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*
Division of Pediatric Nephrology, Stanford University School of Medicine,
Stanford, California
Division of Nephrology, Stanford University School of Medicine, Stanford,
California
Department of Statistics, Stanford University School of Medicine,
Stanford, California
§
Phoenix Epidemiology and Clinical Research Branch, National Institute of
Diabetes, Digestive and Kidney Diseases, Phoenix, Arizona.
Correspondence to Dr. Kevin V. Lemley, Division of Pediatric Nephrology, Room G306, Stanford University Medical Center, Stanford, CA 94305-5208. Phone: 650-723-7903; Fax: 650-498-6714; E-mail: klemley{at}leland.stanford.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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300 mg/d) in half of the
participants by 7 yr of follow-up. The size selectivity of the glomerular
barrier was also investigated using dextran sieving and pore theory. Whereas a
comparison group of macroalbuminuric Pima Indians had an excess of large pores
that served as a macromolecular "shunt," individuals with
microalbuminuria had a shunt size no different from long-term diabetic,
normoalbuminuric control subjects. An abrupt transition from little or no
relationship to a highly significant and positive relationship between
increasing albuminuria and shunt size occurred at an albumin-to-creatinine
ratio of approximately 3000 mg/g. Shunt size in macroalbuminuric individuals
correlated with the extent of foot process broadening. Podocyte foot processes
in microalbuminuric participants were not different from those in control
subjects. In conclusion, although microalbuminuria in type 2 diabetic Pima
Indians often heralds progressive glomerular injury, it is not the result of
defects in the size permselectivity of the glomerular barrier but rather of
changes in either glomerular charge selectivity or tubular handling of
filtered proteins or of a combination of these two factors. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The dipstick test most commonly used to detect proteinuria first becomes positive when albumin concentrations exceed 30 mg/dl, corresponding to a urinary albumin excretion rate of more than 300 mg/24 h. With more sensitive immunochemical assays, however, the upper limit of albuminuria in healthy populations is only approximately 30 mg/24 h (2). The intervening stage (30 to 299 mg/24 h) between normal levels of urinary albumin excretion and dipstick-positive albuminuria is known as microalbuminuria. Microalbuminuria has been shown to predict strongly later development of diabetic nephropathy (3), so persistent microalbuminuria is considered to reflect "incipient" diabetic nephropathy (4).
The foregoing understanding is based largely on studies of individuals with
type 1, insulin-dependent diabetes mellitus
(5). The degree to which
microalbuminuria also portends the development of diabetic nephropathy in type
2 diabetes has been studied less often. In large part, this is because type 2
diabetes, at least in Caucasian populations, usually is detected during the
sixth through ninth decades of life, when other processes that can result in
albuminuria, such as hypertension and renovascular disease, are prevalent. To
circumvent such complicating conditions, we studied the Pima Indians of
Arizona, in whom type 2 diabetes is common and occurs at a relatively young
age (6). Screening of this
population reveals a high prevalence of both microalbuminuria and
macroalbuminuria (i.e., albuminuria 300 mg/24 h)
(6,7,8).
Urinary albumin excretion within the microalbuminuric range in diabetic Pima
Indians is associated with a 10-fold higher risk of progression to overt
nephropathy (9), suggesting
that the development of microalbuminuria in this population is an incipient
form of nephropathy, as it is in type 1 diabetes.
To examine the natural history of microalbuminuria in diabetic Pima Indians, we followed for up to 8 yr the rate of urinary albumin excretion in 43 individuals with microalbuminuria on initial screening. In addition, to investigate alterations in glomerular capillary wall function that may underlie microalbuminuria and its evolution to macroalbuminuria, we used the fractional urinary clearance of neutral dextrans to probe the size-selective barrier. We then analyzed the dextran fractional clearances with a mathematical model of hindered transport through a porous barrier to assess quantitatively the size-selective properties of the glomerular filtration barrier. We also performed a morphometric analysis of the glomerular capillary wall in a subset of these participants to clarify the structural basis for barrier dysfunction. Our findings form the basis of this article.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eighty-seven (39%) of those screened initially were judged to have elevated urinary albumin excretion because the geometric mean of their three A/C ratios was 30 mg/g or more. By comparison, an earlier study (8) of Pima Indians of similar age with normoalbuminuria at study entry and up to 11 to 12 yr of follow-up found a total cumulative incidence of abnormal albuminuria of approximately 42%, with 90% of these cases in the microalbuminuric range.
