples provided by thousands of participants like Paviglianiti, year after year, for over a decade. These precious commodities were plucked out, analyzed, measured, and scanned dozens of times by industrious researchers, many at Penn, looking for markers that may be driving the disease.

And as CRIC has grown up alongside the advent and broadening accessibility of new genetic approaches in the last two decades, researchers have found genes of interest in CKD with an evolving set of methods—from basic genotyping, like family-pedigree studies; to DNA microarray and genome-wide association studies, or GWAS; to epigenetic approaches.

In 2011, what CRIC researchers hail as one of the larger clinical contributions from the study was published in JAMA. From a mineral metabolic marker in plasma, researchers identified a risk factor for end-stage renal disease in patients with relatively preserved kidney function, and for death in patients at all stages of CKD—it’s called FGF-23, or phosphate-regulating hormone fibroblast growth factor 23. But, as with the discovery of arterial stiffness as a risk factor, this finding has not yet moved forward to potential interventions for CKD. Antibody drugs that target FGF-23 exist, and there are ongoing trials in other diseases pursuing the hypothesis that intervening on this marker will ultimately lead to clinical benefit. Thus far, nothing has been put into motion to study it in CKD patients.

A seminal CRIC study published in the New England Journal of Medicine in 2013 found gene variants called APOL1 that could help explain the striking racial disparity in CKD progression. More recent epigenetic studies include an ongoing project led by Katalin Susztak, MD, PhD, a professor of Medicine in the Division of Renal-Electrolyte and Hypertension at Penn. Preliminary data from a study that incorporated both genetic and epigenetic analyses identified novel genes that may be drivers of CKD in diabetics.

The vast span of genetic and epigenetic discovery underscores another strength of CRIC: It’s the repositories at both the NIDDK and Penn—which now have over one million samples split between labs in the Smilow Center for Translational Research and Penn Presbyterian—that make these and future analyses even possible.

“I think CRIC is best characterized as a research platform,” said Anderson, who has co-authored several biomarker papers. “At the beginning of the study when it was being planned and conceptualized, we had no idea that FGF-23 was something that we even wanted to measure. No one knew about APOL1. So just building the capacity for these investigations with the longitudinal follow-up is just an incredible asset of the study. It is really the hallmark of the study that we have stored specimens and the ability to go back to test novel markers.”

New Age

When the clinical research team approached Paviglianiti about remaining enrolled as one of the 3,000 patients who CRIC continues to follow for its new, fourth phase, he said yes without hesitation, just like he did every single phase before it.

The new phase was funded by a recent award of $40 million by NIDDK, including $17 million to Penn, extending it another five years; it will have been running for 22 years when this phase ends.

“I’m hoping they see the trends, so they can piece all this together and even be able to figure out where the trajectory of this goes,” said Paviglianiti, whose disease advanced to stage 4 briefly last year, but is back at stage 3 and being successfully managed today. His initial enrollment in 2003, and his continued enrollment in the newest phase, means he expects to be a CRIC participant for at least 20 years, nearly a third of his life—and longer might be possible if it is renewed yet again.

For this next phase, CRIC investigators will need more frequent data on top of the data they’re already capturing. To do that, its data collection will go mobile. Patients like Paviglianiti will be equipped with small persistent-monitoring devices that strap onto the chest. The device measures physical activity and physiological parameters and generates heart beat data—which can tell researchers over time who may be at highest risk of cardiovascular and other complications.

A second device will allow patients to regularly use finger-stick blood samples to measure their own creatinine so the team can track kidney function over time. That will be in conjunction with monitoring other stressors, like illnesses, medications, or physical activity, for example, that researchers suspect play a cumulative role in kidney function decline. The biggest advantage is the amount of data. Blood and urine collection will go from once—those annual visits Paviglianiti has made each December, for the last 16 years and counting—to at-home collection over 20 times a year. All of these data will then be downloaded and fed to the team.

“Integrating the data is a large challenge, but it is also an opportunity for what I will call global discovery,” Landis said. “We are going to have more frequent and complex detailed measures to add in to what we have done so far…Having it all in the same study allows us to pursue questions we couldn’t before.”

Despite advances in understanding the progression of CKD and several promising discoveries that pointed to interventions, no new therapies to halt the disease have surfaced. The reality is, there is a lot that researchers still need to learn about this highly variable disease. Another hurdle is a lack of funding. Compared to other diseases, overall, CKD research dollars rank fairly low, despite taking more lives than breast or prostate cancer.

To move forward, the CRIC study is using its burgeoning set of data and bringing a new discipline—biomedical informatics—and some of its most sophisticated “big data” approaches into the fold. Many of the same questions about the progression of CKD and CVD apply, but how they’re tackling them will look different. Artificial intelligence and machine learning tools will soon be used to see patterns in a sea of new and old data that traditional approaches can’t identify alone.

“I think the reason why informatics is an important part of this is because, while traditional statistical analyses are very important, and still maintain pride of place here, new machine learning methods can help inform those analyses and lead people to certain directions that they might not have thought of,” said John H. Holmes, PhD, of Penn’s Institute of Biomedical Informatics (IBI), who is leading the informatics piece of CRIC’s latest phase.

As an example of these machine learning methods, imagine a 3D image on a screen. There are several thousand dots clustered together according to different colors and intensities. Some of those clusters are connected by various lines, each, too, with their own different colors and intensities. That visual may sound a bit chaotic, but to informaticians like Holmes, it’s a byproduct of a sophisticated method sorting terabytes to tell a story—or many stories. Approaches like this, called a topological analysis, can reveal commonalities among and between patients by using algorithms that enable researchers to recognize a vast number of data points and then learn what to do with them as they come in.

CRIC researchers will use the tools, for the first time, to not only find unseen traits, but also predict future health states of patients.

“We’ll essentially be mapping the progression of a disease over time—and that mapping has a certain architecture to it, a certain design,” Holmes said. “We can actually get a good sense of, ‘Oh, this person over here is probably going to deteriorate faster than this person over here. Let’s see why.’ And the reason why might be in the link between the two people.”

One cluster, for example, could represent CKD patients of a certain age or stage, with a bright line connecting to other clusters of patients with heart failure or, say, on a certain drug during a period of time. Those connections may represent telling relationships or patterns about subgroups and risks, and why certain patients are progressing.

“We now have the ability to leverage this [persistent] monitoring, where we literally paste a device onto someone’s chest for multiple days, and it generates terabytes of information,” Feldman said. “And then to use informatics to extract from those data meaningful patterns that we can analyze statistically. That is something that we just didn’t even know about back in 2000, when the study was first designed.”

They didn’t know all of the methods—nor all of the questions that the CRIC core teams, and ancillary study teams across the country would come to ask. But day after day, week after week, samples still arrive at the Smilow Center. They are scanned, barcoded, and carefully stored, alongside a million plus samples that arrived before them. And so CRIC continues to grow and await new methods and new questions yet to be imagined.

Read more about CRIC and the Penn Medicine staff behind it at the Penn Medicine News Blog.