Polycystic Kidney Disease: An Unrecognized Emerging Infectious Disease?

[Guest guest]

http://www.cdc.gov/ncidod/eid/vol3no2/miller.htm

Polycystic Kidney Disease: An Unrecognized Emerging Infectious

Disease?

Marcia A. -Hjelle,* J. Hjelle,* ,*

R. Mayberry,† Ann Dombrink-Kurtzman,‡ W. ,‡

Deborah M. Nowak,* and S. Darras*

*University of Illinois College of Medicine at Peoria, Peoria,

Illinois, USA; †East Tennessee State University, City,

Tennessee, USA; and ‡U.S. Department of Agriculture, Agricultural

Research Service, Peoria, Illinois, USA

---------------------------------------------------------------------

-----------

Polycystic kidney disease (PKD) is one of the most common genetic

diseases in humans. We contend that it may be an emerging infectious

disease and/or microbial toxicosis in a vulnerable human

subpopulation. Use of a differential activation protocol for the

Limulus amebocyte lysate (LAL) assay showed bacterial endotoxin and

fungal (13)-ß-D-glucans in cyst fluids from human kidneys with PKD.

Fatty acid analysis of cyst fluid confirmed the presence of 3-

hydroxy fatty acids characteristic of endotoxin. Tissue and cyst

fluid from three PKD patients were examined for fungal components.

Serologic tests showed Fusarium, Aspergillus, and Candida antigens.

IgE, but not IgG, reactive with Fusarium and Candida were also

detected in cyst fluid. Fungal DNA was detected in kidney tissue and

cyst fluid from these three PKD patients, but not in healthy human

kidney tissue. We examine the intertwined nature of the actions of

endotoxin and fungal components, sphingolipid biology in PKD, the

structure of PKD gene products, infections, and integrity of gut

function to establish a mechanistic hypothesis for microbial

provocation of human cystic disease. Proof of this hypothesis will

require identification of the microbes and microbial components

involved and multifaceted studies of PKD cell biology.

Examining the hypothesis that polycystic kidney disease (PKD) is an

emerging infectious disease and/or microbial toxicosis in a

vulnerable population of humans must begin with a review of the

conceptual tools that relate disease etiology and progression to the

identification of microbes, their cellular components, and shed

toxins in affected persons (1). An emerging infectious disease can

be defined, in part, as an existing disease for which microbes are

newly recognized as a causative factor and/or as a factor

contributing to disease progression. The microbe may be 1) present

at the site of a lesion, 2) disseminated throughout the body, or 3)

localized to an anatomic site separate from the primary lesions. In

this third case, one or more toxins released by the microbe into

distribution fluids, usually blood, produce the pathologic effect

(s); this process resembles microbial toxicosis, where the patient

is exposed only to the toxin(s) present in the diet, environment,

and gut microflora. Distinguishing between an infectious disease and

microbial toxicosis is essential if the source of the toxin is to be

removed or reduced. Concurrent infection and microbial toxicosis

(e.g., endotoxicosis, mycotoxicosis) can also occur. Although

mycotoxicosis is commonly understood to refer to the absorption of

small organic fungal toxins, such as aflatoxin, fumonisin, or

trichothecene, detection of toxin in body fluids and tissues is

often difficult. Exposure to toxin may be episodic. For many known

and newly discovered microbial toxins, analytical methods pertinent

to the levels of toxins involved in acute and chronic disease,

especially in vulnerable subpopulations, need to be developed and

verified in human tissues and fluid. We have taken the view that the

presence of signature components of microbial genera/species that

produce toxins may indicate that all components of the microbe,

including its shed toxins, are also present. As a corollary,

absorption of the detectable microbial components, which are often

larger in molecular size than the classical mycotoxins and

endotoxins, should presume the absorption of other components and

toxins from this organism.

On its face, PKD would not appear to be an emerging infectious

disease. Classically viewed as an inherited disease that follows

Mendelian genetics, PKD in its autosomal dominant form affects

600,000 people in the United States at a rate of 1 in 400-1,000

persons; the autosomal recessive form occurs in 1 per 44,000 births.

An acquired form of PKD occurs in patients on renal dialysis. Animal

models of inherited and chemically induced PKD have been described

(2-4). The current consensus is that fundamental anomalies in cell

differentiation or maturation explain the array of anomalies in

cystic renal epithelium. Although cysts can sometimes be detected in

utero, loss of kidney function to the point of endstage renal

disease occurs by the sixth decade in 50% of autosomal-dominant PKD

patients. Despite substantial progress in molecular genetics studies

of PKD, the role of defective gene products in its causation and

progression to end-stage renal disease has remained elusive (3,5,6).

Indeed, additional factors have been proposed as being necessary for

progression of PKD to end-stage renal disease. PKD patients have

higher rates of renal and urinary tract infections and higher rates

of illness and death from infection in general than healthy persons

(7,8). Since these infections in otherwise-healthy-persons do not

lead to PKD, PKD patients could be exhibiting heightened

vulnerability to infection and sensitivity to microbial cell

components and toxins. How such sensitivity and vulnerability might

occur is unknown. The disease does not appear to involve an

immunocompromised state. Thus, alternative possibilities need to be

considered, such as altered colonization of the uroepithelium and

altered bowel structure and colonization.

Microbes and Microbial Components

Werder et al. (9) performed perhaps the critical experiment in

establishing a pathogenic role for microbes in PKD. Using

genetically cystic mice, they found that mice grown under germ-free

conditions survived substantially longer than cystic littermates

grown under ambient conditions.

Gardner et al. (10,11) extended these observations when they

reported that rats fed a chemical cystogen also had lower rates of

renal cystogenesis when raised under germ-free conditions; moreover,

coadministering the ubiquitous cell wall component of gram-negative

bacteria, lipopolysaccharides (LPS), and the chemical cystogen

produced higher rates of cyst formation than using cystogen alone.

Thus, LPS was considered a provocateur of the underlying genetic (or

chemically induced) susceptibility to renal cystogenesis. From this

line of research has emerged the possibility that LPS, either alone

or in combination with other microbial or environmental factors,

promote disease progression, and removal of such provocateurs would

slow the progression of PKD to end-state renal disease.

Endotoxin is the mixture of LPS and other bacterial cell wall

proteins and constituents that is found in bacterial infections

(12). LPS is isolated by solvent extraction of bacterial cell walls;

its chemical purity may vary across preparations. The portion of LPS

responsible for most of its known biological activities is Lipid A;

the carbohydrate side chains also elicit responses in humans (13).

The chemical composition of LPS depends on the genera and species of

bacteria from which it is isolated (13,14). As knowledge of the

structure and biological activities of LPS has increased and merged

with the infectious disease vocabulary based on endotoxin, these

terms are sometimes used interchangeably. For example, the highly

purified LPS used as positive reference material in the Limulus

amebocyte lysate (LAL) assay is called Control Standard Endotoxin.

Even though interchanging the terms LPS and endotoxin may promote

appreciation of microbial involvement in disease, we prefer the term

endotoxin in discussing disease because it respects the structural

and pharmacologic heterogeneity of LPS and mixtures of other

microbial components present in vivo.

