Kidney International, Vol. 63 (2003), pp.
809–825
Pathophysiology of proteinuria
GIUSEPPE D’AMICO and CLAUDIO BAZZI
Division of Nephrology, San Carlo Borromeo Hospital, Milano, Italy
Pathophysiology of proteinuria. Proteinuria is consequence of
two mechanisms: the abnormal transglomerular passage of pro-
teins due to increased permeability of glomerular capillary wall
and their subsequent impaired reabsorption by the epithelial
cells of the proximal tubuli. In the various glomerular diseases,
the severity of disruption of the structural integrity of the
glomerular capillary wall correlates with the area of the glomer-
ular barrier being permeated by “large” pores, permitting the
passage in the tubular lumen of high-molecular-weight (HMW)
proteins, to which the barrier is normally impermeable. The
increased load of such proteins in the tubular lumen leads to
the saturation of the reabsorptive mechanism by the tubular
cells, and, in the most severe or chronic conditions, to their
toxic damage, that favors the increased urinary excretion of
all proteins, including low-molecular-weight (LMW) proteins,
which are completely reabsorbed in physiologic conditions.
Recent clinical studies showed that in patients with glomeru-
lar diseases the urinary excretion of some HMW proteins [im-
munoglobulins G and M (IgG and IgM)] and of some LMW
proteins, ␣1-microglobulin, ␤2-microglobulin, correlates with the
severity of the histologic lesions, and may predict, better than
the quantity of proteinuria, the natural course, the outcome,
and the response to treatment. It is suggested that some patients
have already, at the time of clinical presentation, a structural
damage of the glomerular capillary wall (injury of podocytes)
and of the tubulointerstitium, the severity and scarce reversibil-
ity of which are reliably indicated by an elevated urinary excre-
tion of HMW and LMW proteins.
PHYSIOLOGIC MECHANISMS OF
GLOMERULAR FILTRATION AND TUBULAR
REABSORPTION OF PROTEINS
Determinants of the transglomerular passage of
plasma proteins
Despite the extremely low resistance to water flow, the
glomerular capillary wall very efficiently restricts the pas-
sage of proteins from the blood into Bowman’s space
on the basis of their molecular size, electrical charge,
Key words: proteinuria, glomerular wall permselectivity, urinary IgG,
urinary ␣1-microglobulin, podocytes.
Received for publication July 4, 2002
and in revised form October 16, 2002
Accepted for publication October 18, 2002
 2003 by the International Society of Nephrology
809
PERSPECTIVES IN BASIC SCIENCE
and sterical configuration. Large and negatively charged
molecules are less readily filtered than small and electro-
neutral or positively charged ones. The mechanisms
whereby normal glomerular capillary wall restricts the
transmural passage of large plasma proteins have been
extensively explored in the last 25 years, but a universally
accepted theory has not yet been agreed upon. There is
a consensus that the glomerular wall may be thought of
as containing three barriers in series. Along its route to
this filtration barrier, the filtrate first has to pass through
the open pores of the glomerular endothelium, then
through the highly hydrated collagenous network of the
glomerular basement membrane (a molecular scaffold
composed of tightly cross-linked type IV collagen, lami-
nin, nidogen, and proteoglycans), and finally, through the
filtration slits established by interdigitations of podocyte
foot processes and covered by a complex structure called
“slit diaphragm,” which is anchored at the sides of the
adjacent foot processes [1–6]. Although the fenestrated
endothelium can represent an electrostatic barrier for
negatively charged proteins, and glomerular basement
membrane with its laminae can restrict the traversal of
large plasma proteins and negatively-charged proteins
(due to the presence of heparan sulfate proteoglycans),
recent evidence suggests that the ultimate, more selective
barrier for the majority of proteins resides in the slit
diaphram [7–9]. However, the molecular composition of
this last structure is still largely unknown. The most re-
cent studies tend to confirm the model of substructure
proposed by Rodewald and Karnovsky [10] in 1974 on
the basis of their transmission electron microscopic find-
ings, according to which the slit diaphram is made up of
rod-like units connected in the center to a linear bar,
forming a zipper-like pattern. Nephrin, a molecule spe-
cifically expressed in podocytes, has been suggested by
Tryggvason [8] to be the candidate molecule that might
assemble into a zipper-like isoporous filter structure.
Nephrin molecules extending toward each other from
two adjacent foot processes are likely to interact in the
slit through homophilic interactions and covalent cross-
linking. Other proteins, such as ZO-1 [11], P-cadherin,
and catenins [9], have also been found to be expressed
810 D’Amico and Bazzi: Pathophysiology of proteinuria

in the slit diaphragm and could be involved in the struc-

ture of the adherent junctions of a zipper-like filter [9].
Very recently, two other proteins have been discovered
as components of the slit diaphragm domain of podocytes,
CD2AP, which for its structure probably acts as an adap-
tor linking the actin-based cytoskeleton of podocyte to
nephrin [12], and podocin, a stomatin-like protein that
forms aggregates and organizes lipid rafts [13]. A complex
of nephrin, podocin, and CD2AP is emerging as the
functional unit upon which the slit diaphragm assembles.
These proteins are tightly associated and are embedded
into lipid rafts. Podocin may be critical for the stability
of this complex; its binding dramatically activates the
signaling capabilities of nephrin [13–16]. The basal por-
tion of podocytes is attached to the glomerular basement
membrane through several adhesion proteins. The integ-
rin ␣3␤1 and the dystoglycan complex are the major re-
sponsable for the binding to the extracellular matrix of
such basement membrane [14–16]. ␣3␤1 integrin con-
stitutes stable, static bonds, and is associated on its
cytoplasmic side with paxillin, talin, and vinculin, which
mediate its connections to the actin cytoskeleton. The
dystoglycan complex probably provides an actin-directed
positioning system by which podocytes activity control
the exact spacing of matrix proteins, and thus the poros-
ity and permeability of the glomerular basement mem-
brane [15].
In the last 20 years, numerous studies have been per-
formed, in experimental animals and in humans, based Fig. 1. Glomerular pore size distribution in normal individuals (solid
curves) and in nephrotic patients (dashed curves), according to the two
on the measurement of the fractional excretion (sieving most accepted theoretical models. (A ) The membrane is perforated by
coefficient) of some biologically inert molecules of differ- a single population of cylindrical restrictive pores that have a continuous
ent size, either neutral or charged, used as test transport log-normal distribution of radii (heteroporous model). (B ) The mem-
brane is perforated by a lower distribution of restrictive pores with a
probes. They demonstrated that glomerular capillary log-normal distribution of radii and a parallel upper distribution of
wall, as a whole, can be assumed to be perforated by nonrestrictive dimensionless shunt-like pores (bimodal heteroporous ⫹
cylindrical pores of different diameter. The most used shunt model).

have been for years the dextrans of different size. They

have given useful results for the definition of the porosity
of the glomerular filter. However, like many of the pro-
teins physiologically crossing this filter, they are de-
formable and flexible (differences in molecular configu-
ration may have an important influence on the relative
filtration rates of proteins); therefore, they did not allow
to precisely define the real radius of the pores. More
recently, another inert molecule, Ficoll, has been pre-
ferred for such studies, because it is a rigid spherical
molecule, the configuration of which is not altered during
transglomerular passage. Many different mathematical
models, based on hemodynamic theories, have been pro-
posed in order to explain the results of the studies on the
transport probes. The models most extensively accepted,
called heteroporous models, consider the glomerular
capillary wall as a membrane perforated by pores of
different diameter having a log-normal distribution of
radii [17–29]. A single population of restrictive pores
with a log-normal distribution of radii extending to radii
⬎60 Å (Fig. 1A) has been recently considered the best
fitting model by Ruggenenti et al [28]. In contrast, others
[18, 20, 24, 27], using as transport probe not only dex-
trans, but also Ficoll, have recently concluded that a
bimodal model better fits the description of their results
(Fig. 1B). They hypothesized a prevailing population of
small restrictive pores with a log-normal distribution of
radii, with a mean radius around 45 Å, with a range
between 37 and 48 Å, and a limited number of nonrestric-
tive pores (⬎80 Å), that they called the “shunt” pathway,
that in physiologic conditions is permeated by a very
small fraction of filtrate (only 0.1 ⫻ 10⫺3 on average).
