Molecular and physiological aspects of nephropathy in type I (insulin-dependent) diabetes mellitus

ELSEVIER

Molecular and Ph siological Aspects of Nephropathy in ? ype I (Insulin-Dependent) Diabetes Mellitus Gregory S. Raskin William V. Tamborlane

INTRODUCTION

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ype I, or insulin-dependent diabetes mellitus (IDDM), strikes roughly 15 of 100,000 people under the age of 20 years per year.’ After the age of 20 years, the prevalence of IDDM drops to 5 of 100,000 people per year.* The metabolic problems associated with IDDM are caused primarily by inadequate secretion of insulin by the pancreatic p cells. The day-to-day dangers of diabetes, such as acute hyperand hypoglycemia are well known, and until the 1920s all children afflicted with diabetes died because of such metabolic derangements. In the early 1920s Banting and Best discovered insulin, which was hailed as a cure for diabetes; indeed, insulin therapy is effective in preventing the vicious cascade of rapid dehydration, catabolism, ketoacidosis, and death. Nevertheless, while patients live much longer with insulin treatment, they become susceptible to a variety of diabetic complications that occur in the longer term. These complications include proliferative retinopathy, peripheral neuropathy, macrovascular complications, and nephropathy. First described in 1936 by Kimmelstiel and Wilson,* diabetic nephropathy is one of the most serious complications of diabetes. In fact, 35% of diabetic patients ultimately develop endstage renal disease as a result of their diabetes, and require kidney transplantation or dialysis for survival. 3 Because of the seriousness of this complication, there has been much interest in this area of diabetes research.

Yale University School of Medicine, Departments of Molecular Biophysics and Biochemistry, Pediatrics and the Children’s Clinical Research Center, New Haven, Connecticut, USA Reprint requests to be sent to: Gregory S. Raskin, Yale University School of Medicine, Department of Pediatric EndocrinologyiDiabetes, PO Box 3333, New Haven, CT 06510. ]ournaZ of Diabetes and Ifs Complications

1996;

The causes, pathophysiological mechanisms, and possible prevention of diabetic nephropathy have been the focus of most of the recent research in the field. This paper will address some of the physiological and molecular aspects of nephropathy, with special attention paid to the markers or predictors of diabetic nephropathy, a subject that has been under active study especially in England and Denmark. NATURAL HISTORY OF DIABETIC NEPHROPATHY Renal function in patients who will develop diabetic nephropathy culminating in endstage renal failure has a relatively consistent natural progression. In 1983, Mogensen described a five-stage classification system for diabetic nephropathy.* It is important to note that the adverse effects of IDDM on the kidney primarily involve glomerular rather than tubular function. Normally, the glomerulus filters from 108 to 125 mL of plasma per min .5 While diabetes may ultimately lead to a severe reduction in glomerular filtration rate (GFR), due to obliteration of the glomerular capillaries, the opposite is seen in Stage I renal involvement, the earliest stage of diabetic nephropathy. This stage is characterized by an increase in glomerular filtration to values often exceeding 150 mL/min (hyperinfiltration) in association with slight glomerular enlargement.” Stage II is the “silent stage” of diabetic nephropathy, because there are no clinically detectable changes in renal function, but structural lesions are occurring. This stage may last anywhere from 7 to 15 years. Albumin, the smallest plasma protein, is not usually excreted in the urine, but rather is maintained in the circulatory system. At times, poor metabolic control or exercise may cause an elevation in albumin excretion rate above normal, but this is a reversible abnormality during Stage II.

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As soon as microalbuminuria (albumin excretion rate > 20 pgimin) becomes persistent, the patient has entered Stage Ill, the period of incipient diabetic nephropathy. Incipient diabetic nephropathy is diagnosed when such microalbuminuria occurs in two of three urine samples collected over a period of 6 months.4 While GFR is still above normal in this stage, it may decline to roughly 130 mL/min and blood pressure may be elevated compared to healthy subjects. During the second half of Stage Ill, blood pressure increases by about 3.5% per year. The beginning of Stage IV is signaled by an albumin excretion rate of more than 200 pglmin, a condition known as clinical grade proteinuria. This stage of overt diabetic nephropathy also is characterized by an inexorable decline in GFR from = 130 mL/min to lo-30 mL/ min. In the glomerulus, there is capillary basement membrane thickening and mesangial expansion that squeezes off capillaries, leading to destruction of the glomerulus. Blood pressure during Stage IV increases by about 8% annually, and there is often frank hypertension. In addition, hypercholesterolemia is more common among patients with this stage of renal impairment. Stage V, the final or endstage, is defined clinically by an increase in the excretion of a larger plasma protein, P-2-microglobulin, with an albumin excretion rate of approximately 1000 vglmin; almost all patients in this stage are hypertensive. Most importantly, there is a generalized glomerular closure, and the kidney is unable to excrete urea and other waste products, leading to the clinical state called uremia. The patient will need dialysis or a kidney transplant in order to survive. MICROALBUMINURIA: ADVANCED DIABETIC

