Intermittent diabetic microalbuminuria: Association with blood pressure, glycemic control, and protein intake

IntermittentDiabeticMicroalbuminuria: Associationwith BloodPressure, GlycemicControl,and ProteinIntake Mark E. Cooper, MB BS, Richard C. O’Brien, MB BS, Robin M. L. Murray, MD, Ego Seeman, MB BS, George Jerums, MD

Endocrine Unit, Department of Medicine, Austin Hospital, University of Melbourne, Victoria, A us tralia.

The factors associated with intermittent nilcroalbuminuria were studied over 7 years In 49 Type I and 53 Type II diabetics who had normal initial albumin clearance. Fasting plasma glucose, HbA,, 24 hour urlnary glucose, blood pressure, protein Intake (24 hour urinary urea), and the renal clearance of albumin, transferrin, and IgG, as well as total proteinuria, were assessed every 3-8 months. Fifteen Type I and 11 Type II diabetics had 40 and 31 episodes, respectively, of intermittent microalbuminuria, defined as an albumin clearance >ll nl/sec, without progressing to persistent microalbuminuria. Rises In transferrin and IgG clearance paralleled albumin clearance in both Type I and Type II diabetics. There were no significant changes in blood pressure or glycemlc control during episodes of intermittent microalbumlnuria. However, In Type I dlabetlcs, intermittent microalbuminurla was associated with higher levels of urinary urea excretion. This study raises the possibility that increased protein intake may participate in the development of nephropathy in Type I diabetes. (The Journal of Diabetic Complications 3;2:92-98, 1989.)

INTRODUCTION Persistent and intermittent Albustix@ (Ames Company, Miles Labs, Elkhart, IN) positive proteinuria have been described as separate phases of established diabetic nephropathy.l.2 These phases are preceded by persistent microalbuminuria in Type P-5 and Type II diabetes.6 Intermittent microalbuminuria has been described previously as occurring after cessation of insulin therapy7 and after acute glucose and protein loads.8 It can also occur in association with increased urine flow rates,9 exercise,lO,ll urinary tract infection, cardiac failure, and hypertension.12 However, the occurrence of intermittent microalbuminuria as part of the natural history of diabetic renal disease has not been studied previously. Its relationship to persistent microalbuminuria and the ultimate development of overt diabetic nephropathy is not known. The present study was designed to analyze the relationship of protein intake, blood pressure, and glycemic control to episodes of intermittent microalbuminuria. The study was performed in a group of 49 Type I and 53 Type II diabetics who had normal baseline albumin clearances. This group comprised the majority of a previously described cohort, which exhibited four distinct longitudinal patterns of proteinuria: minimal, intermittent, progressing, and established.13

SUBJECTS

Submitted for publication in March 1988; accepted in revised form in June 1988. Reprint requests: Dr. M. E. Cooper, Endocrine Unit, Austin Hospital, University of Melbourne, Heidelberg, 3084, Victoria, Australia.

92

In 1971, a Diabetes Clinic was set up at the Austin Hospital, and from 1973 to 1979 regular clinic attenders were recruited into an open-ended study of evolving diabetic nephropathy. In this study of the natural history of diabetic nephropathy, the initial plan was to examine renal protein handling in diabetes and to document changes in the selectivity of proteinuria as determined by IgG/albumin and IgG/transferrin clearance ratios. Serial measurements of blood pressure and glycemic control were also made to assess their role in evolving proteinuria. Glycosylated hemoglobin measurements were added when this technique became available in 1978. In 1983, it was decided to study protein intake prospectively and to evaluate protein intake from data obtained