In this study, final assignment to a particular study group was based on the geometric mean of albumin excretion during initial screening and on two subsequent measurements made during urinary clearance studies separated by 6 mo. Participants were classified as having microalbuminuria if at least two of these three A/C ratios were between 30 and 299 mg/g.
Of 53 participants who met this definition, 43 completed at least 40 mo of
follow-up. They compose the MICRO group and are the principal subjects of this
article. Of these, 10 underwent a renal biopsy 3 to 4 yr after study entry.
Thirty-one other participants had macroalbuminuria, defined analogously as an
A/C ratio of 300 mg/g in at least two of the following: the initial
screening (geometric mean of three values) and the subsequent two follow-up
studies. Each of these participants had follow-up of more than 48 mo, and they
form the MACRO group, a comparison group for studies of the mechanism of
proteinuria. Ten of these participants also underwent renal biopsy 3 to 4 yr
after study entry. Some results of the functional and structural studies of
the MACRO group have been reported previously
(11,
12).
There were also two control groups. One was a functional control group composed of 11 Pima Indians who had longstanding type 2 diabetes and normal levels of urinary albumin excretion on screening (NORMO). They underwent an evaluation of glomerular function on a single occasion and serve as a non-nephropathic control group for studies of glomerular hemodynamics and barrier function. The other control group, a structural control group, consisted of 10 Pima Indians who were not included in the original screening study. These individuals had type 2 diabetes of recent onset with a normal A/C ratio. They underwent renal biopsy and an evaluation of glomerular function 3 ± 2 SD yr after detection of type 2 diabetes, and they provide non-nephropathic control values for the morphometric analysis of glomerular structure. Structural and functional features of these groups have also been reported previously (11, 12). They are reported again here as control groups for the MICRO participants.
All participants received medical treatment independent of the study protocol by their usual Indian Health Service physicians. During the first 4 yr of this study, these physicians were asked to avoid, if possible, prescribing to study participants medications that might alter glomerular function, such as angiotensin-converting enzyme (ACE) or cyclooxygenase inhibitors. Only one MICRO participant had received ACE inhibitor therapy by the time of the 4-yr follow-up study. Otherwise, treating physicians were allowed to use calcium channel blockers, diuretics, and ß-adrenergic blockers to control BP. Participants gave informed consent to be studied according to a protocol approved by the following participating institutions: Stanford University, CA; the National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD; and the Gila River Indian Community Tribal Council, Sacaton, AZ.
Study Protocol
Both the MICRO and MACRO groups underwent longitudinal evaluation as
reported elsewhere (10). The
urinary A/C ratio, BP, fasting blood glucose, and hemoglobin A 1C
levels were measured at 3- to 6-mo intervals throughout the study. BP were
measured to the nearest 2 mmHg using a mercury sphygmomanometer with the
participant seated. Diastolic pressure was determined as K5. Blood was drawn
for plasma renin activity determination after the participant had been upright
for 1 h. Serial urinary clearances of iothalamate and para-aminohippuric acid
(PAH) and fractional clearances of albumin (
Albumin) and IgG
were performed at 6- to 12-mo intervals. Of the 43 members of the MICRO group,
39 were followed beyond 48 mo. The total observation period for these
individuals ranged between 60 and 99 mo.
The iothalamate and PAH clearances were not normalized by body surface area
to avoid a spurious downward drift in these values with time as increasing
obesity led to an increment in body surface area. Fractional clearances of
dextrans of graded sizes (32 to 60 
effective
molecular radius) were performed in the MICRO group at the 48-mo examination,
i.e., approximately midway through the period of longitudinal
evaluation. As reported previously
(11), fractional dextran
clearances were also performed at the 48-mo study in the MACRO group and are
presented here again to permit a comparison of glomerular permselective
properties between the MICRO and MACRO groups.
Twenty of the study participants—10 with microalbuminuria and 10 with macroalbuminuria—had a renal biopsy performed between 36 and 48 mo after entry into the study. Participants who underwent renal biopsy were among the first 51 participants in the larger study to volunteer for this procedure. They were not selected on the basis of any clinical or physiologic attributes. A morphometric analysis of the biopsy material was performed to characterize the structure of the glomerular filtration barrier and to relate these findings to barrier permselective function.
