Nephrotoxicity associated with exposure to chemicals, principles and methods for the assessment of (EHC 119, 1991)

Nephrotoxicity associated with exposure to chemicals, principles and methods for the assessment of (EHC 119, 1991)

INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

ENVIRONMENTAL HEALTH CRITERIA 119

PRINCIPLES AMD METHODS FOR THE ASSESSMENT OF NEPHROTOXICITY
ASSOCIATED WITH EXPOSURE TO CHEMICALS

This report contains the collective views of an international group of
experts and does not necessarily represent the decisions or the stated
policy of the United Nations Environment Programme, the International
Labour Organisation, or the World Health Organization.

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acting on behalf of the Commission is responsible for the use which
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(EUR 13222 EN)

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the United Nations Environment Programme,
the International Labour Organisation,
and the World Health Organization, and on
behalf of the Commission of the
European Communities

World Health Orgnization
Geneva, 1991

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WHO Library Cataloguing in Publication Data

Principles and methods for the assessment of nephrotoxicity
associated with exposure to chemicals.

(Environmental health criteria: 119) (EUR ; 13222)

1. Kidney diseases - chemically induced
2. Kidney neoplasms - chemically induced
3. Kidney - drug effects I. Series II. Series EUR; 13222

ISBN 92 4 157119 5 (NLM Classification WJ 300)
ISSN 0250-863X

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CONTENTS

PRINCIPLES AND METHODS FOR THE ASSESSMENT OF NEPHROTOXICITY
ASSOCIATED WITH EXPOSURE TO CHEMICALS

1. SCOPE OF THE HEALTH SIGNIFICANCE OF NEPHROTOXICITY

2. NEPHROTOXICITY

2.1. Target selectivity
2.2. The dynamics of renal injury
2.3. Classification of renal disease
2.4. The epidemiology of nephrotoxicity
2.5. Risk factors for toxic nephropathies
2.5.1. Factors related to renal function
2.5.2. Clinical risk factors
2.5.3. Extrapolation of animal data to man
2.5.4. Risk assessment from nephrotoxicity
studies in animals
2.5.5. Special risk groups in humans
2.5.6. Multichemical exposure
2.5.7. Renal functional reserve
2.5.8. The effects of chemicals on kidneys
with pre-existing renal lesions
2.5.8.1 Nephrotoxicity in the presence
of renal and extrarenal disease

3. KIDNEY STRUCTURE AND FUNCTION

3.1. Renal anatomy
3.1.1. Histology
3.1.2. Enzyme histochemistry and quantification
3.1.3. Immunohistochemistry
3.2. The renal blood supply
3.2.1. Renal haemodynamics
3.3. The nephron
3.3.1. Cellular heterogeneity and cell-cell
interaction
3.3.2. The glomerulus
3.3.3. The proximal tubule
3.3.4. The medulla 58
3.3.4.1 The loops of Henle
3.3.4.2 Collecting ducts
3.3.4.3 The distal tubule
3.3.4.4 The countercurrent multiplier
system and urine concentration
3.3.4.5 The interstitial cells
3.4. Species, strain, and sex differences in renal
structure and function
3.5. Renal biochemistry

3.5.1. Biochemistry and metabolism in the cortex
3.5.2. Biochemistry and metabolism in the medulla
3.5.2.1 The biochemistry of renal
prostaglandins (PG)
3.5.2.2 Lipid metabolism
3.5.2.3 Carbohydrate metabolism in the
medulla
3.5.2.4 Medullary glycosaminoglycan (GAG)
3.6. The metabolism of xenobiotic molecules in the kidney
3.6.1. Oxidases
3.6.1.1 Cytochrome P-450-dependent mixed-
function oxidases (monooxygenases)
3.6.1.2 Prostaglandin peroxidase-
mediated metabolic activation
3.6.2. Conjugation
3.6.2.1 Glucuronide conjugation
3.6.2.2 Sulfate conjugation
3.6.2.3 Glutathione conjugation
3.6.2.4 Mercapturic acid synthesis
3.6.2.5 Amino acid conjugation
3.6.3. Other enzymes involved in xenobiotic
metabolism

4. THE MECHANISTIC BASIS OF CHEMICALLY INDUCED RENAL INJURY

4.1. Immunologically induced glomerular disease
4.2. Direct glomerular toxicity
4.3. Tubulointerstitial disease
4.3.1. Acute interstitial nephritis
4.3.2. Acute tubular toxicity
4.3.3. Chronic interstitial nephritis
4.4. Mechanisms of cellular toxicity
4.5. Factors that modify cellular injury by toxins
4.5.1. Cellular transport and accumulation
4.5.2. Metabolic degradation
4.5.3. Intracellular protein binding
4.5.4. Membrane reactions and pinocytosis

5. THERAPEUTIC AGENTS AND CHEMICALS THAT HAVE THE POTENTIAL TO
CAUSE NEPHROTOXICITY

5.1. Therapeutic agents
5.1.1. Analgesics and non-steroidal
anti-inflammatory drugs (NSAIDs)
5.1.2. Paracetamol and para-aminophenol
5.1.3. Antibiotics
5.1.3.1 Aminoglycosides
5.1.3.2 Cephalosporins
5.1.3.3 Amphotericin B
5.1.3.4 Tetracyclines

5.1.4. Penicillamine
5.1.5. Lithium
5.1.6. Urographic contrast media (UCM)
5.1.7. Anticancer drugs
5.1.7.1 Cisplatin
5.1.7.2 Adriamycin
5.1.8. Immunosuppressive agents
5.1.8.1 Cyclosporin A
5.1.9. Heroin
5.1.10. Puromycin aminonucleoside
5.2. Chemicals
5.2.1. Ethylene glycol
5.2.2. Organic chemicals and solvents
5.2.2.1 Volatile hydrocarbons
5.2.2.2 Chloroform
5.2.2.3 Halogenated alkenes
5.2.2.4 Hydrocarbon-induced
nephrotoxicity
5.2.2.5 Bipyridyl herbicides
5.3. Mycotoxins
5.4. Silicon
5.5. Metals
5.5.1. Lead
5.5.2. Cadmium
5.5.3. Mercury
5.5.4. Gold
5.5.5. Bismuth
5.5.6. Uranium
5.5.7. Chromium
5.5.8. Arsenic
5.5.9. Germanium

6. RENAL CANCER

6.1. Renal tumour classification
6.2. Renal adenocarcinoma
6.3. Upper urothelial carcinoma (transitional
cell carcinoma)
6.4. Experimentally induced renal adenomas and
adenocarcinomas
6.4.1. Background incidence of spontaneous
tumours in experimental animals
6.4.2. Inorganic compounds
6.4.3. Organic molecules
6.4.3.1 Nitrosamines and related
compounds
6.4.3.2 Morphological changes
6.4.3.3 Biochemical changes in cells
6.4.3.4 The mechanistic basis of renal
carcinoma
6.5. Experimentally induced upper urothelial
carcinomas (transitional cell carcinomas)

7. ASSESSMENT OF NEPHROTOXICITY

7.1. In vitro studies
7.1.1. Choice of chemical concentrations
for in vitro studies
7.1.1.1 Proximate and ultimate
nephrotoxicants in vitro
7.1.2. In vitro investigations of
nephrotoxicity
7.1.2.1 Perfusion and micropuncture
7.1.2.2 Renal cortical slice
7.1.2.3 Isolated nephron segments
7.1.2.4 Primary cell cultures
7.1.2.5 Established renal cell lines
7.1.2.6 Subcellular fractions
7.2. In vivo experimental studies
7.2.1. Methods for assessing chemically reactive
nephrotoxic metabolites in animals
7.2.2. Evaluation of glomerular function
7.2.3. Evaluation of tubular functions
7.2.4. Proteinuria
7.2.4.1 Total proteinuria and
electrophoretic pattern
7.2.4.2 Urinary excretion of single
plasma proteins
7.2.4.3 Enzymuria
7.2.4.4 Immunoreactive tissue
constituents
7.2.4.5 Urinary excretion of
prostaglandins
7.2.5. Clinical context
7.2.6. Radiological techniques
7.2.7. Other non-invasive renal assessment

8. DETECTION OF NEPHROTOXICITY IN HUMANS

8.1. Markers of nephrotoxicity
8.1.1. General requirements
8.1.2. Diagnostic value
8.1.3. Prognostic value
8.2. Screening for nephrotoxicity in humans
8.2.1. Glomerular filtration
8.2.2. Tests designed to assess selective
dysfunction
8.2.3. Tests designed to assess tissue damage
8.2.3.1 Enzymuria
8.2.3.2 Immunoreactive tissue
constituents
8.3. Clinical investigations
8.3.1. Invasive techniques
8.3.1.1 Biopsies from humans
8.3.1.2 Autopsy in humans

8.3.2. Tests designed to assess glomerular
filtration and renal blood flow
8.3.3. Proteinuria
8.3.4. Tests designed to assess selective
damage

9. SUMMARY AND CONCLUSIONS

10. RECOMMENDATIONS

REFERENCES

RESUME ET CONCLUSIONS

RECOMMANDATIONS

RESUMEN Y CONCLUSIONES

RECOMENDACIONES

WHO/CEC TASK GROUP ON PRINCIPLES AND METHODS FOR THE ASSESSMENT OF
NEPHROTOXICITY ASSOCIATED WITH EXPOSURE TO CHEMICALS

Members

Professor E.A. Bababunmi, Biomembrane Research Laboratories,
Department of Biochemistry, University of Ibadan, Ibadan, Nigeria
(Vice-Chairman)

Dr P. Bach, Nephrotoxicity Research Group, Robens Institute of Health
and Safety, University of Surrey, Guildford, Surrey, United Kingdom

Professor G. Baverel, Department of Pharmacology, Alexis Carrel
Faculty of Medicine, Lyon, France

Professor W.O. Berndt, University of Nebraska Medical Centre, Omaha,
Nebraska, USA (Chairman)

Dr G. Duggin, Toxicology Unit, Royal Prince Alfred Hospital,
Camperdown, New South Wales, Australia

Dr H. Endou, Department of Pharmacology, Faculty of Medicine,
University of Tokyo, Bunkyo-ku, Tokyo, Japan

Professor R. Goyer, Department of Pharmacology, University of Western
Ontario, Health Science Centre, London, Ontario, Canada

Dr M. Robbins, Tissue Radiobiology Research Unit, Churchill Hospital,
Headington, Oxford, United Kingdom (Rapporteur)

Observer

Dr C. Cojocel, European Chemical Industries Ecology and Toxicology
Centre, Brussels, Belgium

Secretariat

Dr J.C. Berger, Health and Safety Directorate, Commission of the
European Communities (CEC), Luxembourg

Dr E. Smith, International Programme on Chemical Safety, Division of
Environmental Health, World Health Organization, Geneva,
Switzerland

Consultants representing the CEC

Dr A. Bernard, Unit of Industrial Toxicology and Occupational
Medicine, Catholic University of Louvain, Brussels, Belgium^a

Secretariat (contd.)

