Research Updates in Kidney and Urologic Health

NIDDK Marks 50 Years of Research and Discovery; Next Decades Promise More Breakthroughs

NIDDK Support Helped Develop EPO and Treatments for Stone Disease

In its first 50 years, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) has supported innovative studies to expand our understanding of diseases, some leading to life-altering breakthroughs for people. The full range of conditions within NIDDK's purview is too long to list. People with such chronic conditions as diabetes and hepatitis have benefited from NIDDK-sponsored research. The story of NIDDK's involvement in the development of dialysis for treating end-stage renal disease will appear in a future issue of Research Updates. Two examples of the many NIDDK-supported achievements in kidney and urologic health are the development of a synthetic form of the hormone erythropoietin (EPO) and multiple therapies to treat the various metabolic disorders that can lead to urinary stones.

The Search for EPO

Before the discovery, understanding, and synthesis of EPO, people on dialysis in the 1960s suffered from anemia, with hematocrits typically below 22 percent. The therapeutic use of synthetic EPO for people with chronic renal failure was decades in the making.

Nineteenth-century scientists recognized a link between kidney disease and anemia but could not decipher it. In the 1950s, scientists discovered EPO and identified its role in forming red blood cells. In 1957, a team of scientists at the University of Chicago, led by hematologist Dr. Leon Jacobson, published a paper in the journal Nature that pinpointed the source of EPO in the kidney.¹ When hemodialysis in the 1960s began to improve the survival of many patients with end-stage renal disease, the need to find a treatment for anemia in these patients became more urgent.

In the 1970s, NIDDK-supported researchers established a sheep model to explore this problem. John Adamson and Joseph Eschbach induced anemia and uremia in a number of sheep by performing a four-fifths nephrectomy. Sheep were kept alive by dialysis if necessary. The researchers then developed a process for obtaining EPO-rich plasma from healthy sheep given phenylhydrazine to induce hemolysis. By 1980, Adamson and Eschbach showed that injecting anemic and uremic sheep with the EPO-rich plasma would raise hematocrit values. They predicted that finding a practical way to produce sufficient quantities of human EPO would provide an effective treatment for anemia in human patients with end-stage renal disease.

During this same period, another team of NIDDK-supported researchers, led by Eugene Goldwasser, succeeded in obtaining purified human EPO from the urine of patients with aplastic anemia. Goldwasser and his team studied the sample to determine its amino acid composition. Researchers at a biotechnology company used the information provided by Goldwasser's team to discover the EPO gene and began to produce recombinant human EPO.

By 1985, enough recombinant human EPO had been produced for Adamson and Eschbach to conduct the first clinical trial at the University of Washington in Seattle, followed by other trials around the country. In 1989, the Food and Drug Administration (FDA) approved epoetin (the product name for recombinant human erythropoietin) for treating anemia in patients with chronic renal failure. Subsequent studies have shown that increasing patients' hematocrits with EPO decreases mortality² and improves quality of life.³

Although epoetin has proven benefits for patients with ESRD, much remains to be done. The U.S. Renal Data System reports that the average hematocrit for epoetin-treated patients on hemodialysis has increased to 32.4 percent. But many patients still fall below the recommended range of 30 to 36 percent. And the mortality and morbidity for patients with ESRD are unacceptably high. Clearly, research is needed not only to find ways to increase hematocrit values, but also to improve vascular access survival, ensure that dialysis dose and adequacy targets are met, and reduce the host of complications that accompany kidney failure.

New Treatments To Prevent Kidney Stones

Kidney stones are a painful condition that often requires medical intervention and hospitalization. In extreme cases, urinary stones result in life-threatening kidney failure that may require kidney transplantation. In recent decades, scientists have refined surgical techniques for removing kidney stones and developed shockwave lithotripsy, an innovative procedure that breaks up kidney stones so they can pass more easily in urine. But these life-saving and pain-sparing developments did little to increase scientific understanding of how stones develop or provide ways to prevent stone disease.

In the 1970s and 1980s, NIDDK sponsored research into the causes of stones. In susceptible people, recurrent kidney stones may result from dietary habits, such as excessive consumption of salt or animal protein,⁴ or from metabolic abnormalities.⁵ Some stones may result from a combination of factors. For example, a person may have the metabolic disposition to form stones, and diet provides the required biochemical ingredients.