However, even among the supporters of the bimodal
distribution, not everyone accepts the nonrestrictive na-
ture of the large shunt-like pores. A group of investiga-
 D’Amico and Bazzi: Pathophysiology of proteinuria
tors from Lund [25, 26, 29], using a different approach
to the study of glomerular permeability based on the
inhibition of tubular reabsorption in animal models,
agrees on the existence of two populations of pores, the
vast majority being “small pores,” but hypothesizes that
even the second pore population consists of a limited
number of discriminatory “large pores” with a radius 80
to 90 Å. These investigators postulate that, in addition
to the two populations of pores, sporadic “membrane
defects” or “shunts” large enough to allow transport of
very large proteins of the size of ␣2-macroglobulin and
even red blood cells (similar to the non-discriminating
shunt-like pores proposed by the Myers group) could
also exist, and that they should contribute less than 10⫺5
of the total glomerular filtration rate (GFR) in physio-
logic conditions, but could increase in number in patho-
logic conditions (“two pores with a shunt” model).
It is accepted by all investigators that in physiologic
conditions, proteins of the size of immunoglobulin G
(IgG) (molecular radius ⫽ 55 Å) are completely (or
almost completely) restricted from filtration because
their radius is higher than that of all the small restrictive
pores, and the contribution of the shunt pores or large
selective pores is quantitatively irrelevant (Fig. 2A). On
the contrary, the low permeability of the glomerular
capillary wall to albumin (molecular radius ⫽ 36 Å)
cannot be explained in terms of simple restriction by pore
size. The majority of investigators refer the restricted
movement of this protein to the Bowman’s space to some
characteristics related to its negative charge and to the
consequent electrostatic repulsive interactions with the
negative charge of the glomerular capillary wall. It has
been demonstrated experimentally that some transport
probes, if negatively charged, cross the glomerular filter
with a reduced sieving coefficient and that neutralization
of the anionic sites of glomerular basement membrane
by polycations results in increased permeability to such
probes, as well as to albumin. It has been recently calcu-
lated, using uncharged Ficoll as a probe, that, in rats,
albumin has a much lower sieving coefficient compared
with Ficoll of the equivalent size of 36 Å [28] and that
in normal individuals albumin filtration is restricted by
a factor of approximately 100 relative to a 36 Å probe
[24]. However, the role of charge selectivity in permselec-
tivity of glomerular capillary wall has been recently ques-
tioned [22]. The anionic charge of the capillary wall has
been estimated to be much lower than that required to
affect the clearance of some proteins based on charge
repulsion. Even the higher fractional clearance of cat-
ionized dextran in comparison with its anionic counter-
part found in many in vivo experiments has been ex-
plained arguing that this cationic polysaccharide binds
to anionic sites and damages the glomerular capillary
wall, making it leaking [22].
811
Renal tubular handling of filtered proteins
As we have stated, in normal conditions only a fraction
of proteins of intermediate molecular weight, in particu-
lar albumin, the concentration of which in the Bowman’s
space of healthy animals has been calculated to be ap-
proximately 1 mg/dL [30, 31] or even higher [32], and
almost none of the high-molecular-weight (HMW) pro-
tiens arrives in the tubular lumen. On the contrary, the
passage across the glomerular filter of all proteins of
molecular weight lower than 40,000 D and radius lower
than 30 Å, the so-called low-molecular-weight (LMW)
proteins, is almost completely unrestricted in the normal
individuals. All these proteins that arrive in the tubular
lumen are excreted in negligible amounts in the urines
in physiologic conditions because of a very efficient
mechanism of total reabsorption by the epithelial cells
of proximal tubules (Fig. 2A). For ␤2-microglobulin, the
molecular radius of which is 11.8 Å, the block of tubular
reabsorption with lysine in normal rats increased the
clearance by a factor of 450, while the clearance of large
proteins increased only five- to sixfold [20].
This reabsorption of proteins occurs predominantly in
the pars convoluta (S1 and S2 segments) and, to a lesser
extent, in the pars recta (late S2 and S3 segments) of the
proximal tubule. The epithelial cells of these segments
contain an extensive apical endocytic apparatus, con-
sisting of a network of coated pits and small coated
and noncoated endosomes, but also of prelysosomes,
lysosomes, and so-called dense apical tubules involved
in recycling membrane from the endosomes to the apical
plasma membrane. The proteins absorbed at the luminal
membrane are endocytosed and concentrated within ves-
icles at the apical border of tubular cells (endocytic vesi-
cles). These vesicles fuse with acidic organelles that be-
long to the endosomal compartment. The endosomes
containing the segregated proteins then migrate to the
cell interior, where they fuse with the lysosomes, cell
organelles that contain acid hydrolases. Absorbed pro-
teins are completely hydrolyzed within lysosomes and
the resulting aminoacids cross the contraluminal mem-
brane to return to the circulation [33, 34].
Until recently, it was believed that proteins were
largely absorbed by nonspecific absorbptive endocytosis,
the tubular uptake being a high-capacity, low-affinity
transport system. However, kinetic parameters differ sig-
nificantly according to the charge of proteins, and, even
for proteins of similar size and net charge, there is com-
petition for tubular absorption among the various pro-
teins. Moreover, in the case of albumin, in addition to the
high-capacity, low-affinity transport system, which is uni-
versal among all proteins tested, a low-capacity transport
system has been found that is probably operational in
the lower ranges of tubular fluid albumin concentration
[35–37]. There is a growing body of evidence suggesting
812 D’Amico and Bazzi: Pathophysiology of proteinuria

Fig. 2. Schematic representation of transglo-

merular transfer to the tubular lumen, reabsorp-
tion by the tubular cells and excretion in the
urines of plasma proteins of low-molecular-
weight (LMW), intermediate-molecular-weight
[mainly albumin ALB)], and high-molecular-
weight (HMW) in physiologic conditions and
in pathologic conditions characterized by pro-
gressively increasing permeability of the glo-
merular barrier and reabsorptive load of the
tubular cells. (A ) In physiologic conditions,
all LMW proteins and a fraction of albumin
cross the glomerular barrier and are com-
pletely reabsorbed by the tubular cells. (B )
The alteration of the permeability of the glo-
merular barrier is moderate, involving mainly
a loss of restriction to passage of negatively
charged proteins (especially albumin); albu-
min and a small fraction of HMW proteins
reach the tubular lumen and saturate the re-
absorptive capacity of tubular cells, inducing
the loss in the urines of a fraction of LMW
proteins and albumin, together with a very
small fraction of HMW proteins (selective
proteinuria).
D’Amico and Bazzi: Pathophysiology of proteinuria 813

Fig. 2. (Continued) (C ) A more severe dam-

age progressively increases size permeability
of the glomerular barrier, and, due to the satu-
ration of the reabsorptive mechanisms of tu-
bular cells, a greater percentage of HMW pro-
teins is excreted in the urines (nonselective
proteinuria). (D ) Permeability of the glomer-
ular barrier is further increased, and the mas-
sive and protracted reabsorptive load of the
tubular cells induces toxic lesions of these cells
and reduces their reabsorptive capacity; excre-
tion with the urines of all three classes of pro-
teins and, in particular, of LMW and HMW
proteins, is increased, and represents a valid
marker of the severity of the glomerular and
tubular damage.