Dr P. Druet, National Institute of Health and Medical Research
(INSERM), Broussais Hospital, Paris, France^a

Professor A. Mutti, Institute of Clinical Medicine and Nephrology,
University of Parma, Parma, Italy^a

^a Attended 6 December 1989 only.

NOTE TO READERS OF THE CRITERIA DOCUMENTS

Every effort has been made to present information in the criteria
documents as accurately as possible without unduly delaying their
publication. In the interest of all users of the environmental health
criteria documents, readers are kindly requested to communicate any
errors that may have occurred to the Manager of the International
Programme on Chemical Safety, World Health Organization, Geneva,
Switzerland, in order that they may be included in corrigenda, which
will appear in subsequent volumes.

* * *

A detailed data profile and a legal file can be obtained from the
International Register of Potentially Toxic Chemicals, Palais des
Nations, 1211 Geneva 10, Switzerland (Telephone No. 7988400 or
7985850).

PREFACE

The preparation of this monograph was undertaken jointly by the
International Programme on Chemical Safety (UNEP/ILO/WHO) and the
Commission of the European Communities.

A joint WHO/CEC Task Group on Principles and Methods for the
Assessment of Nephrotoxicity Associated with Exposure to Chemicals met
at the National Institute of Public Health and Environmental
Protection, Bilthoven, the Netherlands, from 4 to 8 December 1989. The
meeting was opened by Dr K.A. van der Heijden on behalf of the
Netherlands and the host institute. The Secretariat responded and
welcomed the participants on behalf of the three cooperating
organizations of the IPCS (UNEP/ILO/WHO) and the Commission of the
European Communities. The Task Group reviewed and revised the draft
criteria document and prepared a final text.

The drafts of this Monograph were prepared by DR P. BACH,
Guildford, United Kingdom, PROFESSOR W.O. BERNDT, Omaha, USA and
PROFESSOR R. GOYER, London, Ontario, Canada. During the preparation of
the monograph, many scientists made constructive suggestions and their
contributions are gratefully acknowledged. The photographs in Figure
2 were supplied by Dr N.J. Gregg, in Figure 4 by Professor W. Guder,
and in Figure 14 by the Department of Toxicology, Institute for
Medical Research and Occupational Health, University of Zagreb.
Following the Task Group, Dr P. Bach and Dr M. Robbins collated the
text for IPCS. Dr E. Smith and Dr P.G. Jenkins, both of the Central
Unit, IPCS, were responsible for the overall scientific content and
technical editing, respectively.

ABBREVIATIONS

ADH anti-diuretic hormone

AIN acute interstitial nephritis

ARF acute renal failure

BEA 2-bromoethalamine

BEN Balkan endemic nephropathy

BUN blood urea nitrogen

CIN chronic interstitial nephritis

DBCP 1,2-dibromo-3-chloropropane

DCVC S-(1,2-dichlorovinyl)-L-cysteine

DTPA diethylenetriamine pentaacetic acid

EDTA ethylenediaminetetraacetic acid

ESRD end-stage renal disease

GAG glycosaminoglycan

GBM glomerular basement membrane

GFR glomerular filtration rate

GSH glutathione-SH

H & E haematoxolin and eosin

HCBD hexachloro-1,3-butadiene

HPLC high-performance liquid chromatography

LDH lactate dehydrogenase

3MC 3-methylcholanthrene

NAC N-acetyl cysteine

NADPH reduced nicotinamide adenine dinucleotide phosphate

NNM N-nitrosomorpholine

NSAID non-steroidal anti-inflammatory drugs

PAH p-aminohippurate

PAS periodic acid Schiff stain

PCBD S-(1,2,3,4,4-pentachloro-1,3-butadienyl

PG prostaglandin

PoG proteoglycan

RPN renal papillary necrosis

TEA tetraethyl ammonium

UCM urographic contrast medium

UDP uridine diphosphate

1. SCOPE OF THE HEALTH SIGNIFICANCE OF NEPHROTOXICITY

Over the last 20 years it has become increasingly obvious that
the kidney is adversely affected by an array of chemicals. Man is
exposed to these as medicines, industrial and environmental chemicals,
and a variety of naturally occurring substances. The level of exposure
varies from minute quantities to very high doses. Exposure may be over
a long period of time or limited to a single event, and it may be due
to a single substance or to multiple chemicals. The circumstances of
exposure may be inadvertent, accidental, or intentional overdose or
therapeutic necessity. Some chemicals cause an acute injury and others
produce chronic renal changes that may lead to end-stage renal failure
and renal malignancies. The extent and cost of clinically relevant
nephrotoxicity has only started to become apparent during the last
decade. However, the full extent of the economic impact of chemically
induced or associated nephropathy is difficult to define because the
diagnosis of early injury and the documentation of the cascade of
secondary degenerative changes have not been adequately identified.
Instead most chemically associated renal disease is only identified as
an acute renal failure or as chronic renal failure at a very late
stage when therapeutic intervention is impossible. More importantly at
this stage, the etiology may be obscured by lack of reliable
information on the likely causative agents, the levels and duration of
exposure, and other possible contributing and exacerbating factors. At
present, epidemiological evidence indicates that nephrotoxicity
leading to acute and/or chronic renal failure represents a substantial
financial burden to society (Nuyts et al., 1989). Indeed, there is
some indication that chemical exposure could play a much greater
influence in the very high incidence of end-stage renal disease
encountered in nephrology and dialysis clinics than is currently
considered to be the case.

There are already several examples of this type of chemically
associated disease that went unrecognized for some time. These include
those nephropathies caused by cadmium, other environmental heavy
metals, and, more recently, the organo-metallic compounds used as
therapeutic agents, anti-cancer drugs, cyclosporin, analgesic abuse,
and antibiotics.

Owing to its diverse functions and small mass in relation to the
resting cardiac output that it handles, the kidney is a target both
for chemicals that are pharmacologically active and for toxic
material. The nephron and its related cells perform a diversity of
physiological functions. It is the major organ of excretion and
homeostasis for water-soluble molecules; because it is a metabolically
active organ, it can concentrate certain substances actively. In
addition, its cells have the potential to bioconvert chemicals and
metabolically activate a variety of compounds. There are a number of
other processes described below that establish the potential for
cellular injury. Specific physiological characteristics are localized
to specific cell types. This makes them susceptible to, and the target
for, chemicals. The effect of any chemical on a cell may be

pharmacological, in which case the effect is dose related and occurs
only as long as the concentration of the effector is high enough to be
active. Alternatively, the chemical may cause damage to the cell. The
cell responds to injury by repair and the kidney responds to cellular
lesion by renal and extrarenal adaptation to compensate for loss of
that cell function. Although there is a substantial capacity within
the kidney for repair, there are also several circumstances where
damage may be irreversible. In general, the proximal and distal
tubules and urothelia can be repaired, but the glomeruli and medulla
may have a significantly lower repair facility. It is, therefore,
possible to initiate a series of degenerative changes as a result of
interfering with one or more of the normal physiological processes.

The Environmental Health Criteria monographs normally focus on
industrial chemicals, but at present most of the experimental and
human information on nephrotoxicity is based on therapeutic
substances. These data are most useful because there are animal and
human comparisons for specific chemicals where the levels of exposure
and the nephrotoxicological consequences are well documented. From
these data it has been possible to glean some understanding of
mechanisms of primary injury and the long-term consequences and health
significance. Thus, these compounds are generally well studied, and
the more rational understanding of the mechanism of their
nephrotoxicity in animals and man provides the basis for validating
extrapolation between species and making rational risk assessment.

Most risk assessment decisions are currently based on information
concerning the aminoglycosides, halogenated anaesthetics, several
heavy metals, and lithium, where there is an excellent concordance
between animal data and findings in humans exposed to these agents
(Kluwe et al., 1984; Porter & Bennett, 1989). This has provided some
predictive indication of what will take place in humans exposed to
analogues of these compounds. On the other hand, the demonstration
that the occurrence of light hydrocarbon-related adenocarcinomas is
specific to male rats shows that there are examples where the
molecular understanding of a renal lesion in animals is irrelevant to
humans.

There are also therapeutic agents where attempts to extrapolate
from animals to man have not been as successful. These include
compounds such as cyclosporin, analgesics and non-steroidal
anti-inflammatory agents. It has, however, been possible to develop
some model lesions that parallel those in humans using these
compounds. Generally, different protocols have had to be used, such as
water deprivation and renal injury, but these have in turn provided
the basis for developing improved screening methods for such chemicals
and also for probing the molecular nature of the lesion. There are,
however, a number of chemicals, such as renal carcinogens, mycotoxins,
other natural toxins, and anti-cancer drugs, and some types of lesion,
such as the immunonephropathies, where it has been difficult to
establish good models in animals. A host of chemicals alter glomerular
filtration rate (GFR) or some other aspect of renal function, but the

long-term health significance is still not known and it is uncertain
how to extrapolate such data to man.

2. NEPHROTOXICITY

Nephrotoxicity can be defined as renal disease or dysfunction
that arises as a direct or indirect result of exposure to medicines,
and industrial or environmental chemicals. It is well established that
toxic nephropathies are not restricted to a single type of renal
injury. Some chemicals target one discrete anatomical region of the
kidney and may affect only one cell type. Chemical insult to the
kidney may result in a spectrum of nephropathies that are
indistinguishable from those that do not have a chemical etiology.

2.1 Target selectivity

It has become increasingly apparent that there are a number of
chemicals that may adversely affect one or more of the anatomical
elements of the kidney, such as the glomerulus, proximal,
intermediate, and distal tubules, and medullary, endothelial, and
urothelial cells. Although some of these cell types (such as the
proximal tubular cells) have a marked ability to repair damaged
regions, others, such as the glomerular epithelium and the "type 1"
medullary interstitial cells, do not. It is for this reason that the
dynamic process that follows any renal injury can affect the outcome
of the chemical insult.

2.2 The dynamics of renal injury

The renal response to injury is dynamic, and the kidney adapts to
maintain homeostasis during the cascade of repair and recovery that
follows the primary insult (Bach, 1989). Depending on the type and
frequency of the damage, and the region of the kidney that is damaged,
the organ can respond by a recovery, a reduced functional reserve, or
by a progressive degenerative change. A reduced functional reserve may
play a very important role in sensitizing the kidney to subsequent
renal injury, and an initiated degenerative cascade may either
stabilize or progress to acute or chronic renal failure. It is not
possible to differentiate between a kidney that has totally recovered,
one with a reduced functional reserve, and an organ with early
progressive degenerative change, except in animals where function and
morphology can be assessed under well controlled conditions.

