Chapter 16

Determination of Collagen Content,

Concentration, and Sub-types in Kidney Tissue

Chrishan S. Samuel

Abstract Fibrosis and sclerosis are widely recognized as hallmarks of pro-

gressive renal disease and are caused by the excessive accumulation of connec-
tive tissue, mostly collagen. The detection of collagen content, concentration
(collagen content/dry weight tissue), and sub-types from kidney tissues is
therefore an important part of determining the extent of renal fibrosis in age-
ing and diseased states. This chapter describes a colorimetric-based hydroxy-
proline assay used to estimate total collagen content and concentration. Based
on the method of Bergman and Loxley (8), this spectrophotometric technique
estimates total collagen by measuring the hydroxyproline content of tissue.
The assay relies on the fact that the collagen triple helix is one of the few
proteins that contain the amino acid hydroxyproline. The second part of this
chapter describes the use of sodium dodecyl sulfate (SDS) polyacrylamide gel
electrophoresis (PAGE) to isolate, detect and quantify changes in the soluble
and insoluble interstitial collagen sub-types. This technique complements the
hydroxyproline assay by providing a means of identifying which interstitial
collagens are altered in renal disease.

Keywords Hydroxyproline, Kidney collagen content, Kidney collagen concentration,

Interstitial collagen subtypes

1 Introduction

Fibrosis and sclerosis is the final common pathway in all forms of progressive
renal disease, regardless of etiology (1, 2). The progression of renal fibrosis/
sclerosis is the result of an excess accumulation of connective tissue, primarily
collagen, which is over-expressed by renal vascular smooth muscle cells,
mesangial cells, and interstitial myofibroblasts in response to repeated patho-
logical stimuli and inflammation in the kidney (1, 2). Various collagen sub-types
accumulate in this process, including both so-called interstitial (collagen I, III,
and V) and basement membrane (collagen IV) proteins (3). Measuring changes

From: Methods in Molecular Biology, Vol. 466: Kidney Research 223

DOI: 10.1007/978-1-59745-352-3_16, Edited by: T.D. Hewitson and G.J. Becker
© Humana Press, Totowa, NJ
224 C.S. Samuel

in collagen content, concentration, and composition is therefore fundamental to

our understanding of renal fibrosis.
Several techniques have been established for this purpose. While morphometric
techniques in particular are widely used, they are limited by the fact that they often
only measure changes relative to normal/control tissue, and therefore do not meas-
ure absolute quantities. Such methodologies are therefore often unable to account
for hypertrophy and atrophy of organs.
The spectrophotometric-based hydroxyproline assay is one of the few assays
that allows for the actual quantitation of collagen content and concentration in
several organs, including the kidney. The assay was first described in 1950 (4),
based on the fact that all collagens contain globular domains and share the com-
mon structural motif of triple helical segments. This triple helical structure is
composed of three α- (polypeptide) chains, which range from 10 to 150 kDa per
chain. Each chain consists of a repeating triplet amino acid sequence (Gly-X-Y)n,
where X and Y can be any amino acid, but are often proline and hydroxyproline,
respectively (5). Collagen is one of the few proteins containing the amino acid
hydroxyproline. Thus, based on the absolute quantitation of hydroxyproline
(which represents a fixed percentage of the amino acid composition of collagen
in most mammalian organs (6, 7)), the amount of collagen content and concentra-
tion in tissues can also be derived.
Since its original description (4), this method has been adapted and/or
modified by several investigators to overcome a number of difficulties includ-
ing reproducibility in optimal values between sample replicates, differences
between standard curve values prepared on separate occasions, and interference
by other components (particularly when the hydroxyproline concentration is
low relative to other amino acids). While some of these issues are yet to be
fully resolved, the method of Bergman and Loxley (8) has been established as
one of the better assays for hydroxyproline determination, yielding optimal
hydroxyproline values with reproducible standard curves. In this chapter, a
1:10 scaled-down version of the original Bergman and Loxley method (8) is
described. This method has been used successfully in our laboratory to meas-
ure collagen content and concentration in the normal, ageing, and diseased
kidney (3, 9, 10).
Additionally, this chapter describes the extraction, detection, and quantita-
tion of interstitial collagen sub-types (types I, III, and IV) by sodium dodecyl
sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), based on the origi-
nal procedure described by Sykes and colleagues (11). Again this can been
applied to kidney tissue from experimental (3, 9, 10) or clinical models. The
SDS-PAGE method allows for both quantitation of the relative changes in
interstitial collagen sub-types, and the amounts of soluble and insoluble
(cross-linked) interstitial collagens in several organs, including the kidney.
This technique is particularly useful for studying temporal changes in individual
interstitial collagen subtypes.
16 Collagen Content, Concentration, and Sub-types in Kidney Tissue 225

