Nephrol Dial Transplant, 2023, 0, 1–11

https://doi.org/10.1093/ndt/gfad215
Advance access publication date: 3 October 2023

Tubular toxicity of proteinuria and the progression of

REVIEW
chronic kidney disease

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Zhalaliddin Makhammajanov1 , Abduzhappar Gaipov 2 ,3
, Askhat Myngbay4 , Rostislav Bukasov5 , Mohamad Aljofan1
and Mehmet Kanbay 6
1
Department of Biomedical Sciences, School of Medicine, Nazarbayev University, Astana, Kazakhstan
2
Department of Medicine, School of Medicine, Nazarbayev University, Astana, Kazakhstan
3
Clinical Academic Department of Internal Medicine, CF “University Medical Center”, Astana, Kazakhstan
4
Department of Biology, School of Sciences and Humanities, Nazarbayev University, Astana, Kazakhstan
5
Department of Chemistry, School of Sciences and Humanities, Nazarbayev University, Astana, Kazakhstan
6
Division of Nephrology, Department of Internal Medicine, Koc University, Istanbul, Turkey
Correspondence to: Abduzhappar Gaipov; E-mail: abduzhappar.gaipov@nu.edu.kz

ABSTRACT
Proteinuria is a well-established biomarker of chronic kidney disease (CKD) and a risk predictor of associated disease outcomes. Pro-
teinuria is also a driver of CKD progression toward end-stage kidney disease. Toxic effects of filtered proteins on proximal tubular
epithelial cells enhance tubular atrophy and interstitial fibrosis. The extent of protein toxicity and the underlying molecular mecha-
nisms responsible for tubular injury during proteinuria remain unclear. Nevertheless, albumin elicits its toxic effects when degraded
and reabsorbed by proximal tubular epithelial cells. Overall, healthy kidneys excrete over 1000 individual proteins, which may be
potentially harmful to proximal tubular epithelial cells when filtered and/or reabsorbed in excess. Proteinuria can cause kidney dam-
age, inflammation and fibrosis by increasing reactive oxygen species, autophagy dysfunction, lysosomal membrane permeabilization,
endoplasmic reticulum stress and complement activation. Here we summarize toxic proteins reported in proteinuria and the current
understanding of molecular mechanisms of toxicity of proteins on proximal tubular epithelial cells leading to CKD progression.

Keywords: albuminuria, chronic kidney disease, proteinuria, renal tubular toxicity, tubulointerstitial fibrosis

INTRODUCTION sion, e.g. via toxic effects of filtered proteins on proximal tubu-
lar epithelial cells (PTECs), leading to tubular cell loss and tubu-
Chronic kidney disease (CKD) is characterized by long-term
lar atrophy followed by tubulointerstitial fibrosis, and eventually
functional decline and structural deterioration of the kidneys [1].
ESKD [7]. The presence of excess proteins in the urine follows at
The estimated prevalence of CKD varies between 11% and 13%
least one of the mechanisms: increased levels of low-molecular-
worldwide [2]. The level of glomerular filtration rate (GFR) and
weight proteins in the systemic circulation (prerenal proteinuria);
the extent of proteinuria/albuminuria define the severity of CKD
impaired permselectivity of the glomerulus (glomerular protein-
[3]. Proteinuria refers to elevated urinary protein levels, while
uria); abnormal reabsorption of low-molecular-weight proteins by
albuminuria specifically indicates abnormal albumin loss in the
tubular cells and tubular cell–expressed protein loss (tubular pro-
urine. GFR and albuminuria levels are the excretory and barrier
teinuria); or protein shedding from cells of the lower urinary tract
dysfunction indicators, respectively [1]. In the general population,
(post-renal proteinuria) [8] (Supplementary data, Table S1).
CKD is commonly observed among people over 65 years of age
Prerenal proteinuria is primarily caused by circulating plasma
as episodes and causes of kidney injury accumulate throughout
proteins released due to progressing diseases located outside
life; hence CKD in the elderly is usually multifactorial [2, 3].
the kidneys (Supplementary data, Table S2). The most common
Unfortunately, mortality due to CKD has been increasing over
causes of prerenal proteinuria are plasma cell neoplasms, rhab-
the past 25 years, and it is expected to grow yearly by about 1%
domyolysis and hemolysis [9–11]. Plasma cell neoplasms are re-
[4]. The primary factors associated with increased mortality have
sponsible for the overproduction of monoclonal immunoglobulin-
been identified as lower estimated GFR and higher proteinuria [5].
free light chains that render cytotoxicity on PTECs via different
These factors were further supported by a recent meta-analysis
mechanisms, leading to progressive CKD [9]. During rhabdomyol-
of clinical trials, which revealed that with treatment-induced
ysis, an excessive amount of myoglobin is released from muscle
reduction of proteinuria, every 30% reduction was associated
breakdown, swiftly filtered by the glomerulus and accumulated
with a 23.7% decreased risk of CKD progression to end-stage
in tubules, causing proximal tubular toxicity and acute kidney in-
kidney disease (ESKD) [6]. Notably, this reduction in proteinuria
jury (AKI) [10]. Likewise, massive hemolysis results in the accu-
was independent of the drug classes in the treatments.
mulation of hemoglobin in the systemic circulation and then in
Evidence shows that proteinuria is a potential biomarker and
the glomerular ultrafiltrate. Excess free hemoglobin and released
a risk predictor for ESKD and CKD-associated complications [7].
heme in PTECs exert significant direct cytotoxicity to cells, pro-
Proteinuria is recognized as a significant driver of CKD progres-
moting AKI and/or CKD [11].