When et al. (15) measured putative endotoxin levels by the

classic LAL assay in culture-negative urine samples of male PKD

patients with no clinically apparent infection, 80% contained

detectable LAL-positive material, while the urine samples of healthy

male volunteers did not. In cyst fluid from PKD patients, Gardner et

al. (16) reported LAL-positive material and cytokines typically

induced by LPS. The origins of the renal and urinary endotoxin were

not known; possible sources include the gut microflora, occult

urogenital infections, cryptic colonizations, and abnormal handling

by the liver of the normal daily load of absorbed endotoxin.

Enhanced exposure to endotoxin in the PKD patient is important since

endotoxin plays a fundamental role in human health and disease. In

low doses, endotoxin enhances the immune system, but in larger

amounts it is lethal. In combination with drugs and other

substances, it can cause tissue damage and severe disease; examples

of endotoxin synergy with other substances resulting in disease

include alcohol in cirrhosis, aspirin in Reyes syndrome, and

trichothecene T-2 toxin (a mycotoxin from Fusarium) in

gastrointestinal tract damage (17). Endotoxin can also promote the

translocation of bacteria from the gut into the blood (18). In

susceptible persons, chronic exposure to endotoxin has been linked

to rheumatoid arthritis (19); tentative links with atherosclerosis

have also been reported (20). Although suggestive of endotoxin in

PKD, the LAL methods used above were not specific for endotoxin,

since they were susceptible to false positive and/or negative

results.

Bacterial Endotoxin in Human PKD Renal Cysts

In this report, we describe our recent findings concerning 1)

endotoxin levels recorded after recent improvements in LAL methods,

2) analysis of fatty acids specific for bacterial endotoxin, and 3)

the presence of other microbial components in human cyst fluid. The

LAL assay (Figure 1) is the classic method used to detect the low

levels of endotoxin in biologic fluids and pharmaceuticals.

Both endotoxin (in eight of eight patients) and (13)-ß-D-glucans (in

two of eight patients) were detected when methods based on

differential activation of the two Limulus pathways were applied to

cyst fluids from six autosomal-dominant PKD patients, one autosomal-

recessive PKD patient, and a patient with simple cysts, where 200 ml

from a single simple cyst were recovered (Table 1). None of these

patients exhibited clinical evidence of infection, had been on

hemodialysis, or received human immunologic products before

nephrectomy, which could have accounted for ß-D-glucan reactivity

(26). For six patients (four autosomal dominant, one autosomal

recessive, and one with simple cysts), only endotoxin was detected

in cyst fluids; gel clot end points for both the LAL and

LAL+laminarin assays were equal for each fluid tested, indicating

that the ß-D-glucan stimulated pathway was not activated. When

exposed to polymyxin B, LAL-positive cyst fluids became negative.

This is consistent with the conformational changes that occur when

this antibiotic binds to Lipid A to form a stoichiometric complex

that is inactive in the assay (25). In contrast, kidney tissue from

one male patient (donor 4) and one female patient (donor 5) yielded

cyst fluids that contained separately endotoxin or ß-D-glucan;

however, no single fluid contained both substances (Table 1). Fluids

that were LAL positive in the presence or absence of polymyxin B,

but negative in the presence of laminarin are consistent with the

detection of ß-D-glucan. Donor 6 had been on peritoneal dialysis for

slightly more than 1 year before nephrectomy. Although cyst fluids

were all initially LAL negative, when the incubation time of the LAL

assay was increased from 1 to 2 hours, 4 of 13 cyst fluids were

endotoxin positive, albeit at much lower concentrations than the

fluids from patients not on dialysis. None of the cyst fluids from

donor 6 were positive for ß-D-glucan after longer incubations.

Longer incubation times might also have produced LAL-positive

material in LAL-negative cyst fluids from the other donors;

therefore, we view the percentage of LAL-positive cyst fluids for

each patient as a minimum value.

3)-ß-D-glucans bind to a different component, Factor G, in the

Limulus assay reagent, thereby leading to its activation and

subsequent cascade of reactions to gel formation. The addition of

laminarin, an inhibitor of glucan binding to Factor G, to Limulus

assay reagent blocks reactivity of samples containing glucan (21).

Lipid A binds to and activates Factor C resulting in a cascade of

enzymatic reactions leading to gel clot formation, the positive

endpoint (22). Also present in the classic LAL reagents is Factor G,

which is responsible for a side cascade (i.e., the alternative

pathway). Factor G is stimulated by (13)-ß-D-glucans, associated

primarily with fungal cell walls (23). Addition of laminarin, an

inhibitor of glucan binding to Factor G (24), permits a more

specific measurement of endotoxin-induced gel clot formation and

provides a means of screening for (13)-ß-D-glucans in biological

samples, when used in a differential activation protocol with other

inhibitors and activators. As two internal controls, polymyxin B can

block the LAL reactivity of endotoxin (25), and known quantities of

Control Standard Endotoxin can be added to duplicate samples to

detect inhibition of the assay.

Table 1. Limulus assay results from human kidney cyst fluids

---------------------------------------------------------------------

-----------

Donor No. Patient data No. Cysts

Tested Cysts ET positive (%):

Endotoxin units/mla Cysts ß-D-glucan

positive (%): pg/ml

---------------------------------------------------------------------

-----------

1 ADb 45 yr F 5 3 (60%): 0.48 EU/ml ndc

2 AD 52 yr F 6 4 (67%): 1.71 EU/ml nd

3 AD 48 yr M Ld 9 5 (56%): 1.45 EU/ml nd

Re 11 6 (55%): 2.27 EU/ml nd

4 AD 41 yr F 16 3 (19%): 0.88 EU/ml 3 (19%): 120 pg/ml

5 AD 37 yr M 11 2 (18%): 3.84 EU/ml 6 (55%): 106 pg/ml

6 PDf AD 43 yr F 13 4 (31%): 0.26 EU/ml nd

7 ARg 5 wk M pool(8) 3.84 EU/ml nd

8 SCh 48 yr F 1(200ml) 3.84 EU/ml nd

---------------------------------------------------------------------

-----------

aMean EU/ml or pg (13)ßD-glucan/ml

bAutosomal-dominant polycystic kidney disease (PKD)

cNot detected

dLeft kidney

eRight kidney

fPeritoneal dialysis

gAutosomal recessive PKD

hSimple kidney cyst (not PKD)

Kidneys were from seven patients who had received or were awaiting

renal allografts. Cyst fluid was obtained ex vivo from both surface

(cortex) and sub-surface (medullary) cysts by aseptic aspiration

from seven excised kidneys (post-nephrectomy) of PKD patients;

simple cyst fluid was recovered by radiologically guided needle

aspiration. All kidneys and cyst fluids were collected under an

approved Institutional Review Board protocol. The mean number of

cysts aspirated per kidney was 10 (range 1-21); two kidneys were

obtained from one of the patients with both being used for studies.

Fluid from individual cysts in each kidney were collected

separately, except for the autosomalrecessive PKD patient in whom

cyst volumes were too small (~ 0.1 ml) for individual testing.

Therefore, fluid from eight cysts in close proximity were pooled.