PREDICTOR OF NEPHROPATHY

While most IDDM patients show hyperfiltration and glomerular hypertrophy early in the course of IDDM, only about one-third of patients progress to Stage V renal impairment. Consequently, a major focus of research has been directed toward identification of early markers that would help identify the subset of IDDM patients at greatest risk for this complication. The development of assays to measure small amounts of albumin in the urine was one of the first such tests to be examined. Up until the 197Os, the test for proteinuria was done with a dipstick (Albustix) and could only detect large amounts of protein in the urine. By the time the problem was detected, the patients with IDDM had already reached Stage IV and were nearing endstage renal failure. During the late 1970s and early 198Os, assays became available for the detection of much smaller quantities of albumin in the urine, including radioimmunoassay, enzyme-linked immunosorbent assay, zone immunoelectrophoresis, fluorescence immunoassay, and immunoephelometry. With these new techniques, sub-

clinical increases in urinary albumin excretion rate were found in many IDDM patients; this subclinical but higherthan-normal albumin level is termed microalbunzinuria. Once microalbuminuria was able to be assayed, longitudinal studies were performed to test whether microalbuminuria could be used as a predictor for clinical nephropathy. In the early 198Os, three separate longitudinal studies indeed showed that elevated albumin excretion was associated with progression to more advanced stages of clinical nephropathy. Viberti et al., with a 14-year follow-up study, found that while 80% of IDDM patients with an albumin excretion rate above 30 pglmin developed clinical nephropathy, only 4% of those with an initial albumin excretion rate below 30 pglmin developed clinical nephropathy.h Similar results were subsequently reported by two Danish groups of investigators. 7~xIn patients who proceed to develop nephropathy, the AER increases at roughly 20% per year.y Having demonstrated that microalbuminuria is a marker for clinical nephropathy, attention turned to the pathological mechanism underlying this selective alteration of the renal glomerular capillary barrier. PATHOPHYSIOLOGY MICROALBUMINURIA

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Interestingly, the pathophysiology of albuminuria is different from the pathophysiology of later nephropathy. While latestage renal problems stem mainly from mesangial expansion reducing vascular function, albuminuria is a sieving problem. The kidney does not have a problem getting plasma to the filter; rather, the filter is less selective. Plasma proteins are relatively large molecules that are negatively charged and do not normally pass through the glomerular basement membrane but remain in the intravascular space. These macromolecules are normally conserved in the circulatory system by a size-and charge-selective barrier in the glomerulus. In albuminuria, some change occurs that allows these molecules to leak through the glomerular basement membrane. Hemodynamic Factors. Although hypertension is an independent risk factor for the development of advanced vascular complications in diabetes, systemic blood pressure elevation associated with diabetes is not a sufficient cause for the leakage in albuminuria.‘” Increased intraglomerular pressure [calculated as the filtration factor (FF), the ratio of glomerular filtration rate (GFR = mLlmin11.73 m’) to renal plasma flow (RPF = mLlminil.73 m’)] might explain the increased leakage of albumin across the capillary endothelium. Deckert et al., however, found no difference between the FF+of normo- and microalbuminuric IDDM patients.” One group found higher capillary pressure in the skin of IDDM patients when compared to normal controls, but was not able to show a difference between micro-

