Special Issue: Diabetic Nephropathy
Toxic Immunoglobulin Light Chain Autoantibodies are
Associated with a Cluster of Severe Complications in Older
Adult Type 2 Diabetes
Mark B. Zimering1,2*, Mirkovic N1, Pandya M1, Zimering JH1, Behnke JA3, Thakker-Varia S3, Alder J3, Donnelly RJ4
1Medical Service (111), Veterans Affairs New Jersey Healthcare System, East Orange & Lyons, NJ, USA
2Division of Endocrinology, Rutgers-Robert Wood Johnson Medical School, New Brunswick, NJ, USA
3Department of Neuroscience and Cell Biology, Rutgers - Robert Wood Johnson Medical School, Piscataway, NJ, USA
4Molecular Resource Facility, Rutgers - New Jersey Medical School, Newark, NJ, USA
Mark B. Zimering, Medical Service (111), Veterans Affairs New Jersey Healthcare System, Lyons, NJ 07939, USA, Tel: +908
647-0180 x4426; Fax: +908-604-5249; E-mail: @
Received: January 21, 2016; Accepted: March 05, 2016; Published: March 08, 2016
Zimering MB, Mirkovic N, Pandya M, Zimering JH, Behnke JA, et al. (2016) Toxic Immunoglobulin Light Chain Autoantibodies
are Associated with a Cluster of Severe Complications in Older Adult Type 2 Diabetes. J Endocrinol Diab 3(1): 1-14. DOI: http://dx.doi.org/10.15226/2374-6890/3/1/00141
Aims: To assess neuronal depolarization evoked by autoantibodies in diabetic depression compared to depolarization evoked by autoantibodies
in control patients. To determine whether a subset of severe (late-onset) diabetic complications may be mediated in part by toxic immunoglobulin
light chains that may increase in diabetic nephropathy.
Methods: Protein-A eluates from plasma of 21 diabetic depression patients and 37 age-matched controls were tested for depolarization in
hippocampal or immature neurons. Subsets of depolarizing or non-depolarizing autoantibodies were tested for neurite outgrowth inhibition in
N2A neuroblastoma cells or the ability to modulate Ca2+ release in HL-1 atrial cardiomyocytes or in endothelial cells. The stability of depolarizing
autoantibodies was investigated by heat treatment (56°C x 30 minutes) or following prolonged exposure to the pro-protein convertase, furin. Gel
filtration of active depolarizing autoantibodies was performed to determine the apparent molecular mass of peak neurotoxicity associated with
Results: Diabetic depression (n = 21) autoantibodies caused significantly greater mean depolarization in neuroblastoma cells (P < 0.01)
compared to autoantibodies in diabetic (n = 15) or non-diabetic (n = 11) patients without depression. Depolarizing autoantibodies caused
significantly more (P=0.011) inhibition of neurite outgrowth in neuroblastoma cells than non-depolarizing autoantibodies (n = 10) and they evoked
sustained, global intracellular Ca2+ release in atrial cardiomyocytes or in endothelial cells. A subset of older diabetic patients suffering with a cluster
of nephropathy, non-ischemic cardiomyopathy and/or depression demonstrated the presence of stable light chain dimers having apparent MW of
46 kD and associated with peak neurotoxicity in neuroblastoma cells.
Conclusion: These data suggest that autoantibodies in older adult diabetic depression cause long-lasting depolarization in hippocampal
neurons including adult dentate gyrus neural progenitor cells. The autoantibodies may impair adult dentate gyrus neurogenesis associated with
treatment-refractory depression via several mechanisms including suppression of neurite outgrowth, and alteration of membrane excitability.
Stable, toxic light chain autoantibody components may contribute to a cluster of severe (late-onset) complications characterized by dysfunction in
highly vascularized tissues.
Keywords: Autoantibodies; Diabetes mellitus; Depression; Neural progenitor cells; Light chains; Nephropathy
Diabetic depression can affect up to thirty percent of older
adults with type 2 diabetes mellitus  contributing to substantial
morbidity and mortality . Co-morbid micro-vascular
complications are common in diabetic depression , yet the
underlying mechanism for an association between microvascular
complications and diabetic depression is unclear . In a recent
study, circulating endothelial cell (EC) inhibitory autoantibodies
were increased in type 2 diabetic depression and they inhibited
neurite outgrowth and caused decreased survival in adult dentate
gyrus (DG) neural progenitor cells (NPCs) . Dentate gyrus neural progenitor cells are a unique population of adult progenitor
cells whose proliferation and differentiation (into mature adult
DG neurons) requires excitatory synaptic inputs . In a prior
study, cancer fatigue/depression autoantibodies evoked longlasting
depolarization and suppressed spontaneous synaptic
activity in hippocampal neurons . Since dentate gyrus neural
progenitor cell proliferation and maturation correlated with antidepressant
treatment response(s) , in the present study we
tested a hypothesis that diabetic depression autoantibodies may
contribute to treatment-refractoriness (in part) by causing longlasting
(hippocampal) neuron depolarization.
We now report that plasma IgG autoantibodies in diabetic
depression caused significantly greater neuronal depolarization
than autoantibodies in diabetic or non-diabetic patients without
depression. In a subset of diabetes suffering with nephropathy,
non-ischemic cardiomyopathy and/or depression, depolarization
and neurite-inhibition were associated with highly stable light
chain component (s) capable of forming soluble dimers or
oligomers. These data suggest that circulating IgG autoantibodies
in diabetic depression are depolarizing and that a highly stable
component associated with peak neurotoxicity had properties
corresponding to kappa light chain dimers.