2.3 Classification of renal disease

Classification of renal disease can be based on clinical
manifestations, pathological changes, or etiological agents. WHO has
prepared a number of detailed and illustrated publications in recent
years on the classification of renal disease (WHO 1982, l985, 1987,
1988). The general approach is to subdivide the kidney into major
anatomical components (i.e. glomeruli, tubules and interstitium, and
blood vessels) and to relate these to the major clinical syndromes

characteristic of renal diseases. Table 1 contains a modified
classification of renal disease that focuses on major disorders of the
kidney that may be associated with nephrotoxins. This classification
is consistent with previous WHO publications and textbooks of
nephrology and pathology and provides a framework for discussing the
mechanisms and pathology of nephrotoxicity. It must be appreciated
that nephrotoxic agents may have multiple anatomical targets and that
toxicity manifests itself in more than one clinical syndrome. Further
discussion of renal effects due to specific agents are discussed
below.

2.4 The epidemiology of nephrotoxicity

Putting the health significance of nephrotoxicity into
perspective is difficult because of the diverse array of chemicals
that target different parts of the kidney, the spectrum of disease
consequences, and the many interacting factors. There is also
uncertainty in assessing changes in renal function before they reach
the point where preventive medicine can no longer be practised and
therapeutic intervention may be appropriate.

Many industrial and environmental chemicals and therapeutic
agents have been shown in experimental studies and from acute toxic
exposures to be nephrotoxins, but the extent of their contributions to
the overall incidence of chronic renal failure is not known. Data
extracted from the European Dialysis and Transplant Association
Registry identified only about 4% of patients starting renal
replacement therapy in 1984 as having drug or chemical associated
renal disease. However, nearly 50% of these patients were considered
possible (but not diagnosed) cases of toxic nephropathy (Dieperink,
1989). Of those patients identified as having chemical-related renal
disease, analgesic nephropathy is the most important recognized
outcome. In an analysis of the European Dialysis and Transplant
Association Registry (1986), the prevalence of analgesic nephropathy
was found to vary greatly between countries. It is highest in
Switzerland (18.1%) and Belgium (11.8%) and accounts for over 4% of
patients in Denmark, Germany, Czechoslovakia, and Austria. In 20
countries the prevalence is lower than one patient in every hundred
(European Dialysis and Transport Association Registry, 1986; Wing et
al., 1989). Other specific drug nephropathies recorded less frequently
include those due to cisplatin ( cis-platinum) and cyclosporin A. A
small number of patients had other specific drug or chemical-related
nephropathies.

The role of the toxic agents that may contribute to the 50% of
cases of chronic renal failure of undiagnosed etiology is less
certain. There is opportunity for exposure to a number of chemicals in
the workplace or ambient environment (drugs included) that are
possible nephrotoxins. It has been estimated that there were nearly
four million workers in the USA with potential occupational exposure
to known or suspected nephrotoxins in the 1970s (Landrigan et al.,
1984). The major occupational exposure is to workplace solvents, but

Table 1. Classification of renal disease due to nephrotoxins in humans

1. Immunologically mediated

Antibody mediated

Membranous glomerulonephritis or immune complex type disease

metals (gold, mercury)
D-penicillamine
drugs responsible for a lupus-like syndrome
(hydralazine, procainamide, diphenylhydantoin)

Anti-glomerular basement membrane antibody mediated

organic solvents
hydrocarbons

2. T cell mediated (?)

Nephrotic syndrome with minimal glomerular changes

lithium salts
non-steroidal anti-inflammatory agents

toxic metals and organic compounds, including pesticides, are of great
concern. The well documented occurrence of subclinical nephropathies
in subjects occupationally exposed to nephrotoxins such as lead or
cadmium (see section 5.5), the excess of mortality for renal diseases
in cohorts of workers with previous exposure to these two heavy metals
(Bennett, 1985; Bernard & Lauwerys, 1986), and, more recently, the
suggestion that subclinical renal effects caused by cadmium are early
signs of an accelerated and irreversible decline of renal function
(Roels et al., 1989) should all be noted. The linkage of these risks
to the actual occurrence of chronic renal failure has not, however,
been possible. A review of the end-stage renal disease (ESRD)
population in the USA has shown that at least 19% have a renal disease
of unknown or non-specific etiology (Burton & Hirschman, 1979). If
other diagnostic groups that have uncertain etiologies, such as the
30% with glomerulo-nephritis, 5% with interstitial nephritis of
suspected etiology (lead, analgesics, etc.) and possibly the 8%
diagnosed as having pyelonephritis, are added, it becomes apparent
that the etiology of a large portion of patients with chronic renal
failure is unrecognized or undefined. Environmental factors may have
a previously unrecognized role in the etiology of these lesions.

There are many reasons for the failure to recognize toxic
etiologies (Sandler, 1987). A major reason is that chronic renal
failure develops slowly over a number of years, so that retrospective

identification of a toxic agent requires knowledge of lifestyles,
therapies, or workplace environments that might provide risk factors.
However, such data are generally not available. Unless the drug or
toxic chemical is persistent in tissues, it is usually not possible to
confirm or quantify exposure. The inability to recognize multiple
etiologies or confounding factors adds further complexity to the
problem. There is also lack of uniformity in clinical and pathological
diagnoses. A further complexity is that there is a tendency to
categorize chronic renal failure by a mixture of pathological and
etiological classifications. For example, in one instance a patient
may be classified as having chronic interstitial nephropathy on the
basis of a renal biopsy, whereas another patient with the same
pathology might be classified as having toxic nephropathy due to
exposure to a nephrotoxic chemical because of knowledge of exposure.
In a survey of patients requiring dialysis in Israel, Modan et al.
(1975) found diagnostic inconsistencies between hospital diagnosis,
autopsy reports, and diagnosis made by the study reviewers.
Disagreement was most often seen for chronic glomerulo-nephritis,
chronic pyelonephritis and nephrosclerosis. The stage of the
pathological process or severity influences classification.
Interstitial nephritis tends to be diagnosed more frequently in the
early stages of chronic renal failure, whereas glomerulonephritis is
a more common diagnosis for patients undergoing dialysis. There may
be a rational basis for this in that the scarring in persistent
interstitial nephritis does impede blood supply to the glomerulus.
This could lead to glomerular disease and interstitial nephritis
despite the fact that there are different etiologies or risk factors
for the two conditions.

The identification of chronic pyelonephritis is made more precise
by following established criteria. These include the presence of gross
irregular scarring, inflammation, fibrosis and deformity of calyces
underlying parenchymal scars, predominant tubulointerstitial
histological damage, and relative lack of glomeruli. There is evidence
that some of the chronic interstitial nephritis that is labelled
chronic pyelonephritis is due to something other than bacterial
infection.

Environmental Health Criteria 27: Guidelines on Studies in
Environmental Epidemiology (WHO, 1983) provides guidelines for
obtaining human data concerning the health effects of exposure to
chemical agents. For agents that produce acute renal failure,
long-term follow-up may identify those instances where chronic renal
disease has persisted. For agents that give rise to accidental
poisoning, clinical case reports can provide important information. In
the case of agents where exposure to larger population segments
occurs, information may be obtained by using statistical and
epidemiological methods to investigate possible nephrotoxicity from
such exposures (as compared to a non-exposed control group). Specific
segments of the population that might be at higher risk to a potential
nephrotoxic drug or workplace chemical should be particularly closely
monitored for renal effects.

There are marked differences between the incidence of
analgesic-associated ESRD in different countries and within the same
country (Table 2). This varies from up to 22% in Australia (in 1982)

Table 2. National prevalence of analgesic nephropathy
in patients with end-stage renal failure

% %

South Africa 22 Scandinavia (1979) 3
Switzerland (1980) 20 France (1979) 2
Belgium (1984) 18 USA 2
Australia (1985) 15 United Kingdom (1979) 1
Federal Republic of 13 Italy (1979) 1
Germany (1983) Spain (1979) 0.4
Canada (1976) 3

and in parts of some of the European countries to as low as 0.2% in
the USA. It is generally considered that the withdrawal of phenacetin
has lead to the disappearance of the high incidence of renal papillary
necrosis (RPN) in Scandinavia, Canada, and Australia, but a high
incidence remains in Switzerland, Belgium, and the Federal Republic of
Germany (Gregg et al., 1989). Specific geographical locations may have
analgesic abuse problems such as the Winston-Salem area (USA).
Worldwide variability in the prevalence of analgesic nephropathy has
long been recognized. The correlation of the incidence of this disease
with local analgesic consumption has been demonstrated. However, the
relation between both phenomena is not well established since
comparable consumption data, focussed on the sales of analgesic
mixtures, are not available in most countries. The high frequency
abuse area in Belgium is situated in the north (Fig. 1a), where up to
51% of dialysis patients are analgesic abusers, but this is markedly
lower in the south (Elseviers & De Broe, 1988). In Germany (Fig. 1b)
the highest prevalence is in West Berlin (up to 50%), Hamburg, and
Bremen (Pommer et al., 1986). These data indicate that the prevalence
of this disease has been underestimated on a national basis. There
are indications that the overall prevalence of this disease has also
been underestimated in several other countries. Local well-conducted
studies of the prevalence of analgesic nephropathy showed higher
prevalences than the European Dialysis and Transport Association
Registry. In the Federal Republic of Germany, a prevalence of 13% of
analgesic nephropathy in dialysis patients was found, while the
appropriate European Dialysis and Transport Association data was never
more than 6% (Pommer et al., 1986). In Belgium, the prevalence of
analgesic nephropathy was 18%, whereas the European Dialysis and
Transport Association registered a prevalence of 12% (Elseviers & De
Broe, 1988). The percentage of nephropathies of unknown etiology and
of pyelointerstitial nephritis may also indicate an underestimation of
analgesic nephropathy. These percentages are low in countries with a

high prevalence of analgesic nephropathy (Switzerland and Belgium) and
are high in countries such as Italy and Spain (Wing et al., 1989)
where analgesic nephropathy is considered to be rare. Analgesic
nephropathy progresses silently over a long period, and so the
diagnosis is difficult. In addition, most patients deny being
analgesic abusers, which further confounds diagnosis. Symptoms are
nonspecific until the degenerative cascade affects the cortex, when
renal failure occurs. Moreover, even when the renal failure is
recognized, the diagnosis of analgesic nephropathy remains difficult
unless diagnostic criteria are established.

