Mastery in a Micro World

Mastery in a Micro World

Renal physiologist Pablo Ortiz, PhD, is obsessed with studying the molecular mechanism of salt-sensitive hypertension.

By Melanie Padgett Powers

As Pablo Ortiz, PhD, was about to graduate from college, he spotted a newspaper advertisement for a research lab assistant. The ad was unusual because Ortiz is from Argentina, yet the ad was in English. But Ortiz was fluent in English, thanks to his year as a high school exchange student in Pretoria, South Africa. So, he applied for the position.

Little did he know that job ad would set him on a course to become, not a clinical biochemist as planned, but a renal physiologist. In fact, Ortiz would become so proficient at a specific technique that he is now one of only about 30 scientists in the world who have mastered it.

Ortiz is now division head and Earl Ward Endowed Chair of Hypertension in the Hypertension and Vascular Research Division at the Henry Ford Hospital and an associate professor at Wayne State University in Detroit. He arrived in Detroit in 1999, and the city has been his home ever since. He has been running his own lab for 15 years.

When Ortiz was growing up in Argentina, his father was an endocrinologist, but he also had to make house calls as a family doctor to make ends meet. Sometimes, his young son tagged along, which piqued the boy’s interest in medical sciences. When it was time for college, Ortiz enrolled in a dual bachelor-master’s program in biochemistry at the National University of Córdoba in Argentina.

Garvin’s lab was “the first exposure for me to how difficult it was to study renal physiology and also how complex the work was. I got hooked when I came here and I had to learn all the basic aspects of transport physiology.”

Pablo Ortiz, PhD

That job advertisement Ortiz responded to was from Nestor Garcia, MD, PhD, an Argentinian nephrologist who had trained at Henry Ford Hospital in Detroit and the Mayo Clinic in Rochester, Minnesota. He had returned to Argentina to set up a kidney tubule perfusion research lab. Ortiz got the job and began to learn to dissect and microperfuse kidney tubules. Garcia told him that this would make him stand out as he sought lab positions in the U.S.

After working with Garcia for a year, Ortiz was offered a position in the lab of Jeffrey L. Garvin, PhD, then at Henry Ford Hospital in Detroit. Ortiz enrolled in a PhD program through the University of Buenos Aires that allowed him to do his coursework and dissertation outside the country.

Garvin’s lab was “the first exposure for me to how difficult it was to study renal physiology and also how complex the work was,” Ortiz says. “I got hooked when I came here and I had to learn all the basic aspects of transport physiology.”

Notoriously difficult technique

Tubule perfusion is the gold standard for measuring renal tubular ion transport in the various nephron segments. But the technique is notoriously difficult to learn, and the success rate at the beginning is often very low. It can take up to two years to pick up, with multiple steps in which the trainee can fail and may never get past, never mastering the technique at all.

Most trainees don’t want to spend 18–24 months working on a low-productivity skill they may never truly learn in the end, Ortiz says. It’s tedious and takes tremendous patience.

“You learn your limitations very fast,” he says. “It’s a specific set of people that can do it. You’re fighting a specific mindset. I’ve tried to train other people, and you have to be able to have someone that is going to fight with the method every day for a year until they realize, ‘Yes, I’ve got an experiment.’”

So, why is it so difficult? First, you have to learn to make forceps for microdissection. The tubules, with a diameter of 20–25 microns and a length of about one millimeter, are difficult to identify. There are 15,000 nephrons in each mouse or rat kidney, so when you cut a slice of a kidney and put it in the dissecting scope, you have to learn to identify the nephron segment. Then, it takes long days and many months as you learn to pick up a specific tubule segment that you want to microdissect, one that is not damaged and can be perfused.

At the same time, you’re learning how to make glass micropipettes, for which there is no automated method. “For example,” Ortiz says, “for patch clamping there’s an automated puller machine to make a patch pipette. You can’t do that with a glass pipette for microperfusion, so you have to manually make them with a rotary puller and heat, and you shape and polish the pipette.” It can take up to four months just to learn how to make the pipettes so that you have a usable set.

