The glomerular basement membrane
The glomerular basement membrane (GBM) serves as the skeleton of the glomerular tuft, the interior of which contains the capillaries and mesangium and the exterior is covered with epithelial cells (podocytes). It functions as the effective filtration barrier. Ultrastructurally, the GBM is a trilaminar structure, varying in thickness from 310 to 380hm in adult human males. Chemically, the GBM is a complex structure composed of type IV collagen, laminin, heparan sulfate proteoglycans, fibronectin, and other small components; being similar to the mesangial matrix. The GBM is synthesized both by endothelial and epithelial cells.
In the present study, the focus is on those glomerulopathies, where the basement membrane is involved in the disease process and diagnosis depends on ultrastructural study of basement membrane abnormalities. Membranous glomerulonephritis (MGN) also known as membranous glomerulopathy, is a histopathologic pattern, characterized by epi-membranous and/or intra-membranous immunecomplex deposits and/or variable basement membrane thickness.
In most of the cases of MGN, the etiology is unknown and the pathogenesis uncertain. In a minority of cases, MGN is caused by immune complexes containing exogenous antigens, such as, hepatitis-B antigen (Lin, 1993). The incidence of MGN is worldwide. There is particularly a high frequency in Japanese children (Takekoshi et al 1978) and certain African populations (Seggie et al 1984). MGN accounts for 20% to 30% of all cases of idiopathic nephrotic syndrome in adults (Hayslett et al 1973) and 1% to 9% in children (Remirez et al 1982). So, the present study is designed with a view to explore the ultrastructural abnormalities in glomerular basement membrane.
Minimal change disease accounts for approximately 80% of all cases of idiopathic nephrotic syndrome in children (Habib and Kleinknecht, 1975) and 20% to 30% of adult cases (Korbet et al 1988). On routine light microscopy, glomeruli seem to be normal, inspite of the proteinuria due to changed permeability of GBM. Fusion of epithelial foot processes is seen by electron microscopy (Arakawa, 1971).
Thin basement membrane nephropathy or benign familial hematuria and Alport's syndrome are the other renal diseases that need to be explored at ultrastructural level in our population in addition to histopathological, immunohistochemical and immunopathological examinations. Glomerular basement membrane (GBM) is the main component of the filtration barrier in nephron. Thickness of GBM and its structural components have a vital role in the permiability of the GBM and the same will be explored in the specified cases.
The differential diagnosis of thin basement membrane disease (TBMD) and Alport's syndrome depends on the demonstration of GBM on ultrastructure of renal biopsy specimen (Praga et al 1998, Hinglais et al 1972).
The present study has been designed to explore the renal glomerular basement membrane abnormalities at the ultrastructural level supplimented with immunohistochemistry to characterize the basement membrane components and draw conclusions in a significant number of specified cases of MGN, minimal change disease, TBMD and Alport's syndrome.
REVIEW OF LITERATURE
The kidneys are vital excretory organs that regulate the internal environment of the body. In adult-man, these weigh 120 to 150 gm each and through their vascular tree flows approximately 1200 ml blood per minute, some 25% of the total cardiac output. The vascular and epithelial elements form special functional units, the nephrons, consisting each of a glomerulus and a tubule. The formation of urine is the most obvious function of kidneys apart from water and salt metabolism and acid-base balance.
The special apposition of blood vessels and tubules gives a characteristic gross appearance to the kidney and this organization assures the required degree of function. A renal cortex and medulla can be differentiated on a cut surface of the mammalian kidney. The cortex contains all glomeruli and convoluted tubules. The medulla contains some or all of the parallel descending and ascending tubules, called the loops of Henle, and the collecting ducts.
Most of the methods utilized in the study of renal morphology before and during Bowman's time can be best described as preliminary forms of the modern techniques of microdissection. With the introduction of histologic section and staining methods in the mid eighteen hundreds the attention of morphologists was diverted to the wealth of cellular details provided by the newer technique, so that Huber (1905) was among the few who still used older method of microdissection in their investigation of renal structure. Peter (1909) delineated the architectonic patterns of the normal kidneys of various species as composed of nephrons, a term, which was later introduced by Braus in 1924. Sir Robert Platt in 1952, expressed the change in the attitude which has evolved in the study of renal disease by his statement, "We must think in terms of nephrons rather than kidneys", nephron being the functional unit.
