nmda receptors as potential therapeutic targets in

1

NMDA receptors as potential therapeutic targets in diabetic

nephropathy: Increased renal NMDA receptor subunit expression

in Akita mice and reduced nephropathy following sustained

treatment with memantine or MK-801

HILA ROSHANRAVAN1, EUN YOUNG KIM

1, STUART E. DRYER

1,2

1. Department of Biology and Biochemistry, University of Houston, Houston,

TX, USA

2. Division of Nephrology, Baylor College of Medicine, Houston, TX USA

Running title: NMDA antagonists reduce diabetic nephropathy

Author for correspondence:

Stuart E. Dryer, Ph.D., F.A.S.N.

Department of Biology and Biochemistry

The University of Houston

4800 Calhoun

Houston, TX 77204-5001

[email protected]

713-743-2697

Abstract: 200 words

Text: 4,208 words

7 figures

50 references

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Diabetes Publish Ahead of Print, published online July 7, 2016

2

Abstract

N-methyl-D-aspartate (NMDA) receptors are expressed throughout the kidney and the abundance

of these receptors and some of their endogenous agonists is increased in diabetes. Moreover,

sustained activation of podocyte NMDA receptors induces Ca2+

influx, oxidative stress, loss of

slit diaphragm proteins, and apoptosis. Here we observed that NMDA receptor subunits and

their transcripts are increased in podocytes and mesangial cells cultured in elevated glucose

compared to controls. A similar increase in NMDA subunits, especially NR1, NR2A and NR2C,

was observed in glomeruli and tubules of Akita mice. Sustained continuous treatment with the

strong NMDA receptor antagonist dizocilpine (MK-801) for 28 days starting at 8 weeks of age

reduced 24-hr albumin excretion, mesangial matrix expansion, and improved glomerular

ultrastructure in Akita mice. MK-801 did not alleviate reduced body weight of Akita mice and

had no effect on kidney histology or ultrastructure in DBA/2J controls. The structurally

dissimilar NMDA antagonist memantine also reduced diabetic nephropathy, although it was less

effective than MK-801. Inhibition of NMDA receptors may represent a valid therapeutic

approach to reduce renal complications of diabetes, and it is possible to develop well-tolerated

agents with minimal CNS effects. Two such agents, memantine and dextromethorphan, are

already in widespread clinical use.

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Introduction

More than 40% of end-stage renal disease (ESRD) in the United States can be currently

attributed to diabetes mellitus (1). The pathophysiology of diabetic nephropathy is complex (2-5)

and the reasons why only a subset of diabetic patients experience renal complications are not

well understood. Current mainstay therapies are based on glycemic and blood pressure control,

and renoprotective inhibition of renin-angiotensin signaling systems (4, 5). While these therapies

can slow the progression of diabetic nephropathy, many patients will nevertheless progress to

ESRD (4, 5).

NMDA receptors are a class of cation-selective ionotropic receptors with a high intrinsic

Ca2+

permeability (6, 7). These receptors are heterotetrameric and are assembled from multiple

subunits (NR1, NR2A, NR2B, NR2C, NR2D, NR3A and NR3B), encoded by seven different

genes. Functional NMDA receptors require two NR1 subunits that contain binding sites for the

co-agonist glycine. They also require either two NR2 subunits, which bind multiple endogenous

di-acidic agonists, or one NR2 and one NR3 subunit (8). NMDA receptors were first

characterized in the central nervous system (CNS) but are also expressed in peripheral organs,

including the kidney. There is evidence that renal NMDA receptors play a role in regulation of

blood flow, glomerular filtration, proximal tubule reabsorption, and urine concentration in the

collecting duct (9-14). NMDA receptors can be activated by several endogenous di-acidic

molecules, including L-glutamate, L-aspartate, L-homocysteic acid (HCA), L-quinolinic acid, and

guanidinosuccinic acid, although L-glutamate and L-aspartate are relatively weak agonists for

podocyte NMDA receptors (10, 12). In the CNS, sustained activation of neuronal NMDA

receptors can induce a form of neurodegeneration known as excitotoxicity (15). Sustained

exposure of mouse podocytes to NMDA or HCA evokes a similar phenomenon characterized by

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increased Ca2+

influx, oxidative stress, altered expression of slit diaphragm proteins, and

apoptosis (11, 16, 17).

Renal NMDA receptor NR1 subunit expression is upregulated in the Akita mouse model

of type-1 diabetes (18). In addition, elevated plasma L-homocysteine often occurs in patients

with type-1 or type-2 diabetes and is significantly correlated with renal complications (19-21).

Moreover, genes regulating L-homocysteine metabolism are possible susceptibility loci for

diabetic nephropathy (22-24). L-homocysteine spontaneously oxidizes to the NMDA agonist

HCA, and albuminuria and glomerular damage in mice with elevated plasma homocysteine can

be prevented by treatment with NMDA antagonists (16). Based on those previous studies, we

hypothesized that sustained hyper-activation of NMDA receptors might drive some of the

nephropathy in type 1 diabetes. Consistent with this hypothesis, we show here that sustained

treatment with either of two structurally dissimilar NMDA receptor antagonists, MK-801 or

memantine, reduce nephropathy in the Akita mouse model of type-1 diabetes. Memantine is

widely used clinically for treatment of Alzheimer’s disease, and is currently undergoing a

number of clinical investigations, primarily for neurological disorders (25-28).

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Materials and methods

Cell culture protocols: A mouse podocyte cell line (MPC-5) was propagated and differentiated

as described previously (10, 11). Primary mesangial cells were prepared using standard methods

(29). Briefly, de-capsulated glomeruli from 100-150 g Sprague-Dawley rats (Charles River Inc.)

were isolated using a sieving procedure (30, 31). The glomeruli were then maintained in RPMI-

1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml

streptomycin at 37°C for two weeks. At that time cells that had migrated out of the glomeruli

were approximately 80% confluent. The vast majority of those cells had an elongated spindle

shape as expected for mesangial cells, and they also expressed the mesangial cell marker α-

smooth muscle actin (Supplement Fig. 1). In experiments on high glucose, cultured cells were

exposed for 24 hr to RPMI-1640 medium supplemented with 16 mM glucose (for a final glucose

concentration of 25 mM) or with 16 mM mannitol (a metabolically inert osmotic control).