Laboratory Procedures
The concentrations of albumin and IgG in serum of all participants and in
the urine of nephropathic participants were determined by immunoprecipitation
using a semiautomated nephelometer (Behring Diagnostics, Somerville, NJ). The
concentrations of albumin and IgG in the urine of non-nephropathic
participants were often at or below the lowest standard of the nephelometric
assay (0.2 mg/L) and were determined instead by RIA or enzyme-linked
immunosorbent assay (10). All
samples were stored at -70°C until the day of assay, which was performed
within 30 d of the collection of the sample.
GFR was determined as the urinary clearance of iothalamate. The rate of renal plasma flow was calculated from the urinary clearance of PAH. A high-pressure liquid chromatography (HPLC) system with an ultraviolet light detector was used to assay iothalamate and PAH at 236 nm (#LC-6A Shimadzu Instruments, Columbia, MD). Ultrafiltrates of serum and diluted urine were injected onto a C18 reversephase column (Ultrasphere 5 µ, Beckman Instruments, San Ramon, CA). The mobile phase was 3.5% acetonitrile in 10 mM triethylamine at a pH of 3.0. The flow rate was 1.0 ml/min. Iothalamate and PAH concentrations were determined from the peak areas of each solute, corresponding to column retention times of 14 and 10 min, respectively (13).
Size separation of dextrans in ZnSO4-precipitated urine and
serum was achieved by HPLC using an automated injector (WISP 710, Waters
Corporation, Milford, MA) and two size-exclusion columns in series
(Ultrahydrogel 500 and 250, Waters Corp.). Dextran concentration was measured
using a refractive index detector (#RID-6A, Shimadzu Instruments).
Chromatograms were divided into four slices per minute for the 40-min run
(#4270 integrator, Spectra-Physics, San Jose, CA). The columns were calibrated
with five narrowly dispersed dextran fractions of known molecular weight (9.9,
25.6, 40.4, 53.3, and 72.1 kD; kindly provided by Dr. K. Granath, Pharmacia
Fine Chemicals, Uppsala, Sweden). The molecular radius (rS,
) of each dextran fraction was calculated from
its molecular weight (MW, Daltons) using the following equation:
 |
) for each
2 interval was calculated by dividing the
urine-to-plasma dextran concentration by the urine-to-plasma iothalamate
concentration:
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varied between 7.7% and 12.4%. Creatinine concentrations in urine and serum were determined by a modified Jaffé reaction, glycosylated hemoglobin by agar gel electrophoresis or HPLC, and plasma oncotic pressure by membrane osmometry (Wescor 4400 colloid osmometer; Wescor Inc., Logan, UT). Plasma renin activity was determined with endogenous substrate by RIA (PRA kit, Clinical Assays, Stillwater, MN). The interday coefficient of variation for the renin assay was 11.4%.
Computations Based on Pore Theory
We used a hydrodynamic model of hindered solute transport through
water-filled, cylindrical pores to describe the observed transport of dextrans
across the glomerular capillary wall
(14). In this model, the
capillary wall is represented as a heteroporous membrane perforated by a
population of smaller pores with a lognormal distribution of radii and a
parallel population of large-radius nondiscriminatory pores (shunts). We
selected this representation because, as we reported recently, it best fits
dextran sieving data for normal and proteinuric participants
(15). The lognormal-plus-shunt
model contains three parameters: u, the mean pore radius; s,
a measure of the breadth of the lognormal distribution (ln s is the
SD of the distribution of ln r, where r is pore radius); and
0, the shunt parameter, which reflects the fraction of total
filtrate volume passing through the non-restrictive portion of the membrane.
The approach that we used for calculating these intrinsic membrane parameters
(13) separates their effects
on sieving coefficients from those owing to changes in GFR, renal plasma flow,
oncotic pressure, and/or the glomerular transcapillary hydraulic pressure
difference, 
P. Because P cannot be determined in humans, we
assumed it to be 40 mmHg
(10).
