urine Archives

PMA-based PCR amplifies DNA from only live bacteria in urine

February 1, 2022

The question of why urinary tract infection (UTI) symptoms can persist in some patients who have been seemingly appropriately treated with antibiotics and have negative post-antibiotic urine cultures is one that urologists have long sought to answer.

Experts at Children’s National Hospital have successfully developed propidium monoazide (PMA)-based polymerase chain reaction (PCR) methods that amplify DNA from only live bacteria in urine. The study’s results, published in Frontiers, suggest that non-PMA bound DNA from live bacteria can be present in urine, even after antibiotic treatment.

PMA has been shown to differentiate between non-viable and viable bacteria in various settings. However, its effectiveness in urine has not been previously studied.

The question of why urinary tract infection (UTI) symptoms can persist in some patients who have been seemingly appropriately treated with antibiotics and have negative post-antibiotic urine cultures is one that urologists have long sought to answer.

“One theory is that very low levels of bacteria that don’t show up on cultures may be the cause,” says Michael Hsieh, M.D., director of Transitional Urology at Children’s National and senior author of the study.

Although PCR has previously been used to try and confirm this theory, the use of this method has been criticized because PCR can amplify DNA from dead bacteria (which obviously don’t cause UTI).

The authors developed a PCR test that selectively detects DNA from live bacteria. In a preclinical setting, results show that with the use of antibiotics, cultures collected can be negative but urine can contain DNA from live bacteria, as detected using the PCR test.

“We think something similar can occur in patients and we show some data in the paper confirming the PCR test can work with patient samples,” Dr. Hsieh adds. “I’m excited that we might finally have an explanation as to why some patients have persistent UTI symptoms after antibiotic treatment.”

Namely, he adds, that these patients still have a UTI. This may lead to better follow-up management of patients with UTI. The next step is to confirm the theory in patients.

Directly measuring function in tiny hearts

January 8, 2020

The amount of blood the heart pumps in one minute can be directly measured safely in newborns by monitoring changes in blood velocity after injecting saline, indicates the first clinical study of direct cardiac output measurement in newborns.

The amount of blood that the heart pumps in one minute (cardiac output) can be directly measured safely in newborns by monitoring changes in blood velocity after injecting saline, indicates a paper published online Dec. 17, 2019 in the Journal of Pediatrics and Neonatal Medicine. The research, conducted by Children’s National Hospital faculty, is believed to be the first clinical study of direct cardiac output measurement in newborns.

Right now, cardiac output is measured indirectly in the nation’s neonatal intensive care units (NICU) using newborns’ blood pressure, heart rate, urine output and other indirect measures. However, these techniques can produce imprecise readings in children. And the field lacks a feasible “gold standard” to measure cardiac output in newborns.

The COstatus monitor already uses ultrasound dilution – the expected decrease in the velocity of blood when saline is injected, producing a dilution curve. A Children’s National research team used ultrasound dilution in their small pilot study to gauge the feasibility of directly measuring cardiac output in newborns.

“Infants who stand to benefit most from directly monitoring cardiac hemodynamics are often so sick they already have central venous access,” says Khodayar Rais-Bahrami, M.D., an attending neonatologist at Children’s National and the study’s senior author. “Using the COstatus monitor in these children would enable the clinical team to personalize care based on the newborn’s current hemodynamic status, while introducing minimal fluid during measurements,” Dr. Rais-Bahrami adds.

The COstatus Monitor uses an extracorporeal loop attached to arterial and venous lines to measure cardiac output using ultrasound dilution. The research team injected 1mL/kg of body temperature saline into the loop and performed up to two measurement sessions daily.

The research team recruited 12 newborns younger than 2 weeks old who already had central venous and arterial access. The venous line of the arteriovenous AV loop is connected to the umbilical venous catheter while the COstatus monitor’s arterial line is connected to the umbilical arterial catheter. During measurement sessions, two injections of solution are injected into the venous loop, allowing for two measures of cardiac output, cardiac index, active circulating volume index, central blood volume index and systemic vascular resistance index.