Subjects and Methods
Diabetic depression and control patient subgroups
Informed consent was obtained from all study patients prior
to blood drawing. Patients were enrolled from the diabetes and
endocrinology outpatient clinics at the Veterans Affairs New
Jersey Healthcare System. Autoantibodies obtained in patients
having type 2 diabetes and depressive illness (n = 21), diabetic
controls without depression (n = 15) diabetic nephropathy or
another pathology involving dysfunction in electrically-excitable
cells, i.e. (atrial fibrillation, painful neuropathy, or schizophrenia)
(n = 11) or nondiabetic controls (n = 11) were tested in neuronal
depolarization assays (Table 1). Autoantibodies in five additional
schizophrenia patients including two adult men with autoimmune
type 1 diabetes, an adult man with type 2 diabetic nephropathy,
and two non-diabetic, older adult women breast cancer survivors
were tested in neurite outgrowth and endothelial cell survival
assays (Table 1). The baseline clinical characteristics in the
patients whose autoantibodies were tested for ability to evoke
neuronal depolarization are summarized in Table 2. The other
pathologies subgroup included one non-diabetic patient with
painful neuropathy and atrial fibrillation. Diagnostic criteria and
clinical evaluation in diabetic depression patients was previously
reported . Painful diabetic neuropathy is defined according to
previously reported criteria . Diabetic nephropathy is defined
as urinary albumin excretion ≥ 300 milligrams/ gram creatinine
or urinary protein excretion ≥ 500 milligrams/ gram creatinine.
Non-ischemic cardiomyopathy and kidney disease
Pt 5-A 68-year-old Caucasian male (with family history of
Alzheimer's dementia) who suffered with atrial fibrillation
requiring pacemaker placement, hypertension, diabetic
nephropathy (300 milligram protein/gram creatinine, estimated
glomerular filtration rate (eGFR) 38 mL/ min/ 1.73 m2), painful
neuropathy, depression and multiple vague complaints including
unexplained joint pain, bouts of extreme muscular weakness
and unexplained dyspnea on exertion. Estimated glomerular
filtration rate was stable over a two- year period of observation
and the patient died of unknown causes.
Pt 7-A 64-year-old Caucasian male (with family history of
Alzheimer's dementia) who suffered with atrial fibrillation, left
ventricular hypertrophy, hypertension, diastolic dysfunction and
diabetic nephropathy (800 milligram protein/gram creatinine,
eGFR 47 mL/ min/ 1.73 m2). Estimated GFR declined to 40mL/ min/ 1.73 m2 over a four- year observation period during
which time the patient suffered a cerebellar infarct. Previously,
CT imaging of the brain had revealed moderate, global volume
loss, basal ganglia lacunar infarcts, old ischemic changes in the
right parietal lobe, and mid pons and hypo-densities suggestive
of microvascular angiopathy.
Pt 11-A 55-year-old Caucasian male (with family history
of Alzheimer's dementia) who suffered with recurrent atrial
fibrillation requiring implantable cardiac defibrillator,
hypertension, left ventricular hypertrophy, diastolic dysfunction,
diabetic nephropathy (1.3 gram albumin/gram creatinine, eGFR
65 mL/min/1.73 m2), mononeuritis multiplex, and depression.
The patient developed end-stage-renal disease requiring
hemodialysis and severe left ventricular systolic dysfunction
over a twelve-year period of observation.
Baseline plasma samples were obtained from study
participants prior to the initiation of study procedures.
Protein A chromatography
Protein-A chromatography was carried out as previously
described . The protein-A-eluates fractions consisted of total
IgG isolated from plasma upon low pH elution from the protein-A
column. The active protein-A- eluate caused significant inhibitory
activity in endothelial cell survival.
Endothelial cell survival assay
Bovine pulmonary artery endothelial cells (Clonetics, Inc. San
Diego, CA) were grown in Medium 199 plus 10% fetal calf serum
and endothelial cell growth medium (EGM, Clonetics, Inc., San
Diego, CA). Endothelial cell number assays were carried out as
previously reported . After 48 hours' incubation in the presence
of protein-A-eluate fractions, cells were washed with PBS and
processed for the colorimetric estimation of cell number, i.e. cell
associated acid phosphatase activity, as previously described .
Growth-promoting activity is expressed as a percentage of the
control cell number for cells grown in the absence of protein-Aeluate
Neural progenitor cells
The stem cells were originally isolated from the dentate
gyrus of adult Fisher 344 rats and were obtained from Dr. Fred
Gage at the Salk Institute. Dentate gyrus (DG) NPC was obtained
as previously described [5,10]. NPCs were trypsinized with
0.05% trypsin EDTA and plated at 144,500 cells/35mm dish
in Dulbecco's Modified Eagle Medium (DMEM)/F12 medium
high glucose (Omega Scientific, Tarzana, CA) containing N2
supplement (Invitrogen, Grand Island, NY), L-glutamine (2mM),
penicillin/streptomycin (100 U/ mL) without fibroblast growth
factor 2 (FGF-2). Test protein-A- eluate fractions (5-7 μg/ mL
protein) were added to cells on the third day in vitro (DIV 3) in
Mouse neuroblastoma cells
Mouse neuroblastoma N2A cells were cultured in Dulbecco's
Table 1: Summary of autoantibodies obtained in different patient groups.