814 D’Amico and Bazzi: Pathophysiology of proteinuria
that the absorption of albumin, as well as all other pro-
teins, through the entire length of the proximal tubule
but especially in its pars convoluta, is “receptor-medi-
ated” as opposed to “nonspecific.” It is assumed that
the luminal endocytosis is initiated by ligand binding to
receptors localized in the clathrin-coated pits, followed
by internalization, segregation of ligands and receptors
in early and late endosomes, and directing of ligands
to lysosomes for degradation, while the receptors are
directed back to the apical plasma membrane via dense
apical tubules [34]. Data gathered in recent years indicate
that megalin and cubilin play a crucial role in this process
[38]. Both are multiligand receptors massively expressed
in the proximal tubules (but also in several other epithe-
lia). Although they belong to distinct protein families,
they share a common structural feature, the accumula-
tion of motifs potentially important for ligand binding,
which confers upon them a key property for multiligand
receptors. Both are detectable over the apical endocytic
apparatus: clathrin-coated pits, small and large endo-
somes, and dense apical tubules. Megalin and cubilin
appear to carry on a cooperative function, clearly demon-
strated in the case of the absorption of albumin, where
this apparently redundant interaction results in a very
efficient uptake, even though in the case of transferrin,
cubilin appears to be exclusively involved in the internal-
ization process. Megalin and cubilin, although putative
receptors, do not appear to be very selective, as many
proteins are able to bind to them. Because of this multi-
plicity of ligands, it would probably be more appropriate
to characterize them as “binding proteins” rather than
as “receptors” [34]. A potential role of megalin to signal
into the cell interior to stimulate cell proliferation has
been very recently hypothesized [39].
It is worth stressing that cationic proteins, such as
IgG, have been shown to bind more avidly to the apical
membrane of proximal tubule cells because such mem-
brane is negatively charged. As a consequence, cationic
proteins present in the tubular lumen are more readily
endocytosed than anionic proteins such as albumin
[40, 41]. This competitive mechanism adds another vari-
able that affects the concentration of filtered proteins in
the final urine, and renders the correct calculation of the
relative sieving coefficient of HMW and LMW proteins
and of the selectivity index more difficult, especially in
pathologic conditions.
PATHOPHYSIOLOGY OF ABNORMAL
URINARY PROTEIN EXCRETION IN
GLOMERULAR DISEASES
Increases in the urinary excretion of proteins result
from increases in their filtered load, due to alterations
in the permselectivity of the glomerular capillary wall,
or from defects in their tubular uptake. The majority of
the experimental studies using inert test probes, reported
above, showed that the alteration of the selectivity of
the glomerular capillary wall was a combination of the
loss of charge restriction and size restriction. These stud-
ies did not necessarily show an increase in the mean
radius of the small restrictive pore population, but rather,
in all experiments, demonstrated a spread of their distri-
bution due to the appearance of more and wider large-
pore pathways (either discriminatory or shunt-like pores)
(Fig. 1). According to Tencer et al [42], even for the large
pores, a charge restriction exists in physiologic conditions,
and explains why, especially in membranous nephropa-
thy, the ratio between urinary concentration of neutral
IgG2 and negatively charged IgG4 is significantly re-
duced, indicating a loss of the charge restriction for IgG4.
The reduction in the restrictive properties of the glo-
merular barrier leads to proportionally greater increases
in the filtered loads of albumin and HMW proteins than
of LWM proteins; the glomerular permeability of the
latter is already very high and cannot increase further,
whereas even a small increase in glomerular permeability
to macromolecules leads to very significant increases in
the filtered load of larger proteins.
Increased transglomerular passage and urinary
excretion of intermediate and HMW proteins
A moderate increase in the permeability of the glo-
merular capillary wall is characteristically found in mini-
mal change nephropathy and in some initial stages of
other glomerular diseases (primary focal segmental glo-
merulosclerosis, membranous nephropathy, diabetic ne-
phropathy). The increased transglomerular passage in
the tubular lumen of intermediate-molecular-weight pro-
teins, mainly albumin, is not accompained by a similar
passage of HMW proteins, such as IgG, ␣2-macroglobulin
or IgM. Despite their partial reabsorption by the tubular
cells, a fraction of albumin and intermediate-molecular-
weight proteins escapes the reabsorptive process and
appears in the urine. The ensuing proteinuria is indicated
as “selective” (Fig. 2B). A selectivity index (SI) based
on the comparison of the clearance of IgG, as a marker
of proteins of HMW, and that of transferrin, as a marker
of proteins of intermediate size, had already been elabo-
rated in 1964, and since then patients with an IgG SI of
0.2 or higher have been considered to have a nonselective
proteinuria, whereas patients with a ratio below 0.2 have
been considered to have a selective proteinuria [43]. In
spite of the criticism to the theory of charge restriction,
to which we alluded in a previous section, the majority
of investigators still believe that in these pathologic con-
ditions characterized by selective proteinuria, the impair-
ment of the charge selectivity of the glomerular filter is
prevalent in comparison with the impairment of size.
The injury to the structures of the glomerular capillary
wall impairs their function as a negatively charged elec-
 D’Amico and Bazzi: Pathophysiology of proteinuria
trostatic barrier, permitting the unrestricted transglo-
merular passage of albumin, while the damage respons-
able for the increase of the large nonselective pores
permeating the capillary wall is less marked. A loss of
anionic sites of the glomerular basement membrane has
been documented in minimal change nephropathy [44, 45]
and in the microalbuminuric stage of diabetic nephropa-
thy [46]. In both conditions the study with dextrans has
confirmed a very mild increase of the area of the glomer-
ular filter being permeated by large pores, while the
transglomerular passage of albumin through the small
selective pores is more markedly increased [18, 47–49].
In all the glomerular diseases with nonselective protein-
uria, the variable quantity of proteins of HMW reaching
the tubular lumen is an expression of a variable severity
of the impairment of the size selectivity, in addition to
that of the charge selectivity (Fig. 2 C and D). The sieving
coefficient of large (⬎60 Å) dextrans has been found
to be more increased in patients with focal segmental
glomerulosclerosis, in comparison with patients with
minimal-change nephropathy, in the study already men-
tioned [18]. Another study of the same group [50], com-
paring the sieving coefficients of uncharged dextran and
anionic dextran sulfate in nonproteinuric controls and
in two groups of nephrotic patients, with minimal-change
nephropathy and membranous nephropathy, confirmed
that the permeability to large uncharged dextrans, pres-
ent in all nephrotic patients, was more evident in patients
with membranous nephropathy. The sieving coefficient
of the anionic dextran sulfate of small radius (⬃18 Å)
was definitely lower than that of uncharged dextrans
of similar radius in nonproteinuric controls, but not in
nephrotic patients, indicating a loss of charge restriction
which could explain the magnitude of albuminuria better
than the concomitant increase in filtration of large neu-
tral dextrans.
More recent studies of the same investigators from
Stanford, using Ficoll test probe, have been performed
in membranous nephropathy [24] and IgA nephropathy
[51]. In comparison with dextrans, sieving coefficients
for all restricted Ficoll molecules (probably because their
conformation was not altered during transglomerular
permeation) were uniformly lower than those of dex-
trans in nonproteinuric controls [24]. In patients with
membranous nephropathy, the membrane parameters
measured with Ficoll differed from control in two re-
spects: (1) the distribution of restrictive pores was broad-
ened, the mean radius (36 Å) being shifted to smaller
size (from 44 Å to 36 Å); (2) the magnitude of the area
being permeated by large shunt-like pores was substan-
tially increased, the sieving coefficient for Ficoll of equiv-
alent radius to IgG (54-56 Å) being enhanced above
control by a factor of ⬃20. Since the sieving coefficient
of Ficoll of equivalent radius to albumin (36 Å) did not
differ between the patients with membranous nephropa-
815
thy and control groups, the authors suggested that “mas-
sive immunoglobinuria is attributable to an impairment
of barrier size selectivity, whereas the massive albumin-
uria is due to a defect of charge selectivity” [24]. In patients
with IgA nephropathy [51], whose proteinuria was less
severe than that of patients with membranous nephropa-
thy, the study with Ficoll confirmed that the fraction of
filtrate permeating the large shunt-like pores was signifi-
cantly increased in comparison with non-proteinuric con-
trols. At variance with the results obtained in nephrotic
patients with membranous nephropathy, no differences
from control subjects were found in the mean radius of
the small restrictive pores or the range of distribution
of these pores. There was a significant relationship be-
tween the fractional clearance of albumin and IgG and
the fraction of filtrate passing through large shunt-like
pores in this population of patients [51].