2 Materials

2.1 Hydroxyproline Determination of Collagen

Content and Concentration

1. Liquid nitrogen.
2. Hydrochloric acid 6 M and 0.1 M.
3. Sodium hydroxide.
4. Chloramine T (Sigma, St. Louis, MO, USA) is dissolved in distilled water to
give a final concentration of 7% (w/v) and can be stored for up to 2 weeks at
4°C, in a dark bottle.
5. Acetate/citrate buffer, pH 6.0 (per 100 mL): dissolve 5.7 g sodium acetate.3H2O
(or 3.44 g sodium acetate anhydrous); 3.75 g tri-sodium citrate 2H2O; and 0.55
g citric acid in 38.5 mL of isopropyl alcohol and distilled water. The buffer can
be stored at 4°C up to 6 months.
6. Oxidation buffer is prepared (fresh before each assay) by mixing one part 7%
chloramine T with four parts acetate citrate buffer and should be protected
from light.
7. Ehrlich’s reagent is prepared by dissolving 2.0 g para-dimethylaminobenzalde-
hyde (DMAB; Sigma) in 3.0 mL of 60% (v/v) perchloric acid and can be stored
for up to 2 weeks at 4°C in a dark bottle. For assaying of larger sample num-
bers, multiple amounts of DMAB should be dissolved in the respective multiple
volumes of perchloric acid.
8. Analytical isopropanol reagent is prepared (fresh before each assay) by mixing
3 parts DMAB to 13 parts isopropanol.
9. Hydroxyproline standard is prepared by dissolving cis 4-hydroxy-l-proline
(Sigma) in 0.1 M hydrochloric acid (HCl) to a concentration of 1 mg/mL (w/v),
should be protected from light, and can be stored at –20°C.
10. Kimax™ screw-capped glass tubes (Kimble/Kontes, Vineland, NJ, USA).
11. Disposable cuvettes (Lake Charles Manufacturing, Lake Charles, LA, USA).
12. Lyophilizer.
13. Heating block.
14. Spectrophotometer.
15. Microsoft Excel™ (or equivalent software program for plotting regression line).

2.2 SDS-PAGE Analysis of Interstitial Collagen Sub-types

1. Biorad Protean II™ xi gel system (Biorad, Richmond, CA, USA).

2. Neutral salt extraction buffer containing: 0.05 M Tris-HCl acid pH 7.5, 0.15 M
sodium chloride, 10 mM protease inhibitors N-ethylmaleimide (Sigma), 10 mM
226 C.S. Samuel