The glomerulus is a vascular structure composed of a tuft of specialized capillaries that arise from the afferent arteriole to form lobules and then rejoin the vascular pole to drain into efferent arteriole.
The primary function of the glomerulus is production of the glomerular filtrate, being remarkably selective in this filtration process; almost completely excluding macromolecules into the glomerular filtrate. The mesangial cells appear phagocytic and may clear away macromolecules, which may pass the filter and by actively contracting may also regulate glomerular blood flow.
The scheme allows a listing of structures through which filtration occurs, first of course the filtrate passes through the endothelium, a specialized form, that contains fenestrations, each about 90hm in diameter followed by the glomerular basement membrane (GBM) some 300hm in thickness which is the main component of filtration barrier.
In normal adults the GBM measures 310 to 380 hm in thickness. It is somewhat thinner in children and slightly thicker in males than females (Clapp, 1992).
The GBM is a trilaminar structure composed of a central electron-dense zone, or lamina densa, bordered by two narrow electron-lucent layers, the lamina rara interna and the lamina rara externa. Distal to the basement membranes are the visceral epithelial cells, commonly called podocytes; which possess complex interdigitating processes closely associated with the lamina rara externa. The foot processes are separated from one another by filtration slits and the slit diaphragm bridges across each filtration slit. The mesangium forms a supportive branching framework for the glomerular tuft and consists of mesangial cells embedded in a matrix. The major components of the GBM are type IV collagen, laminin, heparan sulfate proteoglycans, fibronectin and entactin but minor amounts of other proteins such as amyloid P-component, collagen V and VI have also been found (Weber 1992).
At least six different type IV collagen genes have been identified, which encode the a1 to a6 chains respectively of type IV collagen. The a3 (IV) and a4 (IV) chains are located in lamina densa of GBM, whereas, the classic a1 (IV) and a2 (IV) chains are found in the lamina rara interna and the mesangium, This finding suggests that a3 and a4 along with a5 form a network that is different from that consisting of a1 and a2 chains (Hudson et al 1993).
Basic structure of GBM is a three dimensional network of collagen type IV. Monomers of type IV collagen consist of triple helices that are 400hm in length and have globular non-collagenous domains (NCl) at the carboxy terminal ends. At the amino terminal, there is a 7S domain, which is a triple helical rod, 60hm in length. Interaction in 7S domains of three helices or NCl domains of four triple helices results in the formation of dimers or tetramers. Further interactions result in a flexible, nonfibrillar polygonal assembly, which provides mechanical strength to the basement membrane (Timpl and Brown 1996).
The major glycoproteins of the GBM are laminin, fibronectin and entactin. Laminin is the most prominent non-collagenous component found in basement membranes. It consists of three polypeptide chains (A, B1 and B2), two of which are glycosylated and crosslinked by disulfide bridges. Laminin binds directly or via entactin to type IV collagen on one side and to integrin and other cell surface receptors on endothelial and epithelial cells, on the other side (Adler 1992; Cybulsky et al 1992).
The major proteoglycan of GBM is a large heparan sulfate proteoglycan, called perlecan, which is composed of a core protein (400 kd) and three heparan sulfate side chains (glycosaminoglycans). Proteoglycan molecules aggregate to form a meshwork that is kept hydrated and act as an anticlogging agent to prevent hydrogen bonding and adsorption of anionic plasma proteins, in order to maintain an efficient flow of water through the membrane (Kanwar 1984)
CLASSIFICATION OF GLOMERULAR DISEASE.
Glomerulonephritis is an inflammation of the glomerulus, where as glomerulopathy is an all-embracing term for disorders affecting this structure. In glomerular disease other parts of nephron may also be involved, but the diagnosis hinges on the identification of derangement of the normal glomerular configuration. The changes may be recognizable by routine light microscopy, although the findings from immunohistochemistry are often significant. Electronmicroscopy is always informative, and it is sometimes the only means whereby the structural changes can be detected and defined.