Similar studies [28, 29, 47–49, 52, 53] using neutral
dextrans or Ficoll as probes have been carried out in the
various stages of type 1 or type 2 diabetic nephropathy.
These studies showed that the evolution of the disease,
through the microalbuminuric stage, to one of overt pro-
teinuria, represents a continuum of progressively de-
ranged glomerular capillary wall function. As we said,
in the microalbuminuric stage all parameters of size se-
lectivity (mean radius and spread of radius distribution
of restrictive pores, as well as the magnitude of the large
pores) were unaltered or moderately altered. With in-
creasing severity of the nephropathy, in the stage of overt
proteinuria, the number and peak radius of the restrictive
small pores was still unaltered or moderately increased.
The increased filtration of IgG and other proteins of
HMW could be explained with an increased fraction of
filtrate being permeated by large pores. In type 2 diabetes
of Pima Indians, it was calculated, using dextrans as
probes, that an excess of large pores becomes evident
when albuminuria reaches a level of 3 grams per gram
of creatinine [49]. In the same disease in Italian patients,
using the same probes, Ruggenenti et al [28] concluded
that distribution of pore sizes was on average shifted
toward larger pores, as compared to normal controls,
the mean pore size radius being of 5 Å greater (Fig. 1A).
The majority of investigators agreed that, when overt
proteinuria develops in the nephropathy of both types
of diabetes, concomitant changes of charge—and size—
selectivity are likely to contribute to albuminuria. How-
ever, Bakoush et al [29] found a better preserved ratio
of IgG2/IgG4 urinary excretion in European patients
with type 2 diabetes than in those with type 1 diabetes
and argued that in the former group charge selectivity
is less impaired even in the macroalbuminuric stage.
They suggested that in type 2 diabetes even albumin is
lost primarily through the large pore pathway. According
to these investigators, even urinary excretion of IgM, a
protein of molecular weight and radius higher than IgG,
816 D’Amico and Bazzi: Pathophysiology of proteinuria
was more frequently detectable in type 2 than in type 1
diabetes; this difference was explained as reflecting a
more marked increase in the population of the nonselec-
tive shunt-like pores in type 2 diabetes.
Structural-functional correlations have been analyzed
in both diabetic and nondiabetic nephropathies associ-
ated with documented changes in size selectivity.
In overt diabetic nephropathy of Pima Indians, Lemley
et al [49] documented increased glomerular basement
membrane thickness, greater foot process width, and a
decreased number of visceral epithelial cells per glomer-
ulus; at multivariate regression analysis, only foot pro-
cess width was significantly correlated with the increase
in larger size pores.
In nondiabetic glomerular diseases characterized by
prevalent lesions of the glomerular capillary wall, like
membranous nephropathy and focal segmental glomeru-
losclerosis, the changes in charge and size selectivity have
been ascribed to alterations of foot processes and slit
membranes of epithelial cells, especially when efface-
ment of adjacent foot processes or complete detachment
of some foot processes and their slit membranes from
the lamina rara externa of the basement membrane are
detected [27, 54, 55], but also to the alterations of the
structural modeling of the glomerular basement mem-
brane [56, 57]. We have already mentioned the loss of
anionic sites from such basement membrane and from
podocytes, responsible for charge selectivity in some pro-
teinuric diseases. Decreased density of slit processes of
glomerular epithelial cells has been also documented
morphometrically in human disease with nephrotic syn-
drome [52], probably favored by the limited capacity
of mature glomerular epithelial cells to proliferate [58].
Dedifferentation of glomerular epithelial cells in some
proteinuric diseases, especially focal segmental glomeru-
losclerosis, is suggested by the decreased expression of
surface antigens usually present in mature cells [59, 60].
In glomerular diseases with mesangial involvement, such
as IgA nephropathy, data are more controversial. Ike-
gaya et al [61] found that the increase in pore size corre-
lated with mesangial sclerosis and tubulointerstitial dam-
age, more than with the severity of capillary changes.
On the contrary, in their very accurate study, already
quoted, Lemley et al [51] have found that a reduced
number of podocytes/glomerulus was the most signifi-
cant morphometric feature associated with other mark-
ers of increasing disease severity such as loss of size
selectivity of the glomerular barrier, IgG urinary frac-
tional excretion, extent of glomerular sclerosis and de-
crease of GFR; they suggested that, whereas IgA ne-
phropathy is initiated by intramesangial deposition of
IgA, it is the injury to, and loss of, podocytes from the
outer aspect of the glomerular basement membrane that
determines the severity of disease as well as its rate of
progression. In a model of lupus nephritis, the fractional
clearance of albumin and IgG was significantly corre-
lated with the percentage of glomerular basement mem-
brane occupied by dense deposits, by the slit number/
glomerular basement membrane surface and by the thick-
ness of glomerular basement membrane [62]. Finally, in
all human glomerular diseases, when the injury to the
glomerular capillary wall was severe, morphologic de-
fects of glomerular basement membrane have been de-
scribed, such as tunnels [63, 64] or focal areas of rupture
or holes in the wall [65, 66]. They are probably responsi-
ble for the leakage of very large proteins (“shunt path-
way”) and might allow the passage of blood cells.
It must be stressed that not only structural lesions, but
also glomerular hemodynamic changes, affect the perme-
ability of glomerular capillary wall to macromolecules.
Filtration of proteins is influenced not only by the intrin-
sic membrane properties of the glomerular capillary wall,
but also by the other determinants of single-nephron
GFR: the glomerular capillary plasma flow rate (QA), the
glomerular transcapillary hydraulic pressure difference
(⌬P), and the afferent arteriolar plasma protein concen-
tration (CA). Moreover, it is becoming increasingly ap-
parent that hemodynamic alterations, either of pressure
or flow, may directly influence the permeability proper-
ties of the barrier itself, as reflected by changes in the
apparent pore size distribution and/or the concentration
of fixed negative charges on the membrane [67].
The studies in normal rats [67–69], even after infusion
of angiotensin II [70–75], or stimulation of endogenous
angiotensin by renal vein constriction [76], have permit-
ted better characterization of the effect of hemodynamic
changes. It has been found that (1) selective increase in
glomerular plasma flow rate (QA), up to a certain level,
tends to decrease the fractional clearance of macromole-
cules (an effect due to a disproportionately greater in-
crease in the average transcapillary volume flux than
solute flux), while a decline in QA has the opposite effect;
(2) selective increase of transcapillary hydraulic pressure
gradients (⌬P) leads to a decrease of the fractional clear-
ance of macromolecules, even through it induces an in-
crease in diffusion-driven macromolecule flux because it
leads to an even greater increase in water flux; however,
when ⌬P increases markedly, in response to a decline
in plasma flow rate (QA), to maintain a normal single-
nephron GFR, the consequent increase in filtration frac-
tion induces an increase in the fractional clearance of
proteins; (3) intravenous infusion of angiotensin II leads
to proteinuria, concomitant and partially related to hemo-
dynamic changes characterized by a significant decrease
in QA and by a rise in ⌬P, with significant rise in filtration
fraction; the degree of proteinuria is strictly correlated
with the changes of QA and filtration fraction; (4) stimu-
lation of intrinsic angiotensin with renal vein constriction
produces similar hemodynamic changes and proteinuria,
due mainly to increased efferent arteriolar resistance,
 D’Amico and Bazzi: Pathophysiology of proteinuria
⌬P, and filtration fraction; (5) infusion of angiotensin
II or stimulation of intrinsic angiotensin II markedly
increase the fraction of filtrate passing through larger,
nonselective pores; thus, changes in ⌬P play an important
role in glomerular permselectivity since on increase in
⌬P recruits previously unexposed large pores of the non-
selective shunt pathway; and (6) saralasin, an inhibitor
of angiotensin, counteracted the effects of angiotensin
II stimulation by renal vein constriction, induced reduc-
tion of efferent arteriolar resistance and ⌬P almost to
preconstriction levels, normalized filtration fraction, de-
creased proteinuria, and corrected the barrier size per-
meability changes described above [76].