RNA isolation, reverse transcription-PCR, and immunoblot analysis: Total RNA from mouse

renal cortex, mouse podocytes or rat mesangial cells was isolated using the QIA™ shredder and

RNeasy™ mini kit (Qiagen Inc.). Aliquots of total RNA were reverse transcribed using the

ThermoScriptTM

RT-PCR System (Invitrogen). The PCR primers were:

NR1: forward ACTCCCAACGACCACTTCAC, reverse GTAGACGCGCATCATCTCAA

NR2A: forward AGACCTTAGCAGGCCCTCTC, reverse CTCTTGCTGTCCTCCAGACC

NR2B: forward CCGCAGCACTATTGAGAACA, reverse ATCCATGTGTAGCCGTAGCC

NR2C: forward GCAGAACTTCCTGGACTTGC, reverse CACAGCAGAACCTCCACTGA

NR2D: forward CAGCTGCAGGTCATTTTTGA, reverse GGATCTGCGCACTGACACTA

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NR3A: forward CAGAGGGATGAGCCAGAGTC, reverse CTTCCACACGGTTCAGGTTT

β-actin: forward AGCCATGTACGTAGCCATCC, reverse CTCTCAGCTGTGGTGGTGAA

Cycling parameters were 94° for 5 min, 30 cycles of 94° for 30 sec, 55° for 30 sec, 72° for 30

sec, and a final extension at 72° for 10 min. Immunoblot (31-33) was carried out using a panel

of primary antibodies selective for different NMDA receptor subunits (NMDA Receptor

Antibody Explorer Kit™, Alomone Labs).

Mouse models of type-1 diabetes: All animal experiments were conducted according to protocols

approved by the Institutional Animal Care and Use Committee of the University of Houston.

Male DBA/2J mice (strain 000671) and D2.B6-Ins2Akita

/MatbJ mice (strain 007562) from

Jackson Laboratory were given free access to food and water during the time of the experiments.

These Akita mice have been back-crossed onto the DBA/2J background for >12 generations. In

a subset of experiments, 10-week old DBA/2J mice were administered five injections of

streptozotocin (STZ) (40 mg/kg i.p in 0.1 ml of 0.1 M Na-citrate buffer, pH 4.5) or with the Na-

citrate vehicle at 24-hr intervals. Treatment with NMDA antagonists began 15 weeks after the

completion of STZ treatment.

Drug delivery: NMDA antagonists or saline vehicle were delivered continuously using Alzet™

1004 osmotic minipumps (Durect Corporation) implanted subcutaneously in the mid-scapular

area under isoflurane anesthesia. These pumps contained memantine HCl, or MK-801, or 0.9%

saline vehicle. Memantine was delivered at a dose of 0.2 mg/kg/day, whereas MK-801 was

delivered at 0.5 mg/kg/day.

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Assessment of diabetic nephropathy: Prior to initiation of treatment with NMDA antagonists or

vehicle, 8-week old mice were weighed and mean arterial blood pressure was measured by tail

cuff plethysmography for experiments on MK-801 (Kent Scientific Instruments). Mice were

then placed in metabolic cages for 24 hr and urine was collected and albumin quantified

(Albuwell M™ ELISA kit). Animals were weighed every 7 days. After 28 days of drug or

vehicle treatment, mean arterial blood pressure was measured again, a final 24-hr urine sample

was collected for albumin analysis, and animals were sacrificed by CO2 inhalation. Kidneys

were excised and a portion of renal cortex was reserved for biochemical analysis. The rest of the

kidney was used for histological and ultrastructural analysis.

Histology, immunohistochemistry, and electron microscopy: To assess surface expression of

NMDA receptors, podocytes cultured in normal or HG medium were fixed in 4%

paraformaldehyde, rinsed, and blocked. Primary antibodies were rabbit anti-NR1 (Alomone

Labs AGC-001) (1:100) or rabbit anti-NR2A (Alomone Labs AGC-002) (1:100) for 24 hr at 4º.

Those antibodies recognize epitopes on extracellular loops of NMDA receptor subunits. Cells

were not permeabilized prior to exposure to primary antibodies. Secondary antibody was Alexa

Fluor® 488-conjugated anti rabbit IgG (Invitrogen). The surface expression of NR1 and NR2A

were compared with Alexa Fluor® 594-conjugated wheat-germ agglutinin (WGA) (Invitrogen),

a plasma membrane marker. Images were collected on an Olympus FV-1000 confocal

microscope using a Plan Apo N 60X 1.42NA objective. Laser intensity and detector sensitivities

were held constant. Fluorescence intensity for NR1 and NR2A was quantified using

Photoshop®. For in vivo experiments, one of the kidneys was immersion-fixed in 4%

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paraformaldehyde, embedded in paraffin, and 2.5 µm sections were stained with periodic acid-

Schiff’s (PAS). Some paraffin sections were immunostained with antibodies against various

NMDA receptor subunits (anti-NR1 from Abcam), anti-NR2A and anti-NR2B (Thermo Fisher

Scientific), and anti-NR2C (Alomone Labs). Antigens were unmasked, and slides were

quenched, blocked, and incubated with primary antibody overnight at 4°C. Immunostaining was

produced using the Vectastain Elite ABC Kit™ and the DAB Peroxidase (HRP) Substrate Kit™

(Vector Laboratories). Mesangial matrix expansion in PAS-stained sections was calculated from

fractional volume of the mesangium on all of the glomeruli in a section using Adobe Creative

Cloud-Ps CC on PAS-stained slides (34). The percentage of mesangial matrix occupying each

glomerulus was rated on a scale using the following cut-offs: 0-25% = 0; 26-50% = 1; 51-75% =

2; and 76.0-100% = 3. For each mouse, glomerular matrix expansion was evaluated in a

minimum of 15 glomeruli and averaged to obtain a value for that animal. Statistical analysis was

carried out on the mean values from each group of animals, with N = 4 mice per group.