The damage to the glomeruli may take the form of definable morphologic pattern, and sub divisions in the character or distribution of the glomerular lesions are used to classify glomerulonephritis. There is general agreement on the definitions applied to the distribution of glomerular lesions as given below:
Classification of glomerular disease by Distribution
- Classification of disease distribution when many glomeruli are considered.
- Classification of disease, distribution when single glomeruli are considered.
Focal: Disease affecting only some of the glomeruli.
Diffuse: Disease affecting most or all glomeruli.
Segmental: A lesion involving only a part of the glomerulus.
Global: A lesion involving the entire glomerulus.
Membranous glomerulonephritis (MGN)
Membranous Glomerulonephritis (also termed as membranous nephropathy, membranous glomerulonephropathy, and epimembranous nephropathy) is a histopathologic pattern characterized by epimembranous and/or intra membranous immune complex deposits and/or variable basement membrane thickening, without mesangial cell proliferation or infiltration by inflammatory cells. Most cases are idiopathic, but identical glomerular lesions can occur in a variety of conditions including drug reactions (gold, penicillamine, captopril), infections (hepatitis B, malaria, leprosy, syphilis), auto-immune diseases (systemic lupus erythematosus, rheumatoid arthritis) and tumors (carcinoma of the lung, stomach, and breast lymphomas) (Gilbert and Wiggelinkhuizen 1994; Glassock 1992; Hotta et al 1993, Yoshida et al 1994).
MGN accounts for 20% to 30% of all cases of idiopathic nephrotic syndrome in adults (Hayslett, et al, 1973) and 1% to 9% in children (Ramirez et al 1982). The incidence of MGN in different populations varies, there is, particularly a high frequency in Japanese children (Takekoshi et al 1978) and in certain African populations (Seggie et al, 1984), probably related to a high incidence of hepatitis-B infection and parasitic infections. The disease is twice as common in males, and it occurs in adults and children. From 60% to-80% of patients have the nephrotic syndrome at on set and others are usually referred for investigations of asymptomatic proteinuria or an abnormal urine analysis (Donadio et al, 1988 and Schwartz, 1992). The proteinuria is usually non selective, but a highly selective proteinuria is seen in as many as 20% of cases (Coggins et al, 1982, Schwartz, 1992).
In natural history and overall prognosis of MGN can be significantly affected by the underlying disease and the way it was treated. Where the MGN is secondary to drugs, toxic substances, or infections, removal of the etiologic agent will often result in the disappearance of the clinical symptoms and resolution of the renal lesion. Most of the patients with idiopathic MGN, present with chronic proteinuria and recurrent episodes of the nephrotic syndrome persisting over many years. Some 20% to 25% of these patients progress to renal failure terminating in end stage renal disease (Schieppati et al, 1993).
The structural damage to the glomerular capillary wall has been used to define four stages that correlate with the clinical evolution of the disease (Ehrenreich and Churg, 1968). In Stage-I, the glomeruli appear normal by light microscopy and there are no significant changes in the thickness of the basement membrane. In stage-II, the capillary walls are thickened and many sub-epithelial deposits are present, separated by extensions of basement membrane. These deposits do not stain with silver impregnation techniques, but the extensions of basement membrane do, thus creating the impression that the capillary loop is covered by spikes. These spikes have been shown to consist of laminin, not type IV collagen (Fukatsu et al, 1988). In stage-III the deposits are encircled by a newly formed basement membrane. The outer zones of the circles are composed of collagen Type-IV with lesser amounts of laminin (Fukatsu et al 1988).
During the late stage of the disease (Stage-IV), deposits gradually lose their electron density, and the basement membrane becomes vacuolated, folded and thickened. Although the degree of proteinuria does not parallel the stages of the renal lesions, there is a correlation between staging and prognosis.
