Full text of DOI:10.1111/j.1755-6686.2002.tb00208.x

A practical approach to the molecular biology
of kidney diseases:
From basic science to bed side
P. Garrido, E. Vélez1, A. Izquierdo, J. Matthews, Mª. A. Gutiérrez-Tarrés,
P. López Luna, J. Bover2 and R. J. Bosch
Laboratory of Renal Physiology and Experimental Nephrology,
Department of Physiology, Alcalá School of Medicine, University of Alcalá,
Alcalá de Henares
1 Department of Nephrology, Fundación Jiménez Díaz, Madrid, Spain
2 Department of Nephrology, Fundació Puigvert, Barcelona, Spain.
Summary
Nowadays, the term “Molecular Biology” (MB) is generally applied to the biochemical processes that involve genes and the
expression of proteins for which specific genes code. In recent years, astonishing advances have occurred in this field. Cur-
rently, there are many important powerful techniques allowing scientists to study the molecular mechanisms involved in
many human genetic diseases. Furthermore, it is important to underline that the possibilities are not limited to the diag-
nosis and study of these genetic diseases. Indeed, by studying gene expression, MB also allows the molecular study of
many acquired diseases such as viral hepatitis and cancer. Therefore, these major advances in the knowledge of gene biol-
ogy are facilitating the arrival of a new era of gene therapy.
This article will describe the most important techniques currently used in MB. Firstly, techniques involved in recombi-
nant DNA technology will be discussed and these will include the study of DNA and the possibility of identifying the
expression of abnormal genes, e.g. to identify individuals for paternity. Secondly, a description of techniques designed to
study the expression of genes and their regulation will follow and they involve the study of RNA. Thirdly, the impact of
genetic molecular studies as tools for medical diagnosis will be discussed and analysed. Finally, a discussion concerning
the rational basis for gene therapy and its future perspectives is included.
In this article, we have focused on technical or diagnostic aspects ofMolecular Biology. Although Ethics are also an
interesting issue to deal with, theseissues are far beyond the scope of this review.
Key words
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Molecular biology
DNA
RNA
Renal diseases
Genes and the methodical basis for their study
hereditary diseases. Many of the changes in nucleic acid
he gene is the unit of inheritance and it is made up of DNA sequences (mutations) do not imply the development of a dis-
(Deoxyribonucleic acid). Each gene is a nucleic acid ease, but they are instead the basis for human variability.
T
sequence that carries the information representing a specific
The term locus applies to unique positions within a gene
polypeptide (1). A gene is a stable entity, but errors may occur and, in addition, the localization of genetic markers along the
during DNA replication causing changes in the nucleic acid chromosome (2). On the other hand, an allele refers to one or
sequence. These changes are called mutations and the new form more alternative forms of genetic information within a locus,
of the gene will be inherited in a stable manner, just like the coding for the expression of a particular characteristic. Allelism
previous form. The organism carrying the altered gene is called is the consequence of the existence of two (homologous) copies
a mutant; the organism carrying the normal (unaltered) gene is of each inherited chromosome, one from each progeny (or par-
called wild type. From a clinical point of view, DNA analysis is ent) (1,2). The existence of multi-functional genes within a
focused towards the diagnosis of mutations responsible for population are known as polymorphisms.
A disease is considered autosomal dominant if it appears in
heterozygous subjects. In other words, the affected subject
Pedro Garrido is a Biologist. Currently he is
studying for his Ph.D. at the Laboratory of Renal
Physiology and Experimental Nephrology,
Department of Physiology, Alcalá School of
Medicine, University of Alcalá, Alcalá de
Henares, Spain.
inherited a normal copy of a gene from the healthy parent and
a mutated gene copy from the affected parent (3). Autosomal
recessive diseases appear when both parents present a mutated
gene copy. In most cases, the underlying strategy to study DNA
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is based on detecting a certain type of mutation with the best instead of EtBr before electrophoresis. This method relies on the
available technique.
detection of particles by autoradiography.