Number of Patients
Neuronal depolarization assays
Specific patients and their characteristics
Pt 10- recurrent major depression & family history of depression/suicide in three successive generations
Pt 5, 11- CKD, non-ischemic cardiomyopathy
Diabetes without depression
Pt 7- CKD, non-ischemic cardiomyopathy
Diabetic nephropathy (n = 5)
Pt 8- CKD/ESRD & family history of diabetic CKD
Diabetic atrial fibrillation (n = 3)
Schizophrenia (n = 3), co-morbidity
Type 2 diabetes (n = 1)
Adult autoimmune type 1 diabetes (n = 1)
Autoimmune Thyrotoxicosis (n = 1)
Non-diabetic without depression
Neurite-outgrowth/Endothelial cell survival assays
Adult autoimmune type 1 diabetes (n = 2)
Type 2 diabetic nephropathy (n = 1)
Adult female breast cancer survivors (n = 2)
CKD- chronic kidney disease; ESRD-end-stage-renal disease
Table 2: Baseline clinical characteristics and co-morbidities in study patients.
(n = 21)
(n = l5)
Diab Neph/ Other Pathology (n = 11)
(n = 11)
69.3 + 3.9
69 + 10.1
66.1 + 10.4
60.7 + 14.6
34.8 + 8.3*
29.3 + 6.1
36.0 + 8.4*
28.5 + 4.2
8.7 + 1.6*
7.7 + 1.0
7.6 + 1.7
Atrial Fib (%)
Painful Neur (%)
Insulin use (%)
*P< 0.05 from T-test, ^ P< 0.05 from Χ2 test; comparing Diabetic Depression or Diabetic Nephropathy and Other Pathologies to Diabetic control group;
HbA1c- glycosylated hemoglobin; BMI- body mass index. Atrial Fibrillation; Painful Neuropathy; NT- not tested.
modified Eagle's medium (DMEM), containing 10% fetal
bovine serum, (FBS) (Invitrogen, Carlsbad, CA) and penicillin/
streptomycin (100 U/mL and 100 mg/ mL, respectively) at 37°C
and 5% CO2. Cells were plated in 96 well plates for 3 days prior to
membrane depolarization experiments.
N2A cell/ neurite outgrowth
Cells were plated at low density in 35 mm dishes. Next
recombinant human bFGF (10 ng/ mL) in the presence or
absence of human IgG test fractions were added to dishes in
triplicate. Groups of 50–100 cells/ dish were counted 2 days
after the addition of test factors. Neurite outgrowth represents
the percentage of N2A cells expressing more than one neurite. A
neurite is defined as a cell process that is at least 2 cell diameters in length of the cell body. Results are expressed as % N2A cells
expressing neurites (which represent the mean + SD of triplicate
determinations) compared to neurite expression in cells grown
with 10 ng/ mL human bFGF, but without added test protein-Aeluate
Developing hippocampal neurons
Embryonic day 18 (E18) rat hippocampal neurons, obtained
as previously reported , were plated in 96 well, poly-D-lysine
coated plates in (Neurobasal Media containing B27 Supplement
(1 mL/ 50 mL), 0.5 mM Glutamine Solution, Penicillin (10,000
units / mL) / Streptomycin (10,000 μg/ mL) (250 μL/ 50 mL),
1 mM HEPES medium at 100 cells/ mm2. Hippocampal neurons
were exposed to protein- A-eluates on (DIV8) (in depolarization assays) or on DIV13 (for 7 days) in survival assays.
Membrane depolarization assays
After cell attachment, growth medium was removed and cells
were washed and then incubated in modified Tyrode's solution
consisting of: 150 mM NaCL, 3 mM KCL and 30 mM HEPES, 10
mM D-glucose and 2 mM CaCL2, pH 7.4. Test fractions (human
IgG fractions) were added in the presence of 97 nM DiBAC4
(Molecular Probes, Eugene, OR)-a fluorescent voltage-sensitive
dye useful for monitoring changes in membrane potential .
Fluorescence was measured after 5 min or longer at room temp
using a Fluoroskan Ascent FL (VWR, Inc., Franklin, MA); Ex = 485
nm, Em = 538 nm. Results are expressed as percent of change in
gross fluorescence compared to cells to which no test protein-Aeluate
fractions were added.
Furin digests of diabetic protein-A eluates
Two microliters of an aqueous solution containing human
recombinant furin (>2000 units/ mL) was added to 40 μL of
protein-A- eluates (8 μg protein) from each of four diabetic
depression subjects in buffer containing 100 mmol/ L Tris, pH 7.0
and 200 μM calcium. Following 300 minutes incubation at 25°C,
4 μL aliquots of furin-treated or-untreated protein-A-eluates
were added (in quadruplicate to endothelial cells) for testing of
biological activity. The remaining aliquot of furin-treated and–
untreated paired samples was subjected to mass spectrometry.
MALDI mass spectrometry
Purified protein samples were mixed with an equal volume
of a saturated solution of Sinapinic acid (Sigma, St. Louis, MO)
and spotted onto MALDI target plates. Data was collected on the
Bruker Daltronics Autoflex III in the size range of 0 to 30,000
Daltons according to the manufacturer's recommendations. Data
was collected and analyzed using the MALDI Biotyper IVD-CE
workflow (Bruker Daltronics).