When angiotensin-converting enzyme (ACE) inhibi-
tors, and, more recently, angiotensin I (AT1) receptor
antagonists became available as antihypertensive agents,
and their antiproteinuric properties were confirmed in
many experimental models of renal disease [77–83] and
in all human glomerular diseases [52, 53, 58, 84–91], it
was initially hypothesized that their effect on glomerular
capillary wall permeability was due to the correction
of the arterial hypertension and of the intraglomerular
hemodynamic changes induced by the activation of the
renin-angiotensin system (RAS) that we have summa-
rized. However, correction of arterial hypertension using
any other conventional antihypertensive drug does not
influence the extent of proteinuria, the only exception
being the nondihydropiridine calcium channel blockers,
verapamil and diltiazem [92–94]. Moreover, it is becom-
ing evident that even the correction of the intrarenal
hemodynamic alterations existing in proteinuric renal
diseases does not fully explain the antiproteinuric effect
of angiotensin inhibitors and nondihydropiridine calcium
channel blockers. ACE inhibitors or AT1 receptor antago-
nists exert their effect in animal models of nephritis de-
spite unchanged intraglomerular pressure [79], or in ex-
perimental nephritis characterized by a normal capillary
hydraulic pressure [80]. The long-term effect of ACE inhi-
bition on proteinuria in human glomerulonephritis could
not be acutely reversed by intravenous administration of
angiotenin II, despite a dose-related fall in renal plasma
flow and rise in systemic blood pressure, renal vascular
resistance, and filtration fraction [91]. An AT1 receptor
antagonist could prevent the alteration in permselectivity
of the glomerular barrier induced by exogenous angio-
tensin II even in an isolated perfused rat kidney prepara-
tion in which hemodynamic modification had been mini-
mized [95] As for calcium channel blockers, it is well
established that they do not have a stable effect on effer-
ent arteriolar tone and induce only little change in intra-
glomerular pressure and filtration fraction [93, 96, 97].
ACE inhibitors and AT1 receptor antagonists seem
to act directly on the permselectivity properties of the
glomerular barrier. These drugs not only reduce the total
amount of proteinuria, but can partially correct the in-
817
crease in the fraction of filtrate being permeated by large
shunt-like pores [52, 53, 58, 80, 82, 83, 88, 90, 95], and
therefore reduce the fractional clearance of HMW pro-
teins, such as IgG. The same reduction in the clearance
of large dextrans, with parallel reduction of the fractional
clearance of IgG, has been found recently in diabetic
nephropathy after 6 months to 1 year of administration
of the nondihydropiridine calcium channel blocker, dilti-
azem, but not of nifedipine. Only diltiazem induced a
significant immediate reduction of proteinuria that could
not be explained as due to changes in intrarenal hemody-
namic different from those induced by nifedipine [98].
In the case of RAS inhibitors in general, an effect
on partially reversible alterations in the structure and
function of the glomerular capillary wall and of mesan-
gial cells and matrix, induced by intrarenal angiotensin
II, has been hypothesized: rearrangement of junctional
complex of podocyte filtration slits, due to actin polymer-
ization in the cytoskeleton of podocytes [95, 99]; reduc-
tion of the increase in the level of glomerular protein
and cortical mRNA for transforming growth factor-␤
(TGF-␤), and collagen types I and III [81]; and blunting
of the marked increase in cell size of cultured cells ex-
posed to angiotensin II [100]. The fact that the antipro-
teinuric effect of angiotensin inhibitors may persist for
some time after withdrawal of treatment [90] speaks in
favor of a slowly developing improvement of structural
alterations. In the case of nondihydropiridine calcium
channel blockers, an effect on the metabolism of mesan-
gial cells [101, 102] and, in diabetic animals, an attenu-
ated decrease in both glucosaminoglycan and heparan
sulfate of the glomerular capillary wall and mesangial
cells [103], have been described.
Renal tubular handling of an abnormal load
of filtered proteins
In physiologic conditions, HMW proteins are re-
stricted almost completely from reaching the tubular lu-
men because of the impermeability of the glomerular
barrier (Fig. 2A). More controversial is the behavior of
albumin. Everyone agrees that part of it crosses the bar-
rier and arrives to the Bowman’s space, where its concen-
tration is at least 1 mg/dL [30, 31]; however, recent stud-
ies suggest that the filtration of albumin is very partially
restricted by charge and its concentration in the tubular
lumen is even higher [32]. Finally, it is universally ac-
cepted that the passage in the tubular lumen of the pro-
teins of molecular weight lower than 40 kD and radius
lower than 30 Å, the so-called LMW proteins, is com-
pletely unrestricted in physiologic conditions.
When impairment of charge and size selectivity of the
glomerular capillary wall increases the filtration of the
proteins of intermediate and HMW, these proteins com-
pete among themselves, and with the LMW proteins, in
818 D’Amico and Bazzi: Pathophysiology of proteinuria
the process of reabsorption by the epithelial cells of the
proximal tubule. If this mechanism is saturated, some of
the filtered proteins of all sizes, including those of LMW,
appear in the urine (Fig. 2 B to D). Unfortunately, the
fraction of the different proteins that escapes reabsorp-
tion is not proportional to the filtered fraction, since it
is becoming evident that the reabsorption process is not
aspecific and unselective, and that even the charge of
the various proteins influences such processes, IgG being
more easily reabsorbed than albumin because of its posi-
tive charge.
When the load of filtered proteins escaping the glo-
merular barrier is massive and/or protracted, due to
chronic glomerular injury, the epithelial cells of the prox-
imal tubule, subjected to a continuous overload, may
progressively lose their integrity, with impairment of ly-
sosomal function and morphologic changes (enlarge-
ment of the protein absorption droplets and loss of brush-
border structure). As a result, the reabsorption of all
proteins, including the physiologic reabsorption of LMW
proteins, is increasingly impaired, leading to the urinary
excretion of a progressively larger fraction of LMW pro-
teins (Fig. 2 C and D). It has been found that such
impairment of the reabsorption of LMW proteins, in
glomerular diseases with severe and/or protracted pro-
teinuria, correlates with the extent of the injury of the
tubular cells and with the overall tubulointerstitial dam-
age [104–109]. Accumulating evidence suggests that
direct toxicity for the tubular cells of some as yet non-
identified proteins reaching the tubular lumen might be
responsible for the injury of such cells, triggering the
release of a number of proinflammatory cytokines and
growth factors, which cause an inflammatory and fibrotic
response in the interstitium [108–124].
QUANTITY AND QUALITY OF PROTEINURIA
PREDICT THE CLINICAL COURSE AND
THE RESPONSE TO THERAPY IN
GLOMERULAR DISEASES
Predictive value of amount of proteinuria and of
tubulointerstitial lesions
Data accumulated in the last decade have demonstrated
that in all glomerular diseases the amount of proteinuria,
together with the degree of the tubulointerstitial damage,
is the most powerful predictor of the progression to
chronic renal failure [107, 109, 110, 125–129].
The analysis of the pathophysiologic mechanisms of
proteinuria performed in the preceding section gives a
good theoretical explanation of the value of these two
parameters as prognostic indicators. The extent of pro-
teinuria is an indicator of the severity of the alterations
of the glomerular capillary wall and of its permeability,
and, therefore, can be a good marker of the overall sever-
ity of the glomerular damage. The consequent involve-
ment of the tubular cells in the reabsorptive mechanisms
of the proteins reaching the tubular lumen can explain
the injury of these cells and, therefore, the correlation
of the overall tubulointerstitial damage with the severity
and duration of proteinuria.