Minimal Change Disease
Minimal change disease, also known as minimal change glomerulonephritis or 'lipoid nephrosis' as it indicates that the essential lesion is glomerular and the structural changes are inconspicuous. On routine light microscopy, changes are seen in the convoluted tubules, where large amounts of lipids and protein transport droplets, accumulate in the cell cytoplasm. In contrast all the glomeruli appear normal. It accounts for approximately 80% of all cases of idiopathic nephrotic syndrome in children (Habib and Kleinknecht, 1975) and 20% to 30% of adult cases (Korbet et al, 1988). The oedema and proteinuria tend to fluctuate, spontaneous remission and recurrence being common and the prognosis is good (Arneil and Lam, 1967) although a minority of patients develop renal failure with ureamia and hypertension.
Experimental studies in rats using an aminonucleoside called puromycin, which is a glomerular poison, have shown that a nephrotic syndrome can readily be induced with minimal changes. The loss of epithelial foot processes is seen by electron microscopy (Arakawa, 1971). In human disease the toxic material responsible has not so far been identified although there is evidence of T-lymphocytes' dysfunction. Lymphokines could be responsible for the altered permeability of the glomerular capillary wall (Silva and Hogg, 1989)
Thin Basement Membrane Disease (TBMD)
Benign familial hematuria is characterized by glomerular hematuria, non-progressive renal disease, and normal renal function in all affected individuals (Blumenthal et al, 1988). It is often associated with thinning of glomerular basement membranes; so termed as TBMD however, other histopathological pictures have been described (Yoshikawa, 1984) Praga and associates (1998) have reported persistent microscopic hematuria in 100% patients of TBMD, A thickness of 264 nm should be considered as the cut-off point for the diagnosis of TBMD (Steffes et al, 1983 and Tiebosch et al, 1989).
The following criteria is generally used for diagnosis of benign familial hematuria (Blumenthal et al, 1988):
- The presence of haemoglobin or red blood cell casts in the urinary sediment of the patient and in that of his parent, child or full sibling.
- The absence of renal insufficiency in the patient and
- No history of renal failure or auditory abnormalities in family members.
Progressive familial (hereditary) nephropathy is termed as Alport's syndrome. The inheritance is autosomal dominant (Glassock et al, 1986).
The following criteria is generally used for the diagnosis of Alport's syndrome:
- The presence of an abnormal urinary sediment (e.g. red blood cells or haemoglobin casts, waxy casts, oval fat bodies or fatty casts) and renal failure in the index case and
- The presence of an abnormal urinary sediment or renal failure in his parent, child or full sibling.
Apart from the above mentioned features and criteria the diagnosis of Alport's syndrome depends on the demonstration of lamellated GBM on ultrastructural examination of renal biopsy (Spear and Slussler 1972; Hinglais et al 1972). The initial abnormality seen in the GBM of children is a focal or diffuse attenuation, which may persist in females as the only abnormality throughout life. In adult men the GBM is usually thickened three fold to five fold and split into two or more lamellae that branch and rejoin to form a basket weave with irregular spaces, sometimes containing electron dense deposits.
The thickening and lamellation can be focal or diffuse and alternate the region of thinning and discontinuity in which the endothelium and epithelium are apposed (Spear and Slussler 1972). A lamellated GBM sometimes occurs in the healing phase of other glomerulonephropathies, but lamellation is more widespread and more pronounced in Alport's syndrome (Hill et al 1972. ; Hood et al 2000). The current view considers the Aloport's syndrome as a developmental defect in collagen biosynthesis resulting from mutation or deletion of gene (COL 4A5) that encodes for a3/a5(IV), collagen chains (Tryggvason et al 1993)
Ever since its advent in 1934, immunocytochemistry/ immunohistochemistry has developed rapidly as a valuable tool for basic cytological and pathological studies (Marrack, 1934; Childs, 1987). The earliest electron microscopic applications employed ferritin (Singer, 1959) and mercury (Pepe, 1961a & Pepe 1961b) as electron dense markers that could be bound to antibodies. Shortly thereafter, peroxidase and other enzymes were developed as sensitive immunocytochemical labels for both light and electron microscopy studies (Nakane and Pierce 1966). One of the most widely used labels today i.e. colloidal gold was developed in 1971 (Faulk and Taylor, 1971; Frens, 1973).
