Abdul R. Asif

Gender differences in kidney function

Sex hormones influence the development of female (F) and male (M) specific traits and primarily affect the structure and function of gender-specific organs. Recent studies also indicated their important roles in regulating structure and/or function of nearly every tissue and organ in the mammalian body, including the kidneys, causing gender differences in a variety of characteristics. Clinical observations in humans and studies in experimental animals in vivo and in models in vitro have shown that renal structure and functions under various physiological, pharmacological, and toxicological conditions are different in M and F, and that these differences may be related to the sex-hormone-regulated expression and action of transporters in the apical and basolateral membrane of nephron epithelial cells. In this review we have collected published data on gender differences in renal functions, transporters and other related parameters, and present our own microarray data on messenger RNA expression for various transporters in the kidney cortex of M and F rats. With these data we would like to emphasize the importance of sex hormones in regulation of a variety of renal transport functions and to initiate further studies of gender-related differences in kidney structure and functions, which would enable us to better understand occurrence and development of various renal diseases, pharmacotherapy, and drug-induced nephrotoxicity in humans and animals.

Proteomic Analysis of Cellular Response to Osmotic Stress in Thick Ascending Limb of Henle’s Loop (TALH) Cells

Epithelial cells of the thick ascending limb of Henle’s loop (TALH cells) play a major role in the urinary concentrating mechanism. They are normally exposed to variable and often very high osmotic stress, which is particularly due to high sodium and chloride reabsorption and very low water permeability of the luminal membrane. It is already established that elevation of the activity of aldose reductase and hence an increase in intracellular sorbitol are indispensable for the osmotic adaptation and stability of the TALH cells. To identify new molecular factors potentially associated with the osmotic stress-resistant phenotype in kidney cells, TALH cells exhibiting low or high levels of resistance to osmotic stress were characterized using proteomic tools. Two-dimensional gel analysis showed a total number of 40 proteins that were differentially expressed in TALH cells under osmotic stress. Twenty-five proteins were overexpressed, whereas 15 proteins showed a down-regulation. Besides the sorbitol pathway enzyme aldose reductase, whose expression was 15 times increased, many other metabolic enzymes like glutathione S-transferase, malate dehydrogenase, lactate dehydrogenase, α enolase, glyceraldehyde-3-phosphate dehydrogenase, and triose-phosphate isomerase were up-regulated. Among the cytoskeleton proteins and cytoskeleton-associated proteins vimentin, cytokeratin, tropomyosin 4, and annexins I, II, and V were up-regulated, whereas tubulin and tropomyosins 1, 2, and 3 were down-regulated. The heat shock proteins α-crystallin chain B, HSP70, and HSP90 were found to be overexpressed. In contrast to the results in oxidative stress the endoplasmic reticulum stress proteins like glucose-regulated proteins (GRP78, GRP94, and GRP96), calreticulin, and protein-disulfide isomerase were down-regulated under hypertonic stress.

Expression of chloride intracellular channel protein 1 (CLIC1) and tumor protein D52 (TPD52) as potential biomarkers for colorectal cancer.

OBJECTIVES Unequivocal biomarkers are needed to predict susceptibility and progression of colorectal cancer. DESIGN AND METHODS Paired samples of tumor and normal tissue from six patients with colorectal cancer of different localization, pTNM stage and grade were employed in the present study. MS analysis was used to identify differentially regulated proteins after 2-DE separation and densitometric analysis. RESULTS Densitometric analysis revealed differential abundance of 55 spots in tumor as compared to normal tissues. Thirty nine out of 55 spots were unambiguously identified by MS representing 32 different proteins. CLIC1, TPD52 and FABPL were consistently overexpressed (>3-fold, P<0.05) in all tumor tissue samples, while TPM1, TPM2, TPM3, TAGL and MLRN were consistently down-regulated (>3-fold, P<0.05) compared to normal tissue. CONCLUSIONS CLIC1 and TPD52 were significantly (P<0.05) up-regulated in all cases of colorectal cancer investigated, irrespective of localization, pTNM stage and grade of colon cancer highlighting their potential to serve as new biomarkers.

Differential S-nitrosylation of proteins in Alzheimer’s disease

Numerous studies have provided evidence regarding the involvement of protein S-nitrosylation in the progression of Alzheimers disease (AD) pathology and its implication in the formation and accumulation of misfolded protein aggregates. The identification of S-nitrosylated proteins can be a major step toward the understanding of mechanisms leading to neuronal degeneration. The present study targeted S-nitrosylated proteins in AD hippocampus, substantia nigra and cortex using the following work-flow that combines S-nitrosothiol-specific antibody detection, classical biotin switch method labeled with fluorescence dye followed by electrospray ionization quadrupole time of flight tandem MS (ESI-QTOF MS/MS) identification. Endogenous nitrosocysteines were identified in 45 proteins, mainly involved in metabolism, signaling pathways, apoptosis and redox regulation as assigned by REACTOME and KEGG pathway database analysis. Superoxide dismutase (SOD2) [Mn], fructose-bisphosphate aldolase C (ALDOC) and voltage-dependent anion-selective channel protein 2 (VDAC2) showed differential S-nitrosylation signal, not previously reported in AD regions. Extensive neuronal atrophy with increased protein S-nitrosylation in AD regions is also evident from immunofluorescence studies using S-nitrosocysteine antibody. A number of plausible cysteine modification sites were predicted via Group-based Prediction System-S-nitrosothiols (GPS-SNO) 1.0 while STRING 8.3 analysis revealed functional annotations in the modified proteins. The findings are helpful in characterization of functional abnormalities and may facilitate the understanding of molecular mechanisms and biological function of S-nitrosylation in AD pathology.