The other kidney was fixed by immersion in 3% glutaraldehyde/3% paraformaldehyde in

0.1 M sodium cacodylate buffer pH 7.3 for transmission and scanning electron microscopy

(EM). For transmission EM, the samples were post-fixed with 1% cacodylate-buffered osmium

tetroxide for 30 min, and stained with 1% uranyl acetate. The samples were dehydrated,

embedded in LX-112 resin, and allowed to polymerize at 60°C for three days. Ultrathin sections

were stained with uranyl acetate and lead citrate, and examined in a JEM 1010 transmission

electron microscope (JEOL USA Inc.) at an accelerating voltage of 80 kV. The thickness of the

glomerular basement membrane (GBM) was measured using Adobe Creative Cloud-Ps CC

software. For each mouse, and 3-4 different points of GBM from the same glomerulus, and from

a total of 15 glomeruli, were used for measurements to calculate a mean GBM thickness for that

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animal. We also measured foot process width for every foot process in a capillary loop through

multiple glomeruli, with a mean calculated independently for each animal. Statistical analyses of

GBM thickness and foot process width on groups of animals was then carried out with N = 4

mice per group. For scanning EM, fixed samples were washed with 0.1 M sodium cacodylate

buffer pH 7.3, post-fixed with 1% cacodylate-buffered osmium tetroxide, washed in 0.1 M

sodium cacodylate buffer, and then with distilled water. The samples were then treated

sequentially with filtered 1% aqueous tannic acid, distilled water, filtered 1% aqueous uranyl

acetate, and distilled water. Samples were dehydrated, transferred through a graded series of

hexamethyldisilazane, and then air-dried overnight. Samples were coated under vacuum with

platinum alloy to a thickness of 25 nm, and immediately flash carbon-coated under vacuum.

Samples were imaged in a JSM-5910 scanning electron microscope (JEOL USA Inc.) at an

accelerating voltage of 5 kV. Images were quantified by measuring the number of foot processes

per micron in numerous capillary tufts in each animal. A mean was then calculated

independently for each animal. Statistical analyses of the number of foot processes per micron

on groups of animals was then carried out with N = 3-4 mice per group.

Statistical analysis: Data from immunoblot and RT-PCR, based on triplicate measures, are

shown graphically as mean ± SD, and analyzed using Student’s unpaired t-test, with α = 0.05.

Fluorescence intensity in confocal microscopy was also analyzed by unpaired t-test. Data from

animal experiments are presented graphically as mean ± SEM. Quantitative data on renal

phenotypes were analyzed using weighted measures two-way ANOVA. In these analyses the

two independent variables were genotype (Akita vs. DBA/2J) and drug treatment (NMDA

antagonist vs. saline vehicle). A statistically positive result was inferred when F values for the

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interaction between drug effects and genotype, or for drug effects alone, indicated P < 0.05. All

statistical analyses were carried out using the on-line computational packages found at

vassarstats.net.

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Results

Increased NMDA receptor abundance in glomerular cells cultured in high glucose.

Immortalized podocytes (33, 35, 36) were cultured for 24 hr in a medium containing 9 mM

glucose supplemented with 16 mM mannitol, or in a medium containing 25 mM glucose. We

focused on NR1 and NR2 subunits required to form functional receptors that respond to NMDA

and other di-acidic agonists. Using a semi-quantitative RT-PCR procedure, we observed that 24-

hr exposure to high glucose increased the apparent abundance of NR1, NR2A, NR2B and NR2C

subunit transcripts (Fig. 1A, B). The same pattern was observed using immunoblot analysis, i.e.

increased abundance of NR1, NR2A, NR2B and NR2C subunits, but not NR2D subunits (Fig.

1C, D). Increases in NMDA subunit abundance also occurred in primary rat mesangial cells

after 24 hr exposure to high glucose. In mesangial cells we observed increased abundance of

transcripts encoding NR1, NR2B, and NR2C, but not NR2A or NR2D subunits (Fig. 2A, B). By

immunoblot we observed increased abundance of those same subunits (Fig. 2C, D). Thus,

elevated glucose is sufficient to increase the expression of multiple NR1 and NR2 subunits in

two different glomerular cell types. Note that there is a corresponding increase in the expression

of NR1 and NR2A subunits at the surface of podocytes cultured in high glucose, indicating that

these extra receptors are likely to be functional (Supplement Fig. 2).

Increased NMDA receptor subunit expression in the Akita mouse model of type-1 diabetes. In

these experiments we used male Akita mice (D2.B6-Ins2Akita

/MatbJ) that are heterozygous for

the C96Y mutation in the Ins2 gene (37). The control mice in our experiments were wild-type

DBA/2J mice. We examined expression of NMDA receptor subunits in renal cortex from 12-

week old animals. Using a semi-quantitative RT-PCR procedure, we obtained evidence for

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increased abundance of transcripts encoding NR1, NR2A, and NR2C subunits, but not NR2B or

NR2D (Fig. 3A, B). This pattern was also observed by immunoblot analyses of renal cortex of

these 12-week old animals (Fig. 3C, D). NR1, NR2A, and NR2C subunits are already increased

by 7 weeks of age (Supplement Fig. 3), which is well before the full manifestation of

nephropathy in Akita mice. The increase in NMDA receptor subunits is seen throughout in

glomeruli and in tubules (Fig. 4). Using immunohistochemistry we observed marked increases in

the abundance of NR1, NR2A and NR2C in tubules (Fig. 4A). Within glomeruli, we observed

increases in NR1, NR2A and NR2C in Akita mice but the signal appeared to be intense in only

some of the cells (Fig. 4B). Signal intensity is quantified in Fig. 4C, but it bears noting that these

should not be interpreted as a comparison of the abundance of different subunits analyzed with

different antibodies.

Blockade of NMDA receptors reduces progression of diabetic nephropathy in Akita mice.

Sustained activation of NMDA receptors can drive glomerulosclerosis in mice and rats (16, 17).