The micropipette size and setup will depend on what you plan to measure, such as chloride, sodium or water absorption. In a collection pipette, the tiny amount of fluid may only be in the range of 10–15 nanoliters, which you use to measure ion concentration.

On top of all that, tubules die easily, so you have less than 30 minutes to select and microdissect a good tubule. Only after you learn all those steps, do you move on to the microscope. You use one specific pipette to transfer the dissected tubule so that you can actually perfuse it through the lumen with a micropipette of about five to eight microns in diameter.

He encourages students, after they’ve learned basic physiology, to find and zero in on conditions in which a specific area of physiology isn’t working, to study the mechanisms of that disease.

Ortiz toiled away in this micro world by himself, without doing actual experiments, for a year. Even after he celebrated learning the technique, it took multiple experiments before he mastered tubule perfusion. In fact, it was another three years of everyday work before he regularly achieved an 80–90% success rate. Because of all of that, he says, there are only about seven labs in the U.S. and maybe four outside the country that currently do tubule perfusion. But his is one of them.

The kidney’s role in hypertension

Ortiz’ lab research focuses on the ion transporter NKCC2, which is found only in the kidney, specifically in the thick ascending limb. It is essential in sodium and chloride reabsorption and for maintaining water balance. The medication furosemide, which inhibits NKCC2, is given to patients to decrease edema, or fluid buildup.

Furosemide was also sometimes given for the treatment of hypertension, but it is too potent as a diuretic. Ortiz believes that NKCC2 overactivity is generally involved in hypertension. “A lot of previous data that has been generated suggest that the transporter activity is increased in rat models of hypertension,” he says. “Instead of starting to study how to inhibit it better, we started studying why its activity is enhanced in hypertension. In reality, I became obsessed with trying to understand the molecular mechanism of salt-sensitive hypertension.”

When COVID-19 hit the U.S. in spring 2020, Ortiz’s lab, like so many, was forced to shut down and lost several of its animal models. His team quickly pivoted and began working with the translational research center and the infectious diseases and nephrology groups at Henry Ford Hospital to set up a COVID-19 biobank. Ortiz remembers the first time, early in the pandemic, that he heard that the so-called respiratory disease also attacked the kidneys: New York City nephrologists were reporting on Twitter that they had several COVID-19 patients with acute kidney injury (AKI). Doctors in Detroit started noticing the same thing.

“The truth is that every physician became a physiologist because they were trying to figure out what was going on,” Ortiz says. “At the beginning, it was just a respiratory syndrome, but … they realized that they were getting higher rates of secondary disease, including AKI, at a much higher rate than other respiratory syndromes.”

It’s still debatable whether SARS-CoV-2 directly attacks the kidney, but Ortiz believes it does in some cases. “Our data supports it in severe AKI patients, and there are a number of publications that support this, but there are also a number of studies where they have found nothing when they look, with different methods, in the kidney.”

Remaining excited by physiology

Ortiz’s lab has begun its transition back to researching the mechanisms of hypertension and kidney function. As division head, he is also overseeing the challenge of getting all the labs back to full capacity, restarting animal colonies and writing grant submissions. And while there’s not much time for teaching, he fits it in where he can. This fall he taught two graduate courses, advanced renal physiology and advanced cardiovascular physiology at Wayne State.

After students have learned basic physiology, he encourages them to find and zero in on conditions in which a specific area of physiology isn’t working to study the mechanisms of that disease.

“What is happening now is the more we have better tools, the more we discover the level of complexity and integration between organs, and it is mind-boggling,” he says. “You’ll never be able to take physiology out of the medical curriculum. If you do, you stop understanding or teaching how all of the organs are integrated.”

Ortiz tries to reserve Fridays as research days. “My best days are the days that I get to sit down and study and I find something new that I can apply or discuss with a postdoc or a graduate student about how we can incorporate that into our research,” he says. “I like to tinker, so even though I have a heavy administrative load and I have five people in the lab, I try to do experiments once a week, mostly develop new methods. That excites me and keeps me connected with how difficult it is to develop something new.”  

This article was originally published in the January 2022 issue of The Physiologist Magazine.