Infants enrolled in the pilot study underwent up to two measurement sessions per day for up to four days, for a total of 54 cardiac hemodynamic measurements. The newborns ranged from 720 to 3,740 grams in weight and 24 to 41.3 weeks in gestational age.

The infants’ mean cardiac output was 0.43L/min and increased with gestational age. By contrast, the mean cardiac index was 197mL/kg/min and changed little with infants’ increasing maturity – either by gestational age or postnatal age. Two of the study participants were undergoing therapeutic cooling for hypoxic-ischemic encephalopathy and had their measurements taken during cooling and after rewarming.

“Although this study size is small, it demonstrates that this minimally invasive technique can safely be used in newborns to directly measure cardiac hemodynamics,” says Simranjeet S. Sran, M.D., a Children’s National neonatalogist and the study’s lead author. “This technology may allow for more precise and personalized care of critically ill newborns in a range of disease states – real-world utility in NICUs that serve some of the youngest and sickest newborns,” Dr. Sran adds.

The research team notes that direct measurement by ultrasound dilution revealed a stark increase in cardiac index as infants undergoing therapeutic hypothermia were rewarmed, raising questions about whether indirect measures using other technology, such as echocardiography, underestimate hypothermia’s effect on hemodynamics.

In addition to Drs. Rais-Bahrami and Sran, Mariam Said, M.D., also a Children’s National neonatalogist, was a study co-author.

Decoding cellular signals linked to hypospadias

April 23, 2019

“By advancing our understanding of the genetic causes and the anatomic differences among patients, the real goal of this research is to generate knowledge that will allow us to take better care of children with hypospadias,” Daniel Casella, M.D. says.

Daniel Casella, M.D., a urologist at Children’s National, was honored with an AUA Mid-Atlantic Section William D. Steers, M.D. Award, which provides two years of dedicated research funding that he will use to better understand the genetic causes for hypospadias.

With over 7,000 new cases a year in the U.S., hypospadias is a common birth defect that occurs when the urethra, the tube that transports urine out of the body, does not form completely in males.

Dr. Casella has identified a unique subset of cells in the developing urethra that have stopped dividing but remain metabolically active and are thought to represent a novel signaling center. He likens them to doing the work of a construction foreman. “If you’re constructing a building, you need to make sure that everyone follows the blueprints. We believe that these developmentally senescent cells are sending important signals that define how the urethra is formed,” he says.

His project also will help to standardize the characterization of hypospadias. Hypospadias is classically associated with a downward bend to the penis, a urethra that does not extend to the head of the penis and incomplete formation of the foreskin. Still, there is significant variability among patients’ anatomy and to date, no standardized method for documenting hypospadias anatomy.

“Some surgeons take measurements in the operating room, but without a standardized classification system, there is no definitive way to compare measurements among providers or standardize diagnoses from measurements that every surgeon makes,” he adds. “What one surgeon may call ‘distal’ may be called ‘midshaft’ by another.” (With distal hypospadias, the urethra opening is near the penis head; with midshaft hypospadias, the urethra opening occurs along the penis shaft.)

“By advancing our understanding of the genetic causes and the anatomic differences among patients, the real goal of this research is to generate knowledge that will allow us to take better care of children with hypospadias,” he says.

Parents worry about lingering social stigma, since some boys with hypospadias are unable to urinate while standing, and in older children the condition can be associated with difficulties having sex. Surgical correction of hypospadias traditionally is performed when children are between 6 months to 1 year old.

When reviewing treatment options with family, “discussing the surgery and postoperative care is straight forward. The hard part of our discussion is not having good answers to questions about long-term outcomes,” he says.

Dr. Casella’s study hopes to build the framework to enable that basic research to be done.

“Say we wanted to do a study to see how patients are doing 15-20 years after their surgery. If we go to their charts now, often we can’t accurately describe their anatomy prior to surgery. By establishing uniform measurement baselines, we can accurately track long-term outcomes since we’ll know what condition that child started with and where they ended up,” he says.