2.5 Risk factors for toxic nephropathies

The risk of developing a clinically significant nephrotoxicity
depends on pre-existing clinical conditions and may be identified in
specific patient populations. Hypertension, diabetes, cardiovascular
disease, etc. are all thought to have the potential to exacerbate
nephrotoxicity, but many of these conditions have not been
systematically investigated for all types of nephrotoxicity. There are
examples where both chemicals and other disease factors cause a
lesion. For example, sickle cell disease and diabetes can cause renal
papillary necrosis, a condition that is also common in individuals who
abuse analgesics. The risk factors that predispose individuals to
renal papillary necrosis are not clear, and it is also unclear whether
diabetics are at greater risk of developing the lesion if they take
high doses of analgesics. This question cannot be resolved until
better diagnostic criteria are developed to identify the lesion before
it involves the cortex. Risk may also vary for different nephrotoxins.
While Bence-Jones protein excretion considerably increases the risk of
radiocontrast-induced renal injury, the effects of other types of
chemicals in patients with multiple myeloma are not clear. Therefore
recognition of the risk factors is necessary for the understanding and
prevention of renal damage.

There are also many examples of animal data that have not yet
been translated into risk terms in humans. For example, the immature
kidney may be resistant to amino-glycosides (Marre et al., 1980) and
cephalosporins (Tune, 1975), but the reverse is true for other
chemicals such as hexachlorobutadiene (Hook et al., 1983). Other
factors, such as electrolyte and volume changes or an alteration in
the renin-angiotensin system, may affect some types of drug-induced
acute renal failure (Bennett et al., 1983).

Risk factors as a measure of vulnerability to potential
nephrotoxicity that could be caused by drugs and chemicals are not
well defined at present. Clearly, wide variation exists among
individuals and even groups of people. Multiple exposure to toxic
agents and multiple drug usage are certainly factors, but the ability
to estimate risks to multiple exposure is limited. Factors intrinsic
to the nature of renal function and risk factors presented by clinical
disease have been reviewed (Porter, 1989) and are discussed below.

2.5.1 Factors related to renal function

The vulnerability of the kidney to toxicity from exposure to a
particular drug or chemical is the product of several groups of risk
factors. In any one person, multiple factors may be operative. The
nature of normal renal function in itself contributes to the
vulnerability to toxins. The intimate association of the capillary
endothelial surface during the process of ultrafiltration provides
opportunity for direct toxicity. A further contribution to the
glomerular capillary vulnerability is the positive hydrostatic
pressure required for producing the plasma ultrafiltrate. Adding to
this vulnerability is the "hyperfiltration injury" hypothesis proposed
by Brenner (1983) and Brenner et al. (1978, 1982), who showed that
when a nephron ceases to function, the remaining nephrons hypertrophy
and the flow rate per functioning nephron is raised, thus increasing
the exposure to the drug or chemical. A further aspect is the concept
of renal reserve, i.e. fewer functioning nephrons further increase the
vulnerability of the kidney to toxicity.

Another aspect of the structure of the glomerular endothelial
cells that can lead to injury to the kidney is the negative charge of
the filtration membranes. Positively charged ligands can become
electrostatically attached and alter the permeability coefficient of
the glomerulus. In addition, cationic proteins can be sequestered in
the glomerulus and act as "planted antigens", and a circulating
antibody can attach to such antigens resulting in an in situ immune
complex formation; hydro-carbons from petroleum products can act in a
similar way (Ravnskov, 1985). This is but one of a wide variety of
immunologically mediated glomerular injury patterns that have been
identified. This variety is not surprising when one considers the
heterogeneity of the biochemical composition of the glomerulus and the
wide spectrum of antigenic compounds to which the body is exposed
(Glassock, 1986).

Tubular vulnerability to nephrotoxins is related to the nature of
normal tubular function. The medullary countercurrent multiplier
system provides a mechanism for eliminating body waste products while
minimizing body water loss. A consequence is the reabsorption and
recycling of compounds of low relative molecular mass, in particular
urea and neutral toxicants and/or their metabolites, which can
accumulate in the medullary interstitium. Depending on their chemical
properties, they may initiate an inflammatory response through
activation of mediators, a factor that may be relevant in the
pathogenesis of analgesic nephropathy (Mudge, 1982).

The organic acid and base transport systems of the proximal
tubule serve to excrete certain molecular species. Several commonly
used drugs, including the organic acid penicillin, utilize this
mechanism of transport. Toxicants that are involved in these systems
might induce renal injury directly because of high cellular
concentration or by acting as competitive inhibitors to block the
elimination of endogenously produced toxic metabolites. Tubular

mechanisms for acidification may play a part in tubular injury by
drugs or chemicals that induce an acidification defect, e.g., lithium
(Batelle et al., 1982). Drugs or chemicals that are absorbed by
pinocytosis become concentrated in lysosomes where they are subjected
to digestion by hydrolytic enzymes. Some toxicants, however, may
inhibit the hydrolytic process, resulting in drug accumulation and
tubular cell toxicity that may resemble lysosomal storage disease (as
occurs in aminoglycoside nephrotoxicity).

2.5.2 Clinical risk factors

The application of multivariate analysis for investigating
clinical risk factors in the onset of acute renal failure (Rasmussen
& Ibels, 1982) may provide some insight into factors that may increase
vulnerability to nephrotoxicity from drugs and chemicals. The risk
factors summarized in Table 3 show that multiple risk factors coexist
in the majority of patients with acute renal failure. Although age has
been recognized as a factor in a number of studies (Porter, 1989;
Porter & Bennett, 1989), it may simply be a convenient marker for the
change in renal vulnerability that relates to the decline in
glomerular filtration rate (GFR) occurring beyond the age of 50
(Davies & Shock, 1950). The pathological basis for this decline is not
certain but may be related to vascular changes that accompany aging
(Avendano & Lopez-Novoa, 1987). Another possible explanation is that
the kidneys of people over 50 years of age no longer respond to
hypertrophic growth factors. Renal donors aged 50 or more show little
or no functional increase after the loss of one kidney (Boner et al.,
1972). Indeed, renal function reserve declines linearly with time
after 30 years of age (Anderson & Brenner, 1986). Age may also reflect
a loss of the ability of the renal tissue to repair. In young
individuals nephrotoxicity in terms of tubular necrosis may be
compensated for by constant repair, while in older patients this
repair capacity may be diminished, resulting in the clinical
expression of renal injury (Laurent et al., 1988).

Pre-existing renal disease is an obvious risk factor predisposing
to abnormal accumulation and excess blood levels of many nephrotoxic
drugs and chemicals. It is not clear whether sex is a predisposing
risk factor in humans, but male rodents are considerably more
susceptible than females to nephrotoxicity and carcinogenicity from
many environmental toxins (NTP, 1983, 1986, 1987).

Factors such as short-term and high-dose exposure versus chronic
and/or low-dose exposure influence vulnerability via the mode of
metabolism, rate of excretion, etc. Long-term, low-dose exposure to
substances that have a long biological half-life, such as lead or
cadmium, increases risk from these nephrotoxins, but their role as
co-risk factors is not known.

Table 3. Frequency of combined risk factors in 143 patients
with acute renal failure (ARF)^a

Age Hyper- Gout/ Diabetes Renal Diuretics
tension hyper- disease
uricaemia

Age (> 59 years) 30
Hypertension 29 4
Gout/hyperuricaemia 21 18 4
Diabetes 11 6 4 1
Renal disease 18 12 12 6 4
Diuretics 29 27 21 8 13 0

Multiple risks^b 108 63 37 14 13 0

^a Modified from: Rasmussen & Ibels (1982).
^b Significant risk contribution to ARF based on discriminant multiple linear
regression analysis.

2.5.3 Extrapolation of animal data to man

The use of animals has been essential to help define the
molecular basis and the progression of model nephropathies, but it may
be inappropriate to extrapolate animal toxicology data directly to man
because of marked species, strain, dietary, and sex differences. In
addition, there may be differences in dosing levels and regimen and in
the absorption, distribution, metabolism, and excretion of potential
nephrotoxins. There are also very significant differences in renal
structural and functional characteristics in the common laboratory
species used for risk assessment.

Chemical safety assessment has generally been undertaken in
relatively few strains of animals, such as the Sprague-Dawley, Wistar
and Fisher-344 rats. There is limited information on inter-species
comparisons. In addition to assessing the renal differences in each
of the species or strains used, it is necessary to examine extrarenal
differences. Thus, for example, there are marked species differences
in the hepatic handling of chemicals (Smith 1974; Testa & Jenner,
1976) and the metabolic capacity of each of the major organ systems
(Litterst et al., 1975a; Kluwe, 1983). This will have profound
consequences on the amount of a parent chemical and the pattern of
metabolites that reach the kidney. Dietary factors such as
carbohydrate, lipid, and protein intake alter renal function, and the
presence of contaminants and natural toxicants may add to the toxic
burden of the kidney (Bridges et al., 1982).

If the mechanistic basis of a renal injury is clearly
established, it is easier to assess the risk of chemical injury in
man, but such data are at present only available for a few chemicals.

There are relatively few examples of nephrotoxic chemicals where there
is a full profile of information from experimental animals and man,
and in the vast majority of cases data are available only in rats.

In order to extrapolate animal data for risk assessment, each
screening procedure should cover a sensible level of exposure and a
comparable condition to that found in man. The experience gained
should provide a foundation from which a rational basis can be
developed to identify potentially exacerbating risk factors and from
which nephrotoxicity can be reduced. Some lesions can only be induced
in rats with difficulty, and there may be a need to use sensitive
species or strains and/or to adapt certain experimental manoeuvres to
produce a lesion similar to that which occurs in man.

2.5.4 Risk assessment from nephrotoxicity studies in animals

Risk assessment from nephrotoxicity studies in animals has been
best defined for therapeutic agents. Many have been widely tested in
animals as a pre-clinical safety evaluation or used to study the
mechanism of renal injury where there are adverse reactions caused by
these compounds in clinical usage. The risk assessment for a number of
workplace or environmental chemicals has been developed from animal
models that have been used to study the mechanisms of these effects,
especially those of the heavy metals and some of the industrial
organic chemicals.

2.5.5 Special risk groups in humans

The marked variability in the response of any study population to
potentially nephrotoxic compounds establishes clearly that there are
groups at risk. There are a number of factors that could be
responsible for increasing the risk of nephrotoxicity. These include
existing renal disease, loss of renal parenchyma, high protein diet,
chemical exposure, predisposing factors, multiple myeloma, and other
conditions where there is an added level of protein excretion when the
kidney is under an additional work-load.

So far there has been relatively little interest in the
individuals that do not appear to be at risk from exposure to
potential nephrotoxins. While this is generally assumed to be the
result of an absence of predisposing factors, there may well be other
chemical, dietary, or disease considerations that provide a protective
effect. There is experimental evidence to suggest that a pre-existing
streptozotocin-induced diabetes and also poly-aspartic acid protect
against aminoglycoside-induced renal injury, and that fish oil diets
(high in omega-3 polyunsaturated fatty acids) reduce cyclosporin-A
nephrotoxicity. These factors could well be used to reduce the health
impact of nephrotoxicity.