All procedures used pyrogen-free materials in the collection,

handling, and processing of kidneys and cyst fluids and preparation

of commodities and reagents for the Limulus assay. Depyrogenation

was carried out at 250°C for 4 hours, which eliminates both

endotoxin and (13)-ß-D glucans (27). Cyst fluids were stored at -70°

C. Assessment parameters of cysts included color and size; for

fluids, color, turbidity, pH, mg protein/ml, specific gravity, and

blood. A commercial dipstick was used to detect and semiquantitate

cyst fluid pH, specific gravity, and blood (Ames 10 SG Multistix,

Miles, Inc., Elkhart, IN, USA). Blood sensitivity was 0.015-0.062

mg/dL hemoglobin, equivalent to 5-20 intact red blood cells per

microliter. Results were recorded as 1-4+. For specific gravity,

determinations permitted ranged from 1.000-1.030; for pH, 5.0-9.0.

Protein was by the method of Lowry et al. (28).

The LAL gel clot end point assay (22) was performed according to the

manufacturer\'s instructions ( River ENDOSAFE, ton,

SC). The mechanism of the LAL assay is illustrated in Figure 1. To

quantify endotoxin, undiluted samples and serial twofold dilutions

through 1:64 were tested. To detect false positives due to

activation of the alternate (13)-ß-DG pathway, LAL assay reagent

fortified with the glucan inhibitor laminarin was used (23).

Laminarin was courtesy of Dr. J. , River ENDOSAFE,

ton, SC. Laminarin had been rendered endotoxin free by

treatment with 0.2 N NaOH at 50°-60°C for 6 hours; treatment did not

affect activity of laminarin in the LAL assay (23). The standard gel

clot end point method was used, except laminarin was added to the

LAL assay reagent (23). Minimum sensitivity of both assays was 0.03-

0.06 Endotoxin Units (EU)/ml (3-6 pg control standard endotoxin,

CSE; 10 EU/ng of EC-5 (E.coli O133) U.S. Standard endotoxin; for ß-

DG, ³10 pg/ml (29).

Known endotoxin-positive (tap water) and -negative (pyrogen-free

water) samples were run as controls along with a CSE curve. The

endotoxin concentration was estimated by multiplying the maximum

dilution producing a positive test against the sensitivity of lysate

that was verified daily. The content of ß-DG in cyst fluid was

estimated by the difference in titers between the endotoxin specific

assay (LAL+laminarin) and the conventional LAL assay (30). Portions

of all samples were additionally spiked with 0.25 EU CSE and tested

for inhibitory substances. If inhibition was observed in the LAL

assays, samples were boiled for 5 minutes, cooled to room

temperature, and retested. Samples were incubated, in some cases

after boiling, for 30 min at room temperature with polymyxin B (5

mg/assay) before LAL testing. Polymyxin B inhibits the LPS-activated

pathway via Factor C by its direct binding to LPS, which yields a

nonreactive complex (25). An alternative approach was to use 20%

dimethyl sulfoxide (DMSO) to inhibit Factor C directly (23); DMSO

was not used to permit detection of nonendotoxin, but LAL-reactive

materials that were not inactivated by binding to polymyxin B.

However, in our system DMSO did completely block the LAL reaction in

the presence of CSE.

After centrifugation of the cyst fluid, the LAL-positive material

was located only in the supernate for autosomal-recessive- and

simple cyst-derived fluids. For autosomal dominant-derived fluids,

the reactive material was located in the pellet. Thus,

centrifugation of fluids to remove particulate material before the

LAL assay may influence results.

LAL assay inhibition was detected in nearly two-thirds of 73 cyst

fluids tested, including autosomal recessive and simple cyst fluids.

Dilution of cyst fluid through 1:16 was often required to overcome

the inhibitory effect in unboiled specimens. Boiling of entire

fluids or supernates and pellets for 5 minutes eliminated LAL assay

inhibition in virtually all specimens at all dilutions. Exceptions

were some undiluted and 1:2 dilutions of chocolate-colored cyst

fluids, where inhibition was not eliminated in both test and spiked

specimens of autosomal dominant fluids.

The concentrations of endotoxin-specific material in autosomal

dominant cyst fluids was 0.12 to 3.84 EU/ml, with substantial levels

of endotoxin (3.84 EU/ml) also found in a pool of eight cysts from

one autosomal recessive kidney and simple cyst fluid from one donor.

The mean concentration of endotoxin across all of the auto-somal

dominant fluids tested (including those that were LAL negative) was

0.65 EU/ml. Using 10 EU/ng as a conversion factor yields 65 pg

endotoxin/ml cyst fluid; normal plasma levels of endotoxin are <4-5

pg/ml (31). Intravenous injection of 5 EU/kg body weight (350 EU/70

kg) can induce shock in the average adult male (21). We estimate

that a single cystic kidney in an adult male (3-5 kg kidney weight

of which 33% is cyst fluid) contains 648 to 1,080 EU/kidney or about

two lethal doses of endotoxin per kidney. Unexplained fever and

flank pain in PKD patients have been attributed to release of IL-1

from rupture or hemorrhage of cysts (27). On the bases of the high

levels of endotoxin observed in this study and the high sensitivity

of humans to endotoxin, we propose that the release of endotoxin

from the cyst into the peritoneum or blood may be an important

initiator of a cascade of biologic events after leakage or rupture

of renal cysts.

For one donor (donor 3) both kidneys were available for cyst fluid

analysis (nine left and 11 right kidney cysts, respectively). When

the frequency of LAL-positive fluids (endotoxin) was compared, no

significant difference was found (Table 1). However, a threefold

difference in the frequency of inhibitor detection was observed in

left kidney cyst fluids compared with the right kidney (90% vs. 31%,

respectively). Thus, LAL testing of fluids for endotoxin requires

vigilance for both false negative and positive materials.

Signature Bacterial Fatty Acids

If endotoxin is present in renal cysts, fatty acids unique to

endotoxin can be used to confirm its presence. The Lipid A region of

LPS contains on average four 3-hydroxy (3-OH) acyl groups of various

chain lengths (13,14). Acid hydrolysis of LPS releases the 3-OH

fatty acids and other fatty acids. Diverse bacterial genera exhibit

characteristic ratios of fatty acids of various chain lengths and

patterns of hydroxylation; compilations of such signature fatty acid

profiles are available for many genera (14). Initially, aliquots of

five cyst fluids were analyzed in a single blind manner by gas

chromatography-mass spectrometry as described by Mayberry and Lane

(32). 3-Hydroxy (3-OH) fatty acids of nC:12:0 and nC:14:0 carbon

length were detected but not quantified in the three cyst fluids

that were positive in the endotoxin specific LAL assay; they were

not detected in the two cyst fluids that were LAL negative. Thus,

complete concordance of LAL reactivity and signature fatty acid

analysis was observed. 3-OH fatty acids (C:14, C:16) were also

reported in LAL-positive cyst fluids by a separate reference

laboratory (Microbiological Insights, Inc., Knoxville, TN).

Additional chemical analyses of cyst fluid are required before a

classic signature fatty acid profile and linkage to one or more

bacterial genera are possible. Also, we cannot discount the

possibility that 3-OH fatty acids released during degradation of

endotoxin were incorporated into mammalian sphingolipids that were

positive in the LAL assay, thereby accounting for both the presence

of such fatty acids and LAL reactivity in cyst fluids.