phenylmethylsulfonyl fluoride (Sigma), 1 mM benzamidine hydrochloride

(Sigma), and 10 mM EDTA (Sigma). The protease inhibitors are added to pre-
vent pro-collagen degradation during isolation.
3. 0.5 M acetic acid diluted with distilled water.
4. Pepsin (Boehringer Mannheim, Mannheim, Germany).
5. Cyanogen bromide.
6. Ammonium bicarbonate.
7. Formic acid.
8. Nitrogen (BOC Gases, Prahran, Australia).
9. Separating gel I: 5% bis-acrylamide gel containing 0.375 M Tris-HCl pH 8.8,
2 M urea, and 10% SDS (Biorad). For large gels (using the Biorad Protean II
xi gel system) add 150 µL of 10% (w/v) ammonium persulfate (APS; ICN
Biomedicals Inc., Aurora, OH, USA) to aqueous gel contents; de-gas contents
for 5 min by air vacuum; and then add 20–40 µL of TEMED (Sigma) before
allowing gel to set/cross-link for 45–60 min (at room temperature).
10. Separating gel II: 12.5% bis-acrylamide gel containing 0.375 MTris-HCl pH 8.8,
2 M urea, and 10% SDS. APS and TEMED can be added as described in Sect. 2.2.9.
11. Stacking gel I: 3.5% bis-acrylamide gel containing 0.125 M Tris-HCl pH 6.8,
2 M urea, and 10% SDS. For large gels (using the Biorad Protean II gel system)
add 50 µL of 10% (w/v) APS to aqueous gel contents; de-gas contents for 5
min by air vacuum; and then add 10–20 µL of TEMED before allowing gel to
set/cross-link for 35–45 min (at room temperature).
12. Stacking gel II: 4.5% bis-acrylamide gel containing 0.125 M Tris-HCl pH 6.8, 2 M
urea and 10% SDS. APS and TEMED can be added as described in Sect. 2.2.11.
13. Sample-loading buffer: 20% (w/v) sucrose/glycerol containing 0.05 M Tris-HCL
pH 6.8, 2 M urea, 0.1% (v/v) SDS, and 0.1% (w/v) bromophenol blue (Biorad).
14. Running buffer: 25 mM Tris-base, containing 0.192 M glycine and 0.35%
(w/v) SDS.
15. 10% (v/v) mercaptoethanol diluted in running buffer.
16. Coomassie blue-R250: prepare a 0.1% (w/v) solution of Coomassie blue-R250
(Biorad) by dissolving in 45% methanol and 7% acetic acid for at least 60 min;
then filter the solution through two layers of Whatmann 3m filter paper and
chill at 4°C overnight.
17. De-stain containing 20–30% methanol and 7% acetic acid.
18. Densitometer with quantitative software (Biorad GS710 Calibrated Imaging
Densitometer; Biorad).

3 Methods

3.1 Preparation of Samples for Hydroxyproline Assay

1. Isolated kidney tissues should be weighed and can then be either snap-frozen
in liquid nitrogen or stored immediately in dry ice. For analysis of more specific
regions within each kidney tissue (i.e. cortex, medulla, renal pyramids, renal
16 Collagen Content, Concentration, and Sub-types in Kidney Tissue 227

papilla, etc.), these regions should immediately be dissected out in cold phos-
phate-buffered saline (PBS) and weighed before being frozen down.
2. Each whole or regional kidney tissue should then be freeze-dried (lyophilized)
down to dry weight and the dry weight tissue accurately measured.
3. The dried tissues should then be re-hydrated in acetate/citrate buffer for 24 h at
4°C (see Note 1).
4. The rehydrated tissue should then be transferred to Kimax™ screw-capped glass
tubes (Kimble/Kontes) and submerged in 0.5–1.0 mL, 6 M HCl before being
hydrolyzed at 110°C (in a heating block) for at least 18–24 h (see Note 2).
5. The following day, the hydrolyzates should be cooled at 4°C, before being
evaporated to dryness in the presence of a strong base (i.e. sodium hydroxide; to
neutralize the acid) in a lyophilizer (overnight).
6. The dried residues should finally be dissolved in 0.1 M HCl (as was per-
formed for the hydroxyproline standard; approximately 1 mL per 100 mg wet
weight tissue).

3.2 Hydroxyproline Determination (See Notes 3 and 4)

1. All assays should be conducted in Kimax™ screw-capped glass tubes, with the
capacity to hold ≥5 mL of assay material.
2. Assay each sample in duplicate or triplicate by adding 10 µL of kidney tissue
hydrolyzate (in 0.1 M HCl) to 90 µL of distilled water (in a separate Kimax™
glass tube per sample and replicate) (see Note 5).
3. In separate tubes, also establish a standard curve for each experiment by adding
0 (blank), 2, 4, 6, 8, and 10 µL of the 1 mg/mL hydroxyproline standard (in 0.1
M HCl) in separate Kimax™ glass tubes and adjusting the volume of each tube
to 100 µL with distilled water.
4. To each of the standard curve and sample tubes (100 µL), add 200 µL of isopro-
panol, followed by 100 µL of the oxidation buffer (to give a volume of 400 µL).
Immediately vortex all samples and allow them to stand at room temperature for
4 min (± 30 s).
5. Add 1.3 mL of the analytical isopropanol reagent to each tube and vortex each
sample again (to give a volume of 1.7 mL), before tightly capping each tube. All
samples should then be placed for 25 min in a shaking water bath or oven pre-set
to 60°C (see Note 6).
6. The samples should then be cooled at 4°C for approximately 5–10 min (see Note 7).
7. Isopropanol (3.3 mL) should then be added to the cooled samples (to give a final
volume of 5.0 mL), before the samples are thoroughly vortexed and decanted
into disposable cuvettes (Lake Charles Manufacturing).
8. The absorbance of each standard curve sample/sample of interest should then be
obtained at 558 nm in a spectrophotometer, using the “blank” to calibrate each assay.
9. A standard curve can then be generated using Ab558 nm (y-axis) vs micrograms
of hydroxyproline added (x-axis) (Fig. 16.1). A line of best fit, equation (i.e. micrograms of
hydroxyproline = Ab558 nm/arbitrary constant value), and R2-value can then be
obtained (using Microsoft Excel™ software).
228 C.S. Samuel