INTERMITTENTDIABETICMICROALBUMINURIA

over the preceding decade, using urinary urea excretion as an index of dietary protein intake.14 There has been no significant change in the protein content of diets recommended for the treatment of diabetes in Australia from 1970 to 1987.l5 At recruitment, patients were asked to attend every three months for at least 3 years. Patients were enrolled independently of the type, age of onset, or duration of diabetes. Patients were excluded if they already had established renal impairment (serum creatinine > 0.2 mM), known nondiabetic renal disease, or uncontrolled cardiac failure. The presence of other medical illness or therapy unrelated to diabetes was not a reason for exclusion. Classification of patients as Type I or Type II diabetics was according to the criteria of the National Diabetes Data Group.16 Patients were designated as Type I if they had persistent ketonuria prior to insulin therapy, weight loss on presentation, and insulin dependency within one month of diagnosis. All Type I diabetics had fasting C-peptide levels less than 0.05 nmol/L (NOVO lndustri A/S, Denmark; normal range, 0.18-0.63 nmol/L). All other patients were classified as Type II diabetics. All insulin treated patients were receiving subcutaneous insulin once or twice daily. Twenty-four hour urine samples were collected every three months at home in the absence of severe or prolonged exercise and stored frozen in aliquots. Blood was sampled at the end of each collection; urine microscopy and culture were performed on samples from each subject to exclude infection. From an initial cohort of 144 patients, 113 fulfilled the criteria of completing six separate tests of proteinuria and renal function spanning at least 36 months of follow-up evaluation (Figure 1). Eleven diabetics with initially ele-

STUDY PROTOCOL All subjects: 6 estimates of proteinuria and renal function with minimum follow up of 36 months

49 (53)

4 (7)

[initial albumin clearance c 11 n&x]

/ 15 (11) INTEFWnTEWr MICROALBUMINURL4 [two or more episodes of albumin clearance > 11 nl/sec]

EXCLUDE [initial albumin clearance > 11 nlhec]

\ 34 (42) EXCLUDE [one or no episodes of albumin clearance > 11 nYsec]

FIG. 1 Numbers shown are for Type I diabetics, for Type II diabetics in parenthesis.

with numbers

vated albumin clearance (>ll nl/sec, equivalent to a urinary excretion rate of 30 pglmin) were excluded, leaving 49 Type I and 53 Type II diabetics. Subsequently, 13 subjects who progressed to persistent microalbuminuria during the study (three elevated albumin clearances documented over a 12 month period) were excluded. The two groups excluded from the present study correspond to the progressing and established proteinuria groups described previously.l3 From the remaining cohort, a group of diabetics was identified in which each subject had at least two episodes of intermittent microalbuminuria, defined as an albumin clearance of greater than 11 nl/sec, without evidence of progression to persistent microalbuminuria.

MATERIALS

AND METHOOS

The renal clearances of albumin, transferrin, and immunoglobulin G (IgG) were determined after measurement of the individual protein concentrations in urine and plasma. Transferrin, IgG, and urinary albumin assays were performed by a coated tube radioimmunoassay on dialyzed urine and plasma.17 Serum albumin was measured by an autoanalyzer technique. Antibodies to individual proteins were obtained from Silenus Corporation, Melbourne, Australia, and 1z51-labeled tracers were prepared by the Chloramine T method.ls The interassay coefficients of variation were 10.9O/bfor albumin (n = 14), 14.6% for transferrin (n = 15) and 15.5% for IgG (n = 14); the intra-assay coefficients of variation ranged from 4-6% for individual renal protein measurements. The sensitivity of all three assays was adequate to detect specific protein levels in the urine of normal as well as diabetic subjects. The detection limit for albumin was 5ng/ml, and Albustix negative urine samples were therefore diluted l/50 and l/100 in assay buffer prior to assay. Total proteinuria was measured in 24 hour urine samples by the fluoresamine method,lg with an interassay coefficient of variation of 8.8% (n = 14). Albustix measurements were performed on the same specimens and termed positive if greater or equal to 30 mg/dl. Plasma and urinary glucose were measured by a glucose oxidase technique.20 Stable glycosylated hemoglobin was measured by the thiobarbituric acid technique from 1978 to 19812’ and after 1981 by column chromatography preceded by a dialysis step.22 There was a close correlation between the two methods (r = 0.90, n = 35, p < 0.01). Urinary and serum creatinine concentrations were measured by an autoanalyzer technique (Beckman Astra 8). Glomerular filtration rate measured by the ggmTechnetium diethylene triamine pentaacetic acid method23 correlated closely with creatinine clearance (r = 0.80, n = 33, p < 0.01); all patients had at least six measurements of creatinine clearance. Urinary urea was measured by autoanalyzer to assess protein intake, since it has been shown that urinary urea is proportional to dietary protein intake.14 Data for urinary urea excretion in each subject were corrected for 24 hour urinary creatinine excretion on the day of collection, based on the overall mean 24 hour urinary creatinine excretion for that subject. Infected samples were excluded from the analysis. Samples were stored at -20°C to prevent

COOPERET AL.

falsely low urea levels because of urease splitting organisms. Twenty-four hour urinary volume was recorded as a marker of urinary flow rate. Supine blood pressure was measured every three months, and the mean of three measurements over a 10 minute period was determined.