Amino acid sequencing was performed as previously described
 on the Applied Biosystems (Foster City, CA) Procise®494
protein sequencer using standard Edman sequencing. BLAST
(basic local alignment sequence tool) software was used to
search for amino acid homology between observed amino acid
sequence data and known human protein sequences.
HL-1 cell culture
HL-1 atrial cardiomyocytes were developed (and generously
provided) by Dr. William Claycomb (Louisiana State University
Medical Center, New Orleans, LA). They were maintained in 5%
CO2/ 95% air at 37°C in Claycomb media (Sigma, St. Louis, MO)
containing 10% FBS (Biocell, Rancho Dominguez, CA), 100 U/
mL:100 μg/ mL penicillin-streptomycin (Invitrogen, Carlsbad,
CA), 0.1 mM norepinephrine (Sigma, St. Louis, MO), and 2 mM
L-glutamine (Invitrogen, Carlsbad, CA).
Intracellular calcium measurement
HL-1 cells were grown in -ΔDT3 dishes (Bioptechs, Inc., Butler, PA) and loaded with 2 mM Fura-2 acetoxymethyl
ester(Molecular Probes, Eugene, OR) for 30 min at 37°C in
a balanced salt solution (BSS) (140 mM NaCL, 2.8 mM KCL,
2 mM CaCL2, 2 mM MgCL2, 10 mM HEPES, pH 7.2). The cells
were then left for a further 10-minute period in the bathing
solution at room temperature to allow ester hydrolysis to go to
completion. Using a dual-wavelength spectrofluorometer with
excitation wavelengths at 340 and 380 nm and emission at
510 nm fluorescence measurements were performed at room
temperature on the stage of an inverted fluorescence microscope
(Nikon TE200). The fluorescence spectra of a group of cells
were continuously monitored with a PTI spectrofluorometer
(Photon Technology International, Monmouth Junction, NJ). The
release of intracellular Ca2+ was measured following exposure
to 1:25 dilutions of the protein-A eluate or a highly- purified
hydroxyapatite chromatography fraction from patients' plasma.
Size exclusion (gel filtration) chromatography
G75 superfine Sephacryl (Pharmacia, Piscataway, NJ)
equilibrated in 10 mM sodium phosphate, 0.15 M NaCL, pH 7.4
was packed into a 5 x 0.7 cm column (Sigma, St. Louis, MO) having
a volume of 2.4 mL. An aliquot of the protein- A-eluate (0.05 mL)
was added to the column and eluted in 10 mM sodium phosphate,
0.15 M NaCL, pH 7.4 at a flow rate of 0.25 mL/ min, at 20°C. Each
individual collected fraction (0.125 mL) was assayed for effects
on N2A neurite outgrowth.
Hydroxyapatite (HA) chromatography was carried out as
previously described . The protein- A-eluate fraction was
diluted 1:1 in 10 mM sodium phosphate, pH 7.4 (binding buffer)
and then it was applied to the HA column and washed with 10
column volumes of binding buffer. Next the column was eluted
stepwise with equal volumes of 0.05 mM, 0.1 mM, 0.25 mM and
0.4 mM sodium phosphate, pH 7.4. The fraction eluting from HA
with 0.05 M sodium phosphate displayed peak endothelial cell
inhibitory activity and it was tested for effect on Ca2+ release in
HL-1 atrial cardiomyocytes.
Chemicals: Protein- A agarose was obtained from Pierce
Chemical Co., (Rockford, IL). All other chemicals and reagents
were analytical grade.
Protein determinations: Protein concentrations were
determined by a bicinchoninic acid protein assay kit (Pierce
Chemical Co., Rockford, IL).
Statistics: All data are the mean ±1 SD or SE as indicated.
Comparisons were made by Student's t-test for a continuous
variable, or by Chi-square (χ2) test for dichotomous variables
with a significance level, P = 0.05. For a multiple test, however,
the p-value was adjusted by the Bonferonni correction method,
dividing it by the number of comparisons.
Baseline clinical characteristics in study groups
Diabetic depression patients had significantly higher mean glycosylated hemoglobin, and higher baseline prevalence(s) of
nephropathy or painful neuropathy compared to age-matched
diabetic patients without depression (Table 2). Diabetic
depression and patients with nephropathy or a co-morbidity, (i.e.
atrial fibrillation, schizophrenia, painful neuropathy) involving
dysfunction in electrically-excitable cells had significantly higher
mean body mass index compared to age-matched diabetic
patients without depression (Table 2).
Depolarization induced by diabetic depression,
diabetic nephropathy or autoantibodies in other
Autoantibodies in diabetic depression (10 μg/ mL) (n = 21)
(121% vs 106%; P = 0.002) caused significantly greater mean
depolarization in N2A cells compared to identical concentrations
of autoantibodies in diabetes patients without depression (n
= 15) (Figure 1a). Autoantibodies in diabetic nephropathy (n
= 5) (128% vs 106%; P < 0.002) or in patients having another
disorder associated with dysfunction in electrically-excitable
cells (n = 6) (141% vs 106%; P < 0.002) were significantly more
depolarizing than identical concentrations of autoantibodies
in diabetic patients without depression (n = 15) (Figure 1a).