3. Nucleic acid hybridization
Recombinant DNA technology: History
This is the binding of specific DNA or RNA sequences with
great specificity along with their homologous or complemen-
DNA was isolated for the first time by Miescher in 1869; how- tary sequences (2,4). This technique allows the identification of
ever, it was not until 1944 when Avery demonstrated that the specific nucleic acid sequences and it is the basis of Southern
DNA molecules and not the proteins carried the genetic infor- (DNA) and Northern (RNA) blot techniques.
mation during bacterial transformation (1,2). In 1953, Watson
and Crick discovered the existence of DNA in a double-helix
4. Southern blot
form by using X-rays and this finding made it possible for Mar- This technique allows the transferral of DNA from an elec-
mur and Doty to discover the process of DNA renaturation (4). trophoresis gel to a nitrocellulose or nylon membrane (1). It
The renaturation process is generally described as hybridization allows the identification by hybridization of nucleic acid
when nucleic acids from different sources are involved, i.e. sequences using a radioactive-labelled probe. Therefore, the
when one preparation consists of DNA and the other consists of DNA probe detects nucleic acid sequences which are comple-
RNA.
mentary to part or all of the probes. Other related techniques
In 1966 Nirenber, Ochoa and Khorana elucidated the genet- are the Northern blot analysis, which detects RNA, and the
ic code and found that each trinucleotide sequence (codon) Western blot, used to detect proteins.
coded for an amino-acid. Other important advances were
reported by Sanger et al. as well as Maxam and Gilbert who
5. Polymerase Chain Reaction (PCR)
developed rapid DNA sequencing methods (1) and, in addition, The PCR allows amplification of specific DNA segments from
the method to amplify specific DNA sequences, known as Poly- any source, using DNA as a template, the four nucleotides from
merase Chain Reaction (PCR) (5). Nowadays, the introduction which DNA is made, the DNA polymerase enzyme (natural
of automatic DNA sequencing has made the process much eas- occuring enzymes that are responsible for DNA synthesis) and
ier and currently it is possible to isolate, sequence and analyse two oligonucleotide primers capable of recognizing the begin-
DNA quickly and effectively.
ning and the end of the DNA template in order to copy it (5,6).
The in-vitro reaction involves a variable number of repeated
cycles of denaturation, annealing and DNA extension. These
reactions are performed in a PCR thermocycler.
Methods and techniques to study DNA
1. Restriction Enzymes
6. Restriction Fragments Length Polymorphisms (RFLP)
Restriction endonucleases (RE) are enzymes that protect the This more recently developed method is based on the detection
bacteria whilst degrading the DNA carried into the cells by of those variations in DNA sequences that have a change with-
viruses (4). Each enzyme recognises a specific 4-8 base pairs in in their restriction targets (1). The restriction fragments ob-
the DNA and cleaves at its target sequence, thus creating restric- tained are different depending on the allele. This technique has
tion fragments (1). These fragments may then be used to create made it possible to analyse, detect and identify genes associat-
a restriction map which enables comparisons to be made ed with certain diseases and which have never been identified
between different regions of DNA without knowing their by using RFLP markers. It is an example of a genetic linkage
nucleotide sequences. These facts allow the use of restriction map and it enables analysis of large genomes.
maps to represent a linear sequence of the sites at which partic-
ular restriction enzymes find their targets (restriction targets).
7. DNA cloning and genetic engineering
Therefore, this means that different genomic areas can be com- This technique enables the amplification of individual DNA
pared without knowing the sequence of nucleotides that form it sequences. Cloning a DNA fragment allows the production of
(6), a very important fact determining relationships within the indefinite amounts from even a single original molecule (4,6).
human genome.