Increased numbers of receptors could lead to excessive NMDA receptor activation on cells even

if endogenous ligands are not changed. However, there is evidence that diabetes causes

metabolic changes leading to increased levels of circulating agonists in humans and Akita mice

(19-22, 38-42). To examine if sustained NMDA receptor activation contributes to the

progression of diabetic nephropathy, we implanted osmotic minipumps containing the NMDA

antagonist MK-801 or saline subcutaneously into Akita mice and DBA/2J controls at 8 weeks of

age, an age at which there are modest increases in urine albumin excretion. The pumps delivered

a dose of approximately 0.5 mg/kg/day of MK-801 continuously for 28 days. We observed that

saline- and MK-801-treated Akita mice do not gain weight at the same rate as DBA/2J controls

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(Fig. 5A). Reduced weight was actually greater in MK-801-treated Akita mice than in the saline

group. The reason why sustained MK-801 attenuates normal weight gain is not known. In any

case, this pattern suggests that MK-801 does not alleviate the primary metabolic consequences of

type-1 diabetes. In addition, 4-weeks of MK-801 treatment had no effect on blood glucose levels

(data not shown) or blood pressure (Supplement Fig. 4).

As with previous reports (37) we observed markedly increased 24-hr urine albumin

excretion in 12-week old Akita mice compared to DBA/2J controls (Fig. 5B). This was

attenuated in mice that received MK-801 compared to mice that received saline. These data

were analyzed using two-way ANOVA. We observed a significant effect of genotype (P <

0.0001), and a significant interaction effect between drug treatment and genotype (P < 0.05),

indicating that MK-801 reduces renal manifestations of diabetes. MK-801 also reduced

mesangial expansion in Akita mice (Fig. 5C, D). Mesangial expansion is not severe in Akita

mice on a DBA/2J background (37) but is nevertheless reduced in Akita mice that received MK-

801 compared to those that received saline, and the effect was significant based on two-way

ANOVA (P < 0.05). We also observed marked foot process effacement and thickening of the

glomerular basement membrane in Akita mice by 12-weeks of age (Fig. 5E-G). Ultrastructure

was improved in Akita mice that received MK-801 (Fig. 5E-G). There was a significant (P <

0.05) interaction effect between the effects of MK-801 and genotype on GBM thickness and foot

process width, again indicating a therapeutic effect of the drug. MK-801 had no discernible

effect on glomerular ultrastructure in DBA/2J controls. The effect of MK-801 was also seen

with scanning EM (Fig. 6). Thus, we observed marked foot process flattening and

disorganization in Akita mice that received saline (Fig. 6A), and a reduction in the number of

foot processes per micron measured along the long axis of a capillary tuft (Fig. 6B). There were

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still some abnormalities in Akita mice that received MK-801, but overall structure is closer to

normal, and there was a significant (P < 0.05) improvement in the number of foot processes per

micron. In general, effects of MK-801 on renal structure and ultrastructure were greater than

effects on albumin excretion. This was also seen in the low-dose streptozotocin model of type-1

diabetes in DBA/2J mice (Supplemental Fig. 5).

MK-801 is a strong NMDA receptor antagonist that produces profound cognitive and

behavioral effects (25, 43). By contrast, memantine is a structurally distinct NMDA receptor

antagonist that spares a basal level of NMDA receptor-mediated synaptic transmission while

reducing excitotoxicity (25, 43). It is in wide-spread clinical use (26-28). Memantine (0.2

mg/kg/day) or saline were applied using osmotic minipumps starting at 8 weeks of age. As with

MK-801, we observed that memantine did not alleviate lower growth rates (Fig. 7A), but

reduced 24-hr urine albumin excretion in Akita mice (Fig. 7B). The interaction between drug

and genotype on albumin excretion was significant (P < 0.05). Memantine also caused a trend

toward reduction in mesangial matrix expansion (Fig. 7C, D) (P = 0.0743) in Akita mice.

Memantine improved glomerular ultrastructure in Akita mice (Fig. 7E-G). The reduction in the

mean foot process width (effacement) revealed a significant (P < 0.05) interaction effect between

memantine treatment and genotype, indicating a therapeutic effect of the drug (Fig. 7G).

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Discussion

In this study we have tested the hypothesis that renal NMDA receptors contribute to the

progression of diabetic nephropathy in the Akita mouse model of type-1 diabetes. We observed

that elevated glucose causes marked increases in NMDA receptor abundance in two glomerular

cell types implicated in diabetic nephropathy; that there are marked increases in the abundance of

NMDA receptor subunits throughout the kidney of Akita mice; and that sustained inhibition of

NMDA receptors in vivo using two structurally dissimilar antagonists reduces urine albumin

excretion. In addition, MK-801 and memantine improved ultrastructural changes that occur in

glomeruli of Akita mice during early stages of diabetic nephropathy.

A surprising observation in this study is that all known NMDA receptor subunits and

their transcripts can be detected in an immortalized podocyte cell line (MPC-5 cells) and in

primary cultures enriched in rat mesangial cells. High glucose caused robust increases in NR1

subunits in both cell types. These cell types differed in terms of which NR2 subunits were

upregulated by high glucose. Nevertheless, the biochemical pattern in both cell types predicts

marked increases in functional responses to agonists. The overall pattern in podocytes is similar

to that seen in the renal cortex of Akita mice, but it should be noted that the biochemical signal in

kidney cortex extracts is almost certainly dominated by increases in the expression of NMDA

receptor subunits in tubules. Increased NMDA receptor abundance was already present by 7

weeks of age in Akita mice, a time at which nephropathy cannot be discerned using light

microscopic histological methods and urine albumin excretion is only slightly increased. This

suggests that elevated renal NMDA receptors drive the glomerular pathology and are not simply

a response to it.

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Previous studies have shown statistically significant correlations between serum L-

homocysteine and the subsequent appearance of microalbuminuria in patients with diabetes (19-

22). However, the marked induction of renal NMDA receptors in mouse models of diabetes

means that an excessive number of these receptors could be activated even if endogenous

agonists (e.g. HCA and L-quinolinic acid) are present at normal levels. Any increase in

circulating NMDA agonists, which can occur as renal failure progresses (38-42), would cause

even more NMDA receptors to become active.