Glomerular Morphometry
Renal biopsy was performed under ultrasound guidance using a 14-gauge
needle (Tru-Cut; Baxter Healthcare Corp., Deerfield, IL), in a subset of the
participants as a part of a previous study
(12). Biopsy cores were
embedded in Epon and sectioned in their entirety at 2.5-µm intervals. Every
fourth section was mounted and stained with toluidine blue so that the entire
core could be viewed at 10- µm intervals. An average of 92 sections were
examined in each participant. Every other mounted section was photographed,
and a series of prints (approximately 40x) were prepared for use as
photographic maps of the serial sections from each core. Only glomeruli that
were contained entirely within the core—and thus represented completely
by profiles on the serial sections—were studied. Midsections from three
patent glomeruli in each biopsy were re-embedded in Epon and thin-sectioned
for electron microscopy. The following quantities were determined from light
and electron microscopic images using standard stereologic techniques
described previously (12):
foot process width, glomerular basement membrane (GBM) thickness, glomerular
visceral epithelial cell number, and density. Briefly, foot process width was
determined on foot processes overlying the GBM of the filtering capillary
surfaces by dividing the number of overlying slits by the length of the GBM
seen in cross section (subsequently multiplying by 
/4 to correct for
effects of the sectioning angle). GBM thickness was determined on higher-power
electron micrographs (approximately 9400x) using the method of
orthogonal intercepts. Epithelial cell volume density was determined using the
method of Gomez-Weibel
(12).
Statistical Analyses
Progression from microalbuminuria to macroalbuminuria was analyzed as
event-free survival curves by the Kaplan-Meier procedure. The influence of
several categorical variables (gender, presence of retinopathy) and continuous
variables (age, initial serum creatinine concentration, initial GFR and renal
plasma flow, initial HbA 1c, mean arterial pressure (MAP),
screening A/C ratio, duration of diabetes) was examined by a Weibull
regression model. Linear relationships between continuous variables were
examined by least squares linear regression or multiple linear regression. In
the case of piecewise linear regression for the analysis of the relationship
between shunt size and albuminuria in the combined MICRO+MACRO groups, the
best fit with two linear segments was defined as that which minimized the sum
of squared residuals for the equations: log10(0)
= a + b (log10(alb)) +
c (log10(alb/d))+ and
log10(0) = a + b
(log10(A/C)) + c
(log10({A/C}/d))+, where (x)+ = x
for x 0 and (x)+ = 0 for x < 0. The point of transition
from one linear regime to another was at A/C or alb =
d. Logarithmic transformation of variables was used to improve
normality of the distributions for this analysis. Differences between groups
were analyzed by unpaired t test, Mann-Whitney U test, or
Wilcoxon signed ranks test, depending on the distributions.
Scheffé's test was used for multiple
comparisons. Values are reported as mean ± SD or as median (range).
Analyses were made using SPSS Base 8.0 statistical software (SPSS Inc.,
Chicago, IL), SAS version 6.12 (SAS Inc., Cary, NC), or S-PLUS version 3.4.1
(Math-Soft Inc., Seattle, WA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Over the course of the first 4 yr of follow-up, the GFR declined only slightly from baseline values (158 ± 43 to 148 ± 40 ml/min, P = 0.03) in the MICRO group. Renal plasma flow rate declined an equivalent amount (852 ± 202 to 790 ± 174 ml/min, P = 0.03), leaving filtration fraction unchanged. BP did not change significantly with time (92 ± 9 mmHg at baseline to 91 ± 9 at follow-up). Five of 43 and 7 of 43 MICRO participants were receiving antihypertensive medication at baseline and at 4-yr follow-up, respectively. The urinary A/C and IgG/C ratios rose during follow-up: from an initial A/C of 81.0 (8.9 to 806) and IgG/C of 15.6 (4.3 to 244) at the baseline study to an A/C of 136 (15 to 4708) and IgG/C of 21.8 (1.8 to 621) at 4 yr (each P < 0.05). The fractional clearances of albumin and IgG in the MICRO group also increased significantly over the first 4 yr of follow-up. Fractional albumin clearance increased from a median value of 9.3 x 10-5 to 11.5 x 10-5 (P = 0.018). Fractional IgG clearance increased from 5.5 x 10-5 to 11.4 x 10-5 (P < 0.001). Analysis of the natural history of the fractional clearances of albumin and IgG after the 48-mo study was limited because 22 of the 43 MICRO participants were subsequently treated with ACE inhibitors, mostly starting 6 to 7 yr after study entry.