A better understanding of the interplay of diet and metabolic dysfunctions that leads to the formation of kidney stones will improve treatment and provide clues to prevention.

For example, one NIDDK-sponsored researcher, Charles Y.C. Pak, identified 16 separate stone-forming disorders and developed selective treatments for each one. In so doing, Dr. Pak set a record as a single investigator for the number of orphan drugs approved by FDA. In one case, Pak demonstrated that using potassium citrate to increase the levels of urinary citrate in stone formers helped inhibit the crystallization of calcium salts in the urine and the formation of calcium oxalate stones. In 1984, FDA approved the use of potassium citrate for that purpose. A few years later, in 1988, FDA approved alpha-MPG (Thiola), a drug Pak and his team at the University of Texas Southwestern Medical Center developed to treat people with cystinuria.

Dr. Pak's work continued in the 1990s with new insights into dietary factors that may promote or inhibit stone formation. In 1993, he published articles demonstrating that consuming too much salt promoted stone formation⁴ and that drinking orange juice could inhibit it.⁶

Recently, research sponsored by NIDDK has helped identify specific genetic defects responsible for some kidney stone disorders. For example, the genes for two types of primary hyperoxaluria have been identified. In type I, the responsible defective gene is liver-specific peroxisomal alanine:glyoxylate aminotransferase.⁷ In some of those affected, the defect is due to a subcellular mistargeting of the enzyme, resulting in ineffective cytoplasmic or mitochondrial cellular localization. In others, the enzyme is missing altogether.

A second type of hyperoxaluria, type II, is caused by a mutation in the hydroxypyruvate reductase gene, leading to a defective protein. A human kidney chloride channel gene, CLCN5, is disrupted in patients with Dent's disease, a rare X-linked inherited kidney stone disorder.^8,9

Another disorder that leads to kidney stones is cystinuria. Defects in the gene encoding a cystine transport protein have been found in some patients with cystinuria.¹⁰ Finally, a recent report has narrowed the search for the gene defect for absorptive hypercalciuria, another disorder associated with kidney stones, to a region on chromosome 1.¹¹

Identifying the genes responsible for these disorders represents an important breakthrough in our understanding of these and other kidney stone diseases. Many of these findings were facilitated through markers developed by the Human Genome Project and show the power of new approaches that can help identify the genes responsible for, or contributing to, pathogenic processes. Now that the molecular basis for some of these disorders is known, improved diagnostic methods, possibly including prenatal diagnosis,⁷ can be developed. Furthermore, treatment methods specifically targeting the underlying genetic defects, perhaps even gene therapies to correct them, can be developed as well.

References

1. Jacobson LO, Goldwasser E, Fried W, Plzak L. Role of the kidney in erythropoiesis. Nature. 1957;179:633–634. 2. Ma JZ, Ebben J, Xia H, Collins AJ. Hematocrit level and associated mortality in hemodialysis patients. Journal of the American Society of Nephrology. 1999;10:610–619. 3. Levin N, Lazarus JM, Nissenson AR. National cooperative rHu erythropoietin study in patients with chronic renal failure—an interim report. American Journal of Kidney Disease. 1993;22(2, suppl 1):3–12. 4. Sakhaee K, Harvey JA, Padalino PK, Whitson P, Pak CYC. The potential role of salt abuse on the risk for kidney stone formation. Journal of Urology. 1993;150:310–312. 5. Scheinman SJ. Nephrolithiasis. Seminars in Nephrology. 1999;19:381–388. 6. Wabner CL, Pak CYC. Effect of orange juice consumption on urinary stone risk factors. Journal of Urology. 1993;149:1405–1408.