These pathophysiologic mechanisms can also explain
the recent finding that some qualitative characteristics
of proteinuria are better predictors of the progression
to chronic renal failure than its quantity.
Predictive value of selectivity of proteinuria and
urinary excretion of HMW proteins
The assessment of SI, based on the comparison of the
clearance of IgG and transferrin, has found little place
in clinical practice for many decades, since it did not
appear to help in predicting histologic diagnosis or in
determining prognosis, the only exception being the dif-
ferential diagnosis between minimal change nephropathy,
in which proteinuria is always selective, and focal segmen-
tal glomerulosclerosis, in which proteinuria is often non-
selective. However, more recently, Mallick, Short and
Manos [130] showed that, even in membranous nephrop-
athy, the selectivity index could discriminate between
patients who remitted and those whose proteinuria per-
sisted, the former having a SI lower than 0.19 (Table 1).
Woo et al [131] confirmed the predictive value of SI in
98 patients with IgA nephropathy (Table 2). We [132]
calculated the SI in 89 patients with nephrotic syndrome
due to membranous nephropahy, focal segmental glomer-
ulosclerosis or minimal-change nephropathy, but classi-
fied patients in three subgroups instead of two. Since as
many as 59% of membraneous nephropathy patients and
38% of focal segmental glomerulosclerosis patients had
a SI lower than the cut-off value of 0.2 but higher than 0.1,
we classified proteinuria as highly selective (SI ⱕ0.10),
moderately selective (SI ⱖ 0.11 ⱕ 0.20), or nonselective
(SI ⱖ0.21). Although the total amount of proteinuria was
comparable in the three groups, a significant difference in
clinical remission, in progression to chronic renal failure
and in response to therapy was found (Tables 1 and 2).
A correlation between the SI and the semiquantitative
evaluation of histologic lesions showed that the extent
of tubulointerstitial damage in patients with highly or
moderately selective proteinuria was significantly less
severe than in those with nonselective proteinuria, while
global glomerular sclerosis did not show a significant
relationship with SI (Table 3). Bakoush et al [133] have
very recently confirmed in a group of 56 proteinuric
patients with various glomerular diseases that the IgG/
transferrin SI below or above 0.2 is significantly corre-
lated with the severity of histologic lesions (Table 3).
The same group of investigators [26] has proposed a
different measure of SI, based on the comparison of the
clearance of IgM (instead of IgG) to that of albumin (in-
stead of transferrin), on the assumption that the higher
 D’Amico and Bazzi: Pathophysiology of proteinuria 819

Table 1. Predictive value of some qualitative markers of proteinuria for clinical remission in primary chronic glomerulonephritides

Glomerular No. of Predictive value for

Reference (No.) diseases patients Markers of proteinuria tested clinical remission
Mallick et al, 1983 (130) MN 40 IgG/transferrin selectivity index (⬍ vs. ⱖ0.19) Yes
Bazzi et al, 2000 (132) MCN, MN, FSGS 60 IgG/transferrin selectivity index (ⱕ0.10; ⱖ0.11 ⱕ 0.20; ⱖ0.21) Yes
Proteinuria (g/24 hours) No
Bazzi et al, 2001 (135) MN 38 IgG excretion (mg/g urine creatinine) Yes
␣1-microglobulin excretion (mg/g urine creatinine) Yes
Proteinuria (g/24 hours) No
Bazzi et al, 2002 (136) FSGS 29 Fractional excretion of IgG Yes
Fractional excretion of ␣1-microglobulin Yes
Proteinuria (g/24 hours) No
Abbreviations are: MN, membraneous nephropathy; MCN, minimal-change nephropathy; FSGS, focal segmental glomerulosclerosis.

Table 2. Predictive value of some qualitative markers of proteinuria for progression to chronic renal failure in
primary chronic glomerulonephritides

Predictive value for

No. of progression to
Reference (No.) Glomerular diseases patients Markers of proteinuria tested chronic renal failure
Bazzi et al, 2000 (132) MCN, MN, FSGS 60 Selectivity index (IgG/transferrin) Yes
(ⱕ0.10; ⱖ0.11 ⱕ 0.20; ⱖ0.21)
Proteinuria (g/24 hours) No
Bakoush et al, 2001 (26) MCN, MN, IgA nephropathy 84 Selectivity index (IgM/albumin) Yes
Albuminuria (albumin/creatinine index) Yes
Woo et al, 1989 (131) IgA nephropathy 98 Selectivity index (IgG/transferrin) (⬍0.20; ⬎0.20) Yes
Woo et al, 1991 (138) 60 LMW proteins at SDS-PAGE Yes
Bazzi et al, 1997 (139) MN, MPGN, FSGS 82 LMW proteins ⬍20 kD at SDS-PAGE Yes
Reichert et al, 1997 (134) MN 22 IgG excretion (mg/day) Yes
Proteinuria (g/24 hours) No
Reichert et al, 1995 (140) MN 30 ␤2-microglobulin excretion (mg/day) Yes
Proteinuria (g/24 hours) No
Bazzi et al, 2001 (135) MN 38 IgG excretion (mg/g urine creatinine) Yes
␣1-microglobulin excretion (mg/urine creatinine) Yes
Proteinuria (g/24 hours) No
Bazzi et al, 2002 (136) FSGS 29 Fractional excretion of IgG Yes
Fractional excretion of ␣1-microglobulin Yes
Proteinuria (g/24 hours) Yes
Abbreviations are: MN, membranous nephropathy; MCN, minimal-change nephropathy; MPGN, membranoproliferative glomerulonephritis; FSGS, focal segmental
glomerulosclerosis; LMW, low-molecular-weight; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Table 3. Correlations between some qualitative markers of proteinuria and severity of histological damage in
primary chronic glomerulonephritides
Tubular
Glomerular No. of Glomerular interstitial Vascular
Reference, year (No.) diseases patients Markers of proteinuria tested damage damage damage
Bazzi et al, 2000 (132) MCN, MN, FSGS 61 IgG/tranferrin selectivity index No Yes No
(ⱕ0.10; ⱖ0.11 ⱕ 0.20; ⱖ0.21)
Bakoush et al, 2001 (133) MCN, MN, FSGS 56 IgG/tranferrin selectivity index No Yes —
(ⱕ0.20 vs. ⬎0.20)
Bakoush et al, 2001 (26) MN, IgA nephropathy 84 IgM/albumin selectivity index Yes Yes —
(ⱕ0.002 vs. ⬎0.002) (Global sclerosis)
Albuminuria (albumin/creatinine index) No No —
Nagy et al, 1987 (137) IgA nephropathy 45 LMW proteinuria at SDS-PAGE — Yes —
Woo et al, 1991 (138) IgA nephropathy 60 LMW proteinuria at SDS-PAGE — Yes —
Bazzi et al, 1997 (139) MN, MPGN, FSGS 80 LMW proteins ⬍20kD at SDS-PAGE No Yes Yes
Bazzi et al, 2001 (135) MN 48 IgG excretion (mg/g urine creatinine) No Yes No
␣1-microglobulin excretion Yes Yes Yes
(mg/g urine creatinine)
Proteinuria (g/24 hrs) No No No
Bazzi et al, 2002 (136) FSGS 30 Fractional excretion of IgG Yes (% segmental sclerosis) No —
Fractional excretion of ␣1-microglobulin No Yes —
Abbreviations are: MN, membranous nephropathy; MCN, minimal-change nephropathy; MPGN, membranoproliferative glomerulonephritis; FSGS, focal segmental
glomerulosclerosis; LMW, low-molecular weight; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophorosis.