Received: June 16, 2023; Editorial decision: September 4, 2023

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2 | Nephrol Dial Transplant, 2023, Vol. 0, No. 0
Renal proteinuria is the primary proteinuria responsible for
CKD development [7] (Table 1 and Supplementary data, Table S3).
Glomerular proteinuria arises from a dysfunctional glomerular
filtration barrier characterized by impaired charge and size selec-
tivity. On the other hand, tubular proteinuria is caused by dys-
functional tubules, mainly due to impaired endocytic receptors
such as the megalin–cubilin complex [12]. Unlike renal protein-
uria, post-renal proteinuria is a secondary form of proteinuria. It is
usually transient, resulting from infectious, inflammatory and/or
cancerous conditions of the distal genitourinary tract.
Molecular mechanisms for the progression of CKD into ESKD
are primarily associated with tubulointerstitial damage due to the
cytotoxicity of filtered urinary proteins to PTECs, regardless of the
type of glomerular disease, causing proteinuria. In this review, we
discuss the effects of proximal tubular toxicity due to filtered pro-
teins on the progression of CKD in renal proteinuria.
PROTEIN COMPOSITION IN PROTEINURIA
The glomerular ultrafiltrate primarily contains low-molecular-
weight proteins with molecular weights <60 kDa because of
glomerular permselectivity but are efficiently reabsorbed in the
proximal tubule [13]. Among serum proteins, albumin is the only
abundant larger protein that appears in the urine of healthy indi-
viduals at a concentration <30 mg/24 h out of total protein con-
tent of <150 mg/24 h. The intact filtration barrier retains most
serum albumin because of its molecular weight of 69.367 kDa
and a net charge of −15. Despite their high molecular weight,
some albumin molecules can appear in the ultrafiltrate owing
to their ellipsoid shape and flexibility [12]. Nevertheless, the re-
nal tubules efficiently reabsorb most of the albumin from the
ultrafiltrate. Thus, albumin in the final urine is negligible and
accounts for approximately 20% of the total protein. However,
albumin levels may increase up to 300-fold in glomerular pro-
teinuria due to filtration barrier damage and the limited capac-
ity of the proximal tubule to degrade and recover such albumin
from the filtrate [14]. Any disruption of the glomerular filtration
barrier that leads to an abnormal loss of albumin causes selec-
tive glomerular proteinuria (i.e. albuminuria). Additionally, the
presence of other high-molecular-weight proteins results in non-
selective glomerular proteinuria (Supplementary data, Table S1)
[15]. In clinical practice, urinary albumin concentration between
30 and 300 mg/24 h is called microalbuminuria, and >300 mg/24 h
is called proteinuria/albuminuria.
Although albumin is the most abundant serum protein in
the urine, some studies reported thousands of different proteins
as a component of normal urine, which can be detected us-
ing different techniques [16]. Marimuthu’s group identified 1823
proteins [17], while Santucci’s group reported 3429 proteins by
analyzing urine samples of healthy individuals [18]. Generally,
the main protein component of urine consists of 50–100 mg
of uromodulin secreted from cells of the thick ascending limb
of Henle, which makes up about 50% of the total protein and
is hence the most abundant in urine [16]. Normal urine also
contains low amounts of low-molecular-weight proteins and se-
creted proteins from lower urinary tracts, such as immunoglob-
ulin A (IgA) (Supplementary data, Table S4). The most fre-
quently reported proteins in proteinuria are listed in Table 1 and
Supplementary data, Tables S2 and S3, with indications of their
effects on PTECs.
PROXIMAL TUBULAR RECEPTORS AND
PROTEINURIA
Proximal tubules are crucial in maintaining low-protein urine
by an enzymatic breakdown and endocytosis of filtered proteins.
PTECs have extensive apical endocytic apparatus, where reab-
sorbed proteins are endocytosed, concentrated in vesicles and un-
dergo hydrolysis in lysosomes. Megalin, cubilin, amnionless and
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the neonatal Fc receptor for IgG are the primary receptors that
maintain the uptake of proteins from the ultrafiltrate [45]. Me-
galin is a member of the low-density lipoprotein receptor family
and is highly expressed at the apical surface of PTECs [45]. Cubilin
has no transmembrane domain and therefore needs megalin to be
associated with the membrane and for cellular endocytosis with
bound proteins. Cubilin also needs amnionless, a transmembrane
protein, for trafficking to the plasma membrane and correct local-
ization [45].
Notably, acute and chronic diseases, particularly those af-
fecting proximal tubular endocytic receptors, can lead to low-
molecular-weight proteinuria, which, if left untreated, may
progress to ESKD. Like patients with glomerular proteinuria,
many patients with low-molecular-weight proteinuria also show
progressive kidney function decline. Nonetheless, it remains
unclear whether low-molecular-weight proteinuria is a con-
tributor to tubular damage or solely a biomarker of such
damage [46].
Despite the knowledge advancement in tubular endocytic
receptors, little is known about their regulation and roles in
proteinuria. Previous in vitro studies have identified megalin as
a sensor of filtered albumin, determining whether PTECs will be
protected or injured by it [47]. At low doses, albumin seems to
promote PTECs survival by inducing the megalinprotein kinase B
interaction and the phosphorylation of BCL2 associated agonist
of cell death (BAD). Conversely, high doses of albumin reduce
megalin expression and lead to cell apoptosis. Also, in vitro stud-
ies showed that long-term exposure to albumin decreased the
binding and reabsorption capacity of PTECs, suggesting that per-
sistent exposure to albumin can be toxic to endocytic receptors
[48]. A recent study demonstrated that the expression of megalin,
cubilin and the neonatal Fc receptor for IgG was downregulated
in PTECs in an experimental model of protein-overload protein-
uria, while the expression of megalin was also markedly reduced
in kidneys of proteinuric patients [49]. Still, these receptors
were found in high levels in the urine, indicating a potential
shedding from cell surfaces in proteinuria. However, increased
shedding of megalin in urine samples of patients with IgA and
membranous nephropathy was associated with its maintained
expression [50].
Nevertheless, it is worth mentioning that albuminuria caused
by receptor-related genetic kidney diseases, particularly genetic
mutations of cubilin, has been found to be benign and not to lead
to kidney function decline in patients with chronic albuminuria
[51]. This study also showed that albuminuria was developed due
to decreased albumin reabsorption by mutated cubilin, thereby
causing less cellular cytotoxicity.
Overall, proteinuria/albuminuria appears to cause damage to
endocytic receptors, with or without changes in their expression.
However, whether proteinuria/albuminuria directly affects tubu-
lar receptors has not been clarified and thus is a subject for fur-
ther research.
Table 1: Most frequently reported urinary proteins and protein-bound molecules in renal proteinuria and their effects on proximal tubular cells.