The normal glomerular matrix has been studied by immunohistochemical methods at the light and electron microscopy levels and found to contain a number of components including type IV collagen, the glycoproteins, laminin, fibronectin and entactin; and heparan sulfate proteoglycans (Abrahamson et al, 1986; Hood et al, 2000).
In the present study we will use indirect technique involving peroxidase labeled secondary antibodies at light microscopy level and gold-labeled secondary antibodies at electron microscopy level to localize and characterize the basement membrane components. The thickness of glomerular basement membrane will be measured using conventional transmission electron microscopy.
AIMS AND OBJECTIVES
The main aim of this study is to demonstrate renal glomerular basement membrane abnormalities in the terms of ultrastructure and basement membrane components in patients of:
- Minimal change disease
- Idiopathic membranous glomerulonephritis (Idiopathic-MGN)
- Thin basement membrane disease (TBMD)
- Alport's syndrome
The study would include the following objectives:
- Morphometric study at light microscopy level to assess the intraglomerular changes (The gomerular diameter and area, tuft diameter and area, and the area occupied by the capillary wall and mesangium will be worked out).
- Ultrastructural examination by transmission electron microscopy (TEM) to explore the thickness of glomerular basement membrane and other ultrastructural abnormalities.
- Immunohistochemical study at light microscopy and electron microscopy level to demonstrate the glomerular basement membrane components. Fibronectin, laminin, collagen-IV will be localized by the immunohistochemical and immunoelectron microscopy procedures.
MATERIALS AND METHODS
Patients and Renal Biopsies
Patients confirmed to have microscopic hematuria or familial microscopic haematuria, excretion of more than 0.350 gm protein per 24 hour and/or clinical features of nephrotic syndrome will be included in the study. Renal biopsies performed on these patients, attending the Nephrology Department of Postgraduate Institute (PGI), Chandigarh will be taken up for the study.
Every patient included in the study will be Indian citizen with a grand- parent as a full-blooded Indian and informed consent will be obtained at the time of performing percutaneous renal biopsy. Patients negative for Acquired Immunodeficiency Syndrome (AIDS) by ELISA test and negative for antinuclear antibodies by indirect immunofluorescence technique will be selected for study.
Monoclonal antibodies (mouse IgG) to Type-IV collagen and its variants classic and novel collagen chains, fibronectin and laminin available from Dako Corporation, USA will be used for immunohistochemistry and immunoelectron microscopy. Rabbit anti-human antibodies (IgG) to above mentioned components available from Dako Corporation, USA will be used in case of non-availability of mouse monoclonals. Rabbit anti-mouse IgG or goat anti-rabbit IgG (secondary antibodies) conjugated with peroxidase (Dako Corpn.USA) will be used for immunohistochemistry. Secondary antibodies conjugated with gold particles of 10 or 20 hm will be procured from Taab Laboratories, U.K. for use in immunogold staining.
Kidney Tissue Processing
Renal, needle biopsies obtained for histopathological examination would be divided into three portions and processed separately for light microscopy, conventional electron microscopy and immunoelectron microscopy. Some of the cases will be studied retrospectively.
- Processing for Light Microscopy
- Biopsy specimens for light microscopy will be fixed in phosphate buffered 10% formalin, embedded in paraffin wax, sectioned at 2 mm thickness and stained with haemotoxylin-eosin, periodic acid-Schiff's and Silver-methenamine.
- Processing for Conventional Electron Microscopy
- Processing for Immunoelectron Microscopy
The portion of renal tissue (1 x 1 x 1 mm) for electron microscopy will be fixed in 0.2 M Phosphate buffered 3% glutaraldehyde for 2 hour post-fixed in 2% Osmic acid for 2 hour, then dehydrated through graded series of ethanol and embedded in Epon-812 (Taab Laboratories, U.K.). Ultrathin sections will be cut at a thickness of 80 hm and double stained with uranyl acetate and lead citrate and examined under transmission electron microscope (TEM).