820 D’Amico and Bazzi: Pathophysiology of proteinuria
molecular weight of IgM in comparison with IgG could
reveal a more severe disruption of the permeability of
the glomerular capillary wall. Dividing 67 patients with
various primary glomerular diseases (IgA nephropathy,
membraneous nephropathy and mesangial proliferative
glomerulonephritis) into two subgroups, respectively,
with low (ⱕ0.002) and high (⬎0.002) SI based upon IgM
(IgM-SI), they found that at the end of the follow-up
significantly less patients with low IgM-SI had a decline
of creatinine clearance, despite the fact that the initial
average amount of proteinuria was higher in patients with
low IgM-SI (Table 2). In patients with low IgM-SI, the
number of obsolescent glomeruli was significantly lower
and the extent of interstitial fibrosis significantly less
marked (Table 3). No significant difference could be
found when the same parameters were correlated with
albuminuria. At the multiple regression analysis, IgM-SI,
but not albumin excretion rate, appeared to be significant
predictor of decline of renal function (Table 2).
Since the discriminant factor, in the evaluation of the
SI, is the urinary excretion of proteins with the highest
molecular weight, the urinary excretion of IgG has been
used as a marker of severity of the glomerular damage by
some investigators. Reichert, Koene, and Wetzels [134]
evaluated the predictive value for progression of urinary
excretion of IgG, together with total proteins, albumin,
and transferrin, in 22 patients with membraneous ne-
phropathy, nephrotic syndrome, and normal renal func-
tion. Only excretion of IgG and transferrin, but not that
of total proteins, was significantly different in progressors
versus nonprogressors. By multivariate analysis, only
IgG excretion was independently associated with deteri-
oration of renal function (Table 2).
We studied the predictive value of the excretion of
IgG, expressed in milligrams per gram of urinary creati-
nine and logaritmically transformed, in 78 patients with
biopsy-proved membraneous nephropathy [135]. The ex-
cretion of IgG, but not albumin, transferrin, or 24-hour
proteinuria, significantly correlated with the severity of
the tubulointerstitial damage, while no correlation was
found between glomerulosclerosis and any of the pro-
teinuric parameters, including IgG excretion (Table 3).
In one half of the patients, who had a nephrotic syndrome
and a normal renal function at the time of the evaluation
of proteinuria and renal biopsy, and a sufficiently long
follow-up, complete or partial remission was significantly
different (100% vs. 20%) according to a urinary excre-
tion of IgG lower or higher of a cut-off level of 110 mg/g
urinary creatinine (Table 1), as was progression to chronic
renal failure (0% vs. 35%); on the contrary, an amount
of 24-hour proteinuria below or above 5 g did not show
any discriminant value for either remission or progres-
sion (Tables 1 and 2). In the same study [135], 19 patients
treated with immunosuppressive therapy were compared
with 19 untreated patients. There was no difference in
the rate of remission or progression to chronic renal
failure between treated and untreated patients when the
baseline values of IgG were less than the cut-off value.
When IgG excretion values were greater than the cut-
off value, however, a difference between treated and
untreated patients for progression to chronic renal fail-
ure at the limit of statistical significance (10% vs. 60%;
P ⫽ 0.05) was found.
We have concluded a similar study in 50 patients with
primary focal segmental glomerulosclerosis (FSGS) [136]
and in 30 patients with membranoproliferative glomerulo-
nephritis (unpublished observations), with very similar
results (Table 1 to 3). In 29 FSGS patients with nephrotic
syndrome and baseline normal renal function the frac-
tional excretion of IgG, below or above a defined cutoff,
predicted remission in 91% and progression to end-stage
renal failure in 71% of patients, respectively. Further-
more, a significant difference (P ⫽ 0.000) was observed
for therapy responsiveness according to fractional excre-
tion of IgG: 70% of patients with fractional excretion of
IgG ⬍0.025 were responsive to steroids alone, 80% of
patients with fractional excretion of IgG ⱖ0.025 to ⬍0.140
were responsive to steroids and cyclophosphamide in com-
bination, 100% of patients with fractional excretion of
IgG ⬎0.140 were unresponsive to both drugs [136].
In our three cohorts of nephrotic patients (membranous
nephropathy, focal segmental glomerulosclerosis, and
membranoproliferative glomerulonephritis), we have mea-
sured the urinary excretion of another HMW protein,
␣2-macroglobulin, of molecular radius ⬃90 Å, the con-
centration of which is usually extremely low, below the
detectability limits, even in many pathologic conditions
characterized by increased size permeability of the glo-
merular barrier. This very large protein was detectable
in the urines in the totality (focal segmental glomerulo-
sclerosis and membranoproliferative glomerulonephritis)
or great majority (79%, in membranous nephropathy) of
patients in whom the IgG fractional excretion was above
the cut-off levels selected for each of the three diseases,
while it was undetectable in the majority of patients whose
fractional excretion of IgG, even though markedly in-
creased, was below the cut-off levels. These findings sug-
gest that the large pores permitting the transglomerular
passage of HMW proteins in nephrotic patients discrimi-
nate between IgG (55 Å) and ␣2-macroglobulin (90 Å),
as they discriminate, according to Bakoush et al [26],
between IgG and IgM (120 Å). They can be explained
assuming that discriminatory large pores exist, the den-
sity of which declines with their size.
Predictive value of fractional excretion of proteins of
very LMW (“tubular” proteins)
In 1987, Nagy et al [137] studied the proteinuria in
45 patients with IgA nephropathy and found that the
presence of LMW proteins in the sodium dodecyl sulfate-
 D’Amico and Bazzi: Pathophysiology of proteinuria
polyacrylamide gel electrophoresis (SDS-PAGE) pro-
teinuric pattern was significantly associated with higher
values of serum creatinine and was correlated with tu-
bulointerstitial lesions (Table 3). In 1991, Woo et al
[138] characterized proteinuria with SDS-PAGE in 60
patients with the same disease and found that the pres-
ence of LMW proteins was significantly associated with
a higher incidence of chronic renal failure after 6 years
of follow-up (Table 2); the proteinuric patterns were also
correlated with tubular atrophy and tubulointerstitial le-
sions (Table 3). We [139] measured LMW proteins by
SDS-PAGE in 145 patients with proteinuric primary
glomerulonephritis (focal segmental glomerulosclerois,
membranous nephropathy, and membranoproliferative
glomerulonephritis), further distinguishing the molecular
weight according to the presence or absence of LMW pro-
teins of a molecular weight below 20 kD (as low as 10 kD).
We found that the presence of such proteins of very
LMW was significantly associated with a higher serum
creatinine level, more marked proteinuria, and more se-
vere tubulointerstitial damage (Table 3). The presence
at baseline of LMW ⬍20 kD significantly predicted the
progression to chronic renal failure (Table 2). In the 20
patients of this cohort who were treated with steroids
(alone or in combination with cyclophosphamide), re-
sponse to therapy was significantly lower in those with
presence of LMW proteins ⬍20 kD (30% vs. 80%).