Dr. Casella’s research project will be conducted at Children’s National under the mentorship of Eric Vilain, M.D., Ph.D., an international expert in sex and genitalia development; Dolores J. Lamb, Ph.D., HCLD, an established leader in urology based at Weill Cornell Medicine; and Marius George Linguraru, DPhil, MA, MSc, an expert in image processing and artificial intelligence.

NUP160 genetic mutation linked to steroid-resistant nephrotic syndrome

March 26, 2019

DNA strands on teal background

Mutations in the NUP160 gene, which encodes one protein component of the nuclear pore complex nucleoporin 160 kD, are implicated in steroid-resistant nephrotic syndrome, an international team reports March 25, 2019, in the Journal of the American Society of Nephrology. Mutations in this gene have not been associated with steroid-resistant nephrotic syndrome previously.

“Our findings indicate that NUP160 should be included in the gene panel used to diagnose steroid-resistant nephrotic syndrome to identify additional patients with homozygous or compound-heterozygous NUP160 mutations,” says Zhe Han, Ph.D., an associate professor in the Center for Genetic Medicine Research at Children’s National and the study’s senior author.

The kidneys filter blood and ferry waste out of the body via urine. Nephrotic syndrome is a kidney disease caused by disruption of the glomerular filtration barrier, permitting a significant amount of protein to leak into the urine. While some types of nephrotic syndrome can be treated with steroids, the form of the disease that is triggered by genetic mutations does not respond to steroids.

The patient covered in the JASN article had experienced persistently high levels of protein in the urine (proteinuria) from the time she was 7. By age 10, she was admitted to a Shanghai hospital and underwent her first renal biopsy, which showed some kidney damage. Three years later, she had a second renal biopsy showing more pronounced kidney disease. Treatment with the steroid prednisone; cyclophosphamide, a chemotherapy drug; and tripterygium wilfordii glycoside, a traditional therapy, all failed. By age 15, the girl’s condition had worsened and she had end stage renal disease, the last of five stages of chronic kidney disease.

An older brother and older sister had steroid-resistant nephrotic syndrome as well and both died from end stage kidney disease before reaching 17. When she was 16, the girl was able to receive a kidney transplant that saved her life.

Han learned about the family while presenting research findings in China. An attendee of his session said that he suspected an unknown mutation might be responsible for steroid-resistant nephrotic syndrome in this family, and he invited Han to work in collaboration to solve the genetic mystery.

By conducting whole exome sequencing of surviving family members, the research team found that the mother and father each carry one mutated copy of NUP160 and one good copy. Their children inherited one mutated copy from either parent, the variant E803K from the father and the variant R1173X, which causes truncated proteins, from the mother. The woman (now 29) did not have any mutations in genes known to be associated with steroid-resistant nephrotic syndrome.

Some 50 different genes that serve vital roles – including encoding components of the slit diaphragm, actin cytoskeleton proteins and nucleoporins, building blocks of the nuclear pore complex – can trigger steroid-resistant nephrotic syndrome when mutated.

With dozens of possible suspects, they narrowed the list to six variant genes by analyzing minor allele frequency, mutation type, clinical characteristics and other factors.

The NUP160 gene is highly conserved from flies to humans. To prove that NUP160 was the true culprit, Dr. Han’s group silenced the Nup160 gene in nephrocytes, the filtration kidney cells in flies. Nephrocytes share molecular, cellular, structural and functional similarities with human podocytes. Without Nup160, nephrocytes had reduced nuclear volume, nuclear pore complex components were dispersed and nuclear lamin localization was irregular. Adult flies with silenced Nup160 lacked nephrocytes entirely and lived dramatically shorter lifespans.

Significantly, the dramatic structural and functional defects caused by silencing of fly Nup160 gene in nephrocytes could be completely rescued by expressing the wild-type human NUP160 gene, but not by expressing the human NUP160 gene carrying the E803K or R1173X mutation identified from the girl’s family.