] Diab Camp 1996; 10~31-37

and normoalbuminuric patients, except when measuring the microalbuminuric patients against a select subgroup of long-term diabetic patients under extreme metabolic control.12,13 Glomerular hyperfiltration, which has been shown to be a precursor to albuminuria and is often present early in the course of diabetes5 does not cause enough disruption of the normal macromolecular network within the glomerular extracellular matrix to explain the development of albuminuria. As reported by Hostetter, less than one-half of the patients with hyperfiltration developed albuminuria. I4Also, Lervang et al.15did not show a correlation between early hyperfiltration and nephropathy. Thus, there must be some added factor which, when coupled with increased glomerular filtration and renal plasma flow, leads to albuminuria. Alteration in Basement Membrane Function: The Steno Hypothesis. The group at the Steno Diabetes Center in Gentofte, Denmark, has proposed an explanation for the cause of albuminuria in diabetes. The “Steno Hypothesis” contends that there is a change in the glomerular basement membrane, so that it loses some of its negative charge, thereby decreasing the charge selectivity of the basement membrane barrier and allowing more macromolecules to pass through.16 For example, albumin, a negatively charged protein, is normally repelled by the negatively charged glomerular basement membrane. In patients with albuminuria the anionic strength of the barrier is reduced, thus albumin is able to pass through into the urine space. The glomerular basement membrane consists of compressed extracellular matrix between the capillary endothelium and glomerular epithelium. The extracellular matrix contains a web of collagen type IV fibrils, which acts as a size-selective filter by binding to cell adhesion molecules .l’Trapped in the web are the large glycoproteins laminin and nidogen and anionic molecules of heparan-sulfate proteoglycan (HSPG).lR It is the presence of HSPG that confers the negative charge to the glomerular basement membrane. l9 HSPG consists of three heparan-sulfate side chains covalently attached to a monomeric protein core with a molecular weight of 470 kilodaltons. 2oPettersson et al.21 demonstrated that the heparan-sulfate component of the proteoglycan is formed from D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc) units joined in a (GlcAGlcNAc)n structure. The next step is the placement of sulfate groups onto the heparan molecules; it is these sulfate groups that confer the important negative charge to the molecule. The N-sulfation of heparan must occur after the heparan has been deacetylated; appropriately, sulfation occurs via a deacetylated intermediate (GlcNAc + GlcNHs’ + GIcNSO~).~~ The malfunction of these vital steps would create a smaller proportion of sulfated nitrogen atoms, thus decreasing both the

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negative charge of the molecule and the charge selectivity of the glomerular basement membrane. The molecule is further modified by the C-5 epimerization of glucuronic acid residues and the subsequent 0-sulfation of various positions, but these steps have not shown to affect the anionic charge of the molecule. Loss of HSPG has been shown to increase the permeability of the glomerular basement membrane. Van den Born et al.22 showed that a monoclonal antibody against HSPG induced acute, dose-dependent, selective proteinuria in rats, immediately upon injection. The antibody localized in the glomerular basement membrane and the charge selectivity index (clearance IgGl clearance albumin) dropped by at least one-half (depending on the dose of antibody). Vernier et al.2” demonstrated a loss of HSPG in the glomerular basement membrane of IDDM patients with advanced stages of renal damage using electron microscopy, but they were not able to show a loss of HSPG in microalbuminuric patients. One hypothesis for the loss of charge in the basement membrane is that there is a genetic enzymatic dysfunction that leaves a subset of patients with IDDM more susceptible to the adverse effect of poor metabolic control.24 Deckert and his group2* believe that the problem lies in the N-deacetylation of the heparan molecule. The enzyme glucosaminyl N-deacetylase is responsible for this crucial step. Inhibition of this enzyme in streptozotocin-induced diabetic rats indeed produced alterations in the extracellular matrix composition. 25In addition, Unger et al.Zb found that diabetic rats had a lowered N-deacetylase activity when compared to control rats. Finally, Kofoed-Enevoldsen et al.27 found that poor short-term blood glucose control decreased N-deacetylase activity not only in streptozotocin-induced diabetic rats, but also in spontaneously diabetic rats. Thus, that group concluded that N-deacetylase activity is a rapid regulatory response to poor metabolic control, and is not the result of longterm effects. This preliminary finding seems to have major significance: it may explain why strict metabolic control tends to fend off late complications, as demonstrated in the multicenter Diabetes Control and Complications Trial.28 Ion Exchange Systems. Hypertension and diabetic complications are closely linked. Hypertension has been implicated in the acceleration of the downward course of glomerular filtration, along with other complications such as retinopathy and atherosclerosis. In addition, Viberti et al.29 found that a family history of raised blood pressure was more prevalent in diabetic patients with nephropathy than in those without nephropathy Alterations in the sodium-lithium countertransport and sodium-hydrogen antiport systems have been demonstrated in nondiabetic patients with hypertension and