While many studies of early-stage diabetic nephropathy have focused on glomerular and

vascular elements, diabetes produces effects throughout the kidney as a result of hyperfiltration,

proximal tubule hyper-reabsorption, alterations in sodium delivery to distal tubules, and marked

increases in tubular flow rates (44, 45). Indeed, in diabetes, there are sustained changes in

overall regulatory set-points resulting in marked changes in Na+ dynamics in diabetic tubules

(44, 45). An intriguing question is whether NMDA receptor activation leads to sustained

changes in expression of other transport proteins in a wide range of renal cells in manner

reminiscent of biochemical changes in neurons that occur during NMDA receptor-mediated

synaptic plasticity (46).

Excessive activation of CNS NMDA receptors induces Ca2+

-overload and oxidative

stress, leading to neurodegeneration (47). These observations spurred development of several

NMDA antagonists, many of which are effective in animal models of neurodegeneration (25,

43). MK-801 can cause nearly complete inhibition of NMDA receptors resulting in cognitive

and behavioral deficits, and therefore strong NMDA antagonists of this type are not used

clinically. We observed that continuous MK-801 treatment for 28 days reduced nearly every

index of diabetic nephropathy. Importantly, a reduction in albumin excretion and improvements

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in mesangial matrix expansion and foot process effacement were observed with memantine, a

second structurally distinct NMDA receptor antagonist. Memantine blocks the NMDA receptor

pore, although blockade with this drug is less complete than MK-801. Therefore, synaptic

transmission through NMDA receptors is maintained in the presence of memantine, while

excessive activation is eliminated (25, 43). Clinical studies have shown that memantine slows

the progression of cognitive decline in Alzheimer’s disease (26-28). We observed that

memantine reduced albumin excretion, foot process effacement and mesangial matrix expansion

in Akita mice, albeit less than MK-801. Memantine was administered at a dose of 0.2 mg/kg/day.

This dose was chosen to be comparable to the FDA-approved regimen of 20 mg daily that is

recommended for treatment of Alzheimer’s disease (48). However, higher doses of memantine

are well tolerated and may produce additional benefits for patients with Alzheimer’s disease

(48). It is possible that higher doses of memantine might produce greater effects in diabetic

nephropathy, as we saw with MK-801.

The mechanisms whereby NMDA antagonists could be beneficial to renal function in

diabetes are likely to be complex. NMDA antagonists at the doses used here did not affect

systemic mean arterial blood pressure. However, NMDA receptors in renal cortex mediate a

tonic vasodilatory response, and it is possible that inhibition of these receptors produces

hemodynamic effects that help to preserve renal function in the presence of hyperglycemia.

NMDA receptors in collecting ducts are upregulated in response to osmotic challenge (14), and

perhaps this is relevant in the context of polyuria that occurs in diabetes. As mentioned

previously, sustained application of NMDA to cultured podocytes induces Ca2+

influx, oxidative

stress and reduced abundance of slit diaphragm proteins (11). If this occurs in vivo, one would

expect glomerulosclerosis and glomerular dysfunction, as is seen in mouse models of

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hyperhomocysteinemia (16, 17). With continuing loss of renal function, circulating NMDA

agonists such as HCA, guanadinosuccinate, and L-quinolinic acid may increase, thereby

producing a positive feedback loop driving renal pathology.

The major limitation of this study is its reliance on small molecule pharmacological

inhibitors of NMDA receptors, which are not just expressed in kidney, but which are also

expressed in other peripheral organs, including pancreatic β-cells, and throughout the CNS.

While the two inhibitors studied here are structurally dissimilar, it is certainly possible that they

have off-target effects in common. Therefore, it is not possible to pinpoint exactly where

NMDA antagonists are acting to reduce the renal complications in Akita mice, especially since

both agents cross the blood-brain barrier.

On the other hand, this study also suggests possible utility of a pharmacological strategy

based on well-tolerated drugs that are already in wide-spread clinical use. The availability of

usable drugs to block NMDA receptors, such as memantine, contrasts with the future promise of

gene therapies or other as yet unproven strategies that may be required for some other potential

therapeutic targets.

In summary, we have observed that hyperglycemia and diabetes cause marked increases

in the expression of renal NMDA receptors throughout the kidney, and that sustained treatment

with NMDA antagonists reduces the progression of nephropathy in a mouse model of type-1

diabetes. One of the agents tested here, memantine, is well tolerated and in widespread clinical

use for other conditions. It is possible that other clinically used drugs in this class, such as

dextromethorphan, might also be useful. In this regard, the present experiments were carried out

in a mouse model of type-1 diabetes in which there is quite minimal β-cell function. However,

there is evidence that patients with type-2 diabetes have improved insulin secretion and glycemic

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control after treatment with dextromethorphan, in part due to effects on NMDA receptors on

pancreatic islets (49). In addition, a large number of NMDA antagonists that bind to sites on

NR1 subunits have been discovered (50). Many of those have limited ability to cross the blood-

brain barrier. While that would limit their usefulness for most neurological conditions, it could

represent a strategy to strongly inhibit peripheral NMDA receptors without producing cognitive

dysfunction.

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Acknowledgements

Author contributions. HR carried out all analyses of renal structure and function in mouse

models, carried out statistical analyses, prepared figures, and edited the manuscript. EYK

carried out all of the biochemical experiments on podocyte and mesangial cell lines, and

measured NMDA receptor expression in renal tissues, analyzed those data, prepared figures,

and edited the manuscript. SED conceived the project including statistical design,

contributed to figure preparation, and wrote and edited the manuscript.

The High Resolution Electron Microscopy Facility at the M. D. Anderson Cancer Center in

Houston, TX is supported by NIH grant P30-CA-016672. We are grateful to Mr. Kenneth

Dunner, Jr. B.A., A.B., of the M. D. Anderson Cancer Center in Houston, TX, for carrying

out scanning and transmission electron microscopy.

This research was supported by the Juvenile Diabetes Research Foundation Grant

201301041.