“This study identified new genetic mutations that could lead to steroid-resistant nephrotic syndrome,” Han notes. “In addition, it demonstrates a highly efficient Drosophila-based disease variant functional study system. We call it the ‘Gene Replacement’ system since it replaces a fly gene with a human gene. By comparing the function of the wild-type human gene versus mutant alleles from patients, we could determine exactly how a specific mutation affects the function of a human gene in the context of relevant tissues or cell types. Because of the low cost and high efficiency of the Drosophila system, we can quickly provide much-needed functional data for novel disease-causing genetic variants using this approach.”

In addition to Han, Children’s co-authors include Co-Lead Author Feng Zhao, Co-Lead Author Jun-yi Zhu, Adam Richman, Yulong Fu and Wen Huang, all of the Center for Genetic Medicine Research; Nan Chen and Xiaoxia Pan, Shanghai Jiaotong University School of Medicine; and Cuili Yi, Xiaohua Ding, Si Wang, Ping Wang, Xiaojing Nie, Jun Huang, Yonghui Yang and Zihua Yu, all of Fuzhou Dongfang Hospital.

Financial support for research described in this post was provided by the Nature Science Foundation of Fujian Province of China, under grant 2015J01407; National Nature Science Foundation of China, under grant 81270766; Key Project of Social Development of Fujian Province of China, under grant 2013Y0072; and the National Institutes of Health, under grants DK098410 and HL134940.

$2 million NIH grant to study nephrotic syndrome

March 11, 2019

Zhe Han lab 2018

A Children’s researcher has received a $2 million grant from the National Institutes of Health (NIH) to study nephrotic syndrome in Drosophila, a basic model system that has revealed groundbreaking insights into human health. The award for Zhe Han, Ph.D., an associate professor in Children’s Center for Genetic Medicine Research, is believed to be the first ever NIH Research Project grant (R01) to investigate glomerular kidney disease using Drosophila. Nephrotic syndrome is mostly caused by damage of glomeruli, so it is equivalent to glomerular kidney disease.

“Children’s National leads the world in using Drosophila to model human kidney diseases,” Han says.

In order to qualify for the five-year funding renewal, Han’s lab needed to successfully accomplish the aims of its first five years of NIH funding. During the first phase of funding, Han established that nephrocytes in Drosophila serve the same functions as glomeruli in humans, and his lab created a series of fly models that are relevant for human glomerular disease.

“Some 85 percent of the genes known to be involved in nephrotic syndrome are conserved from the fly to humans. They play similar roles in the nephrocyte as they play in the podocytes in human kidneys,” he adds.

Pediatric nephrotic syndrome is a constellation of symptoms that indicate when children’s kidneys are damaged, especially the glomeruli, units within the kidney that filter blood. Babies as young as 1 year old can suffer proteinuria, which is characterized by too much protein being released from the blood into the urine.

“It’s a serious disease and can be triggered by environmental factors, taking certain prescription medicines or inflammation, among other factors. Right now, that type of nephrotic syndrome is mainly treated by steroids, and the steroid treatment works in many cases,” he says.

However, steroid-resistant nephrotic syndrome occurs primarily due to genetic mutations that affect the kidney’s filtration system: These filters are either broken or the protein reabsorption mechanism is disrupted.

“When genetics is to blame, we cannot turn to steroids. Right now there is no treatment. And many of these children are too young to be considered for a kidney transplant,” he adds. “We have to understand exactly which genetic mutation caused the disease in order to develop a targeted treatment.”

With the new funding, Han will examine a large array of genetic mutations that cause nephrotic syndrome. He’s focusing his efforts on genes involved in the cytoskeleton, a network of filaments and tubules in the cytoplasm of living cells that help them to maintain shape and carry out important functions.