2.5.6 Multichemical exposure

At present, there is virtually no information on the effects of
multichemical exposure in man and very little data on the effects of
more than one chemical administered simultaneously in animals.
Simultaneous exposure to several chemicals represents a major
toxicological problem, as man is generally exposed to more than one
substance in medicines, in food, and from environmental factors.
However, most experimental studies have investigated only single
chemicals. Interactions have been studied between mercuric chloride,
potassium dichromate, citrinin, and hexachloro-1,3-butadiene (HCBD)
in vivo and in vitro using a rat model (Baggett & Berndt 1984a,b;
1985). Dichromate potentiates the mercuric chloride effect, i.e. the
effects produced by the combination of metals are always greater than
the sum of the individual effects. There appears to be no simple
kinetic explanation, i.e. no enhanced renal accumulation of the
mercuric ion. The plasma membrane may be a site for the interaction of
these metal ions and could be the preliminary step that leads to
overall renal dysfunction and an ultimately enhanced acute renal
failure. Dichromate-citrinin and dichromate-HCBD interactions have
been demonstrated by alterations in urine flow, glucose excretion, and
transport processes. Some experimental data suggest that a
synergistic interaction may occur in analgesic nephropathy. The
mechanisms that underlie these interactions are not understood, and at
present there is no rational basis to predict them. Experimental
studies have shown that tubular cell injury, induced by
trichloroethylene and carbon tetrachloride, is potentiated by exposure
to polyhalogenated biphenyls, e.g., polychlorinated biphenyls (Kluwe
et al., 1979).

2.5.7 Renal functional reserve

The concept of renal functional reserve is a simple one in which
not all of the nephrons nor all of the cellular functions in a single
nephron are available or used at any one time (Friedlander et al.,
1989). Thus there is a buffering capacity in the kidney that can cope
with short-lived or protracted demands on function that exceed the
normal level. Part of this functional reserve is used to meet the
response to perturbation of the homeostatic system by water or
electrolyte loading. Most of the studies on and understanding of renal
functional reserve relate to changes in glomerular filtration rate and
renal blood flow. It is likely that additional approaches are needed
to test for other types of functional reserve.

2.5.8 The effects of chemicals on kidneys with pre-existing renal lesions

Although it is generally acknowledged that there are several
types of renal lesions that exist as a nephropathy in the general
population at a low but significant level (e.g., nephrotic syndrome),
little is known about how these pre-existing lesions affect the
response of the kidney to subsequent nephrotoxic insults.

2.5.8.1 Nephrotoxicity in the presence of renal and extrarenal disease

Safety screening is conducted on young, disease-free animals,
housed under optimal conditions, fed contamination-free, high-protein
food. By contrast, man is exposed to a variety of dietary and
environmental chemicals and to a poly-pharmacy of both prescribed and
self-administered medications over many years. In addition, screening
is generally undertaken using normal experimental animals. This may be
inappropriate because, with the exception of occupational and
environmental exposure, man is exposed to potentially nephrotoxic
therapeutic substances to treat disease. Pre-existing diseases can
have a profound effect on the direct or indirect response of the
kidney to handling chemicals (Bennett, 1986). There is an increasing
wealth of animal data to demonstrate that common clinical conditions
in man, such as hypertension, renal compromise, and renal ischaemic
injury, exacerbate cyclosporin nephrotoxicity and bacterial endotoxins
in animals and that systemic infection increases the sensitivity of
the kidney to aminoglycoside toxicity (Bergeron et al., 1982).

The role of pre-existing renal lesions on nephrotoxicity is
important, but there are few clear indications of what can be
predicted from existing clinical data and from animal studies.
Diabetes is generally associated with reduced renal function, diabetic
nephropathy, and renal papillary necrosis, and might be expected to
exacerbate chemical-associated nephrotoxicity. Untreated
streptozotocin-induced diabetes, however, protects rats against
gentamicin, low-dose cisplatin, and uranyl nitrate nephrotoxicity
(Teixeira et al., 1982; Vaamonde et al., 1984). Recent studies on the
acute effects of intravenous radio-contrast media on anaesthetized
diabetic rats were inconclusive (Reed et al., 1983, Golman & Almen,
1985, Leeming et al., 1985).

3. KIDNEY STRUCTURE AND FUNCTION

An in-depth review of kidney structure and function is beyond the
scope of this monograph. Only sufficient information will be given to
provide a general background against which nephrotoxicity can be
framed. A fuller insight into the complexities of the kidney in
health, disease, and nephrotoxicity has been described by Valtin
(1973), Orloff & Berliner (1973), Hook (1981), Porter (1982), Bach et
al. (1982, 1989), Bach & Lock (1982, 1985, 1987, 1989), Seldin &
Giebisch (1985), Brenner & Rector (1986).

3.1 Renal anatomy

The two kidneys are situated retro-peritoneally, on either side
of the vertebral column, and process 25% of the resting cardiac output
via an arterial blood supply. Much of the fluid and most of the
solutes in blood are filtered through the glomeruli into the proximal
part of the nephron (the functional unit of the kidney) from which
essential small molecules are reabsorbed. Numerous macro-molecules are
reabsorbed into the tubular cells by an endocytotic process and are
digested in tubular lysosomes. Many organic acids and bases (including
many drugs) are secreted (and reabsorbed) by carrier-mediated
processes located principally in the proximal tubule. There is some
secretion, mainly of waste solutes, from the blood into the distal
part of the nephron, and much of the water in which they are dissolved
is subsequently reabsorbed.

Each kidney is made up of a large number of nephrons, groups of
which unite to continue as collecting ducts or tubules, and these in
turn combine to make up the ducts of Bellini, which exit around the
papilla tip. The papilla opens into the calix, which is in continuity
with the renal pelvis, a funnel-shaped area that narrows to the
ureter. The continued production of urine, together with peristalsis
of the ureter, carries excreted waste to the bladder. The
morphophysiology of the kidney varies markedly between species.
Therefore, a generalized description will be provided, and only the
important differences between the rat (and other common laboratory
animals) and man will be described (Moffat, 1979).

3.1.1 Histology

Renal lesions occur in discrete anatomical regions. This
highlights the need to understand changes in terms of the biochemical
properties of the specifically affected region and its adjacent cells.
While haematoxylin and eosin staining and a number of other routinely
used staining procedures identify nephropathies and renal
degeneration, these are generally based on a relatively non-specific
assessment. The non-specificity of routine histopathology has, in
fact, been the strength of these methods in the preliminary
assessment of chemically induced nephropathies. It may, however, miss
some types of lesions and generally gives little information that can
help identify the mechanistic basis of a lesion.

Histochemical techniques can provide insight into primary and
secondary cellular mechanisms. One aspect of "histochemistry" is the
use of frozen segments of the nephron (Bach et al., 1987) and the
application of fluori-metric or radiochemical assays to measure the
activities and distribution of specific biochemical characteristics.
Microdissection generally fails to give a detailed localization of
these properties in relation to specific or individual cells. In
addition, the technique is difficult to apply to injured renal cells.

The most widely used histochemical approach is based on obtaining
frozen, fixed frozen, or fixed embedded sections of the kidney that
are then used with chromo- or fluoro-phores. These react with a
selected type of material. The types of materials that can be
visualized in section depends on their chemical structure (e.g.,
carbohydrate), enzymic activity (e.g., lactate dehydrogenase),
antigenicity (e.g., specific molecules), or physicochemical
properties (e.g., lipophilicity), some of which are shown in Fig. 2.
This approach also includes the distribution or incorporation of
radiolabelled molecules by their interaction with a photographic film
laid over the section (Bach et al., 1987). Immunohistochemical
techniques using labelled antibodies permit antigens to be localized
at the light microscopical and ultrastructural levels.

These microscopic histochemical techniques provide information on
the distribution at, or within, specific cells and their relative
activities, and have been used to define a variety of characteristics
of the kidney. It is important to stress that each method has its own
inherent strength and weakness, and that it may be difficult to relate
data from tissue sections to absolute biochemical measurements. This
is a consequence of the complex mixture of materials that are present
in tissue sections and the chemical changes that may take place in
these sections, particularly once they have been fixed. While the
biochemical characteristics of tissue in frozen sections are least
likely to be adversely affected, subtle and misleading alterations in
chemical properties can still occur. In addition, treatments to
conserve morphological features (fixation and embedding) alter these
values. It may also be difficult to be certain whether an increased
intensity of staining for a certain substance represents de novo
synthesis, unmasking, or the loss of factors that suppress staining.
These two techniques have proved to be very powerful in providing
information on the biochemical characteristics of cells and the
changes associated with renal injury. The in situ hybridization
techniques, using specific labelled nucleotidic probes, permit the
detection of protein synthesis at a subcellular level.

3.1.2 Enzyme histochemistry and quantification

Histochemical heterogeneity is evident in each part of the
nephron and varies between different species. In addition, the profile
of characteristics within any region of the kidney and nephron may be
related to sex and age. Antibodies are directed against unique or
novel characteristics along the nephron that are present on enzymes,

glycoproteins, or other molecules associated with membranes or soluble
cytosolic constituents. The biochemical characteristic can be
visualized using enzyme, fluoro-phore, or radioactive labels (Bach et
al., 1987). Individual microdissected nephron segments have been
studied to determine the quantitative distribution of selected enzymes
(Table 4). Table 5 lists biochemical and function parameters for the
different nephron segments, showing their individual receptor
sensitivity.

3.1.3 Immunohistochemistry

The use of antibodies raised towards enzymes can help in the
study of isoenzyme distribution and factors affecting changes in their
distribution. For instance, aldolase-B monomers increase in the
proximal tubules of rats during renal maturation, but not in the
distal tubules. In contrast, aldolase-A monomers increase in the
distal tubules but not in the proximal tubules.

Immunohistochemical techniques demonstrate many functional
proteins at discrete locations in the kidney, including the glomeruli
and the tubular basement membrane, but there are few data on changes
in these proteins as a result of nephrotoxicity. A variety of
immunodeposits are associated with glomerulopathies including those
caused by heavy metals, but these appear to be T-cell mediated. They
are assessed by immunofluorescent monitoring in the glomerulus, but
this acts as a passive sieve and may be involved as a secondary
consequence of immunodeposition.