STATISTICAL

ANALYSIS

Individual episodes of elevated albumin clearance (> 11 nl/sec, equivalent to an albumin excretion rate > 30 pg/min) were identified in subjects with intermittent microalbuminuria. Data were pooled before and after each episode and compared with data during episodes. Analysis was performed by Student’s t-test for normally distributed variables. Parameters of proteinuria, 24 hour urinary urea and glucose excretion were not normally distributed and were therefore analyzed after logarithmic transformation.24 Some of the episodes of intermittent microalbuminuria were associated with elevated albumin clearance before and after the peaks. Therefore, episodes with normal albumin clearance before and after the peaks were subjected to a further analysis for evaluation of 24 hour urinary urea excretion. Mean values with standard error of the mean were used, but geometric means and tolerance factors are shown for variables that are not normally distributed. To further evaluate the association of protein intake with albuminuria, a correlation analysis was performed between albumin clearance and urinary urea excretion. This was performed after these nonparametrically distributed variables were normalized by logarithmic transformation. To avoid the effects of potentially confounding variables, such as blood pressure and glycemic control, stepwise multiple regression analysis was performed to identify the relationship between proteinuria and protein intake.25

TABLE 1

Number Sex (M/F) A9e (yr) Disease duration at start of study (vr) Follow-up evaluation (yr) Number of observations Creatinine clearance (ml/min/l.73m2) Weight (kg) Systolic Blood Pressure (mmtig) Diastolic Blood Pressure (mmHg) HbAl (%) Fasting plasma glucose (mg/dl) Urinary glucose (g/d)

TvDe II

49 33/16 29 f 2 6.6 f 1.1

53 29124 57fl’ 5.9 f 1.0

7.7 f 0.3 22 f 2 96f3

6.4 * 0.3t 20 f 1 88 f 3

73 f 2 13of2 81 f 1 10.5 * 0.2 220 + 9 37

77 f 2 147 f 2’ 86f 1’ 10.1 * 0.3 216 + 8 12

Data are represented as mean f SEM, except for urinary glucose, which are shown as medians, p < 0.01, t p < 0.05. Data were derived from observations on individual subjects collected throughout the study period. The normal range for creatinine clearance is 90-150 ml/min/1.73m2 and for HbA, is 5-896. l

Clinical Characteristics of Subjeots with Intermittent Mlcroalbumlnuria TYW

Number of Subjects Number of Episodes Sex (M/F) Age (yr) Disease duration at start of study (yr) Follow up (yr) Number of observations Creatinine clearance (ml/min/l.73m2) Weight (kg) Systolic Blood Pressure (mmlig) Diastolic Blood Pressure (mmfig)

HbAl (%) Fasting plasma glucose (mg/dl) Urinary glucose (g/day) Urinary urea ex;,etion (g/day) a3

1

15

TYW

11

40 817 31 *5 8.8 * 2.8

11 31 813 57 * 2 6.0 f 1.8

8.6 f 0.6 27 f 3 92 f 5

6.5 f 0.7 21 f3 94 f 8

69 f 3 131 f 4 82 f 2 10.5 f 0.4 234 f 14 31 20.1 14.2 32.5

78 f 5 147f3 86 f 2 11.6 f 0.5 248 f 23 42 21.9 16.2 33.8

FormatandsymbolsareasinTablel. Urinaryureaisshownas a median as well as first (al) and third quartiles (03).