Figure 1: A) Diabetic depression (DM Depr), diabetic nephropathy (DM Neph) or other pathologies (Other Path) autoantibodies cause significantly
increased mean depolarization in neuroblastoma (N2A) cells compared to control diabetic (DM) autoantibodies. ** P < 0.002. B) Dose- N2A cell depolarization
response curves in representative autoantibodies from two diabetic depression (solid triangle- red, open diamond- blue line) and one
diabetic control patient (open square, green line). C) Dose- dentate gyrus neural progenitor cell depolarization response curves in representative
autoantibodies from two patients with diabetic nephropathy with (inverted open triangle-blue) or without depression (solid triangle- red line). B-C)
Data are mean, SD was < 10%. * P < 0.05 compared to cells without added autoantibodies.
Figure 1c); half- maximal depolarization occurred at 1.4 or 4.1
μg/ mL concentrations of the Pt 6 or Pt 7 IgG autoantibodies,
respectively (Figure 1c). The dose-depolarization curve in the
Pt 7 autoantibodies was multiphasic suggesting more than one
depolarizing IgG substance occurring at different concentrations
Diabetic depression or nephropathy autoantibodies
induce Ca2+ release in endothelial cells or in HL-1atrial
An overview of the experiments conducted to characterize
a stable low MW IgG component in depolarizing autoantibodies
having peak cell toxicity is shown in Figure 2. We next tested
whether depolarizing autoantibodies affect Ca2+ release in
endothelial cells or in electrically-excitable atrial cardiomyocytes.
Neuronal depolarizing autoantibodies (n = 3 of 3 tested at 1
μg/ mL concentrations) evoked prolonged intracellular Ca2+
release in HL1 atrial cardiomyocytes (e.g. Figure 3b). Potent
endothelial cell inhibitory diabetic nephropathy autoantibodies
caused prolonged suppression of rhythmic spontaneous calcium
oscillations in HL-1 cells (Figure 3c). A purified component (1
μg/ mL) having peak apparent MW (56 kD) corresponding to
IgG heavy chain  caused large increase(s) in intracellular Ca2+
in HL-1 cells (e.g. Figure 3c, arrow to the right). Diabetic and
nondiabetic control patient autoantibodies (n = 3) had little if
any significant effect on HL-1 Ca2+ release (Figure 3a, d) or on
spontaneous Ca2+ oscillations (Figure 3c). Diabetic recurrent major depression autoantibodies caused a ramp-like, persistent
increase in Ca2+ in endothelial cells (Figure 3e) and substantially
decreased survival in hippocampal neurons (arrows, Figure
2f). Mass spectrometry of the Pt 10 recurrent major depression
protein-A-eluate revealed MW peaks (11 kD, 23 kD, 56 kD, 112
kD) corresponding to half-light chain, light chain, heavy chain
monomer or heavy chain dimer, respectively (Figure 3g).
Stability of depolarizing activity in diabetic depression
protein A eluates
In a prior study, long-term storage ( > 6 months) of protein-
A-eluates in a subset of cancer fatigue/depression patients
unmasked latent EC inhibitory activity having apparent MWs
corresponding to IgG light chains (23 kD) or half -light chains
(11.5 kD) . Heating increased EC inhibitory effects from the
protein-A-eluates  suggesting increased toxicity resulted from
light chain-induced aggregation.
Heating (56°C x 30 mins) caused substantial (80%) loss of
depolarizing activity in three of three diabetic depression protein-
A-eluates tested (Diab Depression Pt 1-3, Table 3) suggesting that
in most cases, a complex tertiary protein structure is necessary to
mediate depolarization. Following heat treatment of the eluate
from Pt 5-who suffered with a triad of diabetic depression,
nephropathy, and atrial fibrillation- neuronal depolarization,
was increased by (613%) (Table 3), suggestive of enhanced
toxicity resulting from aggregation in a subset of heat-stable
immunoglobulin light chains.
Figure 2: Outline and brief overview of results in experiments performed to characterize stable peak toxicity in IgG autoantibodies causing increased
Figure 3: Representative non-depolarizing IgG A) had no effect on intracellular Ca2+ release; B) representative depolarizing IgG autoantibodies increased
intracellular Ca2+ release in HL-1 atrial cardiomyocytes. C-D) diabetic nephropathy/left ventricular hypertrophy (Pt 8) IgG interrupted spontaneous
HL-1 calcium oscillations (arrow to the left); D) a highly purified fraction (arrow to the far right) caused a large increase in intracellular
calcium; middle arrow represents wash-out of Pt 8 protein A eluate E) Pt 10 recurrent major depression eluate caused ramp-like increase in Ca2+ in
endothelial cells, F) Pt 10 eluate caused increased neurotoxicity after 7 days incubation with rat hippocampal neurons, arrows indicate degenerated
cell bodies G) mass spectrometry in Pt 10 eluate revealed low MW 11 kD and 23 kD light chain components. A&B) similar results were obtained in
five protein-A-eluates (three depolarizing IgG from diabetic nephropathy (n = 2) or a nondiabetic control patient (n = 1) with hypertension and left
ventricular hypertrophy (B), and two non-depolarizing IgG, both from nondiabetic controls.
Table 3: Effect of heat on depolarizing activity in diabetic depression protein-A- eluates Depolarizing Activity (%)*.