7. Danpure CJ, Rumsby G. Strategies for the prenatal diagnosis of primary hyperoxaluria type I. Prenatal Diagnosis. 1996;16:587–598. 8. Fisher SE, van Bakel I, Lloyd SE, Pearce SHS, Thakker RV, Craig IW. Cloning and characterization of CLCN5, the human kidney chloride channel gene implicated in Dent disease (an X-linked hereditary nephrolithiasis). Genomics. 1995;29:598–606. 9. Lloyd SE, Pearce SHS, Fischer SE, et al. A common molecular basis for three inherited kidney stone diseases. Nature. 1996;379:445–448. 10. Gitomer WL, Reed BY, Ruml LA, Sakhaee K, Pak CYC. Mutations in the genomic deoxyribonucleic acid for SLC3A1 in patients with cystinuria. Journal of Clinical Endocrinology and Metabolism. 1998;83:3688–3694. 11. Reed BY, Heller HJ, Gitomer WL, Pak CYC. Mapping a gene defect in absorptive hypercalciuria to chromosome 1q23.3-q24. Journal of Clinical Endocrinology and Metabolism. 1999;84:3907–3913.

[Top]

New Directions for Research in the Post-Genome Age: Targeting Disease at the Molecular Level

The Human Genome Project is providing a wealth of information that can be applied across the spectrum of human disease, including kidney and urologic conditions. In a recent issue of JAMA, directors of various units within the National Institutes of Health (NIH) were asked to speculate about what their field of medicine would look like in the year 2020.

Francis Collins, director of the National Human Genome Research Institute (NHGRI), answered, "In 20 years, most human diseases will be understood at the fundamental level of molecules; knowledge about genetic control of cellular functions will underpin future strategies to prevent or treat disease phenotypes."¹

New Technologies To Investigate Genome Expression

The overall goal of the Human Genome Project is to sequence all the DNA of the human genome. These sequences include both segments coding for expressed proteins and noncoding segments. Both types of information will be valuable to future investigations in several types of studies.

First, polymorphisms in DNA sequences will be essential tools for family studies to home in on possible genetic contributions to disease. Furthermore, this same approach may be used to identify genes contributing to disease severity or therapeutic resistance. It may be that polymorphisms

• occur within contributing gene(s),
  
  
• are linked to contributing gene(s),
  
  
• are in a regulatory region or regulating gene(s), or
  
  
• are in a nearby noncoding DNA region that affects the expression of regulating gene(s).
  
  

Because of the amount of information available through the Human Genome Project, many thousands of such polymorphic markers will be available for analysis, and the breadth of these investigations will increase. Also, this vast amount of information will eventually allow complex genetic interactions of multigenic traits to be identified.

Second, a new field of functional genomics is emerging. In the new approaches, expressed RNAs are being extensively evaluated for their participation in cellular and disease processes. These studies are usually performed as "arrays," which may be either filter based or chip based. They allow evaluation of changes in the RNA expression of many thousands of genes at once.

However, expressed RNA represents only part of the story. Proteins are responsible for many of the tasks genes perform. The Human Genome Project has so far concentrated on DNA and RNA structure and expression, but proteomics, or the systematic evaluation of protein expression, is also emerging as a field. These studies can be performed either on a large-scale platform, for example by two-dimensional gel electrophoresis, or on a multiplex analysis of individual proteins, for example by micro-ELISA (enzyme-linked immunoassay). Again, the Human Genome Project has raised the bar on these studies so that analysis of individual proteins will be supplanted by analysis and integration of changes in the expression of many proteins at once. The technology for such studies is still emerging but is likely to be swiftly developed in the next phase as the Human Genome Project shifts into analyzing function.

These studies all generate huge quantities of data, which require enormous amounts of analysis. Identifying the important players in a field of many thousands is a daunting task. A new field—informatics—has been born out of the need to evaluate, analyze statistically, and integrate this flood of information.

Impact of Genomic Research in Kidney and Urologic Disease

The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) is supporting research programs that encourage exploration into the genetic components of diseases and development of new technologies such as microarray analysis and serial array of gene expression (SAGE). Around the world, the study of genetic expression and molecular interactions is leading to new insights into many kidney and urologic diseases, including nephrogenic diabetes insipidus² and renal tubular acidosis.³

Chromosome 16

In the 1990s, researchers located and sequenced two genes (PKD1 and PKD2) involved in the dominant form of polycystic kidney disease (PKD). NIDDK-funded researchers Gregory Germino and Stephen Reeders found many of the clues that helped pinpoint the first gene's locus on chromosome 16. ^4-6 Stefan Somlo, another NIDDK grantee, is credited with discovering the PKD2 gene.^7-9

NIDDK continues to sponsor research that focuses on the complex of genetic mechanisms that cause polycystic kidneys. Four NIDDK-supported centers at leading research institutions around the country are coordinating their efforts to identify and study animal models for human PKD, to locate and sequence the elusive autosomal recessive PKD gene, and to understand how protein interactions direct cell formation, or misdirect it in the case of PKD.