For immunoelectron microscopy small blocks (1 x 1 x 1 mm) of renal biopsy will be fixed in 0.1M Phosphate buffer, pH 7.2 containing 0.2% picric acid and 3% glutaraldehyde for 2 hour. The tissues will be dehydrated in graded series of ethanol, infiltrated with LR White (Taab Laboratories, U.K.) embedded in the same at 50° C for 24 hours. (Graber and Kreutzberg, 1985) or in Lowicryl resin (Roth et al, 1981) at low temperature.
Staining Procedures for Light Microscopy
- Haematoxylin and Eosin stain (H&E) (Culling, 1976): Paraffin embedded tissue sections will be deparaffinized in two changes of xylene x 15 minutes each, re-hydrated in descending grades of ethanol and then in tap water, stained with Cole's (1943) haematoxylin for 20 minutes, differentiated in 1% acid alcohol. After bluing of nuclei in Scott's tap water sections will be counter-stained in 1% aqueous eosin for 1-2 minute. Dehydration will be done in ascending grades of alcohol, Finally slides will be cleared in xylene and mounted with dibutylphthalate xylene (DPX) and morphometric evaluation will be done under light microscope.
- Periodic Acid Schiff's Stain (PAS) (Bancroft and Stevens, 1990.; McManus 1946 ): Paraffin embedded sections will be dewaxed in two changes of xylene x 15 minutes each, hydrated in descending grades of alcohol and then in distilled water. Oxidation will be done for 5 minutes with 0.5% aqueous periodic acid, after washing in tap water sections will be washed in distilled water and treated with Schiff's reagent for 15 minutes and then washed in running tap water for 5-10 minutes to develop magenta colour. Sections will be counter stained with Cole's haematoxylin for 30 seconds, washed in tap water, dehydrated in ascending grades of alcohol, cleared in xylene and mounted with DPX.
- Periodic Acid-Methenamine Silver Stain (Jone,1957): Paraffin embedded sections will be brought down to water after the conventional procedure mentioned earlier, rinsed with distilled water, oxidised with 1% aqueous periodic acid for 20 minutes, rinsed with distilled water and then kept in running tap water for 15 minutes, and again rinsed with distilled water and then kept in running tap water for 15 minutes, and again rinsed with distilled water. Now, sections will be placed in Hexamine-Silver bath for 1-3 hours and examined at half hourly intervals after the first hour. When the glomerular basement membrane develops black stain, sections will be rinsed with distilled water and toned in 0.2% Ferric Chloride for 5 minutes, rinsed in distilled water. After fixation in 5% Sodium thiosulphate for 2 minutes, sections will be washed in tap water, counter-stained with hematoxylin and eosin briefly, dehydrated cleared and mounted with DPX. Slides will be examined with light microscope.
Staining Procedure for Ultrastructural Examination
Uranyl acetate and Lead citrate staining(Watson,1958 and Reynolds, 1963): For better contrast double staining with Uranyl acetate and Lead citrate will be done on epon-812 embedded ultrathin sections mounted on copper grids.
Sufficient volume (5 to 7 ml) of 1% (W/V) Uranyl acetate in 50% ethanol will be centrifuged at 1500 rpm for 5 minutes to remove any particulate matter and the supernatant will be decanted into a small covered staining dish, the bottom of which is covered with a piece of lens paper. Grid mounted sections will be transferred to the staining dish, covered and stained for 10 minutes. Grids will be rinsed well in distilled water and stained in similar way with alkaline lead citrate for 5 minutes. After staining grids will be washed in 0.02 N NaOH solution and then with distilled water. Grids will be dried and stored in labeled containers till examination by TEM.