y = 0.1916x
R2 = 0.996
2.5

2
Absorbance 558nm

1.5

0.5

0
0 2 4 6 8 10 12
Hydroxyproline (µg)

Fig. 16.1 A typical standard curve for the hydroxyproline assay, which plots Ab558 nm (y-axis)
versus micrograms of hydroxyproline (cis 4-hydroxy-l-proline) added (x-axis). A line of best fit, equa-
tion y = arbitrary constant/x, and R2 value can be obtained using Microsoft Excel software, as shown.
Using the provided equation, the micrograms of hydroxyproline in each sample of interest can then
be determined by dividing the corresponding Ab558 nm value by the arbitrary constant value

10. Based on the Ab558 nm readings, the generated equation (for each assay) should
then be used to determine the amount of hydroxyproline (between 0 and 10 µg)
in each sample (from 10 µL of starting material). The hydroxyproline values in
duplicates or triplicates of the same sample should then be averaged out.
11. The total hydroxyproline content in each sample can be then extrapolated by
multiplying the amount hydroxyproline (from the 10 µL of starting material)
by the total volume of 0.1 M HCl added to the sample hydrolyzates (see Sect.
3.1.6)/10 µL. For example, if the sample hydrolyzates were dissolved in 1.0 mL
(1,000 µL) of 0.1 M HCl, then the total amount of hydroxyproline in each
sample is calculated by multiplying the amount of hydroxyproline from the 10
µL of assay material by 100 (Fig. 16.2a).

3.3 Determination of Collagen Content and Concentration

(See Note 8)

1. The total (whole/regional) kidney collagen content can then be extrapolated by

multiplying amount of total hydroxyproline content in each sample by a factor
of 6.94, based on the fact that hydroxyproline represents 14.4% of the amino
acid composition of collagen in most mammalian tissues (6, 7) (Fig. 16.2b).
16 Collagen Content, Concentration, and Sub-types in Kidney Tissue 229

Total hydroxyproline content (µg)/

***
200
###
150 *
kidney tissue

100

50

0
Day 0 Day 3 Day 10
a OB UO OB UO

***
###
1200

*
Total collagen content (µg)/

900
kidney tissue

600

300

0
Day 0 Day 3 Day 10
b OB UO OB UO

***
Total collagen concentration (%)/

4
###

3
kidney tissue

2 *

0
Day 0 Day 3 Day 10
c OB UO OB UO

Fig. 16.2 Total hydroxyproline content (a), the corresponding collagen content (b), and collagen
concentration (c) from the kidneys of control wild-type (C57B6Jx129SV) mice (at day 0); and
from the obstructed (OB) and unobstructed (UO)/contralateral kidneys of mice 3 days and 10 days
after unilateral ureteric obstruction. *p< 0.05; ***p< 0.001 versus values from day 0 kidneys;
###p<0.01 versus values from day 10 UO kidneys
230 C.S. Samuel

2. Collagen concentration (in the whole/regional kidney tissue), expressed as a

percentage, can then be calculated by dividing the total collagen content of tis-
sues (see Sect. 3.3.1) by the dry weight tissue (Fig. 16.2c).