Actuarial analysis by Kaplan-Meier of the transition from microalbuminuria to macroalbuminuria is shown in Figure 1. MICRO participants progressed steadily to macroalbuminuria over the period of follow-up. The actual "event" of progression was defined in two ways: the time point at which the A/C ratio first exceeded 299 mg/g and the time point of onset of sustained macroalbuminuria, i.e., the time after which no further A/C ratios less than 300 mg/g were found. These bracketing definitions of the time of transition from microalbuminuria to macroalbuminuria were necessitated by the considerable variability in the rate of urinary albumin excretion caused by factors, such as exercise, hydration status, and posture, that are not directly related to disease progression (4). According to the definition of first development of (possibly transient) macroalbuminuria, 77% (95% confidence interval [CI], 60 to 86%) of microalbuminuric individuals had progressed by 84 mo of follow-up. By contrast, using the definition of progression to persistent macroalbuminuria, 46% (95% CI, 29 to 60%) of individuals had progressed by 84 mo (Figure 1). By the time of the physiologic studies at 48 mo, 11 of the 43 MICRO participants had progressed to persistent macroalbuminuria.
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The Kaplan-Meier survival curves represent the group average behavior and do not adequately reflect the substantial heterogeneity present within the population. For example, a small subset of eight individuals had stable microalbuminuria or even became normoalbuminuric over long-term follow-up (Figure 2, a and b). Several other distinct patterns of progression of albuminuria could also be delineated: slow, steady progression (Figure 2c), long-term stability followed by rapid progression (Figure 2d), and moderately rapid progression through macroalbuminuria to nephrotic-range proteinuria (Figure 2e).
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We performed a regression analysis on survival time to progression to persistent macroalbuminuria on the basis of the Weibull distribution. Variables included in the regression were screening A/C ratio; gender; presence of retinopathy; age; duration of diabetes; initial MAP, GFR, and renal plasma flow; and initial concentrations of HbA1c, total cholesterol, and creatinine. The only significant factors in the regression were MAP with a P = 0.040 and renal plasma flow with a P = 0.054. The initial MAP was significantly correlated with the MAP at 4 yr of follow-up (r = 0.426, P < 0.005), suggesting that an elevated MAP may be a stable characteristic of the subgroup with more rapid progression.
In addition to the regression analysis on the MICRO group as a whole, we separately analyzed the subgroup with the stable or improving patterns of albuminuria (n = 8) illustrated in Figure 2, a and b. We compared them by Mann-Whitney analysis with the remainder of the MICRO group (n = 35) who manifested increasing albuminuria (Figure 2, c through e). The stable subset had a lower initial GFR (120 ± 23 versus 167 ± 42 ml/min, P = 0.002) and renal plasma flow (660 ± 134 versus 903 ± 187 ml/min, P = 0.002), more advanced age (48 ± 9 versus 41 ± 9 yr, P = 0.031), and a lower plasma renin activity (0.64 ± 0.99 versus 1.47 ± 1.63 ng/ml per h, P = 0.02). Plasma renin activity in normoalbuminuric longterm diabetic participants is 1.30 ± 1.19 ng/ml per h. MAP was not different between the stable subgroup and the remainder of the MICRO group (P > 0.85).
Mechanisms of Proteinuria
Fractional clearances of neutral dextrans of graded sizes (dextran sieving
curves) were used to assess the size selectivity of the glomerular
permeability barrier after 4 yr of follow-up. In the macroalbuminuric
individuals, the dextran sieving curves at 4 yr showed a significant loss of
size restriction for larger dextrans compared with long-term normoalbuminuric
control subjects (Figure 3).
This was manifested by a selective increase in the size of the sieving
coefficients of large nearly impermeant dextrans at the high-radius end of the
sieving curve. The sieving curves for the MICRO participants, however, showed
no evidence of impaired size restriction after 4 yr of follow-up (data not
shown). Even when the MICRO group was subdivided into individuals who had
irreversibly progressed to macroalbuminuria by the time of the 4-yr dextran
clearance study (Figure 4;
n = 11) and those who were still at least intermittently
microalbuminuric (Figure 5; n = 32), there was no clear difference in their sieving curves in
comparison with normoalbuminuric control subjects. However, the 11 progressors
did show a trend toward depression of low-radius sieving coefficients and
elevation of high-radius sieving coefficients similar to that observed in the
MACRO group.
