Glomerulonephritis can be induced in WKY rats by immunization with the non-collagenous domain (NC1) of type IV collagen, which is the major component of glomerular basement membranes (GBM), as well as by a single injection of monoclonal antibodies against alpha 4 (IV) NC1. Chondrex provides the NC1 fraction of type IV collagen isolated from bovine GBM for inducing nephritis in rats. Futhermore, rat models of glomerulonephritis and diabetic nephropathy demonstrate comparable albuminuria with human patients, suggesting that rat urinary albumin levels are useful for evaluating disease severity and treatment efficacy. In addition, NC1 fragments purified from mouse and rat renal type IV collagen are supplied for studying autoantibodies to GBM in mice and rats.
Chondrex's rat urinary protein assay kit uses the turbidity method in a 96-well fl at bottom plate, which offers a solution for assaying a large number of rat urine samples within a short time and a wide assay range (Link). This kit requires a minimal volume (less than 10 μl) of urine sample, and is ideal for studying nephritis in various strains of rats that do not secrete a sufficient volume of urine during a 16-hour collection period. Assay results are good
NC1 Fragment of Type IV Collagen
|Product||Quantity||Catalog #||Price (USD)|
|NC1 Fragment of Bovine GBM Type IV Collagen||1 mg, lyophilized||1102||281.00|
|NC1 Fragment of Mouse Renal Type IV Collagen||0.1 mg, lyophilized||1106||303.00|
|NC1 Fragment of Rat Renal Type IV Collagen||0.1 mg, lyophilized||1104||303.00|
Rat Albumin Detection Kit
|Product||Catalog #||Price (USD)|
|Rat Albumin Detection Kit||3020||299.00|
Rat Urinary Protein Assay Kit and Standard
|Product||Catalog #||Price (USD)|
|Rat Urinary Protein Assay Kit||9040||90.00|
|Rat Urinary Protein Assay Standard||90401||56.00|
Induction of Glomerulonephritis in WKY/NHsd Rats (Harlan) by Immunization with the NC1 Fragment of Bovine Type IV Collagen
Female WKY/NHsd rats (Harlan) were immunized with 50 µg of the NC1 fragment of bovine type IV collagen from glomerular basement membrane (GBM) emulsified with CFA (M. tuberculosis, 1mg/ml, catalog # 7008) by subcutaneous injection at the base of the tail on day 0, and received 100 µg of nephritogenic monoclonal antibody, Clone b35 by Intraperitoneal injection. The severity of nephritis was determined by the total amount of proteins secreted in a 16-hour urine collection (from 5:00 PM to 9:00 AM). Rats immunized with the NC1 fraction of bovine GBM type IV collagen, developed mild nephritis with medium proteinuria on day 10, and lasted until day 28. Similarly, the control rats, which received b35, developed mild nephritis within 5 days and lasted at least 14 days. These results indicated that WKY/NHsd strain of rats from Harlan are medium responders to glomerulonephritis induced by immunization with the NC1 of type IV collagen and by IP injection of monoclonal antibodies to the NC1 of type IV collagen.
NOTE: For unknown reasons, mice are generally resistant to GBM autoantibody-mediated nephritis.
Figure 1 - Induction of glomerular nephritis in WKY/NHsd (Harlan, USA) by immunization with the NC1 fragment of bovine GBM type IV collagen
Turbidity Assay and Bradford Assay
The turbidity assay method has been widely used to determine urinary protein levels in human specimens, because it is accurate, easy to use, and economical. However, the current turbidity method requires large sample volumes, and cannot be used for assaying urine specimens from certain strains of rats, because of low urine volumes. Please note that the16-hour urine volume varies significantly from 0.1 to 20 ml depending on rat strains. Furthermore, reading turbidity (OD 450 nm) of individual test tubes using a spectrophotometer is time consuming.
Protein concentrations in urine samples can be determined by Bradford assay methods. However, the turbidity assay method using 3% sulfosalicyclic acid dihydrate is more convenient than the Bradford method because of the wider range (0.5 – 4.0 mg/ml) of the dose response curve (Figure 1). Importantly, regardless of which assay method is used, bovine serum albumin (BSA) cannot be used as a standard. The dose response curve of BSA significantly differs from the dose response curve of serum proteins in the turbidity assay. More importantly, the OD value of globulins by the Bradford assay method is only 70% of the value of BSA. Therefore, a standard protein solution prepared from normal rat serum is ideal for assaying urinary protein levels regardless of the assay method.