Proteomics Approach to Identify the Interacting Partners of Cellular Prion Protein and Characterization of Rab7a Interaction in Neuronal Cells

The present study was undertaken to identify proteins interacting with PrP(C) that could provide new insights into its physiological functions and pathological role. Human PrP(C) was expressed in prion protein-deficient murine hippocampus (HpL3-4) neuronal cells. The PrP(C) along with its interacting proteins were affinity purified using STrEP-Tactin-chromatography, in-gel digested, and identified by Q-TOF MS/MS analysis. Forty-three proteins appeared to interact with PrP(C) in this neuronal cell line. Of these, 15 were already known for their interaction with PrP(C) or PrP(Sc), while 28 new proteins were identified. Interaction of a novel interacting partner of GTPase family-Rab7a, having a suggested role in vesicle trafficking, was further investigated using confocal laser scanning microscopy and reverse coimmunoprecipitation. Both reverse coimmunoprecipitation and immunofluorescence results confirmed potential interaction of Rab7a with the PrP(C). siRNA against the Rab7a gene decreased expression of Rab7a protein, in PrP(C) expressing HpL3-4 and SH-SY5Y cells. This depleted Rab7a expression led to the enhanced accumulation of PrP(C) in Rab9 positive endosomal compartments and consequently an increased colocalization between PrP(C)/Rab9. However, the Rab9 accumulated PrP(C) remained sensitive to proteinase-K digestion. The work described demonstrated for the first time that Rab7a interacts with PrP(C) and highlighted the involvement of endosomal compartments in the trafficking and regulation of PrP(C).

Physiological Role of the Cellular Prion Protein (PrPc): Protein Profiling Study in Two Cell Culture Systems

The physiological role of the cellular prion protein (PrP (c)) is still not fully understood. Current evidence strongly suggests that PrP (c) overexpression in different cell lines sensitizes cells to apoptotic stimuli through a p53 dependent pathway. On the other hand, an expression of PrP (c) in PrP (c)-deficient cells undergoing apoptosis exhibited repeatedly antiapoptotic effects. Therefore, the presence/absence and/or the level of PrP (c) expression seem to be critical for the fluctuation between PrP (c)s pro- and antiapoptotic properties. The present study examined whether an overexpression of PrP (c) itself, without addition of any apoptotic agent, can lead to proteome changes that might account for the higher responsiveness to apoptotic stimuli. Beyond this, we examined whether the sole introduction of PrP (c) into PrP (c)-deficient cells could be sufficient to up-regulate antiapoptotic proteins capable of mitigating apoptosis. For this purpose, we used two cell lines, one expressing [human embryonic kidney (HEK) 293 cells] and the other lacking (mouse neuronal PrP (c)-deficient cells) endogenous PrP (c). Protein profiling following transient PrP (c) overexpression in HEK 293 cells revealed a major PrP (c) involvement in regulation of proteins participating in energy metabolism and cellular homeostasis, whereas transient introduction of PrP (c) into mouse neuronal PrP (c)-deficient cells resulted mainly in the regulation of proteins involved in protection against oxidative stress and apoptosis. In addition, we report for the first time that PrP (c) overexpression influenced the regulation of several proteins known to have contributory roles in the pathogenesis of Alzheimer disease (AD). Revealing the correlation between presence/absence and/or different levels of PrP (c) expression with the regulation of certain cellular proteins might further contribute to our understanding of the complex role of PrP (c) in cell physiology.

Identification of Novel Autoantigen in the Synovial Fluid of Rheumatoid Arthritis Patients Using an Immunoproteomics Approach