Protein
molecular
Origin of proteinuria Tubular reabsorption Urinary proteins as biomarkers for weight
Protein (causes) (mechanism/receptor) Effects on PTECs (mechanism/cell phenotype) pathologies (kDa)

Albumin Renal: glomerular Reabsorption by – Proinflammatory via ROS accumulation – Glomerular and/or tubulointerstitial 69.367
damage and/or megalin–cubilin – Endothelin-1-mediated tubulointerstitial fibrosis and damage and CKD progression
inadequate complex, fluid-phase AKI–CKD progression (progressor of AKI to CKD) [20] – Detection of early-stage CKD [25]
reabsorption due to endocytosis and FcRn – Profibrotic via NOX activation [21] – Risk for AKI/progressive
tubular damage [19] [13] – Lysosome-mediated damage [22] CKD/CVD/ESKD/mortality in diabetic
– Endoplasmic reticulum stress-mediated damage [23] and nondiabetic patients [26]
– Oxidative damage via cadmium-albumin
complex/NOX activation/dysfunctional
mitochondria/lysosomal membrane
permeabilization [24]

Albumin-bound Renal: glomerular and/or Reabsorption is through – Proinflammatory via ROS accumulation [28] NA
fatty acids (e.g. tubular damage albumin endocytosis – Pro-apoptotic [27]
palmitate and and/or fatty acid – Profibrotic [29]
oleate) transporter-2–
mediated endocytosis
[27, 28]
Transferrin Renal: glomerular and/or Reabsorption is mediated – Endothelin-1-mediated tubulointerstitial fibrosis and – Early endothelial dysfunction in 78
tubular damage via cubilin and AKI–CKD progression (progressor of AKI to CKD) [20] diabetic patients at a very early
transferrin receptor 1 – Proinflammatory renal damage [31]
receptors [13] – Cytotoxicity via C3 deposition [30] – Prediction of onset of DKD [32]

IgG Renal: glomerular FcRn-mediated – Endothelin-1-mediated tubulointerstitial fibrosis and – Glomerular damage in diabetic and 150
damage (FSGS) endocytosis [33] AKI–CKD progression (progressor of AKI to CKD) [20] nondiabetic patients with
proteinuric glomerulopathies [34]
– Prediction of onset of DKD [32]

Complement C3 Renal: filtered and/or
locally expressed in
injured tubular cells
NA – Proinflammatory [35] – IgA nephropathy [38] 180
– Reperfusion damage [36]
– Profibrotic and cytolytic via generation of C3a and
C5a anaphylatoxins and membrane attack complex
[37]

Ceruloplasmin Renal: glomerular NA – Cytotoxic via increased release of copper from – IgA nephropathy [38] 132
damage ceruloplasmin under acidic conditions in tubular – Prediction of onset of DKD [32]
lumen [39]

N-acetyl-β-d- Renal: glomerular Not reabsorbed – Hydrolysis of glycoproteins, glycolipids, or – Tubular cell dysfunction and a 130-140
glucosaminidase damage or locally mucopolysaccharides in cells [40] predictor of outcome in primary
secreted due to tubular glomerulonephritis [40]
damage – Risk of kidney disease and
progression in diabetic patients [41]

Fibrinogen Renal: glomerular NA – Tubulointerstitial fibrosis by enhancing renal – Progression of CKD to ESKD [44] 340
damage and/or locally fibroblast proliferation [43]
secreted by injured
tubular cells [42]

CVD, cardiovascular disease; DKD, diabetic kidney disease; FcRn, the neonatal Fc receptor; FSGS, focal segmental glomerulosclerosis; NA, not applicable; NADPH, nicotinamide adenine dinucleotide phosphate; NOX, nicoti-
namide adenine dinucleotide phosphate oxidase.
Z. Makhammajanov et al. | 3

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4 | Nephrol Dial Transplant, 2023, Vol. 0, No. 0

Proximal tubular epithelial cell

Cubilin TGF-β1 TβRII FATP2

FFAs
Megalin Proteins (albumin)