in IDDM patients with advanced nephropathy. It has recently been suggested that such alterations in transport function might also provide an early predictor of IDDM patients at risk for the development of endstage renal failure. Sodium-Lithium Countertransport. The sodium-lithium countertransport (SLC) system is a pH- and temperature-sensitive exchange reaction that still has an unclear physiological role. 30The system is one of the methods that transports sodium across the cellular membrane. Other possible routes include the sodium-potassium pump, an ATP-coupled reaction. The SLC system can be distinguished from the other transport systems by the use of specific inhibitors; for example, the ATPcoupled transporter is inhibited by ouabain, and ion transport that occurs in the presence of ouabain is independent of the ATP-coupled route. The SLC system preferentially transports lithium by a factor of 20. Thus, lithium can be transported against the electrochemical gradient, driven by an oppositely directed electrochemical-potential gradient for sodium.30 Most of the SLC experiments were performed on red blood cells, because this is the most easily obtainable cell type. SLC activity is determined by loading the cells with lithium, and measuring the efflux (in mmol of lithium per liter of red cells per hour) into a sodium-containing, lithium-free medium with atomic absorbance spectrophotometry. In 1980, Canessa et al.“l first showed increased SLC activity in red blood cells of patients with essential hypertension. In 1988, two groups showed similarly increased SLC activity in the red blood cells of patients with IDDM and clinical nephropathy.32 Mangili et al.32 showed that the SLC activity in a group of IDDM patients with diabetic nephropathy was significantly higher than in a group of patients with diabetes but no renal disease and in a group of normal controls. Even more importantly, Krowleszki et al.“” found elevated SLC activity in patients with microalbuminuria and only slight hypertension but no discernible renal impairment.33 Mangili’s group hypothesized that the predisposition to arterial hypertension as indicated by an increase in activity of the SLC system may, in the presence of diabetes, confer susceptibility to nephropathy.32 Thus, in order for a diabetic patient to develop nephropathy, this genetic predisposition may be necessary. Furthermore, a study by Lopes de Faria et al.X has shown SLC to be more strongly correlated than either arterial blood pressure, duration of IDDM or glycemic control to nephropathy (as evidenced by elevated albumin excretion rate). While these studies have shown a distinct correlation between elevated sodium-lithium countertransport activity and diabetic nephropathy, there have been some challenges to this theory. Jensen et al.35 and Elv-

ing et al. 36,37did not find any significant differences in SLC activity in patients with and without diabetic nephropathy. Rutherford et al.‘* did not find any differences among controls, normoalbuminuric IDDM patients, proteinuric IDDM patients, IDDM patients with declining renal function, IDDM patients receiving renal replacement therapy and nondiabetic renal disease patients. The results of these three research groups, however, have been challenged because of inconsistent methodologies used in measuring SLC activity.” Another confounding variable is the effect of hyperlipidemia on SLC. Rutherford et al.40 have shown that hyperlipidemia, per se, can cause increased sodiumlithium countertransport activity. Even though hyperlipidemia is common in IDDM patients, most studies that have shown a correlation between SLC activity and diabetic nephropathy have not taken hyperlipidemia into account. Because the experiments done on the SLC system were performed with nonphysiological amounts of lithium, which exists in only trace amounts in the human body, the clinical usefulness and the physiologic importance of this system has been questioned.*’ The SLC system, however, does have certain similarities to the sodium-hydrogen antiport system, which has a clearly defined physiological role. Several groups have examined the sodium-hydrogen antiport system to determine whether there is a difference in the activity between diabetic patients with and without nephropathy . Sodium-Hydrogen Antiporter Activity: Sodium-hydrogen antiporters operate in all cells by extruding hydrogen cations in a reaction driven by the inwardly directed electrochemical sodium gradient.42 The reaction is reversibly inhibited by amiloride and activated by an acidic intracellular H’ concentration. The sodiumhydrogen antiporter regulates intracellular pH as well as cell volume and growth; it is also involved in the reabsorption of sodium in the proximal renal tubule.43 In diabetes, there are marked pertubations in metabolic and hormonal regulation of several growth factors in addition to insulin. The effect of this dysregulation may cause renal complications in genetically predisposed individuals. 44Enhanced cell growth could lead to the overactivity of the sodium-hydrogen antiporter and to increased tubular sodium reabsorption, which would, in turn, raise renal plasma flow, and lead to glomerular hyperfiltration in order to maintain sodium balance.44 Trevisan et a1.45demonstrated an increased amiloride-sensitive sodium influx and enhanced cell proliferation in fibroblasts of IDDM patients with nephropathy, thus demonstrating an overactivity of the sodium-hydrogen antiporter in this group of patients. Also, sodium-hydrogen antiport activity has been shown