Heated (56°C x 30 minutes)
Atrial fibrillation, without nephropathy
144 + 11
109 + 14
Atrial fibrillation, with nephropathy
5. 70/ M
192 + 3
*Depolarizing activity was determined as change in basal N2A cell fluorescence (after 15 minutes incubation with protein-A-eluates fractions), as
described in Material and Methods.
^P-value from T-test comparing depolarizing activity before and after heat treatment.
Effect of furin treatment on EC toxicity in diabetic
depression protein A eluates
Furin is a ubiquitously expressed pro-protein convertase
which cleaves downstream of multi-basic amino acid sequences,
such as K-X-X-X-K-R ↓  present at the variable-constant
switch region in certain kappa light chains. In a prior study, furin
treatment unmasked highly potent, latent EC inhibitory activity
in diabetic glaucoma and dementia protein-A-eluates . In the
present study, furin selectively activated latent, highly potent EC
inhibitory activity present in the Pt 5 eluate; latent activity was
absent from three other diabetic depression protein- A-eluates
tested (Table 4). Mass spectrometry revealed (23 kD) light chain
peaks in both the Pt 5 and Pt 1 diabetes depression protein-Aeluates
before furin treatment (Figure 4). Several lower MW 8-9
kD peaks were present in the Pt 5 eluate before furin treatment
and they survived prolonged exposure (5 hours) to furin (Figure
Association between diabetic CKD and low molecular
weight IgG- derived peptides
We next subjected the protein-A-eluates from diabetic
patients having depression and/or atrial fibrillation with or
without nephropathy (n = 9) to mass spectrometry. Diabetic
nephropathy patients (n = 4) had significantly lower mean
estimated glomerular filtration rate (eGFR) indicative of chronic
kidney disease (CKD) compared to age-matched diabetic patients
without nephropathy (n = 5) (Table 5). The IgG components in
diabetic nephropathy protein-A- eluates had significantly lower
mean MW (8.0 kD vs 21.0 kD) compared to the IgG peaks in
protein-A- eluates from diabetes without nephropathy (Table 5).
Depolarizing protein A eluates inhibit neurite
Depolarizing autoantibodies in diabetic nephropathy or
diabetic depression patients inhibited neurite outgrowth (in
N2a cells) to a significantly greater extent (72 ± 13% vs. 105 ±
4%, P = 0.011, n = 10) than (identical concentrations of) nondepolarizing
autoantibodies from diabetic patients without
depression (Figure 5a). Schizophrenia plasma autoantibodies
(n = 5) including patients without diabetes (n = 2) or those
having type 1 (n = 1) or type 2 diabetes (n = 2) inhibited neurite
outgrowth to a significantly greater extent (42 ± 12% vs 105 ±
4%, P = 0.000015) than non-depolarizing diabetic autoantibodies from patients without depression (not shown in Figure 5a). The
schizophrenia plasma autoantibodies (n = 5) also substantially
decreased mean EC survival (63 ± 11%) (data not shown in Figure
5a). Heat treatment (56°C x 30 minutes) in diabetic depolarizing
autoantibodies caused an increase in mean neurite-inhibitory
activity (39 ± 15% vs 72 ± 13%, P = 0.046) compared to basal
levels (Figure 5a). Gel filtration of three heat-treated, diabetic
nephropathy/non-ischemic cardiomyopathy protein-A-eluates,
(i.e. Pt 5, 7, 11) revealed that peak neurotoxicity eluted from the
column with an apparent MW of 45.7 kD, corresponding to a κ light
chain dimer. Additional peaks of neurotoxicity having apparent
MWs of ~89 kD appeared in the Pt 7 and Pt 11 eluates, or ~11
kD in the Pt 7 eluate (Figure 5b). Gel filtration of the unheated
protein-A-eluate from a type 1 diabetic patient with co-morbid
schizophrenia and suffering with a seizure disorder, diabetic
nephropathy, and dementia displayed peak neurotoxicity in the
fractions eluting with apparent MWs of ~ 46 kD and 11 kD (not
shown in Figure 4b).
Diabetic depression autoantibodies bind neuralderived
Autoantibodies in diabetic depression displayed significantly
increased binding to PC12 cell-derived HSPG (195 ± 6% vs 149
± 9%; P = 0.004) (Figure 6a) or to HSPG purified from adult DG
neural progenitor cells (320 ± 9% vs 222 ± 16%; P = 0.004) (Figure
6b) compared to autoantibodies in diabetes without depression.
Association between non-ischemic cardiomyopathy/
nephropathy and family history of dementia
An unexpected observation was that patients 5,7,11 whose
plasma contained (heat-inducible) neurotoxic soluble light
chain dimers each had a parent who had died from (late-onset)
Alzheimer's dementia. Evidence that this association may not
have occurred entirely by chance, comes from our preliminary
observation that (3/4) patients having co-morbid nephropathy
and atrial fibrillation had a parent who had died of Alzheimer's
dementia compared to (0/18) (informative) diabetic patients
who lacked co-morbid nephropathy, and atrial fibrillation (75%
vs 0%, n =22; P < 0.001, Chi-square test).
Neural progenitor cells have a high input resistance and
normally reside in a 'vascular niche' accessible to the general
circulation . The present data suggest that diabetic
Table 4: Effect of furin treatment in diabetic depression protein- An eluates on endothelial cell inhibitory activity Endothelial cell activity (%)*.