ECM synthesis

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Enzymes ↑

TIMP-1 and TIMP-2 Rac1

Inflammation Enzymes
• NF-κB Amino acids
NOX • eNOS
Mitochondrion • Cytokines Stress/dysfunction
• VEGF ↑ Increase
↑ROS • MAPK
↑ATP ↑ROS Cyt c Induce
↑Cyt c • JNK/SAPK
Inhibit
Movement

Lysosome Caspase-9-mediated
PTEC death
ERK1/2
↑Actin and fibronectin

Tubular interstitial fibrosis ECM synthesis

Figure 1: Intracellular ROS activation and its effect on PTECs in proteinuria/albuminuria. ROS production is one of the major players in the
pathogenesis of tubulointerstitial injury. Reuptake of massive urinary filtered proteins, particularly albumin, leads to Rac1-mediated NOX activation
and ROS synthesis. Reabsorbed albumin also increases ROS accumulation in the cytoplasm by causing mitochondrial dysfunction. Reabsorbed
albumin is transported to lysosomes, and albumin-bound FFAs are transported further to tubular mitochondria after either albumin degradation into
amino acids in lysosomes or receptor-mediated endocytosis. FFA molecules are also reabsorbed from the ultrafiltrate by FATP2 and transported to
mitochondria. In the mitochondria, FFAs accelerate mitochondrial ROS synthesis leading to the loss of mitochondrial membrane potential and the
release of ROS and Cyt c into the cytoplasm. Cyt c activates caspase-9-pathway-mediated PTEC apoptosis. Accumulated ROS induces the activation of
NF-ĸB, eNOS, proinflammatory cytokines, VEGF, MAPK and JNK/SAPK. Most of these are potent proinflammatory factors as well as induce ROS
generation enabling a vicious cycle of tubulointerstitial injury. Furthermore, ROS is involved in TGF-β-mediated ECM synthesis and expansion that
results in interstitial fibrosis, which can also occur independently of TGF-β1 induction. Notably, the accumulating ECM components need to be
degraded and normalized. However, this regulatory mechanism is broken by albumin-induced TIMP-1 and TIMP-2 which block a family of zinc
metal–dependent enzymes that degrade one or more ECM proteins. In addition, FFAs can promote ECM development by enhancing the expression of
alpha–smooth muscle cell actin and inducing the secretion of fibronectin which is present in tubulointerstitial fibrosis. ATP, adenosine triphosphate;
Cyt c, cytochrome complex; eNOS, endothelial nitric oxide synthase; FATP2, fatty acid transport protein-2; JNK/SAPK, Jun N-terminal
kinases/stress-activated protein kinases; MAPK, p38 mitogen-activated protein kinase; NOX, nicotinamide adenine dinucleotide phosphate oxidase;
Rac1, Rho-related C3 botulinum toxin substrate 1; TIMP-1, tissue inhibitor of metalloproteinase 1; TIMP-2, tissue inhibitor of metalloproteinase 2;
VEGF, vascular endothelial growth factor.

TUBULAR TOXICITY OF FILTERED PROTEINS minimal change disease, disease progression is mild, and patients
Proteinuria increases the production of ROS do not develop ESKD with proper treatment and care. This may be
inside proximal tubular cells explained by the substantially low FFAs content of urinary albu-
min in minimal change disease compared with other nephrotic
Previous studies showed that reuptake of massive albumin trig-
syndromes [55].
gers high levels of reactive oxygen species (ROS) in PTECs (Fig. 1)
Serum albumin is the main carrier protein for FFAs (also
[52]. Albumin activates Rac1, a Rho-family small GTPase, by pro-
named non-esterified fatty acids), which, after passing the in-
tein kinase C in PTECs, leading to NOX, nicotinamide adenine din-
jured glomerular filtration barrier, are endocytosed into PTECs,
ucleotide phosphate oxidase activation and ROS production [52].
and transported to cell mitochondria after albumin degradation
Mechanistically, urinary albumin induces ROS generation by trig-
into amino acids by lysosomes [28]. During proteinuria, cellular
gering mitochondrial dysfunction [53]. Importantly, Ruggiero et al.
uptake of FFAs is also mediated by the apical plasma membrane
argue that albumin-bound free fatty acids (FFAs) cause mitochon-
receptor, FATP2, and FFAs are transported into the mitochondria
drial ROS-mediated PTEC demise, but not albumin itself [53]. In-
to trigger lipoapoptosis [27]. Inside mitochondria, FFAs are utilized
deed, it should be noted that multiple in vitro and in vivo exper-
for ATP generation by the electron transport system, tricarboxylic
iments have claimed that delipidated albumin is not cytotoxic,
acid cycle and β-oxidation [56]. Thus, protein overload results in
but rather albumin-bound FFAs (e.g. palmitate and oleate) cause
the accumulation of abundant FFAs in tubular cell mitochondria,
and exacerbate tubulointerstitial injury during protein overload
and they accelerate mitochondrial ROS synthesis leading to the
of PTECs [27, 53, 54]. In some cases of albuminuria, particularly in
loss of mitochondrial membrane potential [28]. Subsequently, this