(13)-ß-D-Glucans (ß-DG)

In addition to endotoxin, (13)-ß-D-glucans were identified in cyst

fluids from two patients by differential activation of the LAL assay

(Table 1). The range of ß-DG reactive material in cyst fluids was

estimated at 40 to 160 pg/ml. In plasma of healthy persons, levels

of ß-DG are lower than 6.9 pg/ml (26). ß-DG, a ubiquitous

constituent of filamentous fungal and yeast cell walls (33), is

exceeded only by endotoxin in its reactivity in the LAL assay;

glucans lacking a 13 linkage are not LAL reactive (34), nor are

mannans, dextrans, and cellulose (24,26). Although diverse forms of

glucans are components of fungal cell walls, those with (13)-ß

linkages are the most potent in causing hypersensitivity pneumonitis

and other severe pulmonary illnesses (35). Although ß-DGs are

indicative of fungal cell walls, their occurrence alone does not

identify the genera and species of the fungus that produced it. To

confirm the presence of fungal components and determine the source

of ß-DG, serologic tests were performed on three ß-DG positive and

two ß-DG negative cyst fluids from three autosomal-dominant PKD

patients (Table 2). Cyst fluid from donor 6 that was negative for

LAL-reactive material was also free of detectable fungal antigens

and anti- from both kidneys containing ß-DG-positive material

(donors 4 and 5; Table 1) were also positive for fungal antigens.

Fluid from donor 5 also exhibited fungal antibodies; fluid from

donor 4 was not tested for antibodies to fungi.

Table 2. Serologic results of five cyst fluids from three autosomal-

dominant PKD patients tested for fungal antigens or antibodies to

fungal components

---------------------------------------------------------------------

-----------

Donor;Cyst No. LALa Findings Serologic Findings

---------------------------------------------------------------------

-----------

D6;1 Neg. Etb;Neg. ß-DGc Antigens not detected

D6;2 Neg. ET;Neg. ß-DG IgE and IgG reactive with fungi were not

detected

D4;3 Neg. ET;Pos. ß-DG Antigens: Fusarium solani at 1:2 dilution;

Aspergillus galactomannan at >50 ng/ml

D5;4 Neg. ET;Pos. ß-DG Antigens: Aspergillus galactomannan at >50

ng/ml;

Candida albicans serotype A mannan at 10 ng/ml

D5;5 Neg. ET;Pos. ß-DG IgE reactive with Fusarium moniliforme; C.

albicans

(weak positive)

---------------------------------------------------------------------

-----------

aLimulus amebocyte lysate assay

bEndotoxin

cß-D-glucan

Fungal serology was performed on three cyst fluids from donors 4 and

5; (Table 1) with ß-DG positivity and two cyst fluids from donor 6,

which were negative for both endotoxin and ß-DG after extended

incubation in the LAL assay. Fluids were tested for antibody to

Penicillium notatum (Penicillium chrysogenum) and Penicillium

frequentans, Aspergillus fumigatus, Candida albicans (yeast), and

Fusarium moniliforme. The Pharmacia CAP systems RAST FEIA (IgE) and

IgG FEIA were employed (Pharmacia & Upjohn Diagnostics, Kalamazoo,

MI, USA). The tests were modified by substituting cyst fluid for

plasma; interference of fluid with the test was not observed. Tests

to detect fungal antigens were performed by the Centers for Disease

Control and Prevention, Atlanta, GA. To detect Fusarium sp. antigen,

a modified aspergillosis microimmunodiffusion test (36) was used

with experimental rabbit anti-Fusarium solani antibody; kidney cyst

fluid was substituted for plasma. A double-antibody sandwich enzyme

immunoassay was used to detect C. albicans serotype A mannan

concentrations in cyst fluids; specificity of this test in sera is

reported as 100% (37). An experimental ELISA inhibition assay was

used to detect Aspergillus galactomannan ( Hurst, unpub.

protocol) Standard serum reference curves were used for

extrapolating quantities of C. albicans mannan and Aspergillus

galactomannan in cyst fluids. However, these tests were not designed

for cyst fluids, but for serum antigen detection, and must,

therefore, be viewed as estimated quantities.

Fungal serologic tests provided insights into potential sources of ß-

DG. Initial measurements showed Fusarium solani antigen. These

findings suggest the antigen to be from F. solani or a shared

antigen present in other Fusarium species (L. Kaufman, pers. comm.).

IgE (but not IgG) antibody to Fusarium moniliforme was also

detected. Aspergillus galactomannan antigen and Candida albicans

serotype A. mannan were present in selected cyst fluids. Cross-

reactivity of Fusarium with Aspergillus and Penicillium is unlikely

as 1) the Fusarium antibody (prepared against the mycelium) was

cross-absorbed with Aspergillus, and 2) the immunodominant group of

Aspergillus and Penicillium species, galactomannan with a

galactofuranosyl moiety (38), is absent in Fusarium species (39). As

the serologic tests for Fusarium and Aspergillus in cyst fluids are

experimental and have not been evaluated clinically, these data are

indirect presumptive indication of the presence of these fungal

antigens. The paucity of fungal serologic and chemical methods for

detection and identification of emerging fungal pathogens in human

tissues and body fluids and fungal components in the human diet has

been noted by others.

Also detected were IgE antibodies to Candida. IgE antibodies to both

Fusarium and Candida in cyst fluid suggest recruitment of

immunologic defenses against fungi. The site of production of these

IgEs and route of entry into the cyst are unknown; a

hypersensitivity reaction cannot be discounted. In human and

experimental PKD, the severity of cystic disease correlates with the

numbers of circulating neutrophils; neutrophilia is related to

exposure to endotoxin and cytokines, both of which have been

reported in cyst fluids (16). Our finding of fungal antigens and

antibodies raises the additional possibility of leukocyte activation

in PKD by ß-DG and mannan released from fungi, as ß-DG and mannan

have been reported to stimulate cytokine production (40).

Serologic testing is frequently used to detect fungal infection.

Cyst fluids collected from PKD patients at sequential times during

progressive stages of the disease were not feasible. Therefore,

quantitative and/or qualitative changes in antigen or antibody

titers could not be used to determine active fungal infections in

these patients. Although antigens of Aspergillus or Fusarium species

are generally interpreted as apparent or covert infection, another

possibility is the absorption and distribution of fungal components

independent of infection (i.e., mycotoxicosis versus infection).

While Aspergillus enters primarily by inhalation, this route does

not appear to be the predominant source of Fusarium; fewer than 1%

of air samples contained Fusarium species (41). Fumonisin from

Fusarium is present in grains, rice, and corn consumed by humans

(42), with regional variations in levels and the type and processing

of dietary product. Because of their occurrence in the food supply

and their effect on experimental animals, it has been proposed that

fumonisins may contribute to kidney disease in humans (43).