1 Diab Comp 1996; 10:31-37

to be higher in leukocytes of IDDM patients with micro- as well as macroalbuminuria.46 In addition to its role in sodium reabsorption, the sodium-hydrogen antiporter has been shown to increase smooth-muscle-cell Ca2+ levels and to cause smoothmuscle-cell hypertrophy through an increase in cellular pH.47 Both of these effects would contribute to increase in peripheral vascular resistance, and would therefore lead to hypertension. A link between a genetic predisposition to hypertension (which in this case would be due to an elevated sodium-hydrogen antiport level) and nephropathy in diabetic patients has been proposed. Thus, it is possible that an overactive sodium-hydrogen antiporter, in combination with diabetes, could be seen as a marker for those patients with diabetes most likely to develop nephropathy. Indeed, Davies et a1.48showed that the raised sodium-hydrogen antiport activity in the fibroblast cells of patients with diabetic nephropathy persists as the cells were passaged; thus, this overactivity is a heritable component that results from an increased affinity for intracellular H’ ions. This heritable component could be seen as a marker for nephropathy. While the studies mentioned previously have shown a correlation between sodium-hydrogen antiport activity and nephropathy, not all researchers are in agreement as to the usefulness of this antiporter in predicting nephropathy; not all studies have shown a difference between sodium-hydrogen antiport activity in fibroblast cells.49 In the past, the antiporter molecule (the NalH Exchanger, NHE) had been thought to be ubiquitous. Recent studies, however, have found several isoforms of the molecule. In 1991, two isoforms, NHE-1 and NHE-2 were isolated from rabbit ileal villus epithelia cells50 In addition, Orlowski et a1.51found two other related proteins, NHE3 and NHE-4, using an NHE-1 cDNA probe under low-stringency hybridization conditions in a cDNA library consisting of rat heart, spleen, brain, kidney, and stomach cDNA. There is some concern, then, that the sodium-hydrogen antiport activity in various cell types may not be consistent throughout the multiple members of the gene family. Therefore, comparing the different antiport activity levels in leukocyte, fibroblast, and other cell types, may not be as clinically useful as once thought. The sodium-lithium countertransport system and its physiological relative, the sodium-hydrogen antiporter may be useful as markers for diabetic nephropathy, but this remains controversial and an area of continued study. IMPLICATIONS: PREVENTION AND TREATMENT One of the goals of research in diabetic nephropathy is to be able to predict the subset of patients at highest risk for the development of advanced diabetic nephrop-

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athy, so that preventative measures can be focused on this group. For example, the Diabetes Control and Complications Trial has recently shown that strict metabolic control can reduce the risk of microalbuminuria by more than one-third, and was able to reduce the prevalence of albuminuria by more than one-half.2s Such therapy, however, is extremely difficult and increases the risk of hypoglycemia. Thus, it might be useful to direct this treatment toward those patients who are at increased risk for the development of diabetic nephropathy and other complications. Because of the relationship between hypertension and nephropathy, treatment of nephropathy and hypertension appear to be interdependent. In 1976, Mogensen 52 showed that 2 months of antihypertensive treatment lowered the albumin excretion rate of patients with diabetic nephropathy. Later, short-term studies showed that treatment with a specific type of antihypertensive drug, angiotensin-converting enzyme inhibitors (ACE inhibitors), significantly reduced the rate of decline of renal function.53*54 In addition, it has been suggested that treatment with captopril, an ACE inhibitor, actually affects renal hemodynamics in a way that it protects renal function.55 More recently, two studies of IDDM patients with nephropathy showed that treatment with captopril significantly retarded the decline in renal function independently of its effect as an antihypertensive agent.56,57In fact, the Collaborative Study group suggested treatment with captopril even in normotensive IDDM patients with clinically evident nephropathy. Also, as sodium and water retention have been shown to help initiate and aggravate hypertension, combining ACE inhibitors or calciumchannel blockers with diuretics has been shown to be effective in postponing renal insufficiency in IDDM patients with nephropathy.58 Finally, ACE-inhibitor treatment has been shown to prevent progression to proteinuria in normotensive, NIDDM patientss9 The early stages of diabetic nephropathy may be treatable or even preventable by the administration of ACE inhibitors in normotensive, normoalbuminuric IDDM patients who have exhibited markers for diabetic nephropathy. Such drugs are expensive, however, and patients treated with those agents are at increased risk for the development of hyperkalemia and other side effects of the treatment. Once again, the discovery of more accurate predictors for diabetic nephropathy would provide a means of avoiding indiscriminate use of such agents in patients with little risk for developing diabetic nephropathy. ACKNOWLEDGMENT This work was supported by a Robert C. Bates Fellowship from Yale University and NIH grant RR06022. Special thanks to Dr. Giancarlo Viberti, Dr. Carl Erik Mogensen, and Dr.

36

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AND TAMBORLANE

sen T, Kofoed-Enevoldsen: Albuminuria reflects widespread vascular damage. Diabetoloxia 32:219-226,1989.

Hans-Henrik Parving for allowing the primary author access to their laboratories to carry out this project.

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