leads to the release of mitochondrial cytochrome-c along with
ROS into cytosol and activation of caspase-9-pathway-mediated
PTEC apoptosis [57], leading to the loss of functional PTECs in the
kidney (Fig. 1).
Accumulated intracellular ROS induce a variety of signaling
cascades such as nuclear factor-kappaB (NF-κB), Jun N-terminal
kinases/stress-activated protein kinases, p38 mitogen-activated
protein kinase and uncoupling of endothelial nitric oxide syn-
thase, along with increased expression of inflammatory cytokines
and vascular endothelial growth factor [58–60]. Interestingly, most
of these signaling pathways will also increase the generation of
ROS. For example, a dimerized/coupled endothelial nitric oxide
synthase produces nitric oxide; however, an uncoupled form of
the enzyme will lead to the generation of superoxide anion O2 •– ,
the main form of ROS [61].
Altogether, these findings indicate that chronic protein over-
load in combination with lipid moiety substantially increases cel-
lular synthesis of ROS, thereby triggering and exacerbating tubu-
lointerstitial necroinflammation that eventually results in ESKD.
Proteinuria-induced ROS mediate
tubulointerstitial fibrosis development
Wolf and colleagues suggest that albumin upregulates the type II
transforming growth factor-beta receptor (TGF-β) in PTECs; this
can amplify the autocrine effect of proteinuria-induced TGF-β1
[62]. Bondi et al. claim that nicotinamide adenine dinucleotide
phosphate oxidase–derived ROS is involved in TGF-β1-induced ac-
tivation and transformation of fibroblasts into myofibroblasts, a
primary responsible source for extracellular matrix (ECM) syn-
thesis and expansion that results in interstitial fibrosis (Fig. 1)
[21]. Overproduced ROS induces α-SMA-positive myofibroblast ac-
tivation through the ERK1/2 signaling pathway [21]. This ROS-
mediated fibroblast conversion into myofibroblast can occur in-
dependently of TGF-β1 induction [63]. Notably, the accumulat-
ing ECM components are balanced by proteinases degrading ECM
molecules. Still, this counterregulatory mechanism gets impaired
by albumin-induced tissue inhibitors of metalloproteinases-1 and
tissue inhibitors of metalloproteinases-2 which block a family of
zinc metal–dependent enzymes that degrade one or more ECM
proteins at neutral pH [63]. Besides, lipid moiety of urinary al-
bumin has previously been shown to be responsible for ECM de-
velopment in protein-overloaded rats by upregulating the expres-
sion of alpha–smooth muscle cell actin, a marker of myofibroblast
transformation, and inducing the secretion of ECM proteins such
as fibronectin, which is present in tubulointerstitial fibrosis [54].
Recent work in the unilateral ureteral obstruction kidneys model
also demonstrated the roles of receptor-mediated uptake of FFAs
in kidney fibrogenesis [29]. Moreover, a previous human study of
kidney biopsies of patients with heavy proteinuria reported in-
creased production of TGF-β1 in PTECs and increased urine excre-
tion [64], signifying the role of TGF-β1 in tubulointerstitial fibrosis.
Overall, it seems that either directly or indirectly, albuminuria
via lipid moiety causes increased accumulation of ECM protein
and its decreased degradation, cumulatively enhancing the pro-
gression of tubulointerstitial fibrosis.
Proteinuria impairs tubular cell stress resilience
by compromising autophagy
Autophagy is a cellular recycling pathway that increases cell re-
silience to oxidative stress by retrieving biomolecules from redun-
dant or injured cellular components [65]. Autophagy carries out
recycling either non-selectively to maintain cellular homeostasis
Z. Makhammajanov et al. | 5
or selectively to target specific cellular components for degrada-
tion [65]. Examples of selective autophagy processes include mi-
tophagy, which targets damaged or dysfunctional mitochondria,
and lysophagy, which removes damaged or dysfunctional lyso-
somes. Autophagy activation has been suggested to play protec-
tive roles in PTECs early during AKI by increasing the resilience of
tubular cells to ischemic necroinflammation [66]. Nevertheless,
numerous studies showed that persistent or impaired autophagy
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activation could significantly contribute to AKI–CKD transition
and CKD progression in general (Fig. 2) [67].
Proteinuria activates autophagy in PTECs to protect the cells
from injury; however, as proteinuria persists, protein overload
impairs autophagy and renders their demise. Treatment of the
human proximal tubular HK-2 cell line with urinary proteins or
human serum albumin resulted in a dose- and time-dependent
increase in the synthesis of microtubule-associated protein 1
light chain 3-II (an autophagosome marker) [68]. This increase
peaked at 8 hours, and effective autophagic flux was completed
at 16 hours post-treatment, in comparison to the control cells.
In this study, oxidative stress was reported to mediate urinary
protein-induced autophagy activation. This study also demon-
strated autophagy activation induced by proteinuria in rat
protein-overload models, which could be modulated by chloro-
quine and rapamycin. Similarly, other studies also confirmed the
protective roles of protein-induced autophagy in PTECs [69].
Persistent protein exposure to PTECs is tubulotoxic by sup-
pressing autophagy, allowing the accumulation of toxic intra-
cellular molecules, such as long-lived proteins, which may be
cytotoxic on PTECs. In vitro studies demonstrated that treat-
ment of HK-2 and mouse primary proximal tubule cells with
albumin led to a dose-dependent reduction in the formation of
microtubule-associated protein light chain 3-II and autophagic
flux [14]. This effect was observed in the presence of bafilomycin,
an H+ -ATPase inhibitor that hinders microtubule-associated
protein light chain 3-II degradation. Additionally, these cells
exhibited a significantly increased half-life of long-lived proteins,
indicating impaired autophagy. Albumin-induced reduction of
autophagic response was mediated by inducing the activity of
a potent inhibitor of autophagy, the mammalian target of ra-
pamycin, as shown by highly phosphorylated S6K, a downstream
target of mammalian target of rapamycin, after treatment with
amino acids and proteins in vitro [14], suggesting that albumin
impairs autophagy via mammalian target of rapamycin pathway
after lysosomal degradation into amino acids.
Chronic proteinuria can also disrupt selective autophagy, accu-
mulating defective organelles, such as mitochondria, which pro-
mote tubular injury [70, 71]. Recent in vitro and in vivo studies
found that chronic albumin exposure of NRK-52E (rat-derived
PTEC cell line) cells and experimental mice led to impaired NIP3-
like protein X-mediated mitophagy, increasing the accumula-
tion of damaged mitochondria and mitochondrial ROS synthesis
[70]. In addition, these albumin-treated cells exhibited oxidative
stress–dependent cell death [72], and mice exhibited tubular le-
sions and renal dysfunction [70]. Studies of diabetic kidney dis-
ease models showed that defective mitophagy builds up damaged
mitochondria and ROS in PTECs, activating oxidative stress path-
ways and, eventually, tubular cell death [71]. Therefore, the quality
control of mitochondria is essential for the efficient reabsorption
of filtered proteins by PTECs, which require high energy.
These data indicate that autophagy may be critical in the early
stage of proteinuria to protect cells from injury. However, persis-
tent proteinuria probably has markedly cytotoxic effects on PTECs
by inhibiting basal and selective autophagy activities, allowing
6 | Nephrol Dial Transplant, 2023, Vol. 0, No. 0