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Modeling
We used the "lognormal + shunt" model
(14) to analyze neutral
dextran sieving behavior of the NORMO, MICRO, and MACRO groups. The mean pore
radius u in the MACRO group was smaller and the distribution breadth
s was greater than NORMO control values
(Table 1). In addition, the
value for the shunt parameter 0 was significantly greater in
the MACRO group. In the MICRO group as a whole, the size of the shunt
parameter was not significantly greater than in the NORMO group. It is
interesting that despite their clearly abnormal level of albuminuria,
participants in the MICRO group who had not developed persistent
macroalbuminuria by 48 mo had virtually identical shunt magnitudes to the
normoalbuminuric control subjects: 0 of 0.00089 ±
0.00030 versus 0.00088 ± 0.00041, respectively. There was no
significant relationship between the shunt magnitude and the fasting blood
sugar concentration at the time of study (data not shown).
When the MICRO and MACRO groups were combined, however, a clear transition
was seen in the relationship between proteinuria (fractional albumin clearance
or A/C ratio) and shunt magnitude (Figure
6). One apparent outlier was removed from the 74 participants of
the combined group for this analysis. For lower levels of
albuminuria—albeit levels still clearly in excess of fractional albumin
clearances found in normal participants (< 5 x
10-5)—there was only a weak linear relationship between shunt
magnitude and albuminuria. When the fractional clearance of albumin
(Albumin) exceeded a threshold value of approximately 0.006,
two orders of magnitude above the upper limits of normal, a strong positive
linear relationship became apparent between log(Albumin) and
log(0) (r = 0.764). A similar phenomenon was seen
when shunt magnitude was regressed on the logarithm of the A/C ratio. In this
case, the development of a strong positive linear relationship between ln(A/C)
and log(0) occurred at an A/C ratio of approximately 3000
mg/g.
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In the MACRO group, the fractional clearances of both albumin and IgG were
correlated with the magnitude of the shunt (log-log regression: r =
0.75, P < 0.001; r = 0.77, P < 0.001). By
contrast, no such relationship between 0 and the fractional
clearances of albumin or IgG was evident in the MICRO group.
Morphologic data on a subset of the participants were available from a previous study (12). These data were analyzed to investigate the structural basis for the defect in glomerular size selectivity (Table 2). In this analysis, the control group consisted of newly diagnosed diabetic participants with normoalbuminuria. GBM thickness was least in the control participants (428 ± 118 nm), was slightly greater in the MICRO group (506 ± 75 nm), and was significantly greater in the MACRO group (623 ± 162 nm). Foot process width was also greater in the MACRO group than in the other two groups (845 ± 192 nm versus 650 ± 73 and 634 ± 149 for new-onset controls and MICRO, respectively). The number of visceral epithelial cells per glomerulus was significantly less in the MACRO group than in the new-onset group (355 ± 79 cells/glomerulus versus 476 ± 101); the number in the MICRO group was intermediate at 438 ± 85. A similar trend was seen in the visceral epithelial cell density (42 ± 10 cells/106 µm3 versus 89 ± 19 and 69 ± 21). The only structural index showing a significant difference between new-onset controls and the MICRO group was the visceral epithelial cell number density (P = 0.04).
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We examined the relationship between the shunt parameter
0—calculated from the lognormal + shunt
model—and several quantitative structural indices characterizing the
glomerular capillary wall in individuals who underwent renal biopsy. In the
MACRO group, the only significant association with shunt magnitude by
multivariate regression was that with foot process width
(Figure 7; r = 0.69,
P = 0.027). There was a borderline significant univariate correlation
of 0 with GBM thickness (r = 0.58, P =
0.082). Shunt magnitude tended to increase with increases in either of these
two parameters. No such relationships were discernible in the MICRO
participants (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Natural History of Microalbuminuria
At baseline, the GFR was 28% higher in the 43 diabetic Pima Indians with
microalbuminuria than in nondiabetic Pima Indians. Over the ensuing 4 yr, the
GFR and renal plasma flow fell only slightly, declining 6% and 7%,
respectively. This relative stability of renal function contrasts with the 34%
decline in GFR over 4 yr among diabetic Pima Indians with macroalbuminuria at
screening (11). Despite the
negligible changes in GFR, many of the participants who initially were in the
microalbuminuric range developed persistent macroalbuminuria (A/C ratio
300 mg/g) over the course of the next 8 yr. Progression to macroalbuminuria
was not, however, a uniform process (Figure
2). A small subset of participants had stable urinary albumin
excretion or even fell into the normoalbuminuric range during long-term
follow-up. Others manifested a gradual increase in albuminuria; still others
had a very rapid rise to nephrotic levels, with or without a variable period
of stability. The factors that account for this heterogeneity of patterns of
progression in proteinuria have yet to be fully elucidated, but the
participants with stable or improving A/C ratios were older and had
significantly lower initial renal plasma flows, GFR, and plasma renin
activities than those who progressed.