“Right now, we don’t really understand the cytoskeleton of podocytes – highly specialized cells that wrap around the capillaries of the glomerulus – because podocytes are difficult to access. To change a gene requires time and considerable effort in other experimental models. However, changing genes in Drosophila is very easy, quick and inexpensive. We can examine hundreds of genes involving the cytoskeleton and see how changing those genes affect kidney cell function,” he says.

Han’s lab already found that Coenzyme Q10, one of the best-selling nutrient supplements to support heart health also could be beneficial for kidney health. For the cytoskeleton, he has a different targeted medicine in mind to determine whether Rho inhibitors also could be beneficial for kidney health for patients with certain genetic mutations affecting their podocyte cytoskeleton.

“One particular aim of our research is to use the same strategy as we employed for the Coq2 gene to generate a personalized fly model for patients with cytoskeleton gene mutations and test potential target drugs, such as Rho inhibitors.” Han added. “As far as I understand, this is where the future of medicine is headed.”

Which micro-organisms lurk within urine?

February 11, 2019

Treating nephrotic-range proteinuria with tacrolimus in MTP

February 5, 2019

Mitochondria are the cell’s powerplants and inside them the MTP enzymatic complex catalyzes three steps in beta-oxidation of long-chain fatty acids.

In one family, genetic lightning struck twice. Two sisters were diagnosed with mitochondrial trifunctional protein (MTP) deficiency. This is a rare condition that stops the body from converting fats to energy, which can lead to lactic acidosis, recurrent breakdown of muscle tissue and release into the bloodstream (rhabdomyolysis), enlarged heart (cardiomyopathy) and liver failure.

Mitochondria are the cell’s powerplants and inside them the MTP enzymatic complex catalyzes three steps in beta-oxidation of long-chain fatty acids. MTP deficiency is so rare that fewer than 100 cases have been reported in the literature says Hostensia Beng, M.D., who presented an MTP case study during the American Society of Nephrology’s Kidney Week.

The 7-month-old girl with known MTP deficiency arrived at Children’s National lethargic with poor appetite. Her laboratory results showed a low corrected serum calcium level, elevated CK level and protein in the urine (proteinuria) at a nephrotic range. The infant was treated for primary hypoparathyroidism and rhabdomyolysis.

Even though the rhabdomyolysis got better, the excess protein in the girl’s urine remained at worrisome levels. A renal biopsy showed minimal change disease and foot process fusion. And electron microscopy revealed shrunken, dense mitochondria in visceral epithelial cells and endothelium.

“We gave her tacrolimus, a calcineurin inhibitor that we are well familiar with because we use it after transplants to ensure patient’s bodies don’t reject the donated organ. By eight months after treatment, the girl’s urine protein-to-creatinine (uPCR) ratio was back to normal. At 35 months, that key uPCR measure rose again when tacrolimus was discontinued. When treatment began again, uPCR was restored to normal levels one month later,” Dr. Beng says.

The girl’s older sister also shares the heterozygous deletion in the HADHB gene, which provides instructions for making MTP. That missing section of the genetic how-to guide was predicted to cause truncation and loss of long-chain-3-hydroxyacl CoA dehydrogenase function leading to MTP deficiency.

The older sister was diagnosed with nephrotic syndrome and having scar tissue in the kidney’s filtering unit (focal segmental glomerulosclerosis) when she was 18 months old. By contrast, she developed renal failure and progressed to end stage renal disease at 20 months of age.

“Renal involvement has been reported in only one patient with MTP deficiency to date, the older sister of our patient,” Dr. Beng adds.

Podocytes are specialized cells in the kidneys that provide a barrier, preventing plasma proteins from leaking into the urine. Podocytes, however, need energy to function and are rich in mitochondria.

“The proteinuria in these two sisters may be related to their mitochondrial dysfunction. Calcineurin inhibitors like tacrolimus have been reported to reduce proteinuria by stabilizing the podocyte actin cytoskeleton. Tacrolimus was an effective treatment for our patient, who has maintained normal renal function, unlike her sister,” Dr. Beng says.