FATP2 Cubilin
FFAs
Megalin Proteins (albumin)
Proximal tubular epithelial cell

Autophagosome

? mTOR

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Rac1 NOX
Lysosome
Mitochondrion

↓ ↓ ↑ROS ?
↑
↑ATP ↑ROS Cyt c
Kidney Inflammation
repair Apoptosis
↑pH ↓Degradation Lysosome
ER
↓Fusion ↓Mitophagy Cyt c ↑Ca
↓ ROS
Autophagosome
Calpain CHOP

↓ mTOR
PTEC death
Inflammation

↑
↑ROS

Tubular interstitial fibrosis ECM synthesis

Cathepsins Unfolded/long-lived proteins ↑ Increase Inhibit

Stress/dysfunction Amino acids Induce Movement

LC3-II

Figure 2: Proteinuria-induced dysfunctional autophagy, lysosomal membrane permeabilization and endoplasmic reticulum stress. Proteinuria initially
activates autophagy in PTECs by increasing ROS generation to protect cells from injury. However, prolonged exposure of filtered proteins to PTECs
starts to render toxic tubular effects by suppressing autophagic activities (e.g. mitophagy, basal autophagy and lysophagy), allowing the accumulation
of defective organelles such as mitochondria and toxic intracellular molecules such as long-lived proteins, which promote progressive tubular injury.
Accumulated defective mitochondria increase intracellular ROS and induce PTEC death, leading to tubulointerstitial inflammation, fibrosis and
nephron loss. Reabsorbed albumin can inhibit basal autophagy by activating mTOR after lysosomal degradation into amino acids, thereby allowing the
accumulation of long-lived proteins in the cytoplasm. Furthermore, massive proteinuria leads to an increase in the number and mass of lysosomes,
but it results in decreased lysosomal enzymatic and degradation activities. Massive proteinuria also overwhelms lysosomal pathways and leads to LMP
allowing the release of lysosomal ROS and cathepsins into the cytoplasm. In the cytoplasm, cathepsins cause PTEC death. In addition, LMP may cause
a decline in autophagosome clearance of defective lysosomes. Moreover, proteinuria leads to ER stress via at least two molecular processes, including
increased ROS and intracellular calcium levels in PTECs. Subsequently, ER stress causes calpain and/or CHOP pathways–mediated PTEC death. ATP,
adenosine triphosphate; CHOP, CCAAT/enhancer-binding protein homologous protein; Cyt c, cytochrome complex; ER, endoplasmic reticulum; FATP2,
fatty acid transport protein-2; LC3-II, microtubule-associated protein light chain 3-II; LMP, lysosomal membrane permeabilization; mTOR, mammalian
target of rapamycin; NOX, nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase; Rac1, Rho-related C3 botulinum toxin substrate 1.

accumulation of toxic intracellular molecules such as long-lived
proteins/protein aggregates and damaged organelles such as mi-
tochondria, thus promoting progressive tubular injury and CKD.
Lysosomal membrane permeabilization
A lysosome is a cellular membrane-enclosed organelle that car-
ries out the degradation of cellular components and macro-
molecules delivered through either endocytosis or autophagy [73].
Lysosomes are abundant in free iron because of the degradation
of iron-containing proteins. This elevates ROS inside lysosomes
via the iron-mediated generation of strongly reactive hydroxyl
radicals from lysosomal hydrogen peroxide, promoting lysoso-
mal membrane damage [74]. In addition, massive proteinuria
likely overwhelms lysosomal pathways and results in a lysoso-
mal rupture in PTECs, promoting tubular injury (Fig. 2) [22]. In
support of this notion, Liu et al. conducted in vitro and in vivo
experiments and found that lysosomal mass and amount were
considerably elevated in HK-2 cells and PTECs of patients with
nephrotic syndrome in response to protein overload. However,
lysosomal enzymatic and degradation activities were decreased,
as seen by impaired cathepsin-B/cathepsin-L activities and acid-
ification of lysosomes [75]. Additionally, in HK-2 cells, there was
evidence of cathepsin-B leakage into the cytoplasm. Moreover, in