RESULTS Clinical Characteristics The clinical characteristics of the study cohort, excluding subjects with initially elevated albumin clearance, are shown in Table 1. The mean duration of the study was 7 years (range, 3-12 years), and the mean number of observations was 21 per subject (range, 6-40). Type II diabetics were older and had higher systolic and diastolic blood pressures but had similar levels of glycemic control. The clinical characteristics of subjects with intermittent microalbuminuria are shown in Table 2. There were 40 episodes of intermittent microalbuminuria in 15 Type I and 31 episodes in 11 Type II diabetics. The number of episodes of increased albumin excretion expressed as a percentage of the total number of observations was 14% in Type I diabetics and 18% in Type II diabetics. There

Clinical Characteristics of Total Study Cohort Tvoe I

TABLE 2

were no significant differences in age, duration of disease, duration of follow-up period, number of observations, creatinine clearance, blood pressure, or glycemic control between subjects with intermittent microalbuminuria and the rest of the cohort.13 In 20 age-matched nondiabetics, many of whom were related to subjects within this cohort, we were unable to detect episodes of microalbuminuria.

Analysis of Episodesof Intermittent Microalbuminuria All the protein clearances were elevated during episodes of intermittent microalbuminuria compared with values before and after each episode, as shown in Figure 2. Approximately fourfold elevations of albumin and transferrin clearance occurred during intermittent microalbuminuria. with approximately twofold elevations of IgG clearance, in both Type I and Type II diabetics. Seven of 40 episodes of albuminuria in Type I and four of 31 episodes in Type II diabetics were Albustix positive. In Type I but not Type II diabetics there was a significant

95

INTERMITTENT DIABETIC MICROALBUMINURIA

f =

600 20

8 8 I t c 0 B t

400 10 200

n ”

Albumln

Trantferrin

W

Total Proteinurla

600

.. ..

600

400

200

Albumin

Transferrln

W

Total Proteinuria

FIG. 2 The open columns represent the geometric means of specific protein clearances during episodes of intermittent microalbuminuria. The hatched columns represent the geometric means of the pooled data for specific protein clearances immediately before and after episodes of intermittent microalbuminuria. Tolerance factors of the geometric means are represented by vertical lines. Normal ranges for albumin, transferrin and IgG clearances, were <5.5, <5.3, and C4.8 nllsec. respectively (mean + 2SDJ. p < 0.05, ‘*p < 0.01. l

elevation in total proteinuria during intermittent microalbuminuria. In both types of diabetics, the IgG/albumin clearance ratio decreased during episodes of intermittent microalbuminuria (Type I, -33 f 13%, p < 0.01; Type II, -37 IfI 7%, p < 0.01). Glycemic control (serum glucose and glycosylated hemoglobin), blood pressure, renal function, and urine volume showed no significant differences during intermittent microalbuminuria (Table 3). There was a large variation in 24 hour urinary urea excretion (Table 2), but

TABLE 3

there was no significant difference between Type I and Type II diabetics. Twenty-four hour urinary urea excretion was significantly increased during intermittent microalbuminuria in Type I but not Type II diabetics, when compared to the mean of two measurements immediately before and immediately after each episode (Figure 3). In a further analysis, data were excluded if either the pre- or the post-episode albumin clearance exceeded 11 nl/sec, leaving 28 and 13 episodes in Type 1 and Type II diabetics, respectively. This analysis confirmed the increase in urinary urea excretion during intermittent microalbuminuria (pre- and post-episode VS. during episode: Type I, 16.2 vs. 22.6 g/24 hours, p < 0.05; Type II; 21.4 vs. 21.3 g/24 hours, n.s., medians shown). As outlined above, a correlation analysis was performed between albumin clearance and urinary urea excretion. In Type I but not Type II diabetics this confirmed a significant association between protein intake and albuminuria (Type I, r = 0.31, p < 0.01; Type II, r = 0.03, ns.). To determine if the association of urinary urea with albuminuria is independent of glycemic control and blood pressure, stepwise multiple regression analysis was performed, confirming the association in Type I but not Type II diabetics (Type I, F = 5.8, p < 0.05; Type II, F = 1.1,n.s.).