American Society of Nephrology’s Kidney Week presentation

“Treatment of nephrotic-range proteinuria with tacrolimus in mitochondrial trifunctional protein deficiency

Hostensia Beng, M.D., lead author; Asha Moudgil, M.D., medical director, transplant, and co-author; Sun-Young Ahn, M.D., MS, medical director, nephrology inpatient services, and senior author, all of Children’s National Health System.

How our bladder’s microbiota affect health

May 30, 2018

The presence of bacteria such as Staphylococcus in the urine is linked to the incidence and severity of urge urinary incontinence as well as treatment success.

About half of the cells in our bodies aren’t really “ours” at all. They’re the microbiota: The vast array of microorganisms that live in our gut, skin, oral cavity and other places. Decades ago, researchers thought that these organisms simply happened to colonize these areas, playing only a tangential role in health, for example, helping to break down food in the intestines or causing cavities. More recent work has revealed the incredibly complex role they play in diseases ranging from diabetes and schizophrenia.

The bladder is no exception. Just a single decade ago, the bladder was thought to be a sterile environment. But that view has shifted radically, with more sensitive cultivation methods and precise 16S rRNA gene-sequencing techniques revealing a significant bladder microbiome that could have an enormous impact on pediatric urologic diseases. These findings have opened brand new fields of research aimed at clarifying the role that the bladder’s microbiome plays in common urological diseases that affect children, according to a review article published online Feb. 22, 2018, by Current Urology Reports.

“There is a growing appreciation for the role of diverse bacteria in contributing to improved health as well as triggering disease processes or exacerbating illness,” says Michael H. Hsieh, M.D., Ph.D., director of the Clinic for Adolescent and Adult Pediatric Onset Urology (CAPITUL) at Children’s National Health System and study senior author. “Already, we know that probiotics and dietary modifications have the potential to play powerful roles in preventing urinary diseases that commonly occur among pediatric patients,” Dr. Hsieh says. This underscores the importance of conducting even more studies to improve our understanding and to identify new therapies for health conditions that resist current treatment options.”

The review conducted by Dr. Hsieh and co-authors highlights the effects of the microbiome on a number of urologic diseases that affect children, including:

Urinary tract infection A number of studies point to the association between decreased microbial diversity and the incidence of what is commonly called urinary tract infection (UTI) or “dysbiosis.” This relationship suggests that using probiotics to replace or supplement antibiotics could favorably alter the urinary microbiome. Future research will focus on the pathophysiological role of the microbiome to determine whether it can be manipulated to prevent or treat UTIs.
Urge urinary incontinence While data vary by study, the presence of bacteria in the urine, especially certain bacterial species – such as Gardnerella, Staphylococcus, Streptococcus, Actinomyces, Aerococcus, Corynebacterium and Oligella – are linked to the incidence and severity of urge urinary incontinence (UUI) as well as treatment success. Most studies find an association between greater genitourinary biodiversity and reduced incidence and lessened severity of UUI as well as improved treatment response. Future research will focus on further clarifying this relationship.
Urolithiasis Calcium oxalate stones, the most common type of kidney stone, have a microbiome that differs from the urinary microbiome leading researchers to question whether the stone’s own bacterial makeup could help to predict recurrence of future kidney stones. What’s more, Oxalobacter formigenes, a gram-negative bacterium, lowers oxalate levels in the blood and are associated with a 70 percent reduction in the risk of kidney stones forming. In an experimental model, fecal transplants with the full microbiome represented had a pronounced and persistent effect on oxalate production. Patients who receive some antibiotics often have reduced rates of formigenes colonization. However, the bacteria are resistant to amoxicillin, augmentin, ceftriaxone and vancomycin, which could point to preferential use of these antibiotics to stave off disease and ward off kidney stone formation.

Additional authors include Daniel Gerber, study lead author, The Georgetown University School of Medicine and Health Sciences; and Catherine Forster, M.D., study co-author, Children’s National.

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