3.4 Preparation of Samples for SDS-PAGE

1. Isolated whole/regional kidney tissues should be treated as described in Sect.

3.1.1 (see Note 9).
2. Kidney tissues should be powdered/chopped finely in the presence of liquid
nitrogen and then placed into the neutral salt buffer (0.5–1.0 mL buffer/pow-
dered kidney tissue, which extracts the newly synthesized soluble collagen) for
24 h at 4°C (with gentle agitation) (see Note 10).
3. Samples should then be centrifuged at ∼12,000×g for 45 min at 4°C (in a
Beckman or Sorvall ultracentrifuge; or in an Eppendorf centrifuge with 2-mL
Eppendorf tubes). The neutral salt supernatants can be frozen in dry ice and
stored at –80°C or can be lyophilized down and 1) hydrolyzed in 6 M HCl (see
Sect. 3.1.4) before being re-suspended in 50–100 µL of 0.1 M HCl (see Sect.
3.1.6) for hydroxyproline determination; or 2) re-suspended in Tris/HCl, pH 6.8
and sample loading buffer for SDS-PAGE analysis.
4. The neutral salt pellets can then be further extracted with 0.5 M acetic acid
(which extracts the newly cross-linked soluble collagen) for 24 h at 4°C (with
gentle agitation).
5. Samples should again be centrifuged and the acetic acid supernatants treated as
described in Sect. 3.4.3.
6. The acetic acid pellets should also be freeze-dried/lyophilized down to dry
weight (overnight).
7. One milligram of dry-weight acetic acid pellet should then be digested with 1:10
enzyme:substrate pepsin (which extracts the mature cross-linked insoluble collagen).
The pepsin solution should originally be prepared as a 1 mg/mL (w/v) solution
in 0.5 M acetic acid before being allowed to dissolve at 4°C for 60 min with
gentle agitation. Following this 60-min period, a 1:10 dilution of the 1 mg/mL
pepsin stock should be made with 0.5 M acetic acid (providing a 0.1 mg/mL pepsin
solution); and 1 mL of this 0.1 mg/mL pepsin stock solution should be used to
digest the insoluble collagen from 1 mg of dry weight acetic acid pellet for 24 h
at 4°C (with gentle agitation). If required, larger volumes of 0.1 mg/mL pepsin
can be used to extract the mature insoluble collagen from larger amounts of the
lyophilized acetic acid pellet, as long as the enzyme:substrate ratio remains
the same (see Notes 11 and 12).
8. Samples should again be centrifuged and the pepsin-digested supernatants
treated as described in Sect. 3.4.3.
9. The final pepsin insoluble pellet can be lyophilized and stored at –80°C (over
long-term periods) or can be cleaved with cyanogen bromide (CNBr) using the
methods of Scott and Veis (12 , 13), which digests the remaining insoluble
16 Collagen Content, Concentration, and Sub-types in Kidney Tissue 231

kidney collagen (which is neutral salt, acetic acid, and pepsin insoluble). The
procedures detailed in Sect. 3.4.10–3.4.15 should be carried out in a fume
cupboard.
10. The lyophilized pepsin-digested pellets should be dissolved in 100 mM ammonium
bicarbonate, pH 8.0 (50 µL) for > 30 min (to inactivate the pepsin).
11. Bubble 98% formic acid with nitrogen (to stop any oxygen from entering the
system; as oxygen will interfere with the CNBr reaction).
12. Weigh CNBr in a pre-weighed plastic tube and cap tightly.
13. Dissolve CNBr at 70 mg/mL in nitrogen-saturated 98% formic acid for 10 min.
Smaller/larger amounts of CNBr and nitrogen-saturated 98% formic acid may
be used as long as the ratio of CNBr:formic acid remains constant.
14. Digest samples at about 1 mL of 70 mg/mL CNBr per 1 mg protein for 4 h with
gentle agitation.
15. This reaction can be terminated by drying samples down in a Speedvac centri-
fuge under vacuum. Alternatively, CNBr and formic acid can be removed under
a stream of nitrogen.
16. Wash CNBr-digested pellets with 0.5 M acetic acid and lyophilize to remove
any residual CNBr.
17. Re-suspend washed CNBr-digested pellets with Tris-HCl, pH 6.8 and sample-
loading buffer for SDS-PAGE analysis, or hydrolyze the pellet with 6 M HCl
(see Sect. 3.1.4) before re-suspending it in 100–200 µL of 0.1 M HCl (see Sect.
3.1.6) for hydroxyproline determination.