DISCUSSION This study is the first to describe the associations of intermittent microalbuminuria occurring as part of the natural history of diabetes. Definition of the role of intermittent microalbuminuria as either a phase or as a predictor of the ultimate development of overt diabetic nephropathy requires further long-term studies. In our own study of 13 subjects who have progressed from normal to persistent microalbuminuria, episodes of intermittent microalbuminuria were documented in five subjects.13S*6In these five subjects, 14 discrete episodes of increased urinary albumin excretion were detected before the development of persistent microalbuminuria.26 It is possible that intermittent microalbuminuria precedes persistent microalbuminuria in an analogous manner to the progression from intermittent proteinuria.2s27

to persistent Albustix

positive

Blood Pressure, Glycemic Control and Renal Function in Intermtttent Mkroatbumtnurta

Pressure (mmHg) Diastolic Blood Pressure (mmHg) Fasting plasma glucose (mg/dl)

Systolic Blood

HbAl (%) Urinary glucose (g/day) Creatinine clearance (ml/min/l.73m2) Urinary volume (ml/day) Format and symbols are as in Table 1.

during episode 136 f 4 85 f 3 225 f 16 10.1 f 0.3 (0-::53) 95 f 4 1907 f 189

pre/post episode

238 f 11 10.1 f 0.2

during episode 147f3 85 f 2 182 f 16 10.5 f 0.4

146f3 67 f 2 187 f 11 10.4 f 0.2

(1 -Z22) 93 f 3 1918 f 121

(z75) 101*7 1931 f 149

(O-E74) 96f4 1921 f 110

‘episbde 130f2 81 f 1

96

C9OPER ET Al.

urlmy

urea (g/24 hn)

20

I

<-L

c :1 I

l

TYPE I n=40 n=60

TYPE n=31

II n=62

FIG. 3 The open columns represent the geometric means for 24 hour urinary urea excretion during episodes of intermittent microalbuminuria. Hatched columns represent the geometric means of pooled data for 24 hour urinary urea excretion immediately before and after episodes of intermittent microalbuminuria. The tolerance factors of the geometric means are represented by vertical lines. lp < 0.05.

In the present study, episodes of intermittent microalbuminuria were associated with elevations of transferrin and IgG clearances and total proteinuria (Figure 2). This would justify the use of the term microproteinuria to describe protein excretion in these patients. However, there was a significant reduction in the selectivity index calculated as the IgG/albumin clearance ratio during episodes of intermittent microalbuminuria. This is consistent with the previously documented alteration in selectivity in diabetics with Albustix negative proteinuria2* and with the onset of persistent microalbuminuria,2g which may be related to the loss of negative charge on the glomerular fifter.30 However, the transient nature of intermittent microalbuminuria is difficult to reconcile with a hypothesis based purely on structural factors, suggesting that other variablessuch aschanges in glomerular pressure and flow may be involved. Although it is theoretically possible that proteinuria may be caused by renal tubular mechanisms, previous studies of diabetic microalbuminuria have shown that increases in albumin and IgG excretion are not accompanied by increases in p2 microglobulin excretion.31J2 Raised p2 microglobulin excretion in association with microalbuminuria has only been documented in recently diagnosed diabetics33 or in longer term diabetics after deliberate reduction or interruption of insulin therapy.’ This evidence suggests that intermittent microalbuminuria is probably of glomerular origin. This study was unable to detect a change in creatinine clearance (assessed over 24 hours) during episodes of intermittent microalbuminuria, but it is possible that short-term changes in glomerular filtration rate were not detected by the technique used. However, a recent study suggests that Type I diabetics may have an altered renal hemodynamic response to protein loading.a4 In that study, glomerular filtration rate decreased in diabetics, whereas it increased in control subjects in response to a protein challenge. In another study, moderate protein restriction in Type I diabetics without microalbuminuria