Conversely, higher initial values for renal plasma flow and MAP were predictive of early progression to macroalbuminuria. Taken together, these relationships suggest that higher glomerular perfusion rates and pressures promote injury to the glomerular barrier. Worthy of note is that despite the heterogeneity that we observed in the courses of individual participants, Kaplan-Meier analysis suggests that the majority of microalbuminuric patients progress to macroalbuminuria by approximately 8 yr of follow-up.
Studies in Pima Indians (8) and in Danish patients (16) with type 2 diabetes have identified age, duration of diabetes, HbA1c, serum cholesterol, and the initial level of urinary albumin excretion (even within the normal range) as risk factors for progression from normoalbuminuria to either micro- or macroalbuminuria. The present study examined the somewhat different question of whether the rate of progression of MICRO participants to persistent macroalbuminuria was influenced by these factors. As stated above, the only covariates that influenced progression to persistent macroalbuminuria in this study were BP and renal plasma flow rate, suggesting that different mechanisms may be responsible for the initiation of renal injury and for the tempo of the injury process once it is established. Elevated BP was reported previously (10) to predict more rapid progression of albuminuria in Pima Indians with normo- and microalbuminuria, as well as in other type 2 diabetic populations (17).
Mechanisms of Proteinuria
Despite the elevated urinary albumin excretion in microalbuminuric
participants, the magnitude of the nonselective shunt pathway for the passage
of macromolecules through their glomerular barrier was not different from that
in normoalbuminuric control subjects. The absence of a size-selectivity defect
has been noted before in type 1 diabetic patients with early diabetic renal
disease (18,
19). In participants who
already were manifesting overt nephropathy (MACRO), however, the shunt
magnitude did exceed control values. In addition, when the MICRO and MACRO
groups were analyzed together, an abrupt transition was apparent in the
relationship between shunt magnitude and fractional clearance of albumin
within the macroalbuminuric range (A/C ratio, approximately 3000). This
suggests that at least two distinct mechanisms are responsible for abnormal
albuminuria over the ranges of Albumin seen in
microalbuminuric and macroalbuminuric participants: in the lower range of
Albumin, the shunt seems to make little or no contribution to
the development of albuminuria. The lack of a significant contribution of the
shunt pathway to the development of microalbuminuria in type 2 diabetic
patients may have several explanations.
First, the dextran sieving technique may not be sensitive enough to detect
changes in size permselectivity at low levels of albuminuria (<300 mg/24
h). Accuracy of the dextran sieving method has, in fact, recently been called
into question (20). In
contrast to the findings of Hemmelder et al.
(20), however, the
coefficients of variation of the urine-to-plasma dextran concentration ratios
in our laboratory using HPLC and on-line refractometry show consistently low
values (
12.5), even for dextrans with radii of 50 to 60
. This nominal degree of assay variation can
be effectively overcome by studying a large number of patients, as was done in
the present report. Figure 6,
in fact, shows little trend in shunt magnitude change over several orders of
magnitude in albumin fractional clearance. Thus, it seems likely that no
significant changes in size selectivity of the glomerular filter are present
at the earlier stages of diabetic nephropathy. Second, also consistent with
Figure 6, the contribution of
the defect in size selectivity to microalbuminuria may be small enough that it
is overwhelmed by other processes present in these patients. Only when
proteinuria reaches the macroalbuminuric level does the effect of the shunt
begin to dominate other influences. Other factors that could conceivably
contribute to microalbuminuria are changes in the tubular reabsorption of
filtered proteins and alterations in charge-based restriction at the
glomerular filter (19).