PTECs from patients with various causes of nephrotic syndrome,
both cathepsin-B and lysosome-associated membrane protein-1
showed leakage, indicating lysosomal membrane permeabiliza-
tion. These effects were induced by oxidative stress following ex-
posure to urinary proteins [75]. Subsequently, depending on the
damage and release of its contents, including cathepsins, lyso-
somal membrane permeabilization triggers either apoptosis or
necrosis of cells [74].
In addition, lysosomal membrane permeabilization releases
ROS into the cytoplasm, exacerbating oxidative stress and lead-
ing to cell death [74]. Therefore, proteinuria-induced damaged
lysosomes can be an important contributor to the progression
of CKD. Notably, defective lysosomes must be eliminated by
lysophagy along with recovery of reduced degradation activity
via lysosome biogenesis to maintain the survival of PTECs [76].
However, lysosome depletion in the form of lysosomal membrane
permeabilization may cause defective lysosome clearance and
autophagy flux decline [76], thereby accumulating damaged lyso-
somes and increasing apoptotic pathways. These suggest that ef-
ficient lysosomal function is essential in the physiology of protein
reabsorption; however, protein overload of lysosomes disrupts
this degradation process and causes lysosomal membrane
permeabilization, resulting in lysosome-dependent cell death
and impaired autophagic flux, thereby promoting nephron loss,
interstitial inflammation and CKD.
Endoplasmic reticulum stress
The endoplasmic reticulum is essential in protein synthesis and
folding, lipid synthesis, and calcium storage. However, it can
become stressed when the demand for protein folding exceeds its
capacity. Studies suggest that proteinuria leads to endoplasmic
reticulum stress via at least two molecular processes, including
increased ROS and intracellular calcium levels in PTECs (Fig. 2)
[77]. Endoplasmic reticulum stress is characterized by dysfunc-
tional endoplasmic reticulum and accumulation of unfolded
proteins in the endoplasmic reticulum lumen [78], which triggers
an adaptive response called unfolded protein response [79]. If this
response is insufficient or sustained due to the toxicity of chronic
proteinuria, it fails to overcome endoplasmic reticulum stress and
triggers apoptotic pathways to eliminate damaged cells that even-
tually exacerbate tubulointerstitial injury [77]. Ohse et al. demon-
strated, using immortalized rat PTECs and an animal model of
massive proteinuria, puromycin aminonucleoside nephropathy,
that albumin exposure to PETCs markedly increased endoplasmic
reticulum stress proteins, including glucose-regulated protein 78
and oxygen-regulated protein 150, which led to calpain-mediated
caspase-12 activation and thus endoplasmic reticulum stress–
induced cell apoptosis [23]. Later, it was shown that albumin-
induced unfolded protein response causes tubular apoptosis and
lesions by Lipocalin-2 modulation via activating transcription
factor 4 in corporation with CCAAT/enhancer-binding protein ho-
mologous protein in experimental proteinuric mice [77]. Notably,
the inhibition of endoplasmic reticulum stress by 4-phenylbutyric
acid protected kidneys of mice from proteinuria-induced renal
lesions and Lipocalin-2 overexpression, and thus inhibited CKD
progression [77]. Furthermore, endoplasmic reticulum stress–
associated pro-apoptotic pathways may enter a vicious cycle of
causing irreversible cell death by inducing mitochondrial calcium
increase, dysfunction and pro-apoptotic factor secretion [80].
Overall, tubular toxicity of proteins leads to severe endoplasmic
reticulum stress that substantially contributes to tubular cell
loss and tubule atrophy during CKD progression.
Z. Makhammajanov et al. | 7
Cytotoxicity of complement proteins and
complement activation
Excessive proteinuria with complement activation in the kidney
tubules is one of the major drivers of tubulointerstitial damage
[81]. PTECs are susceptible to complement-mediated cell dam-
age due to negative or low expression of complement suppres-
sors such as membrane cofactor protein, complement receptor
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1, decay accelerating factor or CD59 on the apical region of the
cells [82]. Complement molecules are absent in the glomerular
ultrafiltrate, but they appear during proteinuria and can activate
the alternative pathway of the complement system [83]. Recent
evidence suggests that urinary properdin, a positive regulator of
the alternative pathway, together with filtered other complement
molecules, is responsible for the initiation of complement activa-
tion through binding to PTECs and functioning as a docking sta-
tion for the assembly of alternative pathway C3 convertase on the
apical surface of proximal tubules [84].
Additionally, properdin strongly stabilizes C3 convertase and
thus enhances alternative pathway amplification, leading to the
generation of C3a and C5a anaphylatoxins and membrane at-
tack complex (Fig. 3) [37]. Consequently, complement activation
may contribute to CKD progression with different mechanisms
that eventually lead to interstitial fibrosis. Abbate et al. found that
abnormal accumulation of C3 in the tubular lumen by protein
overload rather than locally expressed C3 triggered proteinuria-
induced tubulointerstitial inflammation and cell damage in a
murine model of protein-overload proteinuria [35]. An animal
study conducted on C3a receptor and/or C5a receptor–deficient
mice with transplanted kidneys from Crry–/– C3–/– mice reported
that the absence of C3a receptor significantly protected kidney
function via considerable reduction of inflammatory cell infiltra-
tion, tubulointerstitial injury and inflammation [85]. In this study,
complement receptor 1–related gene/protein-y (Crry) is a rodent-
specific complement inhibitor that was knocked out along with C3
to prevent embryonic lethality due to uncontrolled complement
activation [85]. Furthermore, experiments using primary and sec-
ondary human cultures demonstrated that C3a induces the ex-
pression of type I collagen and TGF-β1 in PTECs, suggesting that
C3a may be an essential player in interstitial fibrosis and CKD pro-
gression [86, 87].
Moreover, a previous study with C5aR−/− MRL mice supports the
hypothesis that C5a can contribute to tubulointerstitial inflam-
mation and fibrosis development [88]. Similar to C3a, C5a may
also facilitate tubulointerstitial fibrosis formation via direct ef-
fects on interstitial fibroblasts [89]. Unlike the two anaphylatoxins,
the insertion of membrane attack complex into the PTEC mem-
brane upregulates the synthesis of ROS, tumor necrosis factor–
alpha and interleukin 6, which altogether cause tubulointerstitial
damage and collagen synthesis associated with tubulointerstitial
fibrosis development [90, 91]. Also, the urinary membrane attack
complex is a potent inducer of PTEC cytolysis which disrupts the
cellular actin network [91].
In addition, other filtered plasma proteins can also facilitate
complement activation in the tubular lumen. For example, apical
exposure of urinary transferrin on PTECs upregulates cellular
biosynthesis of complement C3 and stimulates its secretion
[30]. Besides, urinary albumin assists with properdin-mediated
assembly and docking of C3 convertase at the surface of PTECs
by disturbing cell binding with factor H, a crucial regulator of
alternative pathways [92]. Thus, proteinuria-mediated comple-
ment activation in the tubular lumen is one of the main drivers
of tubular injury, interstitial fibrosis and CKD progression.
8 | Nephrol Dial Transplant, 2023, Vol. 0, No. 0