Drs. Stuart Dryer, Eun Young Kim and Hila Roshanravan take responsibility for the contents

of this article.

Portions of this work appeared in abstract form in oral and poster presentations at the annual

meetings of the American Society of Nephrology (November, 2015) and Experimental

Biology (April, 2016).

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Figure legends

Figure 1. Exposure to high glucose increases expression of NMDA receptor subunits in cultured

mouse podocytes (MPC-5 cells). A, Representative results of RT-PCR showing increased

abundance of transcripts encoding NR1, NR2A, NR2B and NR2C subunits in cells cultured for

24 hr in a medium containing 25 mM glucose (HG). There was no change in NR2D. Control

cells (Con) were cultured in medium containing 9 mM with 16 mM mannitol as an osmotic

control. B, Densitometric analysis of three repetitions of the experiments shown in A. Bar

graphs represent mean ± SD. In this and subsequent figures, asterisks indicate (P < 0.05). C,

Immunoblot analysis showing increased abundance of NMDA receptor subunits in podocytes

cultured in HG compared to control (Con). D, Densitometric analysis of three repetitions of the

experiments shown in C. Bar graphs represent mean ± SD. Statistical analyses in this figure

were by Student’s unpaired t-test.

Figure 2. Exposure to high glucose increases expression of NMDA receptor subunits in primary

cultures of rat mesangial cells. A, Representative results of RT-PCR showing significantly

increased abundance of transcripts encoding NR1, NR2B and NR2C subunits, but not of NR2A

or NR2D, in cells cultured for 24 hr in HG medium compared to cells cultured in normal glucose

(Con). B, Densitometric analysis of three repetitions of the experiments shown in A. C,

Immunoblot analysis showing increased abundance of NMDA receptor subunits in primary

cultures of rat mesangial cells cultured in HG. D, Densitometric analysis of three repetitions of

the experiments shown in C. Statistical significance was assessed by Student’s unpaired t-test.

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Figure 3. Increased expression of NMDA receptor subunits in renal cortex of Akita mice. A,

Representative results of RT-PCR showing increased abundance of transcripts encoding NR1,

NR2A, and NR2C subunits (but not in NR2B or NR2D) in renal cortex in 12 week-old Akita

mice compared to 12-week old DBA/2J control mice. B, Densitometric analysis from N = 4

mice per group. Bar graphs represent mean ± SD. C, Immunoblot analysis showing increased

abundance of NMDA receptor subunits in Akita mice compared to DBA/2J controls. D,

Densitometric analysis of analyses from N = 4 mice per group. Bar graphs represent mean ± SD.

Data were analyzed by Student’s unpaired t-test.

Figure 4. Immunohistochemistry suggests increased abundance of NMDA receptor subunits

throughout the kidney of 12-week old Akita mice. Immunohistochemistry was carried out in

paraffin sections. Negative control sections shown at the top were not exposed to a primary

antibody. A, Note especially large increases in NR1, NR2A and NR2C in renal tubules. B,

Signal in glomeruli for NR1, NR2A and NR2C. Primary processes are visible in some of the

cells within glomeruli. C, Staining intensity per square micron in the whole kidney (top) and

within glomeruli (bottom). Statistical analysis is by unpaired t-test.

Figure 5. The NMDA antagonist MK-801 reduces progression of nephropathy in Akita mice.

Continuous drug exposure at 0.5 mg/kg/day or treatment with saline vehicle was carried out

using osmotic minipumps starting at 8-weeks of age in Akita mice and DBA/2J controls. A,

Reduced weight gain in Akita mice was not alleviated by MK-801. Bar graphs represent mean ±

SEM in this and all subsequent figures. B, Reduced 24-hr albumin excretion in Akita mice that

received MK-801 for 28 days compared to saline-treated controls. The effect of genotype (F =

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52.15, P < 0.0001), and an interaction effect between drug treatment and genotype (F = 5.2, P =

0.0283) were significant by two-way ANOVA with N = 9-10 mice per group. C, Representative

PAS-stained sections of saline-treated and drug-treated Akita mice and DBA/2J controls. D,

Mesangial matrix expansion expressed as mesangial score shows modest increase in 12-week old

Akita mice that received saline. This increase did not occur in Akita mice that received MK-801.

An interaction effect between drug and genotype on this outcome is significant by two-way

ANOVA (F = 5.33, P = 0.0396, N = 4 mice per group). E, Representative transmission electron

micrographs of mice that received MK-801 or saline. Note extensive foot process effacement

(red arrowheads) and GBM thickening (asterisks) in Akita mice that received saline.

Ultrastructure is markedly improved in Akita mice that were treated with MK-801. F, GBM

thickness is increased in Akita mice treated with saline but not in Akita mice that received MK-

801. An interaction between the effects of MK-801 and genotype (F = 6.22, P = 0.0373, N = 4

mice per group) on GBM thickness was significant by two-way ANOVA. G, The effect of MK-

801 on podocyte foot process width at the contact with the GBM, a measure of foot process

effacement, was significant based on interaction effect om two-way ANOVA (F = 21.66, P <

0.0016).

Figure 6. Scanning EM of glomerular capillary loops in control and Akita mice at 12 weeks of

age. A, Scanning EM show marked disorganization of foot processes along surface of

glomerular capillary in Akita mice. Note flattening of foot processes, especially in areas within

white lines. Foot process morphology is closer to normal in Akita mice that received MK-801.

Treatment with MK-801 has no effect on glomeruli in DBA/2J mice. B, Mean number of

podocyte foot processes per micron in scanning EM images of glomeruli from DBA/2J and Akita

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mice subjected to saline or MK-801 treatment. With podocyte foot process effacement and

disorganization, there are fewer foot processes per micron. Note that MK-801 causes significant

improvement in Akita mice, based on significant interaction effect by two-way ANOVA (F =

5.04, P = 0.0376).

Figure 7. The NMDA antagonist memantine reduces progression of nephropathy in Akita mice.