Compared with the study of PKD genes, the search for genetic factors in the development of diabetic nephropathy is in its early stages. Scientists have determined that chronic renal failure caused by diabetes tends to cluster in families. Siblings of people with diabetic nephropathy are 3 to 10 times more likely to develop the condition than other people who have the same kind of diabetes and the same level of hyperglycemia.

NIDDK has awarded grants to several researchers who are studying genetic samples from pairs of siblings with diabetes. In some of the pairs, both siblings have nephropathy; in others, only one has it. Scientists hope that studying these samples will reveal genetic traits unique to the people who develop nephropathy. Once a gene has been identified, located on a chromosome, and sequenced, scientists can learn more about how it functions and search for ways to correct its actions. NIDDK's Familial Investigation of Nephropathy of Diabetes (FIND) program encourages the application of new gene sequencing techniques to the study of kidney function and kidney failure.

What the Future Holds

By the year 2020, it has been predicted that "designer drugs based on a detailed molecular understanding" of "common illnesses like diabetes and hypertension" will be available.¹ Diabetes and hypertension, which are the leading causes of end-stage renal disease, are only two of the many conditions of interest to NIDDK's Division of Kidney, Urologic, and Hematologic Diseases. Other conditions with important genetic and molecular components include stone disease, prostate disorders, diabetes insipidus, renal tubular acidosis, urinary tract infection, interstitial cystitis, and enuresis. Developing new and better treatments by understanding these conditions at the molecular level requires continuing commitment to the kinds of research that NIDDK is supporting.

References

1. Goldsmith MF. 2020 vision: NIH heads foresee the future. Journal of the American Medical Association. 1999;282(24):2287–2290. 2. Matsamua Y, Uchida S, Kondo Y, et al. Overt nephrogenic diabetes insipidus in mice lacking the CLC-K1 chloride channel. Nature Genetics. 1999;21:95–98. 3. Karet FE, Finberg KE, Nelson RD, et al. Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness. Nature Genetics. 1999;21:84–90. 4. Reeders ST, Bruening MH, Davies KE, et al. A highly polymorphic DNA marker linked to adult polycystic kidney disease on chromosome 16. Nature. 1985:317;542–544. 5. Reeders ST. Multilocus polycystic disease. Nature Genetics. 1992;1:235–237.

6. Germino GG, Weinstat-Saslow D, Himmelbauer H, et al. The gene for autosomal dominant polycystic kidney disease lies in a 750-kb CpG-rich region. Genomics. 1992;13:144–151. 7. Kimberling WJ, Kumar S, Gabow PA, Kenyon JB, Connolly CJ, Somlo S. Autosomal dominant polycystic kidney disease: localization of the second gene to chromosome 4q13-q23. Genomics. 1993;18:467–472. 8. Deltas C, Peters DJM, Somlo S. PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein. Science. 1996;272:1339–1342. 9. Qian F, Germino FJ, Cai Y, Zhang X, Somlo S, Germino GG. PKD1 interacts with PKD2 through a probable coiled-coil domain. Nature Genetics. 1997;16:179–183.

[Top]

NIDDK and Collaborative Network Seek Recruits for Chronic Prostatitis Cohort Study

Doctors in the Chronic Prostatitis Collaborative Research Network are enrolling patients to participate in the Chronic Prostatitis Cohort (CPC) Study, funded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). Researchers expect to continue recruitment through October 2001 and to enroll more than 600 men in the study.

CPC is the first large, multicenter study designed to gather well-defined, detailed clinical information on chronic abacterial prostatitis, a condition that leads to pain in the genital area and lower back, usually accompanied by frequent and urgent urination. Other symptoms may include burning or pain during voiding or ejaculation.