Proteinuria
C3a, C5a, MAC

Proximal tubular
epithelial cell

C5a C3a MAC Properdin

Complement molecules

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C3 convertase
↑ TGF-β1 Collagen ↑ROS Proteins
Cytolysis
↑TNF-α Factor H
↑IL-6
↑ Increase
Induce
Inhibit
Fibrosis Inflammation
Movement

Figure 3: The roles of proteinuria-mediated activated complement products in PTECs. Complement molecules are not filtered from the glomerulus but
during proteinuria, these molecules appear in the tubular lumen and can activate the alternative pathway of the complement system. Properdin along
with filtered other complement molecules leads to complement activation through binding to PTECs and functioning as a docking station for the
assembly of alternative pathway C3 convertase. Properdin stabilizes C3 convertase and enhances AP amplification leading to the generation of C3a
and C5a anaphylatoxins and MAC. C3a promotes tubulointerstitial fibrosis by inducing the synthesis of collagen and TGF-β1 in PTECs. C5a promotes
tubulointerstitial fibrosis by TGF-β1 induction. MAC can contribute to the development of tubulointerstitial fibrosis by inducing collagen synthesis. In
addition, MAC induces ROS, TNF-α and IL-6, as well as promoting tubulointerstitial injury and cytolysis of PTECs. Furthermore, filtered plasma
proteins can facilitate tubular complement activation except for filtered complement components. For example, urinary transferrin induces C3
expression and biosynthesis in PTECs, which is secreted into the tubular lumen. Besides, urinary albumin assists with properdin-mediated assembly
and docking of C3 convertase at the surface of PTECs by inhibiting tubular cell binding with factor H. IL-6, interleukin 6; MAC, membrane attack
complex; TNF-α, tumor necrosis factor-alpha.

CONCLUSION AUTHORS’ CONTRIBUTIONS

Proteinuria is a sensitive biomarker of CKD progression. Protein-
uria can be nephrotoxic directly due to toxicity to PTECs and in-
directly by increasing metabolic stress. Therefore, identifying and
establishing a list of toxic proteins with their state of toxicity on
PTECs can pave the way for identifying reliable specific biomark-
ers in the early stages of CKD and improves our understanding of
disease progression. The most reported molecular mechanisms
in proteinuria that promote kidney tubular damage, interstitial
inflammation and fibrosis include the increase of intracellular
ROS, autophagy dysfunction, lysosomal membrane permeabiliza-
tion, endoplasmic reticulum stress and complement activation.
Of note, these mechanisms are consequences of increased reab-
sorption of filtered proteins and/or FFAs bound to albumin. Thus,
further research is needed to elucidate the effects of reducing
the metabolic workload of PTECs by blocking endocytic receptors
and whether blocking protein reabsorption suppresses protein cy-
totoxicity and halts CKD progression. In addition, it remains to
be studied whether blocking protein uptake into PTECs can elicit
similar nephroprotective effects as blocking uptake of sodium and
glucose with sodium-glucose cotransporter 2 inhibitors, e.g. with
cubulin antagonists.
SUPPLEMENTARY DATA
Supplementary data are available at ndt online.
FUNDING
The authors of this review acknowledge funding from the
Nazarbayev University Collaborative Research Program (CRP) for
2020–2022 (Funder Project Reference: 091019CRP2105).
All authors contributed to researching data for the article, dis-
cussing the article’s content, writing the article, and reviewing or
editing the manuscript before submission.
DATA AVAILABILITY STATEMENT
No new data were generated or analyzed in support of this
research.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interests.
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