Continuous drug exposure at 0.2 mg/kg/day or treatment with saline vehicle was carried out

using osmotic minipumps starting at 8-weeks of age in Akita mice and DBA/2J controls. A,

Reduced weight gain in Akita mice was not alleviated by memantine. B, Reduced 24-hr

albumin excretion in Akita mice that received memantine for 28 days compared to saline-treated

controls. The interaction between drug and genotype on albumin excretion was significant (F =

4.58, P = 0.0403 with N = 10 mice per group). C, Representative PAS-stained sections of saline-

treated and drug-treated Akita mice and DBA/2J controls. D, Mesangial matrix expansion

expressed as mesangial score shows modest increase in 12-week old Akita mice that received

saline. This increase did not occur in Akita mice that received memantine. Memantine also

caused a reduction in mesangial matrix expansion (F = 3.82, P = 0.0743, N = 4 mice per group)

in Akita mice. E, Representative transmission electron micrographs of mice that received

memantine or saline. Note reduction in the severity of effacement in Akita mice that received

memantine. F, The effect of memantine on GBM thickness, in Akita mice was not statistically

significant (P > 0.1) by two-way ANOVA with N = 4 mice per group. G, The effect of

memantine on podocyte foot process width at the contact with the GBM, a measure of foot

process effacement, was significant based on interaction effect in two-way ANOVA (F = 18.66,

P < 0.0025).

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0

2

4

6

NR

1/A

ctin

Con HG

0

2

4

6

0

2

4

6

0

2

4

6

NR

2A

/Act

in

NR

2B

/Act

in

NR

2C

/Act

in

NR

2D

/Act

in

Con HG 0

2

4

6

Con HG Con HG

Con HG

b.

0

2

4

6

NR

1/A

ctin

N

R2

A/A

ctin

0

2

4

6

0

2

4

6

0

2

4

6

0

2

4

6

NR

2B

/Act

in

NR

2C

/Act

in

NR

2D

/Act

in Con HG Con HG

Con HG Con HG

Con HG

d.

a.

NR1

NR2A

NR2B

NR2C

NR2D

Actin

Con HG Con HG

NR2A

Actin

NR2B

Actin

NR2D

Actin

NR2C

Actin

NR1

Actin

c. Con HG Con HG

Fig. 1

∗ ∗

∗

∗

∗ ∗

∗

∗

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a.

NR1

NR2A

NR2B

NR2C

NR2D

Actin

Con HG Con HG

0

2

4

6

Con HG

NR

1/A

ctin

0

2

4

6

Con HG

0

2

4

6

NR

2A

/Act

in

NR

2B

/Act

in

Con HG

0

2

4

6

Con HG

Con HG 0

2

4

6

NR

2C

/Act

in

NR

2D

/Act

in

b.

Con HG

NR1 Actin

NR2A

Actin

NR2B

Actin

Con HG

NR2C

Actin

NR2D

Actin 0

2

4

6

Con HG

0

2

4

6

Con HG

0

2

4

6

Con HG

0

2

4

6

Con HG

NR

2A

/Act

in

NR

2B

/Act

in

NR

2C

/Act

in

NR

2D

/Act

in

0

2

4

6

Con HG

NR

1/A

ctin

c. d.

Fig. 2

∗

∗

∗ ∗

∗

∗

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0

2

4

6

8

0

2

4

6

8

0

2

4

6

8

0

2

4

6

8

0

2

4

6

8

NR

1/A

ctin

N

R2

A/A

ctin

NR

2C

/Act

in

NR

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/Act

in

NR

2D

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in

DBA/2J Akita DBA/2J Akita


DBA/2J Akita

b.

c.

DBA/2J Akita

DBA/2J Akita

NR

2A

/Act

in

DBA/2J Akita

NR

2B

/Act

in

DBA/2J Akita N

R2

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DBA/2J Akita

NR

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in

NR

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d.

a.

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NR2B

NR2C

NR2D

Actin


NR2A

Actin

NR2B

NR2C

NR2D

Actin

Actin

Actin

NR1

Actin

Fig. 3

0

2

4

6

0

2

4

6

0

2

4

6

0

2

4

6

0

2

4

6

∗ ∗

∗

∗ ∗

∗

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Negative Control

Fig. 4

DBA/2J Akita

NR2B

NR2A

NR1

DBA/2J Akita

NR2C

a. b.

50 µm 50 µm 50 µm 50 µm

0

50

100

150

NegativeControl

NR1 NR2A NR2B NR2C

IHC

sta

inin

g in

ten

sity

(µ

m2)

in

Wh

ole

kid

ney

DBA Akita

01020304050

NegativeControl

NR1 NR2A NR2B NR2C

IHC

sta

inin

g in

ten

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(µ

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ulu

s DBA Akita

* *

*

* *

*

*

c.

(no primary antibody)

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24

-hr A

lbu

min

excre

tio

n (m

g)

0

200

400

600

800

1000

DBA/2J Akita

Saline

MK-801

Sa

line

M

K-8

01

Akita DBA/2J

*

Akita DBA/2J

Sa

line

M

K-8

01

a. b.

e.

f.

c.

15

17

19

21

23

25

27

29

0 7 14 21 28

DBA/2J-SalineDBA/2J-MK801Akita-SalineAkita-MK801

Bo

dy w

eig

ht

(g)

Days of treatment d.

Fig. 5

50 µm

500 nm

0

1

2

3

DBA/2J Akita

Me

sa

ngia

l S

co

re

Saline

MK-801

0

50

100

150

200

250

DBA/2J Akita

GB

M w

idth

(nm

)

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*

*

*

0

200

400

600

800

1000

DBA/2J Akita

Foo

t p

roce

ss w

idth

(n

m)

Saline

MK-801

*

g.

of 41 Diabetes

DBA/2J Akita S

alin

e

MK

-801

1 μm

a.

b.

Fig. 6

0

1

2

3

4

5

6

DBA/2J Akita

Nu

mb

er o

f fo

ot

pro

cess

es /

1 µ

m

Saline

MK-801

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a. b.

24

-hr A

lbu

min

excre

tio

n (m

g)

0

200

400

600

800

1000

DBA/2J Akita

Saline

memantine

c.