117 + 5
112 + 12
94 + 10
117 + 15
94 + 6
100 + 5
Pt 5, Atrial Fib, nephropathy, FH of dementia
104 + 10
65 + 8
*Endothelial cell survival after 2 days incubation with protein-A-eluates fractions was determined as described in Materials and Methods: ^P-value
from T-test comparing activity before and after furin treatment. Fibrillation, FH- family history
Table 5: Association between diabetic nephropathy having reducing glomerular filtration rate and the presence of lower molecular weight peak plasma IgG light chain components
Depression and/or Atrial fibrillation
(N = 5)
(N = 4)
70.4 + 3
68.3 + 4
Mean eGFR (mL/min/1.73 m2)
91.8 + 23
38.1 + 21
Average peak MW (kD)*
21.2 + 7.7
8.0 + 0.96
Amino acid sequence homology to kappa light chain**
100% (n = 1)***
*average peak molecular weight (MW) was determined by mass spectrometry as described in Materials and Methods.
**Amino acid sequence homology to human kappa light chain variable sequence was determined as reported in Materials and Methods.
***Pt 8: a 62-year-old man with diabetic nephropathy progressing to end-stage-renal-disease who had family history of diabetic chronic kidney disease.
eGFR- estimated glomerular filtration rate.
depression autoantibodies which were active at concentrations
near the reported affinity constant (20 nM) of HSPG  may
target cell surface or matrix-associated HSPG altering dentate
gyrus neural progenitor cell membrane potential and excitability.
Depolarizing autoantibodies also caused significant neurite
outgrowth inhibition suggesting involvement of more than one
mechanism capable of impairing dentate gyrus neural progenitor
cell maturation and perhaps leading to reduced adult dentate
gyrus neurogenesis which is a hallmark of treatment-refractory
The precise mechanism underlying autoantibody-induced
depolarization is unknown. Lysophosphatidic acid is a serum
depolarizing factor which caused depolarization and RhoAmediated
neurite retraction by activating a subfamily of G proteins
which couple to multiple downstream signaling pathways
. Diabetic nephropathy/non-ischemic cardiomyopathy
autoantibodies were similarly pleiotropic, causing depolarization,
neurite retraction, intracellular Ca2+ release and suppression
of spontaneous Ca2+ oscillations, the latter which are required
for rhythmic firing of cardiac action potentials in HL-1 atrial
cardiomyocytes . Taken together, these data suggest possible
involvement of unknown G proteins in the mechanism linking
autoantibodies with neuronal depolarization, atrial arrhythmias,
cardiac hypertrophy or other diverse cellular effects.
Light chain aggregation causes enhanced cell toxicity in
systemic light chain amyloidosis (AL disease)  and in the present study may have resulted (in part) from heating diabetic
nephropathy/cardiomyopathy autoantibodies. In myelomaassociated
Fanconi syndrome, intact light chain variable domains
having MW 11-12 kD precipitate in proximal renal tubular
epithelial cells . Fanconi syndrome light chains are derived
from a restricted subclass of Vκ (I) germ line genes (e.g. O2/O12,
O8/O18) which can resist lysosomal protease degradation (in
proximal renal tubular epithelial cells) as a result of mutation(s)
involving (nonpolar) amino acid residues at critical solventexposed
position(s) in complementarity determining region,
CDR1, (e.g. amino acid positions 30-32) [22,23]. These germ line
genes encode variable domains having dibasic amino acid residues
in the variable-constant switch region  which may serve as
a preferred site for furin-mediated cleavage . In contrast,
wild- type light chains which lack such nonpolar residues at the solvent- exposed region in CDR1 undergo proteolytic attack
at solvent-exposed polar residues (in CDR1). Cleavages within
CDR1 (amino acid 30) and in the variable-constant switch region
(amino acid 107) are predicted to generate 8-9 kD MW fragments
which may represent a normal byproduct of renal tubular (kappa
light chain) metabolism. Based on these observations, peak
neurotoxic soluble light chain dimers having apparent MWs of 46
kD may arise (in part) through aggregation in protease-resistant
(11.5kD) fragments derived from a restricted subset of κ LC germ
line variable sequences. Assuming simple Mendelian inheritance
of certain restricted germ line kappa variable genes, our chance
observation that three patients harboring toxic soluble light chain dimers had a first-degree relative who had suffered from
late-onset Alzheimer's dementia suggests that additional forms
of neurotoxicity may result from soluble toxic kappa light chain
Figure 4: Mass spectrometry of A) diabetic depression protein- A-eluates, before or after furin treatment or in B) diabetic subsets without or with
nephropathy or chronic kidney disease, as described in Table 5.
Diabetic major recurrent depression autoantibodies which
were comprised (in part) of 11.5 kD MW half- light chains caused
persistent, large increase (s) in intracellular Ca2+ in endothelial
cells and were highly toxic in hippocampal neurons. The kinetics
of endothelial cell Ca2+ release (on-response) was slower than in
autoantibodies comprised of intact IgG or heavy chain dimers and
the absence of a detectable off-response suggests a mechanism
involving Ca2+ overload leading to cell death. Highly potent endothelial cell inhibitory activity previously reported in diabetic
nephropathy protein- A-eluates was associated with light chains
having reduced affinity for heparin Sepharose columns .