Tucker and colleagues (21) examined the pathogenesis of microalbuminuria in a model of streptozotocin-induced diabetes in the rat. They demonstrated an initial increase in glomerular filtration of albumin after 1 wk. This did not translate into pathologic albuminuria, however, because of an offsetting increase in tubular reabsorption of filtered albumin. Only after 7 to 10 wk of diabetes did albuminuria reach the pathologic range, as a result of persistence of increased glomerular filtration of albumin in conjunction with impaired tubular reabsorption. Other studies in animals have suggested that up to 90% of the filtered load of albumin normally is reabsorbed in the proximal tubule (22, 23). If the same relationship were to hold in humans, then abolition of tubular reabsorption alone could result in up to 300 mg/d of albuminuria (based on a normal albumin excretion rate of up to 30 mg/d). In addition, endocytotic uptake of albumin by proximal tubular cells in culture is regulated by phosphatidyl inositide 3-kinase (24). In proximal tubule cell culture, insulin stimulates production of phosphatidyl inositol-(4)-phosphate, phosphatidylinositol-(4,5)-bisphosphate, and phosphatidylinositol-(3,4,5)-triphosphate—all products of phosphatidyl inositide 3-kinase—suggesting that insulin may increase albumin endocytosis, possibly by stimulating interaction of clathrin-coated pits with the endosome compartment. The development of insulin resistance in type 2 diabetes may therefore adversely effect tubular albumin uptake and lead to excretion of a larger fraction of the albumin that is filtered at the glomerulus.
Last, a loss of charge-dependent restriction of macromolecule filtration at the glomerulus may also play a role in the development of microalbuminuria, although, for technical reasons, this phenomenon is difficult to evaluate in humans. A decrease in charge-dependent restriction could be due to decrements in the anionic components that compose the negatively charged electrostatic "barrier" of the glomerular capillary wall. Alternatively, glycosylation of circulating albumin may shift its net charge toward a more neutral range. An increase in the charge microheterogeneity of serum albumin—with an increase in the isoelectric point—has been described in adolescents with diabetes (25). The effects of albumin glycosylation are likely to be complex, however. For example, cationization of albumin changes its spatial configuration and may thus also influence its size-based exclusion by the capillary wall (26). In addition, cationization of albumin could increase its binding to the negatively charged brush border of proximal tubule cells, thereby increasing its reabsorption (22).
In the MACRO group, the magnitude of the shunt pathway correlated modestly with the thickness of the GBM and strongly with the foot process width. Both an increase in GBM thickness and widening of the foot processes will decrease the intrinsic hydraulic permeability of the glomerular barrier and, in turn, the glomerular ultrafiltration capacity (27). Studies of experimental glomerular injury (28) and human nephrotic syndrome (29) have revealed an inverse relationship between ultrafiltration capacity and glomerular permselectivity. The basis of this relationship is unclear, but it may be related to adaptive increases in intracapillary BP in the setting of decreased ultrafiltration capacity. Conversely, the podocyte's response to decreased ultrafiltration capacity may involve adaptive changes that compromise the ability of the filtration slit diaphragm to restrict the passage of macromolecules.
We conclude that although microalbuminuria may exist for years in individuals with type 2 diabetes mellitus, a significant size defect in the glomerular barrier first appears only after transition to the macroalbuminuric stage, a stage characterized by a rapidly progressive decline in permselective and filtration functions (11). Other studies have suggested that this stage is also characterized by a critical loss in the complement of podocytes within the glomerulus (12). The present findings are consistent with the possibility that developing podocyte insufficiency and ensuing alterations in podocyte foot processes or filtration slits could contribute to the progressive loss of glomerular size selectivity that occurs after the incipient, microalbuminuric form of nephropathy evolves to overt, macroalbuminuric nephropathy in type 2 diabetes mellitus.
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) and long-term normoalbuminuric diabetic controls (n = 11,
). Lines are best-fit splines. The MACRO participants show a significant
elevation of the sieving curve at its large-radius end as well as a tendency
toward depression at the low-radius end. Error bars represent one SD. ESR,
Einstein-Stokes radius. * P < 0.05.
) groups (n = 73). Piecewise linear regression lines for the
log-log relations are shown. An abrupt transition in the nature of the
relationship between the two variables is apparent at a fractional albumin
clearance equal to approximately 0.006 or an A/C ratio approximately 3000.
Above these values, there is a strong positive linear relationship between the
parameters. This suggests that increasing albuminuria in the lower range is
due to factors other than decreased glomerular size permselectivity. The
dashed horizontal lines represent the 25th and 75th percentiles of the