ß-D-Glucans and mannans are shed by fungi during growth (26,39) and

thus, like bacterial endotoxin, can potentially be distributed

through blood and lymph. Diverticular disease and other anomalies in

the structural and functional integrity of the gut may occur in up

to 80% of PKD patients (2,44). In addition to diminished barriers to

the absorption of gut-derived endotoxin, the outpouchings

(diverticula) become overgrown by minor subpopulations of gut

microflora (45), thereby generating increased quantities of unusual

mixtures of potentially absorbable microbial components.

Diverticular disease (44) and acquired renal cystic disease (46)

occur in patients on hemo- and peritoneal dialysis. To complete the

symmetry of this line of reasoning, outpouchings of the renal tubule

in PKD are described as both cysts and diverticula (2). Direct

measurement of intracyst pressure and elasticity of the basal lamina

have led to the rejection of the obstruction hypothesis (i.e., a

physical balloon inflation) of cystogenesis in favor of a cell-

mediated restructuring of the basal lamina coupled with electrolyte

transport into the cyst to expand intracyst volume (47). Although

diverticular disease is thought to be a pressure-driven lesion, gut

diverticula in PKD and dialysis patients may also involve a cellular

lesion further promoted by pressure. Thus, diverticula in kidney and

gut are associated with microbial components. It is not known if the

processes of renal cystogenesis, formation of gut diverticula, and

the walling off of infecting material are all facets of the same

defense response in humans. Endotoxin in relatively high

concentrations was found in fluid from a simple cystic kidney (Table

1); intestinal or biliary obstruction and urinary tract infections

have been associated with simple kidney cysts (2). Although

endotoxin has been proposed as being associated with \ " high sodium

cyst fluids\ " (47), we observed endotoxin in cyst fluids of low and

high sodium content (data not shown).

Relevant to our study is the report of the capacity of ß-DG to prime

cell systems, which results in sensitization to bacterial endotoxin

and infection (48), an example of ß-DG-endotoxin enhancement; ß-DG

and endotoxin also demonstrate a strong synergistic effect on

macrophages (49). In addition, there have been reports of synergy

between mycotoxin and endotoxin (50).

Detection of Fungal DNA

To identify fungal DNA as evidence of past or current fungal

infection and/or the absorption and distribution of fungal

components to kidney cysts, polymerase chain reaction (PCR) methods

were used to amplify and thus detect fungal DNA in PKD cyst fluids

and kidney tissue. Six cyst fluids from three patients and two

samples of kidney tissue from two autosomal-dominant PKD patients

were tested; all were positive for fungal DNA. A sample of normal

human kidney was negative for fungal DNA (Figure 2 A-C). Because

culture confirmation was not achieved from the cyst fluids, expanded

studies with species-specific probes are warranted.

Figure 2. Amplification results of normal and PKD kidney tissue and

cyst fluids with universal fungal primers ITS 1 and NL 4. 2A: DNA

from healthy human kidney tissue diluted 1:10, 1:100 and 1:1,000

(lanes 1-3); control fungal DNA, A. tamarii (lane 4); negative

control (lane 5); 1 kb ladder (lane 6); arrow indicates migration

front. 2B (NL 4) and 2C (ITS 1): two cyst fluids, donor 6, negative

for detectable endotoxin and ß-DG (lanes 1 and 5); two cyst fluids,

donor 4, positive for ß-DG (lanes 2 and 3) and positive for Fusarium

solani antigen (lane 3); two cyst fluids, donor 5, positive for ß-DG

(lanes 4 and 6); two PKD kidney tissues, donor 5 (lane 7) and donor

4 (lane 8); negative control (lane 9). Large arrows in 2B and 2C

point to 560 bp molecular weight marker; small arrows point to

product bands.

Polymerase Chain Reaction

Specimens for PCR included 1) normal human kidney tissue (0.46 g)

from a 7-year-old girl who died of a head injury; the kidney was not

transplantable because of the presence of a hematoma but was

otherwise normal; the section used for PCR excluded the area of

hematoma. 2) 0.5 g of cystic tissue, devoid of cyst fluid, from each

of two autosomal-dominant PKD patients (donors 4 and 5, Table 1). 3)

multiple cyst fluids (0.5-1.0 ml each; donors 4, 5, and 6). The

positive control consisted of DNA extracted from A. tamarii (NRRL

26010) plus primer. The CTAB extraction method for fungal genomic

DNA was employed. The universal oligonucleotide primer pair ITS 1

and NL4 for fungi, as previously described (51), was used; NL 4 is

the reverse primer. PCR master mix (37) with 1.5 mM MgCl2 and

primers in a final reaction volume of 100 ml was subjected to the

following thermal cycler parameters (Perkin Elmer Thermo Cycler 1):

96°C—30 sec; 53°C—30 sec and 72°C—30 sec for 40 cycles, followed by

a final 7 min extension at 72°C and soak at 4°C. The presence of the

expected molecular weight PCR products (600 bp) was confirmed by

ethidium bromide staining after separation on a 1.0 % agarose gel.

The amplified products were compared visually for similarity on the

basis of the presence or absence of bands; variations in band

intensity were not considered.

A-C represent the banding patterns obtained by agarose gel

electrophoresis of healthy kidney tissue, cystic kidney tissues, and

cyst fluids following PCR amplification with the universal fungal

primer pair, ITS 1 and NL 4. Amplified fungal products were not

detected in the negative PCR controls (2A, lane 5; 2B and C, lanes

9) or normal human kidney tissue (2A, lanes 1-3). In marked

contrast, distinct bands of the predicted size of each fungal primer

used ( ~ 600 bp; 45) were detected in all cystic kidney tissues and

fluids examined from the three patients (2B-C); minor fragments were

not detected. The migration patterns were consistent with the

positive fungal DNA control (2A, lane 4).

Although fungal DNA might have been anticipated in tissues and

fluids from PKD kidneys showing ß-DG or positive serologic findings,

fungal DNA was also found in PKD cyst fluid from donor 6 (2B and C:

Lanes 1 and 5), which lacked these components. The ß-DG positive

cyst fluids tested from donors 4 and 5 are in Lanes 2 and 3 and 4

and 6, respectively. The fluid depicted in Lane 3 also had positive

serologic results for F. solani antigen; serologic testing was not

done on the other fluids assayed by PCR. Each cyst fluid must be

considered an independent sample, requiring direct measurement;

assumption of cyst content on the basis of other fluids within the

same kidney is not valid (16). Fungal DNA was also detected in

kidney tissue from donors 4 and 5 (Figure 2B and C; Lanes 8 and 7).

Differences were noted between the two primers used; ITS 1 yielded

amplified products in all PKD samples while NL 4 amplified less

well, being more concordant with the detection of ß-DG. The

amplicons are presumptive evidence of fungal DNA.

Despite substantial effort, we have not been able to culture either

bacteria or fungi from PKD cyst fluids by axenic methods, but fungi

were detected in cultured epithelial cells from PKD kidney tissue

(see below); methods appropriate to culture L-forms or other cell-

wall-defective microbes were not performed. Gram stains of cyst

fluid supernates and pellets did not show intact bacteria. Electron

microscopy also did not show intact bacteria, but occasionally, a

field consistent with fungal cell walls was observed (not shown);

intact fungi were not observed. Nonetheless, it was only from PKD

kidneys where ß-DG was identified in cyst fluid that fungal

organisms were frequently isolated from PKD epithelial cell

cultures. Other human and animal cells isolated and propagated in

the laboratory by the same reagents and work spaces were free of

fungal growth. The fungi were identified as belonging to the genera

Paecilomyces and a new species of Penicillium (manuscript in

preparation). Fungi have been reported, albeit infrequently, as

etiologic agents in renal infections in PKD (8). Given all of the

above, it is possible that viable fungi were present in the PKD

renal tissue, but only their remnants were in the cyst fluid.