Histochemistry can also be used to show the distribution of a
number of oxidative enzymes. Cytochrome P-450 mixed-function oxidase
activities have been shown immuno-histochemically to be localized in
the proximal tubule, particularly in the S₂ and S₃ segments in the

Table 4. Distribution of enzymes in individual nephron segments
in various animal species

Enzyme Relative activity^a Animal

Specific to the glomerulus

Adenosine deaminase rat

Specific to the proximal tubule

Glucose-6-phosphatase S₁>S₂>S₃ rat
Fructose-1,6-bisphosphatase S₁<S₂>S₃ rat
Phosphoenolpyruvate carboxykinase S₁>S₂>S₃ rat, rabbit
Fructokinase S₁=S₂<S₃ rat
Fructose-1-phosphate aldolase S₁=S₂>S₃ rat
Glycerokinase S₁=S₂>S₃ rat, rabbit
Glycerol-3-phosphate dehydrogenase S₁=S₂<S₃ rat
Glutamine synthetase S₃ rat
Alanine aminotransferase S₁=S₂<S₃ rat
Gamma-glutamyltraspeptidase S₁<S₂<S₃ rabbit
Gamma-glutamyl-cysteine synthetase S₃ rat
Glutathione-S-transferase S₁<S₂<S₃ rabbit
Cytochrome P-450 S₁=S₂>S₃ rat, rabbit
Alanine aminopeptidase S₁<S₂<S₃ rat
Alkaline phosphatase S₁=S₂=S₃ rat
Leucine aminopeptidase S₁<S₂<S₃ rat
D-Amino acid oxidase S₁=S₂<S₃ rat
L-Hydroxy acid oxidase S₁=S₂<S₃ rat
Fatty-acyl-CoA oxidase S₁=S₂<S₃ rat
Choline oxidase S₁=S₂<S₃ rat
25(OH)-D₃-1alpha-hydroxylase S₁<S₂=S₃ rat
(D₃ deficient)
rabbit (fetus)

Table 4 (contd).

Enzyme Relative activity^a Animal

Relatively specific to the proximal tubule

Glutamate dehydrogenase S₁=S₂>S₃ rat, rabbit
Malic enzyme S₁=S₂<S₃ rat
Trypsin-type protease S₁>S₂>S₃ rat
ß-D-Galactosidase S₁=S₂<S₃ rat
N-Acetyl-ß-D-glucosaminidase S₁=S₂>S₃ rat
Xanthine oxidase S₁>S₂>S₃ rat
Superoxide dismutase S₁>S₂>S₃ rat

Specific to the lower nephron

Hexokinase MTAL=CTAL>DCT>CCD=MCD rat, rabbit
Phosphofructokinase MTAL=CTAL=CCD=MCD rat, rabbit
Pyruvate kinase MTAL=CTAL<DCT<CCD=MCD rat, rabbit
Kallikrein CNT rabbit

Relatively specific to the lower nephron

Fructose 1,6-bisphosphate aldolase MTAL=CTAL>DCT rat
Glycerol dehydrophosphate CCD<MCD rabbit
dehydrogenase

Table 4 (contd).

Enzyme Relative activity^a Animal

Relatively specific to the
lower nephron (contd.)

Lactate dehydrogenase MTAL=CTAL-DCT=CCD rat, rabbit
Aspartate aminotransferase MTAL=CTAL>DCT rat
Citrate synthase MTAL=CTAL=DCT>CCD rat
Isocitrate dehydrogenase (NAD⁺) MTAL=CTAL=DCT=CCD rat
Na,K-ATPase MTAL<CTAL<DCT>CCD rat

^a CCD = cortical collecting duct; CNT = connecting tubule; CTAL = cortical thick ascending
limb of Henle's loop; DCT = distal convoluted tubule; MCD = medullary collecting duct;
MTAL = medullary thick ascending limb of Henle's loop;
S₁ = early proximal tubule; S₂ = middle proximal tubule; S₃ = late proximal tubule.

rat and rabbit. Large numbers of peroxisomes containing D-amino acid
oxidase and catalase are localized in the S₃ portion of the proximal
tubule, but they are absent from the glomerulus and the distal
nephron. There is little immuno-histochemical data on the distribution
of molecules that are likely to protect renal cells from the effects
of reactive intermediates. Ligandin or glutathione- S-transferase B
is located in the proximal tubule of both animals and man and in the
thick limb of the loop of Henle in man. Catalase activity is greatest
in the proximal tubule (where it is localized in the peroxisomes),
less in the distal tubule, and very low in the glomerulus. Glutathione
has been shown by histochemistry to be localized in the proximal
convoluted tubule. However, there are some uncertainties as to what is
being assessed, since the reaction measures sulfhydryl groups and not
only glutathione. The distribution of at least one superoxide
dismutase isoenzyme shows a marked species difference between the dog
and rat, but is localized in the proximal tubules in both (Bach et
al., 1987).

3.2 The renal blood supply

Each kidney is supplied by a renal artery (a branch of the
abdominal aorta), which divides to form several interlobar arteries
(Fig. 3). These in turn give rise to the arcuate arteries, which run
between the cortex and medulla parallel to the kidney surface. Many
cortical radial arteries arise from the arcuate vessel and pass
through the cortex. Here a small amount of blood reaches the surface
to supply the kidney capsule, but most of the blood flow is directed
through branches that form the afferent arterioles to the glomeruli.
Each afferent arteriole breaks up to form the capillary plexus of the
glomerulus; this is drained into the efferent arteriole. The efferent
arterioles form two types of capillary networks

* In the "mid" and "superficial" cortical regions, they form the
peritubular capillaries surrounding proximal and distal tubules
(in the superficial regions some peritubular capillary networks
interlace the nephron from which they were derived, but such an
association appears to be the exception rather than the rule).

* In the juxtamedullary region (and some mid-cortical areas in man)
each efferent arteriole is directed into the medulla, where it
branches into the vasa recta bundles. Each bundle consists of up
to 30 descending vessels, the peripheral vessels of which give
rise to a highly branched capillary network in the outer medulla.
The core of the vasa recta bundle continues to the inner medulla
where it terminates in a capillary network (Beeuwkes, 1980).

The walls of all peritubular capillaries in the kidney are made
up of a thin fenestrated endothelium resting on a basal lamina. The
capillaries in the cortex generally open into the cortical radial
vein, from which blood flows via the arcuate vein to the renal vein
and finally to the inferior vena cava. The capillary plexuses in the

Table 5. Summary of nephron heterogeneity^a

Anatomical region^b Biochemical features^c Functional features

Glomerulus SF<JM Renin:(SF>JM) GFR:(SF<JM)
epithelium Adenosine-AC Mesangial contraction
(AII, histamine)
endothelium Histamine-AC (intra-mesangium)
mesangium Serotonin-AC (extra-mesangium)
ANP-GC ROM generation (intra-
and extra-mesangium)
ET-PGE2 (intra-mesangium)
AVP-PGE2 (intra- and extra-mesangium)
AII-PGE2 (intra- and extra-mesangium)

Proximal tubule Glucose carrier: J-glucose:
S₁, S₂, S₃ (brush border) (S₁>S₂,S₃)
Gluconeogenesis
(S₁>S₂>S₃) J-V:(S₁>S₂>S₃)
Cytochrome P-450: 1,25(OH)₂D₃ synthesis
(S₁<S₂>S₃)
NADPH-cytochrome c reductase: J-V decreased by ANP
(S₁<S₂>S₃)
Ammoniagenesis P-Cl/P-Na:(SF>JM)
(S₁>S₂>S₃)
PTH-AC(S₁>S₃) PAHsecretion:
(S₁<S₂>S₃)
Adenosine-AC J-aminoacid:
(S₁>S₂,S₃)

Henle's loop
Thin: DTL<ATL AVP-AC(+ATL,-DTL) P-water:(DTL>ATL)
P-NaCl:(DTL<ATL)
P-urea:(DTL<ATL)

Table 5 (contd).

Anatomical region^b Biochemical features^c Functional features

Thick: MTAL>CTAL AVP-AC:(MTAL>CTAL) AVP stimulation of
J-Cl: (MTAL>CTAL)
PTH-AC:(MTAL<CTAL) PTH stimulation of
J-Ca: (MTAL<CTAL)
SCT-AC:(MTAL>CTAL)
Tamm-Horsfall glycoprotein
PGE2 synthesis J-NaCl:(MTAL>CTAL)
(MTAL>CTAL) PG inhibition of J-Na:
(+MTAL, -CTAL)
EGF synthesis
J-Na decreased by ANP

Distal tubule
DCT:single cell type SCT-AC:(+DCT) SCT suppression of
Vt:(DCT)
CNT:multiple cell PTH-AC:(+CNT) PTH stimulation of
types J-Ca: (CNT)
AVP-AC:(+CNT) AVP suppression of
Vt:(CNT)
ISO-AC:(+CNT) ISO suppression of
Vt:(CNT)
Kallikrein:(+CNT)
Aldosterone binding Vt:(DCT < CNT)
K-secretion:(DCT<CNT)?
Collecting duct system
two cell types AVP-AC:(CCD > OMCD)
CCD (P.cell>I.cell) ISO-AC:(CCD > OMCD) Vt:(CCD > OMCD)
J-Na and J-V decreased
by ANP

Table 5 (contd).

Anatomical region^b Biochemical features^c Functional features

OMCD (P.cell>I.cell) PG-AC:(CCD < OMCD) P-urea:(CCD,OMCD<IMCD)
PGE2 synthesis
(CCD<OMCD<IMCD)
Aldosterone binding
Adenosine-AC (CCD>OMCD)
ANP-GC (CCD<OMCD<IMCD)
IMCD J-V decreased by ANP

^a Based on data obtained from the rabbit and rat kidney.
^b Parts of the nephron: ATL = ascending thin limb of Henle's loop; CCD = cortical collecting
duct; CNT = connecting tubule; CTAL = cortical thick ascending limb of Henle's loop; DCT =
distal convoluted tubule; DTL = descending thin limb of Henle's loop; I.cell = intercalated
cell; IMCD = inner medullary collecting duct; JM = juxtamedullary nephron; MCT = medullary
collecting duct; MTAL = medullary thick ascending limb of Henle's loop; OMCD = outer
medullary collecting duct; P.cell = principal cell; S₁ = early proximal tubule; S₂ = middle
proximal tubule; S₃ = late proximal tubule; SF = superficial nephrons.
^c Hormone effects on nephron receptors: AII = angiotensin II; AC = adenylate cyclase; ANP =
atrial natriuretic peptide; AVP = arginine vasopressin; GC = guanylate cyclase; EGF =
epidermal growth factor; ET = endothelin; ISO = isoproterenol; PG = prostaglandin; PTH =
parathyroid hormone; ROM = reactive oxygen metabolites; SCT = salmon calcitonin.
Transport substances: PAH = para-aminohippurate; J-x = flux of substance x; J-V = flux of
fluid volume; P-x = permeability for substance x; Vt = transcellular voltage.

medulla drain into the ascending vasa recta, which join the arcuate
veins. The arterial branches are terminal, without anastomoses. In
contrast, the veins are richly anastomosed.