Unlike bacterial prostatitis, which can be readily diagnosed by the presence of infection, the abacterial form produces no signs of infection that can be detected using routine clinical methods. Abnormalities in prostatic secretions provide the only objective sign of the condition. Chronic pelvic pain is the main subjective symptom.

The study will start with a data-collection phase in which the researchers will document symptoms, possible risk factors, and medical histories. In this phase, researchers will examine blood, prostate fluid, semen, and urine. One goal of this phase will be to explore possible relationships between chronic prostatitis, urethral and bladder inflammation, and other chronic pain disorders. The second phase will focus on standardizing a treatment protocol for chronic prostatitis. Results are anticipated after September 2002.

Men interested in participating may contact the following centers for more information.

Clinics

California Mark S. Litwin, M.D., M.P.H. University of California, Los Angeles Contact: Yining Xie Phone: 310–222–3819 Canada J. Curtis Nickel, M.D. Queen's University, Kingston, Ontario Contact: Joe Downey, B.Sc., M.Sc. Phone: 613–545–2894 Illinois Anthony J. Schaeffer, M.D. Northwestern University, Chicago Contact: Gwen Haggis, R.N. Phone: 312–908–7022 Maryland Richard B. Alexander, M.D. University of Maryland, Baltimore Contact: E. Bronwyn Byron Phone: 410–328–0801 Massachusetts Michael P. O'Leary, M.D., M.P.H. Brigham and Women's Hospital, Boston Contact: Judy Spolarich-Kroll, B.A., or Debra Rhodes, M.D. Phone: 617–732–7223 Pennsylvania Michel A. Pontari, M.D. Temple University, Philadelphia Contact: Linda Kish Phone: 215–707–3783 The Prostatitis Foundation Mike Hennenfent, Patient and President Phone: 309–325–7184 (messages and faxes) Email: Mcapstone@aol.com Internet: www.prostate.org

[Top]

Allen Spiegel, M.D., Named Director of NIDDK

The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) welcomed a new director last November. Allen M. Spiegel, M.D., is an internationally recognized endocrinologist whose research on signal transduction has helped define the genetic basis of several diseases.

Harold Varmus, M.D., who was then director of the National Institutes of Health (NIH), welcomed Dr. Spiegel. "I am very pleased

Allen M. Spiegel, M.D.

that Allen Spiegel, one of the Nation's most distinguished medical scientists, will be assuming leadership of NIDDK," said Dr. Varmus in announcing the appointment. "NIDDK is responsible for addressing some of the most important chronic and seemingly intractable diseases facing us today."

Dr. Spiegel has participated in a collaborative effort with colleagues at NIDDK and the National Human Genome Research Institute at NIH to clone the tumor suppressor gene that, when mutated, causes the inherited disease multiple endocrine neoplasia type 1 (MEN1), as well as a number of sporadic endocrine and other tumors. The collaborative group is now studying the structure and function of the MEN1 gene and its encoded protein, menin.

"Throughout my career, I have tried to forge strong links between fundamental science and clinical medicine. Now, I am enthusiastic about being able to do this on a larger scale," Dr. Spiegel said.

Dr. Spiegel comes to the top NIDDK post from his position as scientific director of NIDDK. For the past 9 years, he has led one of the largest and most productive on-site research programs on the NIH campus. As the director of NIDDK, he will oversee a staff or 900 employees and an annual budget of $1 billion.

After graduating cum laude from Harvard Medical School in 1971, Dr. Spiegel completed an internship and residency in internal medicine at Massachusetts General Hospital in Boston. He has received numerous awards for his accomplishments, most recently the 1998 Edwin B. Astwood Lecture Award from the Endocrine Society and the 1996 Komrower Memorial Lecture Award from the Society for the Study of Inborn Errors of Metabolism.

Outgoing NIDDK director Phillip Gorden, M.D., is returning to the NIH Clinical Center as chief of the section on clinical and cellular biology in the NIDDK Diabetes Branch, where he will resume his research on disorders of the insulin receptor, insulin secretion, and severe insulin resistance. Dr. Gorden served as director for 13 years and was honored last fall with a symposium, "The Human Face of Science."

[Top]