15

17

19

21

23

25

27

0 7 14 21 28

DBA/2J-Saline

DBA/2J-Memantine

Akita-Saline

Akita-MemantineBo

dy w

eig

ht

(g)

Days of treatment

f.

DBA/2J

Sa

line

Akita

me

ma

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Fig. 7

d.

Sa

line

m

em

antine

Akita DBA/2J

50 µm

500 nm

0

1

2

3

DBA/2J Akita

Me

sa

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co

re Saline

memantine

0

50

100

150

200

250

DBA/2J Akita

GB

M w

idth

(nm

)

Salinememantine

*

0

200

400

600

800

1000

1200

DBA/2J Akita

Foo

t p

roce

ss w

idth

(n

m)

Saline

Memantine

*

g.

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Online Appendix

Supplemental figures

Supplement Figure 1. Immunoblot analysis showing robust expression of α-smooth muscle

actin (α-SMA) in primary cultures of rat mesangial cells (MC) but not in differentiated

immortalized mouse podocytes (Podo). Different panels show results from three different

preparations of primary mesangial cultures.

Podo MC Podo MC

α-SMA

Actin

Podo MC

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Supplement Figure 2. Confocal images showing expression of NMDA receptor subunits on

the surface of podocytes cultured for 24 hr in normal and elevated glucose. Primary

antibodies are directed against epitopes on extracellular loops of NMDA receptor subunits.

Staining was carried out cells that were fixed but not permeabilized. Laser and detector

settings on the confocal microscope were held constant for all of these samples. Green

fluorescence signal is from NMDA receptor subunits. Red fluorescence is from tagged

wheat-germ agglutinin (WGA). A, Staining for NR1 subunits of cells in control medium and

high-glucose (HG), using conditions described for Fig. 1. B, Relative fluorescence intensity

of NR1 staining for cells cultured for 24 hr in control and HG media. Asterisks indicate P <

0.01 by unpaired t-test. C, Staining for NR2A subunits. D, Relative intensity of NR2A

staining of podocytes cultured in control and HG conditions. Asterisks indicate P < 0.05 by

unpaired t-test.

Con HG

NR

1

Flu

ore

sce

nce

In

ten

sity

(A

U)

0

10

20

30

40

*

0

10

20

Con HG

NR

2A

Flu

ore

sce

nce

In

ten

sity

(A

U)

*

NR1 WGA Merged

Con

HG

NR2A WGA Merged

Con

HG

a. b.

c. d.

of 41 Diabetes

3

Supplement Figure 3. Increased abundance of NR1, NR2A and NR2C subunits (but not

NR2B or NR2D) detected by immunoblot analysis of renal cortex extracts in 7-week old

Akita mice compared to 7-week old DBA/2J controls. Bar graphs show mean ± SD.

Asterisks indicate P < 0.05 (unpaired t-test).

NR2A

NR1

Actin

Actin

DBA/2J Akita

DBA/2J Akita

0

2

4

6

DBA/2J Akita

DBA/2J Akita

0

2

4

6

DBA/2J Akita

DBA/2J Akita

NR

2A

/Act

in

0

2

4

6

NR

2B

/Act

in

NR2B

Actin

NR

1/A

ctin

DBA/2J Akita

DBA/2J Akita 0

2

4

6

NR

2C

/Act

in

NR2C

Actin

DBA/2J Akita

0

2

4

6

DBA/2J Akita

NR2D

Actin

NR

2D

/Act

in

∗

∗ ∗

of 41Diabetes

4

Supplement Figure 4. Mean arterial blood pressure measured by tail cuff volume pressure

recording during experiments in Akita or STZ-treated mice and the DBA/2J controls. There

was no significant change in mean arterial pressure at any point in our studies as a result of

surgery, MK-801 treatment, or genotype.

0

20

40

60

80

100

120

140

Control-Saline Control-MK-801 STZ-Saline STZ-MK-801

Me

an

blo

od

pre

ssu

re

Before injectionBefore SurgeryAfter 28 days treatment

0

20

40

60

80

100

120

DBA/2J-Saline DBA/2J-MK-801 Akita-Saline Akita-MK-801

Me

an

blo

od

pre

ssu

re

Before Surgery

After 28 days treatment

of 41 Diabetes

5

of 41Diabetes

6

Supplement Figure 5. The NMDA antagonist MK-801 reduces progression of nephropathy in

the low-dose streptozotocin (STZ) model of type-1 diabetes in DBA/2J mice. Continuous

drug exposure at 0.5 mg/kg/day or treatment with saline vehicle was carried out using

osmotic minipumps starting 15 weeks after completion of the STZ protocol. A, Reduced

body weight in STZ-treated mice was not alleviated by MK-801. There was a trend towards

reduced body weight in MK-801-treated control mice. All mice were the same body weight

prior to initiation of the STZ protocol (pre). B, Trend towards reduced albumin excretion in

STZ diabetic mice that received MK-801 for 28 days is not statistically significant by two-

way ANOVA. Note that diabetes was more advanced at the time of initiation of MK-801

treatment in the STZ model compared to our experiments in the Akita model. C,

Representative PAS-stained sections of Akita mice and DBA/2J controls that received saline

or MK-801 for 28 days. D, Mesangial matrix expansion expressed as mesangial score is

increased in STZ-treated DBA/2J mice compared to non-diabetic controls that received

vehicle. MK-801 reduced this effect. Mesangial matrix expansion in the STZ model was

more severe than in the Akita model. Mesangial score was reduced in STZ mice that

received MK-801 based on significant interaction effect in two-way ANOVA (F = 5.82, P =

0.0328, N = 4 mice per group). E, Representative transmission electron micrographs of mice

that received MK-801 or saline. Note reduction in the severity of effacement and reduced

GBM thickness in STZ mice that received MK-801. F, The effect of MK-801 on GBM

thickness in STZ-treated diabetic mice was statistically significant (F = 88.51, P < 0.0001, N

= 4 mice per group) by two-way ANOVA.

of 41 Diabetes

nmda receptors as potential therapeutic targets in

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