There is a well-defined structural relationship between the vasa
recta bundles and the nephrons in the outer medulla, at least in the
animals that have been studied. A central core (consisting of
descending and ascending vasa recta) is surrounded by a peripheral
layer consisting of a closely intermingled ascending vasa recta and
the descending thin limbs of the loops of Henle. Between these bundles
are the thick ascending limbs of the loops of Henle, some descending
limbs, and the collecting ducts. Within the bundles both ascending
and descending vasa recta are in intimate contact with each other
(rather than with the same type of vessel). There are more ascending
vessels, all of larger diameter, than descending ones, and this
increased volume capacity relates to the removal of excess water from
the interstitium and the maintenance of the medullary osmotic gradient
shown in Fig. 4.

Many of the "major" and "minor" blood vessels in the kidney have
either smooth muscle cells as an integral part of their structure, or
other cells that may have a contractile function. Thus most of the
intrarenal vascular system has both adrenergic and cholinergic
innervation. Intrarenal blood flow, the factors which alter it, and
its effects on renal function are poorly understood. Although there
appear to be the facilities for a direct and effective perfusion of
the medulla from the arcuate artery, this does not seem to occur
(Moffat, 1979; Beeuwkes, 1980).

3.2.1 Renal haemodynamics

The measurement of total blood flow through the kidneys can be
measured relatively easily using modern techniques (Grunfeld et al.,
1971; Pearson, 1979). Defining the zonal blood flow has given some
conflicting results, but assessing regional blood flow within the
kidney is fraught with difficulties and is subject to varied
interpretations (Grandchamp et al., 1971; Grunfeld et al., 1971;
Pearson, 1979; Aukland, 1980; Knox et al., 1984). There are, however,
consistent data (derived from a number of fundamentally different
techniques) to show that intrarenal blood flow is greatest in the
cortex (80-85% of total renal flow) and that it decreases through the
juxtamedullary region to less than 10% of the total renal flow in the
medulla. It must be stressed that, although the medulla is poorly
perfused in comparison to the rest of the kidney, it is, nonetheless
(because of the 25% resting cardiac output and therefore abundant
renal blood flow), a well-perfused tissue. According to Thurau (1964),
the medullary blood flow is about 15 times that of resting muscle and
the same as that of the brain. In addition, the capillary volume
fraction of the medulla is more than twice that of the renal cortex
(Beeuwkes, 1980). Despite this, there is considerable variation in
tissue pO₂ in the kidney; a marked decrease in pO₂ levels is seen
with increasing tissue depth (Brezis et al., 1984).

3.3 The nephron

The kidney is divided into three main regions, cortex (outer),
medulla (inner), and pelvis (Fig. 5). Within the cortex arise the
renal corpuscles, defined as superficial, midcortical or
juxtamedullary depending on the anatomical location of the renal
corpuscle in the cortex. The nephron is the functional unit of the
kidney and consists of a continuous tube of highly specialized
heterogeneous cells, which show sub-specialization along the length of
nephrons and between them. There are marked structural and functional
differences between the nephrons arising in the cortex and those
arising in the juxtamedullary regions. The total number of nephrons
varies between different species and within any one species as a
function of age. The macroscopic differentiation of the kidney into
distinct zones arises not only from the regional vascularity but also
from the way different functional parts of the nephron are arranged
within the kidney. A more detailed account of the ultrastructure of
the morphologically definable regions of the nephron and their
functional inter-relationship has been provided by Moffat (1981,
1982), Bohman (1980), and Maunsbach et al. (1980). Recently the
nephron nomenclature has been standardized by the Renal Commission of
the International Union of Physical Sciences (Kriz & Bankir, 1988).
This is summarized in Figures 3, 5, and 6, and Table 6.

3.3.1 Cellular heterogeneity and cell-cell interaction

There are well over 20 morphologically different cell types
(based on light microscopy alone) in the kidney, and when
histochemical and immunohistochemical methods are applied to renal
tissue sections the diversity of cell types is even more apparent. The
spectrum of biochemical (and structural and functional)
characteristics in these cells demonstrates the very marked
heterogeneity that is the hallmark of the kidney. It is well
established that the expression of many of these biochemical
characteristics is an integral of the functions of that particular
region of the kidney, and there is the potential to change the
expression of these characteristics in terms of the demands on the
kidney. These include both water and electrolytes, dietary factors,
and chemicals with pharmacological and toxic effects, or may be as a
result of chemical and other types of injury. More importantly, the
characteristics of a cell may make it either resistant or sensitive to
the target selective toxicity of a chemical.

3.3.2 The glomerulus

The glomerulus forms the initial part of the nephron and
functions as a relatively poorly selective macro-molecular exclusion
filter to the hydrostatic pressure of the blood. The number of
glomeruli is, in general, related to the mass of the species, and the
size of each glomerulus depends, among other factors, on the
environmental water balance. Three anatomically distinct types of
glomeruli can be identified: those in the superficial cortex, which
are part of the superficial nephrons; those arising in the midcortical
area; and those ofjuxtamedullary origin, which continue as nephrons
that loop down into the medulla. The structure of the glomerulus is
complex (Fig. 7) and has only been defined using scanning and
transmission electron microscopy (Maunsbach et al., 1980; Moffat 1981,
1982).

The glomerular "tuft" is made up of a number of capillary
branches that arise from the afferent arteriole, anastomose, and drain
to the efferent arteriole. There are also communicating vessels
between the branch capillaries. The fenestrated endothelium cannot
prevent plasma molecules from leaving the lumen, but a negatively
charged cell coat imparts some selective permeability. The capillaries
are in direct contact with the glomerular basement membrane (or basal
lamina), which, when viewed under the electron microscope, can be
divided into three layers: the lamina rara interna on the endothelial
side; the central lamina densa; and the lamina rara externa, which is
in direct contact with the epithelial cells (the podocytes). The basal
lamina contains collagen (mostly Type IV) and sialic acid and is rich
in glycosaminoglycans, mainly heparan sulfate (Kanwar & Farquhar,
1979), which provides a strongly anionic macromolecular filtration
barrier.

The capillary tuft (ensheathed in its basal lamina) is surrounded
by a number of podocytes, each of which gives rise to several primary
processes (trabeculae). These in turn give rise to secondary
processes, and, finally, to numerous tertiary foot processes that are
embedded in the lamina rara externa.

The foot processes of one podocyte interdigitate with those of an
adjacent epithelial cell for adjacent trabeculae. The surfaces of the
podocytes are covered by a strongly anionic cell coat that extends to
the spaces between the foot processes. It is through these spaces that
the glomerular filtrate reaches the lumen of Bowman's space. Thus, the
podocyte provides a structural support for the basal lamina and may
also serve to provide additional anionic forces for the process of
biological ultrafiltration. It has been suggested that podocytes may
have phagocytic properties and undergo contraction (Moffat, 1981).

Table 6. Summary of nomenclature of segments and cells of the renal tubule (From: Kriz & Bankir, 1988). A continuous serpentine
arrow ( ) means that the transition between the two structures is gradual. An interrupted serpentine arrow ( ) means that the
transition is gradual in some species, abrupt in others. Abbreviations marked by a star were introduced by Morel and coworkers
(Kidney Int 9: 264, 1976). They mean: DCTa = Distal convoluted tubule, initial portion; DCTb = Distal convoluted tubule, bright
portion; DCTg = Distal convoluted tubule, granular portion; DCTI = Distal convoluted tubule, light portion; CCTg = Cortical
collecting tubule, granular portion; CCTI = Cortical collecting tubule, light portion

Micro-
Anatomical Main division Subdivisions Segmentation Abbre- Cell types Other frequently used
terms viation denominations

Proximal PROXIMAL pars convoluta Proximal S 1 cells
convolution TUBLULE or Convoluted S 1 - segment PCT P 1 segment PT
convulated part Tubule

S 2 - segment S 2 cells P 2 segment

pars recta Proximal
or Straight S 3 - segment PST P 3 segment PR
straight part Tubule S 3 cells

Loop of INTERMEDIATE pars descendens Descending DTL DTL cells
Henle TUBULE or Thin of short loops Type 1 Short Descending Thin Limb of
descending part Limb of long loops Henle's loop (SDL)
upper part Type 2 Long Descending Thin Limb,
lower part upper part (LDL_u)
Type 3 Long Descending Thin Limb,
lower part (LDL_l)
pre-bend segment ATL cells
pars of ascendens
or Ascending Thin Limb ATL Type 4 Tal Thin Ascending Limb
ascending part (of long loops only)
ascending part

Table 6 (contd.)

Micro-
Anatomical Main division Subdivisions Segmentation Abbre- Cell types Other frequently used
terms viation denominations

DISTAL pars recta Distal Medullary D MTAL DST or Tal MAL Thick Ascending Limb of
TUBULE or Straight straight S cells Henle's Loop
straight part Tubule part T mTALH Medullary Thick Limb
or Cortical o CAL Cortical Thick Limb
Thick straight r CTAL cTALH (incl. Macula Densa)
Ascending part Macula Densa T MD MD MD
Limb postmacular A cells
segment L DCTa* early
Distal pars convoluta DCT cells distal Distal Tubule
convolution or Distal Convoluted Tubule DCT (+ IC cells) DCTb* tubule
convoluted part

COLLECTING CONNECTING TUBULE CNT cells
SYSTEM CNT + DCTg* late Connecting
IC cells CCTg* distal Segment
Collecting COLLECTING CD cells tubule
duct DUCT Cortical Collecting Duct CCD = principal
cells DCTl* Initial
Collecting
Tubule
= light cells CCTl* Cortical
+ Collecting
IC cells Tubule (CCT)
= intercalated
cells
= mitochondria-
rich cells Outer Medullary Collecting
Outer Medullary OMCD = carboanhyd- Tubule (OMCT)
Collecting Duct rase-rich cells
= dark cells
CD cells Inner Medullary Collecting
Inner Medullary IMCD = prinicpal Tubule (IMCT)
Collecting Duct cells Papillary Collecting Duct
(PCD) or ducts of Bellini

The axial regions of each glomerulus contain mesangial cells.
Information on the structure and possible functions of mesangial cells
has been reviewed by Moffat (1981). In brief, they undergo contraction
and may thus control glomerular blood flow via biogenic amine or
hormonal control. Of equal importance is the observation that these
cells take up large molecules (such as colloids, immune complexes, and
protein aggregates), which may eventually be disposed of via the renal
lymphatic system.

The driving force for filtration is provided by the glomerular
capillary hydrostatic pressure (which is controlled mainly by the
vascular tone of the afferent and efferent arterioles), minus both the
plasma osmotic pressure and the hydrostatic pressure in the Bowman's
space. The resulting "effective filtration pressure" across the basal
lamina is about 1.2-2.0 kPa (10-15 mmHg). Selective filtration is
achieved primarily on the basis of size restriction by the basement
membrane, which impedes the passage of macromolecules with an
effective radius greater than 1.8 nm and completely prevents the
filtration of macromolecules with an effective radius greater than 4.5
nm. In addition the presence of fixed negative charges on the
endothelial, epithelial, and basement membranes hinders the filtration
of anionic macromolecules while facilitating the passage of cationic
macromolecules. The selectivity of filtration is, in part, a
consequence of the anionic nature of the basement membrane, which
blocks or slows the passage of negatively charged or neutral
macromolecules and leaves those carrying a cationic charge and small
molecules (irrespective of charge) to pass unimpeded.