Emerging human awareness of infectious disease may well describe our

experience in this study of PKD.

Infectious Disease, Microbial Toxicosis, and Sphingolipid Biology

Emerging knowledge of sphingolipid biology and PKD\'s vulnerability

to microbial toxins offer an opportunity for a fresh look at PKD. As

Spiegel and Merrill (52) have noted, sphingolipids fall into two

broad categories, both of which are altered in PKD (53,54). Complex

sphingolipids (i.e., ceramide with a carbohydrate or phosphocholine

head group on carbon-1) interact with growth factor receptors and

the extracellular matrix and adjacent cells and act as binding sites

for gram-positive and -negative bacteria and microbial toxins. In

the second category of sphingolipids, ceramide sphingoid bases

(i.e., sphingosine, sphinganine, phytosphingosine) and their 1-

phosphates modify the activity of protein kinases and phosphatases,

ion transporters, and a growing number of signal transduction

processes. However, the dynamics of human sphingolipid biology occur

within the context of a microbe-dominated environment.

Thus, we propose a third category called \ " microsphingoids,\ "

sphingolipid-like molecules of microbial origin that mimic or

antagonize the actions or metabolism of human sphingolipids.

Examples include, but are not limited to, various mycotoxins (42,55-

57), bacterial sphingolipids (58), and endotoxin (59,60). In this

report, we have shown the presence of endotoxin and fungal antigens

or antibodies to at least Fusarium, Aspergillus, and Candida in

human PKD tissues and fluids; from human PKD cells in vitro we have

encountered Paecilomyces and Penicillium. These fungi produce

mycotoxins that inhibit multiple enzymes required for sphingolipid

biosynthesis (55; Figure 3) and alter the activity of various

elements of signal transduction cascades, such as protein

phosphatases, calmodulin, and GTP-binding proteins (56,61). Such

mycotoxins are also found in the human diet, which is itself an

emerging concern in food safety (42,43,55). Rietschel et al. (13)

noted the similarity of LPS to glycosphingolipids. and

Kolesnick (59) have reviewed the structural similarity of the Lipid

A region of bacterial LPS with ceramide and the ability of LPS to

mimic the ceramide-induced alterations in protein kinase and

phosphatase activities. Endotoxin is also reported to alter the

structure of mammalian sphingolipids (62) and to initiate cytokine-

mediated cascades that generate ceramide, sphingosine-1-phosphate,

and lysosphingolipids, all of which exhibit biologic activity

(13,63). Not to be ignored are bacterial sphingolipids formed by

genera such as Bacteriodes, which represent approximately 30% of the

gut microflora (58). The role of bacterial sphingolipids and their

metabolites in human biology and their bioavailability in disease

are poorly understood. The term \ " microsphingoid\ " is intended to

highlight the contribution of microbes to sphingolipid biology in

human health and disease.

Figure 3. Sites of impact of endotoxin and mycotoxins on

sphingolipid metabolism. In PKD, renal sphingolipid formation is

altered. Such compromised sphingolipid pathways would be expected to

be vulnerable to these highly potent microbial toxins, especially

during chronic exposure within renal cysts.

Could pertubations in sphingolipid biology caused by genetic

anomalies and/or \ " microsphingoids\ " account for both infantile

autosomal recessive and adult onset forms of kidney cystic disease?

Hannun (64) has reviewed the role of sphingolipids as \ " biostats\ "

that regulate diverse cellular programs executed in response to

various stimuli. Such biostatic regulation could account for both

acute and chronic changes in cellular behavior and differentiation

in the kidney (65). Calvet et al. (2,66) have proposed two models to

explain the immaturity or dedifferentiated state of epithelial cells

found in hereditary and acquired cystic kidneys: In infantile cystic

disease, the epithelial cells never reach terminal differentiation

and are trapped in an immature state, while in adult forms of cystic

disease, toxin-induced injury to an initially mature renal

epithelium is followed by inability of the epithelium to recover to

a fully differentiated state. In genetically cystic cpk/cpk mice (a

model for autosomal recessive PKD), Deshmukh et al. (53) reported

altered levels of ceramide and complex glycosphingolipids in kidney

tissue from cystic mice, but not in their phenotypically normal

littermates. Lower levels of ceramide and sulfatide, but higher

levels of glucosyl- and lacto-sylceramide and ganglioside GM3, were

measured. Our research in infantile and adult human PKD kidney

tissue and cyst fluids showed no detectable free sphinganine, the

primary sphingoid base formed during de novo biosynthesis of

mammalian sphingolipids (54). Thus, anomalies in sphingolipid

biology exist in cystic kidney tissue.

It is difficult to separate concepts of sphingolipid biology from

considerations of infectious disease, microbial components, and

\ " microsphingoids\ " in PKD. Potential consequences of alterations in

glycosphingolipids on the surface of PKD cells include enhanced

microbial colonization due to the availability of complex

sphingolipids as binding sites. Binding of microbes or their

components to such sphingolipids may even promote cystogenesis, as

binding to complex sphingolipid has been reported to cause changes

in differentiation and morphology of cells in vitro (67). Altered

biosynthesis of sphingoid bases affects the ratios of sphinganine to

sphingosine with consequences to signal transduction (61) and may

even alter the antimicrobial environment, as sphingoid bases are

reported to have direct antifungal and antibacterial activity (68).

The levels of sphingosine in human cyst fluid (Hjelle, unpub.) are

comparable to the levels that induce a state of cytoresistance

(cellular dedifferentiation) in kidney cells in vitro (69). In

addition to products of mammalian sphingolipid metabolism, endotoxin

in relatively high amounts was found in cyst fluids from infantile

and adult forms of PKD and in simple cysts (Table 1). Could

\ " microsphingoids\ " or microbial toxins injure and then prevent

repair of renal epithelial cells? Data from diverse scientific

disciplines support this possibility.

LPS influences nephron formation (70) and renal cystogenesis (11).

Fumonisins are potent nephrotoxins (43,55), alter repair mechanisms

in kidney cells in vitro (71), and induce apoptosis (72,73). Renal

cysts are occasionally reported after chronic exposure of rodents to

fumonisins (74). Rates of programmed cell death are abnormal in PKD

kidney tissue (75,76); ceramide is a pivotal messenger in apoptosis

(64). In PKD, altered processing and sorting of cell membrane

proteins and secretory material occurs (2-4); sphingolipids

influence processing, sorting, and movement of membranes and ion

transporters (64,65), as does fumonisin in kidney cells in vitro

(77). In PKD, various electrolyte transport systems are altered (2).

cAMP-mediated electrolyte fluxes were proposed as important in cyst

formation (3). Fumonisins are reported to activate cAMP response

elements (78). Cyst fluid contains uncharacterized substances that

promote cystogenesis in vitro (3). Although LPS isolated from

Escherichia coli did not induce the full array of anticipated

cystogenic responses in kidney cells in vitro, the structure,

potency, and array of elicited biologic responses of endotoxin

depend on the genera and species of bacteria from which it was

isolated (13). Coupled with the presence of fungal components, cyst

fluid likely represents a complex mixture of \ " microsphingoids\ " and

toxins that may change over time with dynamic

consequences to cyst formation. The heterogeneity of cyst volume and

content of growth factors and cytokines in adult PKD has been noted

(3,16).