A clone is defined as a large number of cells or molecules all
identical to an original ancestral cell or molecule (1). Once any
particular segment of DNA has been cloned, its properties can
2. Gel electrophoresis
The negative charge of nucleic acids allows the study of DNA be characterized.
molecules by gel electrophoresis, based on separation according
Cloning technology involves the construction of novel DNA
to size. The most common gel used is agarose and it allows the molecules by joining sequences from different sources. The
different molecules to pass through, separating them along the product is often described as recombinant DNA, and the over-
way. The addition of ethidium bromide (EtBr) – an intercalating all techniques as genetic engineering.
agent – stains the gel and allows gel visualization under ultra-
violet light when it is bound to DNA (1). A more sensitive
method involves the incorporation of a radioisotope (e.g. 32P) The DNA sequence is virtually always looked on as the prima-
8. DNA sequencing
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ry objective once a gene has been cloned (6). This method dis- the viral load of the later can even be quantified by the so-called
closes the DNA nucleotide sequences so that it aids in the pre- quantitative PCR after renal transplantation. Slowly-growing
diction of the protein amino-acid sequence for which they code. mycobacterias such as Mycobacterium tuberculosis maybe
detected by PCR techniques much faster. Other bacteria, virus,
parasites can also be detected by MB techniques (Toxoplasma
Techniques used to study gene expression (RNA)
sp., Plasmodium sp., Bordetella pertussis and so on and so
forth).
1. Nucleic acid hybridization (RNA) northern blot
An important advantage of molecular analysis is that it does
As mentioned before, this is the same technique as Southern not require an immune response (production of antibodies) (8)
blot but it uses RNA instead of DNA. It demonstrates both qual- and thus, a considerable amount of time can be saved. In addi-
itatively and quantitatively the presence of RNA (gene expres- tion, molecular analysis may be carried out on almost any type
sion) coding for certain proteins (1).
of sample obtained from the patient e.g. blood, tissues, hair, etc.
2. cDNA synthesis. Reverse Transcription (RT)
B. Molecular biology applications using tissue biopsies
The use of the inverse (reverse) transcriptase retroviral enzyme The widespread use of tissue biopsies over the last few decades
allows scientists to carry out the synthesis of cDNA. The cDNA has allowed scientists to better describe both diseases and their
is DNA which has been made by using mRNA (messenger RNA) pathophysiologic pathways. It was not until the advent of PCR
as a template (4). As indicated by its own name, it is the reverse that it was possible to apply MB techniques to human biopsies,
process to transcription.
because it required a quantity of nucleic acids impossible to
obtain to run the previously available methods. In recent years,
many studies have been carried out involving MB studies on tis-
sue biopsies or blood samples in a wide range of human dis-
3. Reverse Transcription
and Polymeraese Chain Reaction (RT-PCR).
This is the most powerful, sensitive and versatile technique eases.
used at the present time to study gene expression both qualita-
tively and quantitatively. It can be used to detect either the pres-
C. Hereditary kidney diseases
ence or absence of a gene (6) and it differs from regular PCR A cyst is a cavity lined by epithelium and filled with liquid or
that a reverse transcription (RT) of a specific target from either semi-solid material (3). Renal cysts may develop from the
total RNA or mRNA is performed before any PCR amplification. glomerular capsule along the length of the renal tubule by her-
itable, developmental or acquired processes. A kidney may con-
4. RNAse Protection Assay (RPA).
tain a single or many cysts. The term “Polycystic kidney disease”
This technique involves RNA hybridization in solution (4). includes two genetically distinct outcomes (9), namely, autoso-
When the DNA forms a hybrid with its homologous chain, it mal dominant (ADPKD) and autosomal recessive polycystic
can not be degraded by the activity of the ribonucleases present kidney disease (ARPKD).
in the assay and therefore it becomes possible to study the qual-
itative and quantitative expression of certain genes.