3.5 SDS-PAGE Analysis of Interstitial Collagen Sub-types

1. Prepare 1× running buffer and chill at 4°C overnight.

2. Set up a Biorad Protein II™ xi (large) gel system for SDS-PAGE analysis (as the
larger gel system allows for better separation of the collagen chains) (see Note 13).
3. Prepare the 5% separating gel (for neutral salt, acetic acid, and pepsin-soluble
collagens) or 12.5% separating gel (for CNBr-digested collagens). Mark large
plates 3.5 cm from the top (or 1.0 cm from the bottom of the sample wells).
Also mark the plates 2.2 cm and 8.0 cm from the top of the 5% separating gels
or 11.0 cm from the top of the 12.5% separating gels. Separating gels can be
prepared a day before the addition of stacking gels; but should be covered with
1:3 ratio of Tris-HCl, pH 8.8:running buffer (10 mL), once set, and stored at
4°C overnight. The following day, gels can be thawed to room temperature while
the stacking gel is being prepared.
4. Prepare the 3.5% stacking gel (for neutral salt, acetic acid, and pepsin-soluble
collagens) or 4.5% stacking gel (for CNBr-digested collagens) with 10- to 15-
well sample combs (Biorad), which can hold up to 150 µL (15-well) to 200 µL
(10-well) of sample. Once the stacking gel is set and combs removed, the sam-
ple wells should be washed three to six times with 1× running buffer (to equili-
brate the samples with the running buffer).
232 C.S. Samuel

5. Heat neutral salt-, acetic acid-, pepsin-, and CNBr-extracted samples in Tris/
sample loading buffer at 60–65°C for 10 min before loading (with equivalent
amounts of total protein) onto gels.
6. Once the gels tanks are filled with the appropriate amount of running buffer,
run gels at a constant 25 mA through the stacking gel and 40 mA through the
separating gel. For CNBr gels, samples should be run until they reach the 11.0-cm
mark from the top of the 12.5% separating gel. For neutral salt/acetic acid/
pepsin gels, samples should be run and treated as described in Sects. 3.5.8–3.5.10
(see Note 14).
7. For neutral salt/acetic acid/pepsin gels: once samples have reached the 2.2-cm
mark from the top of the separating gel, the gel should be stopped and the run-
ning buffer from the top of the gel and sample wells removed.
8. Ten percent (v/v) β-mercaptoethanol (diluted in running buffer) should then be
added to the top of the sample wells of the separating gel for 90–120 min in a
fume cupboard. This step is completed to separate the type I collagen α(α1(I)]-
chains from the type III collagen α(α1(III)]-chains. Under non-reducing condi-
tions, these chains would normally migrate to the same position, making it
difficult to distinguish them apart; however under reducing conditions (with
β-mercaptoethanol), the type III collagen trimers/higher polymers are cleaved
and the released α1(III) monomers migrate slightly more slowly than the α1(I)
chains (see Note 15).
9. After 90–120 min the β-mercaptoethanol should be removed from the sample
wells and replaced with 1× running buffer, so that the samples can be run (at
40 mA) to the 8.0-cm mark from the top of the separating gel.
10. Once all gels have been completed, they should be removed from the gel appa-
ratus and stained overnight (in chilled Coomassie blue-R250; ∼200 mL/large
gel) at 4°C.
11. Gels can then be de-stained (at room temperature) until the background stain is
removed and the type I, III, and V collagen chains are visible (Fig. 16.3) and then
washed in distilled water (to stop the de-stain reaction) (see Notes 16, 17, and 18).
12. For quantitation of the interstitial collagen monomers, dimers, and trimers
(Fig. 16.3), the individual collagen chains can be analyzed by densitometry
(using a Biorad GS710 Calibrated Imaging Densitometer and appropriate
quantitative software; which allows for the quantitation of collagen bands from
wet gels). Alternatively, gels can be soaked in 3% (w/v) glycerol for 30–45 min
before being dried in gel-drying film (Promega, Madison, WI, USA) and then
quantitated by densitometry.

4 Notes

1. After whole/regional kidney tissues are lyophilized for measurement of dry weight and rehy-
drated, they may be de-fatted in a mixture containing 2:1 chloroform:methanol for 24 h at 4°C
before being re-hydrated for 24–48 h (at 4°C) and then hydrolyzed in 6 M HCl (see Sect. 3.1.4).
This step is more suited to organs (such as the skin) that have a high fat content.
16 Collagen Content, Concentration, and Sub-types in Kidney Tissue 233

β11
β12

α1(III)

α1(V)

α2(V)

α2(I)

α1(I)

Fig. 16.3 The pepsin-digested (mature cross-linked) interstitial collagens from the obstructed
kidney of a wild-type (C57B6J) mouse, 10 days after unilateral ureteric obstruction. Shown are
the type I collagen monomers, identified by the α1(I) and α2(I) subunits/chains; type III collagen
monomers, identified by the α1(III) chains; and type V collagen monomers, identified by the
α1(V) and α2(V) subunits/chains. Also shown are type I collagen dimers, β11: dimers of two
α1(I) subunits; β12: dimers of α1(I) and α2(I) monomers; and trimers (γ)