Concepts of infection and \ " microsphingoids\ " appear to converge

with the expanding knowledge of PKD gene products. The structures of

the PKD1 (polycystin) and PKD2 gene products share homology with a1E

subtype of voltage-activated, calcium channels (3,6) and a sea

urchin protein likely involved in calcium fluxes during

fertilization (79). LPS alters voltage-activated, calcium channels

(80) and osmoregulation (81) in mammalian cells. The PKD1 gene

product also contains regions of homology with ligand and receptor

domains putatively involved in binding to adjacent cells and

extracellular matrix (3); one such domain is similar to C type

lectins that can bind microbes (82). Tissues, including kidney and

gut, expressing polycystin in adult PKD, may exhibit a relatively

greater leakiness to normal molecular and particulate traffic, as

seen in kidney cysts (3) and susceptibility to diverticulosis

(3,44). Polycystin also shows homology with apoprotein from low

density lipoprotein. Because LPS and sphingolipids bind to and are

transported by serum lipoproteins, the low-density lipoprotein

binding site in polycystin may also enable accumulation and/or

transport of \ " microsphingoids.\ "

Secondary mutations in PKD genes appear to be required for clonal

cyst formation (3,5), as is a loss of renal tissue through

dysregulation of apoptosis (76). Alterations in the expression of

the bcl-2 gene product in mice results in polycystic kidneys and

dysregulation of apoptosis (83). Although infection increases

translocation of the bcl-2 gene in human lymphoid tissue (84), it is

not known if infection in PKD patients causes such a dysregulation

of bcl-2 in kidney tissue.

Regarding infection and sphingolipids, ceramide is emerging as a

pivotal molecule in the immune system (63) and fumonisins are

reported to alter immune function (85). It is ironic that our

findings potentially link Fusarium to PKD, as fumonisin is used as a

molecular tool to study sphingolipid metabolism and signal

trandsduction in PKD. Not to be overlooked are the classic bacterial

sphingomyelinases encountered during infection and released from gut

microflora (e.g., Staphylococcus aureus and Clostridium perfringens)

that can stimulate ceramide formation by hydrolysis of

sphingomyelin. Thus, the vulnerability of PKD patients to infection

may be related, in part, to anomalies in sphingolipid biology that

influence 1) \ " biostatic\ " mechanisms of cell regulation, 2) the

structure of the plasma membrane and the function of PKD gene

products, 3) the availability of glycosphingolipids as binding sites

for microbes and their components, 4) the bioavailability of

microbial components present in the gut, and 5) antimicrobial

defenses in general. Such vulnerability is likely influenced by

repeated courses of antimicrobial therapy that provide selection

pressure for colonization with a modified microflora. The extent to

which sphingolipid biology in PKD is influenced by genetic defects

rather than microbial factors is yet to be defined.

Anomalies caused by the PKD gene defect(s) alone cannot explain

cystogenesis. As shown by Werder et al. (9), raising genetically

cystic mice in a germ-free environment essentially eliminated

cystogenesis and increased survivorship to nearly 100% over that of

littermates raised in ambient environment for 18 months. Even in

cyst fluid from infantile PKD, relatively high levels of endotoxin

were found (Table 1). This suggests that microbial toxins are

available early in this disease. Prenatal exposures to toxins and

\ " microsphingoids\ " are unknown. Although our finding of fungal DNA

in eight of eight samples of autosomal-dominant PKD tissue and cyst

fluids examined suggests an intimate association of fungal exposure

to renal cysts, a contributing multifaceted microbial toxicosis

involving diet and gut microflora cannot be excluded. By itself, the

finding of microbial components at the site of lesion does not prove

a causal role for the microbe(s) in disease progression (1).

However, a working hypothesis can be formed on the basis of evidence

that microbes promote progression of the primary disease and

components of the microbes act on mammalian biology to cause effects

plausibly related to the known pathophysiology of the disease.

Although there is an established body of knowledge that PKD is a

genetic disorder, our data indicate that bacterial endotoxin, ß-DG,

and likely other microbial components are available within the

kidney to provoke cystogenesis. We have provided chemical and

advanced LAL assay-based evidence of bacterial endotoxin and ß-DG in

human PKD cyst fluids. From cyst fluids and PKD kidney tissue we

have provided evidence of fungal DNA and cell components by

serologic testing and PCR with universal fungal primers. We have

integrated these findings into a working hypothesis based on

emerging knowledge of PKD gene products, altered sphingolipid

metabolism in PKD, the effects of LPS on renal cystogenesis, the

mimicry of ceramide by LPS and the effects of mycotoxins on

mammalian sphingolipid biology and signal transduction, the

occurrence of infection in PKD, and the impact of altered gut

permeability and microbial colonization on progression of PKD. Is

PKD a genetic disease promoted by microbial influences? Tests of

this multifaceted hypothesis require awareness of a breadth of

issues drawn from numerous scientific disciplines. Identification of

the microbes and microbial components involved will require a

concerted analysis using highly sensitive and specific methods. As

awareness of the importance of sphingolipid biology in health and

disease grows, so will the appreciation that microbial influences

will need to be considered in pharmacologic studies that seek to

manipulate ceramide and complex sphingolipid biochemistry in

disease. The ubiquitous and highly potent bacterial endotoxin is

again one of the usual suspects examined as provocateur of disease;

in this case, endotoxin\'s modus operandi of coopting the signal

transduction machinery of ceramide to cause chronic disease may

ultimately be revealed.

Acknowledgments

This research was supported by grants and gifts from the Methodist

Medical Center Foundation; the Children\'s Miracle Network Telethon;

and the Campus Research Board, University of Illinois. We thank Dr.

Jim for his advice, counsel, and support during the endotoxin

experiments and preparation of the manuscript; Dr. Kerry O\'Donnell

for analysis of specimens by PCR for fungal DNA; Dr.

on (Candida mannan), Dr. Leo Kaufman (Fusarium antigen), and

Mr. Hurst (Aspergillus galactomannan) for fungal serologic

tests for antigens; and Dr. Hun-Chi Lin for fungal serologic tests

for antibodies. We also thank R. Lane and V. Acuff,

for the use of, and advice regarding, the gas-chromatograph-mass

spectrometer.

Dr. -Hjelle is professor of Microbiology and Diplomate,

American Board of Medical Microbiology.

References

Fredricks D, Relman D. Sequence-based identification of microbial

pathogens: a reconsideration of Koch\'s postulates. Clin Microbiol

Rev 1996;9:18-33.

ez RR, Grantham JJ. Polycystic kidney disease: Etiology,

pathogenesis, and treatment. Dis Mon 1995;41:698-765.