Autosomal dominant polycystic kidney disease
(ADPKD)
Molecular basis of certain kidney diseases:
Diagnostic implications
ADPKD is a common inherited disorder transmitted by a dom-
inant gene and characterized by multiple cyst development and
From a clinical point of view, the initial objective of molecular growth in the kidney leading to end-stage renal failure around
studies is to be able to diagnose the genetic implications of dis- the fifth-sixth decade of life (3). It affects 1 of every 1000 newly
eases in order to give appropiate counselling and treatment to born children. It also affects other organs such as the liver,
the patient. When the genetic anomaly is totally identified, it brain, heart valves, etc. demonstrating the systemic nature of
may be studied using DNA analysis and biochemical methods the disease. As any autosomal dominant disease, an individual
(7). Following this sort of analysis, PCR may be used if short with ADPKD must have the defective gene on one of a pair of
repetition markers are present, otherwise RFLP is the preferred autosomal chromosomes. The PKD-1 gene, that is responsible
method of identification.
for the 85% of cases in adults, has recently been identified and
it was found to be nearer to the 1-alfa-globin gene and to one
of the tuberous sclerosis genes on chromosome 16 p. This
A. Infectious illnesses
Almost all organisms contain DNA or RNA. Therefore, they all PKD-1 gene encodes for a protein known as polycystine 1. The
can be theoretically detected by PCR or RT-PCR, respectively, as absence of this protein affects cell-cell and cell-matrix interac-
far as their sequences are known. These methods are very tion and facilitates cyst formation. The PKD-2 gene (the second
expensive and only several of them have gained wide clinical gene implicated in the disease) is located on chromosome 4,
use. Thus, MB techniques are routinely used for the detection and it encodes for a protein (polycystine 2) which is involved in
of several viruses such as HCV, HBV, HIV and CMV. For instance voltage dependent calcium channels. Both genes also differ in
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clinical grounds, and although heterogeneicity and even intrafa- transfer genes to cell lines in culture. Basically, cells are incu-
miliar variability is common in ADPKD, end-stage renal disease bated with DNA of the gene and then this DNA can be incor-
is reached later in patients affected by the PKD-2 mutation.
porated to the cellular genome using calcium phosphate or
electricity, which increases the cellular membrane permeability
to DNA (4). Using these techniques it is possible to obtain the
expression of the transferred gene (10).
The biological gene transfer methods use viruses, mainly
adenovirus and retroviruses. Retroviruses are RNA virus which
Autosomal recessive polycystic kidney disease
(ARPKD)
This rare, recessive type of inherited polycystic kidney disease possess the enzyme inverse transcriptase. This enzyme is also
is usually discovered in infants; however, young adults may used to carry out the in vitro synthesis of cDNA (1,7). The
develop the disorder. Thus, it is known as ‘infantile’ polycystic retrovirus penetrates into the cells through a membrane recep-
kidney disease in its classical form ARPKD is a disorder whose tor. Initially, the RNA virus is transcribed into cDNA, which
genetic defect has been localizated in chromosome 6 (3), in turn, enters the nucleus and is integrated in the cellular
although the protein for which encodes is still unknown and genome, a process directed by the viral enzyme integrase. The
current diagnosis is mainly undertaken by ultrasonography.
viral genome (provirus) is inherited in a Mendellian fashion and
passes on into the daughter cells. As the provirus codes for its
promoter (region of the gene that regulates its transcription),
Hereditary nephritis
The Alport syndrome (one of the most common hereditary the infected cells express the viral gene and synthesize viral par-
nephritis) is a progressive glomerulopathy frequently associated ticles indefinitely, this being the innoculous cycle for the infect-
with loss of auditory capacity and crystalline alterations (3,8). ed cell (11).
The primary change in almost all cases resides in the non-colla-
In recent years, successful techniques have been developed
gen domains (NC1) of type-IV collagen, which is usually locat- and they have been used in conjunction with a range of differ-
ed in the X chromosome. Using Southern blot techniques and ent experimental approaches; one of them involves gene trans-
DNA probes, it is possible to identify several mutations within fer in kidney tissue. Currently, it is possible to transfer gene
the syndrome. Nowadays, some probes detecting a single muta- markers. Cytokine genes have already been transferred in
tion have been developed; however, no routine methods for experimental animals as well. Therefore, this area gathers an
molecular diagnosis are yet available.
increasing scientific interest and it is currently an area of con-
tinuous expansion.