3.3.3 The proximal tubule

The proximal tubule is found only in the cortex or subcortical
zones of the kidney. Anatomically each proximal tubule can be divided
into the convoluted portion (pars convoluta) and the shorter straight
descending portion (the pars recta), which then continues to become
the descending limb of the loop of Henle. It may be sub-divided, by a
number of morphological and functional features, into three segments,
S₁, S₂, and S₃.

The proximal tubule plays a decisive role in maintaining
homeostasis. This is achieved when sodium and chloride ions flux from
the tubule lumen to the peritubular capillaries under the control of
a number of processes such as nonspecific electrophysiological
gradients and selective active transport mechanisms. Water follows the
ions by osmotic effects. In addition, hydrostatic pressure,
attributable to the presence of both proteins and glycosamino-glycans
(Wolgast et al., 1973), contributes to water movement from the
epithelial cell to the interstitium and thence, by an osmotic
gradient, into the capillaries (Valtin, 1973). The flux of ions within
the proximal tubule, including the absorption and secretion of

HCO₃^- and H⁺ and the "lumen trapping" of ammonium ions, controls
renal acid-base regulation (Valtin, 1973).

Those proteins that have passed from Bowman's capsule (a
significant amount of albumin in the case of normal rats) are
reabsorbed in the proximal tubule by pinocytotic removal from the base
of the microvillous brush border into the epithelial cells. The
vesicles thus formed combine, form protein-filled vacuoles, and fuse
with lysosomes, from which the digestion products of the protein
diffuse, eventually, to the capillary system or are used in the
metabolic processes of the cell.

There are, in addition, other absorptive and secretory
mechanisms. These include the co-transport process that reabsorbs
glucose and the secretion of both acidic and basic organic compounds
(Valtin, 1973; Orloff & Berliner, 1973; Brenner & Rector, 1986;
Berndt, 1989).

3.3.4 The medulla

The medulla differs from the cortex (Fig. 5 and Fig. 8) both at
the macroscopic and at the microscopic levels. This region can be
divided into the outer medulla (which is made up of the thin
descending and the thick ascending limbs of the loops of Henle,
collecting ducts, the vasa recta, and a dense capillary network) and
the inner medulla, the free part of which is referred to as the
"papilla" (although some researchers apply that name only to the apex
of this region). The inner medulla contains the thin limbs of the
loops of Henle, collecting ducts, the vasa recta, and a diffuse
network of capillaries. Packed into the spaces between these
structures are interstitial cells embedded in a matrix rich in
glycosaminoglycans.

The collecting ducts terminate as the ducts of Bellini around the
tip of the papilla. Whereas the mouse, gerbil, rat, guinea-pig,
rabbit, dog, cat, and primate kidneys have only a single papilla, the
pig and man have multi-papillate kidneys. There are between 9 and 20
papillae in each human kidney (Burry et al., 1977), of which there are
two anatomically distinguishable types. The conical non-refluxing
papillae, where the surface orifices of the ducts of Bellini are
slit-like, close when there is an increase in the "back-pressure" of
urine from the bladder and so prevent intrarenal reflux when reflux
occurs from the bladder. These papillae occur predominantly in the
mid zone. The refluxing papillae occur predominantly in the polar
regions, and, as they have flattened tips, the collecting duct
orifices are wide and prone to retrograde flow of urine into the
tubules during vesico-ureteric reflux (Ransley & Risdon, 1979). The
microscopic and ultrastructural features of the medulla have been
described by several researchers (Moffat, 1979, 1981, 1982; Bohman,
1980; Maunsbach et al., 1980).

3.3.4.1 The loops of Henle

The loops of Henle may be divided into two populations on
anatomical grounds. Short loops penetrate no further than the outer
medulla. The proximal tubule and thick ascending limb are closely
associated in the cortex, but in the medulla the descending limb is
intimately related to the ascending vasa recta, and the ascending limb
to the collecting duct. The association of the ascending and
descending limbs of the loop of Henle with the vascular system or with
the collecting ducts provides a multi-dimensional network in which
solutes or water may undergo countercurrent exchange. These exchanges
may either provide a shunt that excludes selected solutes (and water)
from the inner medulla or, alternatively, solutes (e.g., sodium
chloride and urea) may be trapped in this zone. This exclusion of
water and trapping of sodium chloride, urea, and osmolytes helps
maintain the osmotic gradient along the inner medulla. In long loops
(the length is proportional to the renal concentrating potential), the
loop of Henle penetrates the inner medulla. Only about a third of the
ascending and descending limbs of long loops lie together; in the
other instances the ascending limbs are nearer to collecting ducts
than to descending limbs.

3.3.4.2 Collecting ducts

Collecting ducts consists of three identifiable segments, which
lie, respectively, in the cortex, the outer medulla, and the inner
medulla. These segments demonstrate different permeabilities to water
and osmolytes. The difference in permeability may be related to the
presence of two cell types, the intercalated and collecting duct (or
principal) cells.

3.3.4.3 The distal tubule

The distal tubule connects the thick ascending limb of the loop
of Henle to that part of the collecting duct which originates in the
cortex. The distal tubules are in-involved in both ion and water
reabsorption, but play a much less significant role than the proximal
tubules. The underlying mechanisms responsible for reabsorption
appear, in essence, to be similar to those already outlined. The major
differences include a stronger Na⁺ gradient against which to "pump",
the ability to reabsorb sodium without reabsorbing water, the
controlling effects of anti-diuretic hormone (ADH) and aldosterone
(among other mediators), and the very limited (or lack of) protein
reabsorption. The secretion of potassium ions appears to be under the
control of an active transport mechanism, the regulating factors of
which are many and complex (Valtin, 1973; Orloff & Berliner, 1973;
Brenner & Rector, 1986).

3.3.4.4 The countercurrent multiplier system and urine concentration

Less than 1% of the glomerular filtrate leaves the kidney as
urine (unless there is a state of diuresis), the remainder having been
reabsorbed. The process of urine concentration is complex and depends
(at least in part) on the countercurrent multiplier system, which
establishes a steep osmotic gradient along the inner medulla. The high
osmolality is a consequence of the differential permeability of the
limbs of the loops of Henle and the collecting ducts to water and
ions. The thick ascending limb is thought to have an active mechanism
which transports chloride and sodium out of the lumen and into the
interstitium, but the limb remains impermeable to water. As a
consequence the osmolality decreases in this part of the tubule (the
diluting segment). The descending limb, on the other hand, is freely
permeable to water, but probably not to sodium ions. The high ion
concentration in the interstitium would draw water out of the
descending limb, increasing the osmolality towards the turn of the
U-loop. This is augmented by urea and other osmolytes that leave the
collecting ducts and enter the ascending limb via the interstitium,
thus being recirculated to the medulla.

The collecting ducts regulate the final urine concentration by
controlling the amount of water that is reabsorbed. The passage of
water out of the ducts is thought to be mediated largely by cyclic
adenosine-monophosphate (cAMP), the synthesis of which is stimulated
by ADH, which increases the permeability of the luminal cell membrane
to water. Osmotic effects draw the water out of the cell (through the
basement membrane) into the hyperosmotic interstitium. In the absence
of ADH the collecting duct is thought to be impermeable and relatively
little water is reabsorbed from it. The interstitial osmotic gradient
is assumed to be maintained by the effective removal of water via the
ascending vasa recta, which have both a greater radius than the
descending vasa recta and are about twice as numerous. The
countercurrent exchange associated with the loops of Henle arising
from cortical nephrons offers an important "barrier" zone, which is
thought to facilitate solute trapping in and solvent exclusion from
the inner medulla, and thus helps to maintain the hyperosmolality in
this "compartment".

There are a number of other factors that control, alter, or
contribute to urine concentration. Medullary blood flow is complex, as
are the factors controlling it. Increased blood flow rates will
decrease the efficiency of countercurrent exchange in the outer
medulla, as a consequence of which the high osmotic gradient in the
inner medullary compartment will be "washed out", and urine will not
be concentrated. Diuresis is associated with increased blood flow
rates (Earley & Friedler, 1964, 1965; Chuang et al., 1978).

A unique feature of the vasa recta is their permeability to
macromolecules, a consequence of which is that the medulla contains a
large pool of albumin. The factors controlling the rapid turnover of
this milieu are poorly understood. It is generally assumed that
(together with the glycosaminoglycans) these proteins provide an
interstitial osmotic pressure that facilitates water reabsorption (see
Brenner & Rector (1986) for a fuller discussion and list of
references).

3.3.4.5 The interstitial cells

Interstitial cells occur in most organs. Three types of
interstitial cells have been described in the medulla of the rat
kidney (Bohman, 1980). Type I cells are the most abundant and
represent the typical renal medullary cells. Type 2 medullary
interstitial cells are generally round and lack lipid droplets, while
Type 3 cells correspond to the pericytes. Types 2 and 3 are sparsely
distributed and are often overlooked between the tubules, ducts, and
blood vessels. In the inner medulla, however, Type I cells are
numerous and especially prominent because they are set in a dense
matrix of glycosaminoglycans (previously referred to as
mucopolysaccharides or acidic mucopolysaccharides).

The medullary interstitial cells have been described by Moffat
(1979, 1981, 1982), Bohman (1980), and Maunsbach et al. (1980). The
number of cells and the amount of matrix substance occupies 10-20% of
the tissue volume in the outer medulla, and 40% near the apex of the
inner medulla (Bohman, 1980). The cells, which are arranged in a
regular pattern perpendicular to the tubules and vessels, are
irregular in shape and have many long slender processes. These come
into close contact with adjacent interstitial cells, capillaries, and
the limbs of the loop of Henle, but there is no such relationship with
the collecting ducts.

One of the most characteristic features associated with the Type
I cells is the presence of lipid inclusion droplets, which occupy at
least 2-4% of the total cell volume. The lipid content is largely
triglycerides, with variable amounts of cholesterol esters and
phospholipids. A number of conditions have been described where there
are marked changes in the size and number of lipid droplets. The
pathophysiological significance of these changes is difficult to
interpret because of varied experimental approaches, species
variation, and contradictory reports (Bohman, 1980).

3.4 Species, strain, and sex differences in renal structure and function

There are important differences between the renal structure of
animals and man that may have a direct effect on the interpretation of
toxicological data (Stolte & Alt 1980, 1982; Mudge 1985). The kidney
varies greatly in structure and function between different species and
strains, and there are also more