Rheumatoid arthritis (RA) is a chronic, autoimmune and inflammatory joint disease with a poorly understood etiology. Despite widespread diagnostic use of anti-citrullinated protein antibodies and rheumatoid factor proteins there is a strong demand for novel serological biomarkers to improve the diagnosis this disease. The present study was aimed to identify novel autoantigens involved in rheumatoid arthritis (RA) pathogenesis through immune-proteomic strategy. Synovial fluid samples from clinically diagnosed RA patients were separated on two-dimensional gel electrophoresis (2-DE). Samples from patients with non-RA rheumatisms (osteoarthritis and trauma) were used as controls. Immunoreactive proteins were spotted by Western blotting followed by identification through Q-TOF mass spectrometer analysis. Forty Western blots were generated using plasma from ten individual RA patients and 33 reactive spots were identified, 20 from the high molecular weight (HMW) gel and 13 from the low molecular weight (LMW) gel. Among the 33 common immunogenic spots, 18 distinct autoantigens were identified, out of which 14 are novel proteins in this context. Expression analysis of five important proteins, vimentin, gelsolin, alpha 2 HS glycoprotein (AHSG), glial fibrillary acidic protein (GFAP), and α1B-glycoprotein (A1BG) by Western blot analysis using their specific antibodies revealed their higher expression in RA synovial fluid as compared to non-RA samples. Recombinantly expressed GFAP and A1BG protein were used to develop an in-house ELISA to quantify the amount of autoantibodies in the RA patients. RA patients revealed an increase in the expression of GFAP and A1BG in the plasma as compared to osteoarthritis patients. Therefore, GFAP and A1BG can be proposed as potential new autoantigens of diagnostic importance for RA subjects. Further characterization of these proteins in rheumatoid arthritis will be helpful in understanding the role of these proteins in the disease pathogenesis providing new diagnostic tool with better specificity and accurate detection of the disease.

Proteome profiling in murine models of multiple sclerosis: identification of stage specific markers and culprits for tissue damage.

The identification of new biomarkers is of high interest for the prediction of the disease course and also for the identification of pathomechanisms in multiple sclerosis (MS). To specify markers of the chronic disease phase, we performed proteome profiling during the later phase of myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis (MOG-EAE, day 35 after immunization) as a model disease mimicking many aspects of secondary progressive MS. In comparison to healthy controls, high resolution 2 dimensional gel electrophoresis revealed a number of regulated proteins, among them glial fibrilary acidic protein (GFAP). Phase specific up-regulation of GFAP in chronic EAE was confirmed by western blotting and immunohistochemistry. Protein levels of GFAP were also increased in the cerebrospinal fluid of MS patients with specificity for the secondary progressive disease phase. In a next step, proteome profiling of an EAE model with enhanced degenerative mechanisms revealed regulation of alpha-internexin, syntaxin binding protein 1, annexin V and glutamate decarboxylase in the ciliary neurotrophic factor (CNTF) knockout mouse. The identification of these proteins implicate an increased apoptosis and enhanced axonal disintegration and correlate well the described pattern of tissue injury in CNTF −/− mice which involve oligodendrocyte (OL) apoptosis and axonal injury. In summary, our findings underscore the value of proteome analyses as screening method for stage specific biomarkers and for the identification of new culprits for tissue damage in chronic autoimmune demyelination.

Secretion of ERP57 is important for extracellular matrix accumulation and progression of renal fibrosis, and is an early sign of disease onset.

Summary Renal fibrosis is characterized by excessive accumulation of extracellular matrix (ECM), which compromises organ function by replacing normal organ tissue. The molecular mechanisms leading to renal fibrosis are not fully understood. Here we demonstrated that TGF&bgr;1, AGT or PDGF stimulation of renal cells resulted in endoplasmic reticulum (ER) stress followed by activation of the protective unfolded protein response pathway and a high secretory level of protein disulfide isomerase ERP57 (also known as PDIA3). The TGF&bgr;1-induced impairment of ER function could be reversed by treatment with BMP7, suggesting a specific involvement in renal fibrosis. A clear correlation between the degree of fibrosis, ER stress and the level of ERP57 could be seen in fibrosis animal models and in biopsies of renal fibrosis patients. Protein interaction studies revealed that secreted ERP57 exhibits a strong interaction with ECM proteins. Knockdown of ERP57 or antibody-targeted inhibition of the secreted form significantly impaired the secretion and accumulation of ECM. Moreover, ERP57 was excreted in the early stages of chronic kidney disease, and its level in urine correlated with the degree of renal fibrosis, suggesting that the secretion of ERP57 represents one of the first signs of renal fibrosis onset and progression.

Cloning of the pig renal organic anion transporter 1 (pOAT1).

A pig kidney cDNA library was screened for the porcine ortholog of the multispecific organic anion transporter 1 (pOAT1). Several positive clones were isolated resulting in two alternatively spliced cDNA clones of pOAT1 (pOAT1 and pOAT1A). pOAT1-cDNAs consist of 2126 or 1895 base pairs (EMBL Acc. No. AJ308234 and AJ308235) encoding 547 or 533 amino acid residue proteins with 89, 87, 83 and 81% homology to the human, rabbit, rat, and mouse OAT1, respectively. Heterologous expression of pOAT1 in Xenopus laevis oocytes revealed an apparent K(m) for [3H]PAH of 3.75 +/- 1.6 microM. [3H]PAH uptake mediated by pOAT1 was abolished by 0.5 mM glutarate or 1 mM probenecid. Functional characterization of pOAT1A did not show any affinity for [3H]PAH. In summary, we cloned two alternative splice variants of the pig ortholog of organic anion transporter 1. One splice form (pOAT1) showed typical functional characteristics of organic anion transporter 1, whereas the second form appears not to transport PAH.