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TitleComparative in vitro analyses of the effect of immunoglobulin light chain and fatty acid free
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Page 1

Comparative in vitro analyses of the effect

of immunoglobulin light chain and fatty

acid free albumin on proximal tubular

epithelial cells-involvement of megalin

phosphorylation



Thesis submitted for the degree of Doctor of Philosophy at the

University of Leicester

By



Dalia Muhammed Alammari

(BSc, MSc)





Department of Infection, Immunity and Inflammation

University of Leicester

December 2015

Page 2

I

ABSTRACT

Comparative in vitro analyses of the effect of immunoglobulin λ light chain and fatty
acid free albumin on proximal tubular epithelial cells-involvement of megalin

phosphorylation



Dalia Alammari

Kidney disease is a major challenge for health care systems, and the prevalence is

increasing. Proteinuria is a hallmark of progressive renal dysfunction and describes the

pathological excess of plasma proteins in urine, mainly albumin.

Multiple Myeloma is a cancer of plasma cells that leads to excessive presence of free

light chain protein (FLC) in blood. Renal failure due to overproduction of FLC and the

associated light chain proteinuria occurs as a result of decreased renal function or as a

direct toxic effect on the proximal tubular cells (PTCs) by excessive protein. Proteins

are normally reabsorbed by endocytosis via megalin receptor that binds proteins and

mediates their uptake. Exceeding the proximal tubular epithelial cells (PTECs)

reabsorption capacity might trigger inflammation detrimental to the kidney. In

proteinuric nephropathy the cytoplasmic tail of megalin (MegCT) is phosphorylated

after interaction between proteins and megalin on the PTECs, which activates signalling

cascades that regulate the phosphorylation.

An in vitro proteinuric model was established using HK2 cells (a proximal tubular

epithelial cell line derived from normal human kidney) treated with high concentrations

of essentially fatty acid free human serum albumin (FAF-HSA) or lambda light chain

(𝜆-LC) isolated and purified from the urine of a myeloma patient, to induce cellular
damage. The potential pathogenic role for FAF-HSA and 𝜆 -LC on HK2 cells was
examined. Also, renal toxicity that comes from the intracellular signalling through

phosphorylation of MegCT was addressed by utilising antibodies directed against

specific phosphorylation site (PPPSP) of the intracellular portion of megalin in HK2

cells stimulated with different concentrations of FAF-HSA and 𝜆-LC, so-called pre-
stimulated HK2.

In vitro analyses showed (i) a detrimental effect of FAF-HSA and 𝜆-LC on viability of
HK2, (ii) phosphorylation of the cytoplasmic tail of megalin in pre-stimulated HK2

cells. (iii) Production of inflammatory cytokines and H2O2 generation, activation of

autophagy process and increase in several kidney biomarkers/ injury mediators, which

are involved in different pathways in response to protein overload. All these reasons are

likely to contribute to direct PTECs injury and kidney failure in patients.

Potentially these mechanisms may be attractive for drug development to benefit patients

with kidney failure and help to inhibit the progression of proteinuric nephropathy and as

such may save lives.


http://en.wikipedia.org/wiki/Protein
http://en.wikipedia.org/wiki/Urine

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While activity levels of control cells remain stable for 24 and 48h, there is an increase of

activity for HK2 (+/-GF) at 72h, suggesting increased proliferation at this time point in the

presence of the EGF cocktail. The effect, however, is not sufficient to inhibit the toxic

effect of high doses of FAF-HSA. In addition, HK2-GF shows a significant protection from

the FAF-HSA induced toxic effect compared to HK2 at all-time points, with no decrease in

the number of the cells in the first 24 and 48h and a significant decrease at 72h with the

higher concentration of FAF-HSA. In contrast, the HK2 cells show a significant decrease

from the first 24h with the higher concentration of FAF-HSA. For example, with

(30mg/ml) FAF-HSA the HK2 cells showed significant reduction from the initial 24h

incubation up to 72h, however, FAF-HSA with the same concentration had no effect on

HK2-GF cells at 24 and 48h but a significant decrease at 72h.

Next, HK2 (+/-GF) cells were sub-cultured and stimulated with -LC (1, 5 and 10mg/ml in

a serum free medium) for 24, 48 and 72h. Un-stimulated cells in a serum-free medium were

used as controls for each time point.-LC at 10mg/ml was found to significantly impair

HK2-GF cell viability after 24h of incubation when compared with un-stimulated cells. By

contrast, after 48 and 72h of stimulation with a range of -LC between (1 and 10 mg/ml),

cell viability decreased compared to the control (figure 5.3 A).

Also, -LC showed evidence of a toxic effect in the first 24h incubation time of HK2 cells,

however, there was a significant decrease in cell viability after 48 and 72h of incubation

time with all -LC concentrations (1-10 mg/ml) compared with the control (figure 5.3 B).

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Figure (5.1): Effects of FAF-HSA overload on cell viability measured using MTT

assay in HK2 (+/-GF) cells. The HK2 (+/-GF) cells were cultured at density

(2,500 cells/well) for 24h and treated with ascending concentrations of FAF-HSA

(5, 10, 20 and 30mg/ml) in serum free media. After incubation for 2, 4 and 6h

mitochondrial activity, indicative of cell viability, was determined and expressed

as photometrical densities of the solubilised end product, formazan. Exposure to

FAF-HSA showed on change in cell viability for HK2 (+/-GF) cells. Un-treated

cells were used as control. The data are represented as means of triplicates ± SD

(n = 3) (Unpaired t test p < 0.05vs. control), C: control sample.



2h

4h

6h

C
5

1
0

2
0

3
0

C
5

1
0

2
0

3
00.0

0.1

0.2

0.3
HK2-GF

HK2

O
D

(
5
5
0
n

m
)

mg/ml

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