was associated with a decrease in glomerular filtration rate, which was similar to that seen in control subjects.35 These conflicting data on the effects of alterations in protein intake over hours or weeks upon glomerular filtration rate in diabetic and normal subjects make it difficult to predict the effect of spontaneous alterations in protein intake on the natural history of renal function in diabetes. In the present study, the only significant association with intermittent microalbuminuria was 24 hour urinary urea excretion, which is a marker of dietary protein intake.36J7 There was a large range in urinary urea excretion in the present study, suggesting a wide variation in the protein content of diets consumed by Australian diabetics. The values for urinary urea excretion were similar to those found in a previous study of the effects of dietary protein modification on renal function in a group of Type I diabetics.zB In that study, levels of urinary urea excretion were considered to reflect average dietary protein intake in Type I diabetics. Throughout the study, there were no significant alterations in the recommended protein intake for Australian diabetics.‘5 The half life of urea in man is 5-9 hours,3g which suggests that 24 hour collections of urine reflect dietary protein intake during the preceding day as well as the day of urine collection. The failure to find an association between high urinary urea levels and intermittent microalbuminuria in Type II diabetics cannot be readily explained. Higher levels of blood pressure and the presence of nephrosclerosis or lower urinary tract disease are possible confounding factors that could be associated with intermittent albuminuria in older Type II diabetics. Our own recent studies suggest that early renal dysfunction in Type II diabetics differs from the process observed in Type I diabetics, perhaps because of the presence of factors other than diabetic nephropathy.40 Another possible explanation is that the renal effects of a protein load are different in Type I diabetics. If this were so, increases in albuminuria in response to protein loads would not be expected in Type II diabetics or in normal subjects. In recent studies in normal subjects, acute protein loading resulted in an increase in glomerular filtration rate, but no increase in urinary albumin excretion.41 Acute protein loading studies of this sort have not been reported in Type II diabetics, but it is possible that decreasing renal mass with age may be a determinant of the renal response to a protein load.42 The present results are consistent with a relationship between dietary protein intake and nephropathy in Type I diabetes. Such a relationship has been suggested in experimental studies in which high protein diets have accelerated renal disease in streptozotocin diabetic rats.43 Clinical studies have also confirmed the role of protein intake by showing that low protein diets reduce proteinuria in patients with advanced diabetic nephropathy44 as well as in patients with microalbuminuria.45 In the present study, urinary urea levels in Type I diabetics during episodes of microalbuminuria exceeded levels before and after the episodes by approximately 100 mmoV24 hours. It is of interest that urinary urea excretion in patients with renal disease treated by long-term protein restriction has also

INTERMITTENTDIABETICMlCROALf3UMlNURlA

been shown to differ by about 100 mmoV24 hours from values recorded in control patients.46 If the intermittent microalbuminuria described in this study represents an early phase of diabetic nephropathy, it would follow that avoidance of high protein intake or modest dietary protein restriction should be considered as an early step in the treatment of diabetic renal disease. It has been suggested that high protein intake increases albuminuria by causing a rise in intraglomerular pressure.43 It is therefore logical to consider therapeutic modalities in diabetic nephropathy that lower intraglomerular pressure. These options include not only a low protein diet, but also angiotensin converting enzyme inhibition.47 Subjects with intermittent microalbuminuria as a group did not have higher blood pressure, altered creatinine clearance, or worse glycemic control than the remaining subjects in the cohort. In addition, episodes of intermittent microalbuminuria were not associated with variations in glycemic control, blood pressure, or urinary volume. In a previous study, tight glycemic control did not influence albuminuria in 12 subjects with intermittent Albustix positive proteinuria.2 However, the importance of glycemic control as a cause of transient episodes of microalbuminuria cannot be excluded by the results of the present study. In a previous report, deterioration in metabolic control was associated with an increase in urinary albumin excretion.7 Studies of the temporal relationship between spontaneously occurring alterations in glycemic control or blood pressure and intermittent Albustix positive proteinuria have not yet been performed. However, previous workers have documented the association of a small rise in blood pressure with persistent microalbuminuria.48,49 At this stage, the possibility cannot be excluded that intermittent microalbuminuria is not specifically related to diabetic nephropathy. If this was so, episodes of intermittent microalbuminuria may merely reflect the large variability of albuminuria that has been reported in short-term studies in normal subjects50 and in diabetics with incipient nephropathys’ as well as in studies in long-term diabetics with established nephropathy.5’ Further longitudinal follow-up study of these patients will determine the prognostic significance of intermittent microalbuminuria in diabetes.

ACKNOWLEDGMENTS We wish to thank Professor advice and Mrs. J. Holland tance.

John J. McNeil for statistical for expert secretarial assis-

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41.

42. 43.

44.

45.

46.

47.

48.

49.

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51. 52.

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