Staining Procedures for Immunohistochemistry
Immunohistochemical staining to localize fibronectin, laminin, and collagen-IV will be done as per the manufacturer's instructions for immunohistochemical staining kits, based on the method of McDermott et al (1995). For light microscopy staining kits will be purchased from Dako Corporation, USA and for immunoelectron microscopy, 10 or 20 hm gold particles conjugated to rabbit anti-mouse IgG or goat anti-rabbit IgG (Taab Laboratories, U.K.) will be used. Same primary anti-human antibodies (Dako Corporation, USA) will be used for immunohistochemical staining for light microscopy and colloidal gold labeled staining for immunoelectron microscopy.
- Immunostaining for Light Microscopy (Dako Corporation, USA, Kits)
- Immuno-gold Staining for Immunoelectron Microscopy (Johnson and Bettica, 1989)
Paraffin embedded sections will be brought down to water following conventional procedure as mentioned in earlier techniques. Quenching of endogenous peroxidase activity will be done by treating the sections with 0.03% Hydrogen Peroxide. After washing with 0.2M Phosphate buffered saline pH 7.2 sections will be incubated with an appropriately characterized and diluted mouse monoclonal antibodies against basement membrane component, followed by incubation with labeled polymer for 30 minutes each time or as specified by the manufacturers. Staining will be completed by incubation with 3,3'-diaminobenzidine (DAB+) substrate chromogen. Counter staining will be done in a bath of aqueous hematoxylin, dehydration in ascending grades of alcohol, clearing in xylene and finally slides will be mounted with DPX.
Five cases from each group will be taken up for ultrastructural localization of basement membrane components, immuno-gold staining will be performed on 80 hm thin LR white embedded (Graber and Kreutzberg, 1985) or Lowicryl embedded (Roth et al., 1981) sections mounted on the dull side of freshly cleared (Glacial acetic acid for 3 minutes, absolute ethanol for 1 minute and water washes), uncoated nickel grids and overnight heated at 50° C.
The method used for on grid immunolabeling will be comprising two steps, etching and the immuno-gold procedure.
- Etching Protocol: Etching will be done by floating the grids, section side down on millipore filtered water for 5 minutes and then for 60 minutes, on saturated aqueous Sodium metaperiodate (Sigma, 0.1 g/ml, freshly prepared and centrifuged; Bendayan and Zollinger, 1983) followed by three x 5 minutes washes on water. Next, the grids will be floated on 3% Hydrogen peroxide for 5 minutes (Moriarty, 1973) and water washed three times. The entire procedure will be done with 0.5 ml of each solution in a covered depression dish, which will be continuously and gently agitated on a rocker during washes.
- Immuno-gold Protocol: (Grids will be floated section side down throughout on drops placed on parafilm or 'dental wax' in a moist chamber; and washing will be performed in rocking depression dishes as for etching. Grids will be briefly drained and the edge blotted between each change of solution). Grids will be floated on 25 ml drops of 5% normal rabbit serum for 30 minutes. Thereafter grids will be placed on 25 ml drops of primary antibody (rabbit anti-human or mouse monoclonal antibody) for overnight in the refrigerator. Four washings of 5 minutes each will be done with 1% Bovine Serum albumin (BSA, Sigma) in Phosphate buffered saline pH 7.2. Then grids will be floated on 25 ml drops of gold reagent (10 or 20 hm gold particles, conjugated to goat anti-rabbit IgG or rabbit anti-mouse IgG as per primary antibodies used; Taab Laboratories, U.K.) for 2 hour. Grids will be washed on 1% BSA three times for 5 minutes each and then washed with PBS for 5 minutes (3 times). Grids will be dried and stored in labeled containers till examination by TEM.
Measurement of Basement Membrane Thickness
Glomerular basement membrane thickness will be measured using a magnification and illumination system (Osawa et al 1966; Hood et al, 2000). At least 50 measurements of basement membrane thickness will be made at random on electron micrograph (magnification x 15000) of individual renal tissues from patients and normal controls. Results will be compared using student's 't' test.
The material to be used for this study will be constituted by renal biopsy done on patients for diagnostic purposes only. No patient will be subjected to the biopsy procedure for the purpose of this study. Informed consent will be taken from patients for the purpose of renal biopsy, as per the usual practice in the department of Nephrology. Control biopsies will be taken from autopsy material only.
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