Reichert, Koene, and Wetzels [140] and our group [135–
136] confirmed the value of LMW proteins as sensitive
markers of proximal tubular function, and therefore of
tubulointerstitial damage and progressive disease, mea-
suring directly the urinary excretion of single proteins
of this class. Reichert, Koene, and Wetzels [140] mea-
sured the urinary excretion of ␤2-microglobulin in 30
patients with membranous nephropathy, nephrotic syn-
drome, and normal renal function and found that pro-
gression to chronic renal failure was significantly more
frequent when its excretion was greater than 500 ng/min
(Table 2). In 48 patients with membranous nephropathy,
we [135] found that the excretion of ␣1-microglobulin
was significantly associated with the extent of tubulo-
interstitial damage, global glomerular sclerosis, and
arteriolar hyalinosis (Table 3). By contrast, no correla-
tion was found between 24-hour total urinary protein
excretion, albumin, or transferrin, and any of the histo-
logic features. By multiple regression analysis, in a larger
population of 78 patients with membranous nephropa-
thy, we found a significant correlation between urinary
␣1-microglobulin excretion and that of IgG, but not of
albumin or total urinary protein excretion [135]. A
separate analysis, in the subgroup of patients who had
nephrotic syndrome and normal renal function at the
time of evaluation of proteinuria, revealed that those
with a very high urinary excretion of ␣1-microglobulin
(higher than a cut-off level of 33.5 mg/g urinary creati-
821
nine) progressed more frequently to chronic renal failure
(Table 2) and had a lower rate of clinical remission (Ta-
ble 1). As reported above, in the same population of
patients urinary excretion of IgG was also significantly
correlated with remission and progression to chronic re-
nal failure, while an amount of 24-hour proteinuria below
or above 5 g did not show any discriminant value either
for remission or for progression. By multiple logistic re-
gression analysis ␣1-microglobulin, but not IgG or 24-hour
proteinuria, was significantly associated with progression
to chronic renal failure. Only in patients with ␣1-micro-
globulin excretion above the cut-off level, a significant dif-
ference in the rate of progression to chronic renal failure
was found between treated (steroids and immunosup-
pressors) and untreated patients (17% vs. 100%). A simi-
lar study in 50 patients with primary focal segmental
glomerulosclerosis [136] and in 30 patients with idio-
pathic membranoproliferative glomerulonephritis (un-
published data) gave comparable results (Tables 1 to 3).
CONCLUSION
Two major mechanisms are responsable for the abnor-
mal urinary excretion of proteins that characterizes all
glomerular diseases, with or without nephrotic syndrome.
The first is an increased charge and size permeability of
the glomerular capillary wall, leading to the transglomer-
ular passage of albumin and of proteins of HMW that
usually do not cross the glomerular barrier. The second
is the consequent impairment of the mechanism of reab-
sorption of all proteins and, in particular, of the LMW
proteins by the epithelial cells of the proximal tubule,
due to increased work and/or toxic injury deriving from
the increased load of the abnormally filtered proteins in
the tubular lumen.
The quantity and the molecular weight and radius of
the proteins that reach the tubular lumen increase progres-
sively with the increasing severity of the disruption of
the structural integrity of the glomerular capillary walls,
with the consequent impairment of the selectivity of the
“barrier” to the transglomerular passage of proteins. In
the less severe cases with selective proteinuria, albumin
(molecular weight ⫽ 69 kD; molecular radius ⫽ 36 Å)
is the prevalent abnormal protein (Fig. 2B). With the
escalating severity of the glomerular lesions, increasing
amounts of proteins of larger molecular weight cross
the glomerular capillary wall, and the amount of IgG
(molecular weight ⫽ 150 kD; molecular radius ⬃55 Å)
in the tubular lumen increases progressively (Fig. 2C).
In the most severe cases, even proteins like ␣2-macro-
globulin (molecular weight ⫽ 720 kD; molecular radius ⫽
90 Å) and IgM (molecular weight ⫽ 900 kD; molecular
radius ⫽ 120 Å) reach the tubular lumen (Fig. 2D). It is,
therefore, theoretically correct that the SI of proteinuria
based on IgM excretion, or the fractional urinary excre-
822 D’Amico and Bazzi: Pathophysiology of proteinuria
tion of IgG, may be a better marker of the severity of
the damage of the glomerular capillary wall than the
overall amount of proteinuria. The clinical studies re-
ported above, and summarized in the Tables 1 to 3,
confirm this assumption.
In all the primary glomerular diseases with prevalent
damage of the glomerular capillary walls and proteinuria
in the nephrotic range (membranous nephropathy, focal
segmental glomerulosclerosis, and membranoprolifera-
tive glomerulonephritis), urinary excretion of IgG or
IgM, but not 24-hour proteinuria, predicted both clinical
remission and progression to chronic renal failure and
was correlated with the histologic lesions, especially tu-
bulointerstitial damage.
The increased transit in the tubular lumen of large
amounts of proteins of HMW induces a progressively
increasing damage of the epitelial cells of the proximal
tubule and the activation of the network of cytokines
and growth factors that induces interstitial infiltration
and fibrosis, primarily responsible for the progressive
deterioration of renal function. It is, therefore, theoreti-
cally justified that the presence of great fractions of pro-
teins of very LMW, which escaped tubular reabsorption,
such as ␣1-microglobulin (molecular weight ⫽ 31 kD) or
␤2-microglobulin (molecular weight ⫽ 11.8 kD), in the
urine may be a more reliable marker of the severity of
the tubulointerstitial damage than the overall amount
of proteinuria. The clinical studies reported above, and
summarized in the Tables 1 to 3, confirm even this as-
sumption.
Considering the great number of factors that intervene
during the course of chronic glomerular diseases and
that may affect the evolution of the damage and the
natural history, it is surprising to find that the amount
of HMW and LMW proteins detected in the urine at the
beginning of the observation period is a rather accurate
predictive marker of the subsequent outcome. This find-
ing suggests that there is a point of irreversibility of the
anatomic damage, both at the level of the glomerular
capillary wall and of the tubulointerstitium, of which the
amount of HMW and LMW proteins is a very reliable
indicator. If such “point of no return” has been already
reached at the time of the first qualitative characteriza-
tion of proteinuria, during the subsequent clinical course
a reduction in the fractional clearance of HMW and
LMW proteins, marking an improvement of the disrup-
tion of the permselectivity of the glomerualr filter, can
be considered an improbable event.
It has been shown by us [135] and by others [133] that
the fractional excretions of IgG, an HMW protein, and
of ␣1-microglobulin, a LMW protein, are significantly
correlated in proteinuric glomerular diseases, probably
because an increased transit of the HMW proteins in
the tubular fluid exerts an adverse effect on the tubular
cells, resulting in an increased urinary excretion of both
HMW and LMW proteins. However, the concomitant
measurement of both these urinary markers is clinically
justified in our opinion, since IgG is more related to the
glomerular damage, and ␣1-microglobulin to the tubulo-
interstitial damage. This damage is consequent but not
necessarily strictly correlated with the ongoing glomeru-
lar injury. The fact, reported above, that the fractional
excretion of IgG is a better predictor of clinical remission
than fractional excretion of ␣1-microglobulin in patients
with membranous nephropathy, while the latter better
predicts the progression to chronic renal failure than the
former, appear to confirm that the predictive role of
these two parameters can be different. However, at the
regression analysis in membranous nephropathy and fo-
cal segmental glomerulosclerosis, their contemporary
evaluation did not show any additional value for pre-
dicting the clinical outcome in comparison with each of
them considered separately (unpublished data). We still
measure the fractional excretion of both these markers
as a routine in all proteinuric glomerular diseases, and
recommend doing so in the clinical practice, especially
when no morphologic data from biopsy are available.
The impact of the baseline fractional excretion of IgG
(but not of the 24-hour proteinuria) on the therapeutic
response to a prolonged cycle of steroids, given alone
or in association with cyclophosphamide in our patients
with focal segmental glomerulosclerosis, deserves some
commentary. Only when the fractional excretion of this
marker of HMW proteins was below a defined cut-off
limit, was a total or partial response obtained in the
majority of patients. A possible explanation of these
results is that therapy is efficacious only when the density
of largest pores (of which the fractional excretion of
IgG is a good marker) is in the normal range or only
moderately increased. As observed by Ikegaya et al [61],
steroids and immunosuppressors may correct the size
barrier defect, only within a limited range of disruption
of the integrity of the glomerular capillary wall.
ACKNOWLEDGMENTS
This work was supported by the European Union grant QLGC1-
2000-00619 and by the Associazione per la Ricerca in Nefrologia.
Reprint requests to Giuseppe D’Amico, M.D., F.R.C.P., Division of
Nephrology, San Carlo Borromeo Hospital, Via Pio II, 3 – 20153 Mi-
lano, Italy
E-mail: giuseppe.damico@oscb.sined.net
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