Since HSPG is known to promote light chain oligomerization ,
endothelial cell- or neuronal-associated HSPG may play a key
role in the assembly of light chain fragments into toxic oligomers
(in-vivo) and may have accounted in part for the lag phase in
the on-response of Ca2+ release following exposure to 11.5 kD
MW, half-light chain-containing major recurrent depression
The physiologic factors regulating activation of latent
autoantibody-associated light chain toxicity are unknown. In
Figure 5: A) Heat treatment (56 x 30 mins) in depolarizing protein- A-eluates from diabetic nephropathy/ nonischemic cardiomyopathy or depression
caused gain in N2A neurite outgrowth inhibition;
B) elution profile of neurite-inhibitory activity in the heat-treated diabetic nephropathy/nonischemic cardiomyopathy protein- A-eluates following
gel filtration on Sephadex G75 as described in Methods. A) Data are mean + SE; * P < 0.05 comparing activity in depolarizing eluates before and after
heating; or comparing (depolarizing vs non-depolarizing) eluates before; or after heating. B) Similar results were obtained following gel filtration of
the heat-treated Pt 11 protein-A-eluate or unheated protein-A-eluate from a diabetic schizophrenia patient with nephropathy and dementia.
a prior study, furin treatment of diabetic glaucoma and/or
dementia autoantibodies unmasked latent potent endothelial
cell toxicity  as was evidenced following furin treatment in
the Pt 5 protein-A-eluate in the present study. Furin is expressed
at the cell surface and in recycling endosomes in capillary
endothelial cells and it plays an important role in normal cardiac
development via activation of latent growth factors and proteases
. For example, shear stress upregulates endothelial cell
expression of furin and the pro-inflammatory, fibrogenic cytokine
transforming growth factor-β . Another possible mechanism
for increased light chain toxicity in diabetic nephropathy having
reduced glomerular filtration rate is as a result of substantially
increased polyclonal free κ light chains which may circulate at
30-fold higher concentrations  than the concentrations of
pathogenic monoclonal κ light chain in systemic light chain
Amyloidosis (AL) disease. A restricted subset of highly toxic κ light chains which comprises only ~1- 3% of polyclonal light
chain sequences might contribute to systemic pathophysiology,
e.g. non-ischemic cardiomyopathy, in diabetic chronic kidney
disease as a consequence of substantially increased overall free
kappa light chain concentration. Organ involvement in a subset
of diabetic cardiomyopathy/nephropathy patients expressing
toxic immunoglobulin light chains (Pts 5, 7, 11) mimicked the
distribution, (heart, kidney, peripheral nerve, lung and spleen)
found in systemic light chain Amyloidosis (AL) disease. Of
interest, Patient 7 suffered from non-traumatic splenic rupture
which is unusual in diabetes, but not uncommon in systemic (AL)
Amyloidosis. Yet amyloid deposits could not be demonstrated
either on histologic examination of the affected spleen or
following subsequent abdominal fat biopsy suggesting that
earliest toxicity may be mediated by soluble light chain dimers
which may not necessarily progress to the later stage of forming insoluble amyloid fibrils.
Figure 6: Diabetic) depression autoantibodies display increased binding to purified HSPG antigens.
A) derived from PC12 cells or B) NPC cells compared to control diabetic Diab control autoantibodies. A-B) Data are mean + SE.
Schizophrenia is associated with an increased family history of
autoimmune diseases including thyrotoxicosis or type 1 diabetes
. One hypothesis is that birth trauma in diabetic mothers
or maternal passage of brain-reactive autoantibodies causes
abnormalities in fetal neurodevelopment . Nearly all of the
adult patients in our study suffered with chronic schizophrenia,
making it difficult to ascertain whether autoantibodies having
both potent endothelial cell-inhibitory, and neurite outgrowthinhibitory
properties may have preceded the onset of
schizophrenia or were mere bystanders. The BB rat is a genetic
strain of spontaneous autoimmune diabetes in which antiendothelial
cell autoantibodies precede the development (and
are thought to contribute to pathophysiology) in autoimmune
diabetes . It is possible that the early onset in a subset of
genetically-susceptible, autoimmune diabetes-prone individuals
of anti-endothelial cell autoantibodies having additional neuritesuppressive,
and depolarizing properties might adversely affect
hippocampal neurodevelopment leading to mood and cognitive
Our study was limited by having only a small number of
adult male diabetic patients who experienced a cluster of disease
complications associated with toxic immunoglobulin light chains.
More study in a much larger, diverse population is needed
to test for association(s) between 'excitotoxic' depolarizing
autoantibodies and a cluster of non-ischemic cardiomyopathy/
nephropathy or neurodegenerative disorders. Since the diffusion
of bis-oxonol dyes (e.g. DiBac4) across the cell membrane
following depolarization requires several seconds to occur, we
could not assess the rapid phase of kinetics associated with IgGinduced
In summary, depolarizing IgG autoantibodies were
increased in plasma in subsets of diabetes in association
with disorders in which sustained depolarization may have a
role in pathophysiology, e.g. depression, nephropathy, atrial
fibrillation, painful neuropathy or schizophrenia. More study
is needed to determine whether immunoglobulin light chain
dimers comprised of specific kappa variable sequences might
provide a biomarker(s) useful in identifying subsets of diabetes
at increased risk for a cluster of severe complications involving
heart, kidney, lung, peripheral nerve or brain.
Supported by a grant from the Veterans Biomedical Research
Institute (East Orange, New Jersey) to MBZ.
Conflict of Interest
The authors report no conflict of interest that would affect
the objectivity of the present findings.
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