X-linked Nephrogenic Diabetes Insipidus (NDI).
It is also important to point out that gene therapy not only
From a clinical point of view, the most frequent form NDI is includes single techniques of gene incorporation but also tech-
acquired. Hereditary NDI is a much less frequent disease trans- niques suppressing the expression of potentially pathogenic
mitted by X chromosome, and it is caused by a mutation in the genes. To date, two techniques have been developed allowing
V2 receptor gene (3). Recently, a second hereditary recessive the suppression of gene expression (1,3). Firstly, the so-called
NDI form has been identified. This mutation is located in a kid- ‘antisense’ technique, where complementary sequences are
ney cellular water channel known as aquaporine-2, and it employed in an opposite direction to that of the nucleic acids
obstructs the normal water traffic through the tubular cells present. This enables hybridization with homologous
(3,9). Currently, it is possible to prenatally diagnose women to sequences within the cellular genome, thus obstructing genetic
detect carriers of the disease in order to administer treatment.
expression. Secondly, enzymes that can degrade specific RNA
molecules (called ribozymes) are used. These enzymes facilitate
the suppression of the gene product for which they code.
Pathologies which can benefit from techniques which sup-
Wilms tumor (Nephroblastoma)
This disease entails an embrionic renal tumor that originates by
abnormal differentation of mesenquimal cells (3) and it is com- press gene expression include several forms of glomeru-
mon in childhood. It occurs either in the absence or the pres- lonephritis. It has recently been proven that cytokines act as
ence of an abnormal tumor supressor gene.
mediators of lesions in some experimental forms of glomeru-
lonephritis (3). Border and Cabbage demonstrated that by
administering antibodies against the transforming growth factor
beta (TGF-b), it was possible to suppress the effects of the
platelet growth factor as well as interleukin-1 in other experi-
Molecular biology and therapy: Perspectives
In the last decade, many techniques have been developed with- mental models of glomerulonephritis (3).
in genetic engineering allowing the transfer and incorporation
Finally, it may be possible to apply these techniques to
of genes into cells, tissues, organs and even to all the cells of an oncology in the very near future. Immunotherapy is one exam-
organism (transgenic animals). Furthermore, genetic engineer- ple. The transfer of the interleukin-2 gene to tumour cells in
ing has enabled the suppression of gene expression by inhibit- rats has already been accomplished, and it has demonstrated
ing the synthesis of the protein for which the gene codes.
that this genetic transfer effectively allows the recognition of the
It is possible to find physical and biological methods for tumour cells and ultimately their destruction by the rat’s own
gene transfer (3). Physical gene transfer methods are used to immunological system. The National Institutes of Health in the
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United States has stated that it currently has approximately
Acknowledgements
60 projects involving research into human genetic therapy, This work was partially supported by grants from the Programa
highlighting oncology among them and including Hodgkin’s Sectorial de Promoción General del Conocimiento from the
disease, leukaemia, ovarian cancer, and melanoma. Other Spanish Ministerio de Educación y Cultura (PB 98-0700-C02-
research areas include cystic fibrosis, familial hypercholes- 01), the Consejería de Educación de la Comunidad de Madrid
terolemia, bone marrow transplants and immunodeficiency (08.6/0038./2000) and the Spanish Society of Nephrology.
syndromes, to name but a few.
In summary, the current use of MB techniques has a major
impact on the administration of treatment and it has also the
ability of preventing many of the worldwide fatal diseases
before they can affect individuals within the population.
Received: October 2001
Reviewed: November 2001
Revised: January 2002
Accepted: February 2002
Address for correspondence
Esperanza Vélez
Department of Nephrology
Fundación Jiménez Díaz
Avda. Reyes Católicos, 2
28040 Madrid
Spain
email: evelez@mi.madritel.es
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