2. During the hydrolysis step with 6 M HCl, it should be ensured that the Kimax glass tubes fit
into the heating block (with no gaps between the external surface of the glass tube and the
heating block; as this will affect the rate of hydrolysis).
3. The amount of hydroxyproline in collagen may vary between species and organs studied and
should be checked before any calculations on collagen content and concentration are per-
formed (see refs. (6, 7) as a guide).
4. Since the measurement of collagen content and concentration is extrapolated from the absolute
hydroxyproline values of kidney tissues, other measures of collagen content and/or subtypes
(by Western blotting, immunostaining, or SDS-PAGE analysis of interstitial collagen chains)
are also recommended to confirm the findings of the hydroxyproline assay.
5. Ten to 20 µL of sample (hydrolyzate) is recommended for this assay as using larger amounts
(up to 100 µL) does not provide a linear outcome (author’s unpublished observations).
6. While optimal yields of hydroxyproline (color reaction) will be obtained when samples are
heated at 60°C for 25 min, these optimal yields will be lost if samples are heated for longer
periods.
7. Samples need to be cooled down to stop the color reaction.
234 C.S. Samuel

8. If whole kidney tissues are used for analysis, then either collagen content or collagen concen-
tration can be used as appropriate measures and comparisons between treatment groups.
However, if only regional kidney tissues are used, then only collagen concentration should be
used for comparisons between groups, as this measure corrects for the dry weight of the par-
ticular region of tissue used. It should be noted that collagen concentration is best calculated
from the dry weight tissue (which represents the extracellular matrix material, primarily col-
lagen); whereas deriving collagen concentration from the wet weight tissue is not as accurate,
due to the fact that the water content of tissues may vary between organs and diseased
conditions.
9. As with Note 1, whole/regional kidney tissues may be de-fatted in a 2:1 mixture of chloro-
form:methanol for 24 h at 4°C before being subjected to extraction with the neutral salt
buffer.
10. Kidney tissues can be diced finely/powdered with a mortar and pestle or tissue chopper.
11. The pepsin solution should always be prepared fresh and dissolved in 0.5 M acetic acid, 1 h
before use. If required, a second extraction of the mature insoluble collagen chains by pepsin
can be performed, before CNBr treatment of pepsin-digested pellets.
12. All extractions, particularly with pepsin, should be carried out with gentle agitation, as more
vigorous agitation may lead to the autolytic cleavage of the pepsin itself and hence, decreased
efficiency of the pepsin in digesting the mature insoluble collagens.
13. SDS-PAGE analysis of the interstitial collagen chains can be achieved with Biorad mini-gels,
but will result in poorer separation between the individual collagen -chains (see Fig. 16.3).
14. Due to the longer time required to run these larger SDS-PAGE gels, the gel running buffer
should be chilled at 4°C overnight before use and the gel tanks cooled in ice or connected to
a gel cooling system or the gels themselves run at 4°C to ensure a linear migration of all col-
lagen bands. Once gels are heated, the outer samples (that are closer to the electrodes) will
migrate at a slower rate compared with samples loaded in the middle of each gel.
15. Differences in the migration rate of the type III collagen a1(III) chains, under reducing condi-
tions with b-mercaptoethanol, may be observed between species, with greater differences,
between the a1(III) and a1(I) chains, being observed in the rat, mouse, calf, and rabbit than
in humans (11).
16. Templates for CNBr-cleaved peptides from type I, II, III, and V collagen can be found in refs.
(12–14).
17. As stated above, hydroxyproline assays can be used to quantitate the amounts of soluble and
insoluble (cross-linked) collagens from the neutral salt-, acetic acid-, pepsin-, and CNBr-
digested extracts/pellets, once they are lyophilized and re-suspended in 0.1 M HCl.
18. A limitation of this technique is that it cannot be used to identify collagen IV chains by peptide
digestion. Thus, other techniques such as immunohistochemistry or Western blotting may be
required for the detection of collagen IV.

Acknowledgements The author is supported by Career Development Fellowships from the

National Health & Medical Research Council (NHMRC) of Australia and the National Heart
Foundation of Australia (NHFA).

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