Grantham JJ. The etiology, pathogenesis, and treatment of autosomal

dominant polycystic kidney disease: recent advances. Am J Kid Dis

1996;28:788-803.

Carone FA, Bacallao R, Kanwar YS. The pathogenesis of polycystic

kidney disease. Histol Histophathol 1995;10:213-21.

Brasier JL, Henske EP. Loss of the polycystic kidney disease (PKD1)

region of chromosome 16p13 in renal cyst cells supports a loss-of-

function model for cyst pathogenesis. J Clin Invest. In press.

Mochizuki T, Wu G, Hayashi T, Xenophontos SL, Veldhuisen B, Saris

JJ, et al. PKD2, a gene for polycystic kidney disease that encodes

an integral membrane protein. Science 1996;272:1339-42.

Schwab S, Bander S, Saulo K. Renal infection autosomal dominant

polycystic kidney disease. Am J Med 1987;82:714-8.

Sklar A, Caruana RJ, Lammers JE, Strauser GD. Renal infection in

autosomal dominant polycystic kidney disease. Am J Kidney Dis

1987;10:81-8.

Werder AA, Amos A, Nielsen AH, Wolfe GH. Comparative effects of

germfree and ambient environments on the development of cystic

kidney disease in CFWwd mice. J Lab Clin Med 1984;103:399-407.

Gardner KD Jr, Evan AP, WP. Accelerated renal cyst development

in deconditioned germfree rats. Kidney Int 1986;29:1116-23.

Gardner KD Jr, WP, Evan AP, Zedalis J, Hylarides MD, Leon AA.

Endotoxin provocation of experimental renal cystic disease. Kidney

Int 1987;32:329-34.

Munford R, Hall C, Lipton J. Biologic activity, lipoprotein-binding

behavior and in vivo disposition of extracted and native forms of

Salmonella typhimurium. Clin Invest 1982;70:877-88.

Rietschel ET, Kirikae T, Schade FU, Mamat U, Schmidt G, Loppnow H,

et al. Bacterial endotoxin: molecular relationships of structure to

activity and function. FASEB J 1994;8:217-25.

Wilkinson SG. Gram-negative bacteria. In: Rutledge G, Wilkinson SG,

editors, Microbial Lipids, Vol. 1. New York: Academic Press,

1988:431-57.

MA, Prior RB, Horvath FJ, Hjelle JT. Detection of

Endotoxiuria in polycystic kidney disease patients by the use of the

Limulus Amoebocyte lysate assay. Am J Kid Dis 1990;15:117-22.

Gardner KD Jr, Burnside J, Elzinga L. Cytokines in fluids from

polycystic kidneys. Kidney Int 1991;39:718-24.

Nolan JP. Intestinal endotoxins as mediators of hepatic injury--an

idea whose time has come again. Hepatology 1989;10:887-91.

Deitch E, Berg R, Specian R. Endotoxin promotes the translocation of

bacteria from the gut. Arch Surg 1987;122:185-90.

Mimura Y, Yamanaka K, Kawabata R, Inoue A, Sasatomi K, Koga H, et

al. Lipopolysaccharide in rheumatoid arthritis. Journal of Endotoxin

Research 1996;3:17.

Muhlestein JB, Hammond EH, Carlquist JF, Radicke E, Thomson MJ,

Karagounis LA, et al. Increased incidence of Chlamydia species

within the coronary arteries of patients with symptomatic

atherosclerotic versus other forms of cardiovascular disease. J Am

Coll Cardiol 1996;27:1555-61.

Raetz CRH, Ulevitch RJ, SD, Sibley CH, Ding A, CF.

Gram negative endotoxin: an extraordinary lipid with profound

effects on eukaryotic signal transduction. FASEB J 1991;5:2652-60.

Prior RB. The Limulus amoebocyte lysate test. In: Prior RB, editor.

Clinical applications of the Limulus amoebocyte lysate test. Boca

Raton (FL): CRC Press; 1990;27-36.

Zhang GH, Baei L, Buchardt O, Koch C. Differential blocking of

coagulation-activation pathways of Limulus amoebocyte lysate. J Clin

Microbiol 1994;32:1537-41.

Obayashi T, Tamura H, Tanaka S, Ohki M, Takahashi S, Kawai T.

Endotoxin-inactivating activity in normal and pathological human

blood samples. Infect Immun 1986;53:294-7.

on DC, s DM. Binding of polymyxin B to the lipid A

portion of bacterial lipopolysaccharides. Immunochem 1979;13:813-8.

Miyazaki T, Hohno S, Mitsutake K, Maesaki S, Tanaka K, Hara K. (1-3)-

ß-D-glucan in culture fluid of fungi activates Factor G, a Limulus

coagulation factor. J Clin Lab Anal 1995;9:334-9.

Levi ME, Eshaghi N, JW, Elkind C. Fever of unknown origin

following an upper gastrointestinal series in a patient with

polycystic kidney disease. S Med J 1995;88:769-70.

Lowry O, Rosebrough N, Farr A, Randall R. Protein measurement with

the Folin phenol reagent. J Biol Chem 1951; 193:265-75.

Douwes J, Doekes G, Montijn R, Heedrik D, Brunekreef B. Measurement

of ß (1-3)-glucan in occupational and home environments with an

inhibition enzyme immunoassay. Appl Envir Microbiol 1996;62:3176-82.

Miyazaki T, Kohno S, Mitsutake K, Yamada H, Yasuoka T, Malsaki S, et

al. Combination of conventional and endotoxin-specific Limulus tests

for measurement of polysaccharides in sera of rabbits with

experimental systemic Candadiasis. Tohoku J Exp Med, 1992;168:1-9.,

Sturk A, VanDeventer S, Wortel C. Detection and clinical relevance

of human endotoxemia. Zeitschrift fuer Medizinische Laboratoriums

diagnostik 1990;31:147-58.

Mayberry WR, Lane JR. Sequential alkaline saponification/acid

hydrolysis/esterification: a one-tube method with enhanced recovery

of both cyclopropane and hydroxylated fatty acids. J Microbiol

Methods 1993;18:21-32.

Yoshida M, Roth RI, Grunfeld C, Feingold KR, Levin J. Soluble (1-3)-

ß-D-glucan purified from Candida albicans: biologic effects and

distribution in blood and organs in rabbits. J Lab Clin Med

1996;128:103-14.

Ikemura K, Ikegama K, Shimazu T, Yoshioka T, Sugimoto T. False

positive results in Limulus test caused by Limulus amoebocyte lysate-

reactive material in immunoglobulin products. J Clin Microbiol

1989;27:1965-8.

D. (1-3)-ß-D-Glucan. In: Rylander R, s R, editors.

Organic dusts: exposure, effects, and prevention. Boca Raton (FL):

Publishers, 1994;83-5.

Kaufman L, Reiss E. Serodiagnosis of fungal diseases. In: Rose N,

deMacario E, Fahey J, Friedman H, Penn G, editors. Manual of

clinical laboratory immunology. Washington (DC): American Society

for Microbiology, 1992:506-28.

Innis M, Gelfand D. Optimization of PCRs. In: Innis M, Gelfand D,

Sninsky J, White T, editors. PCR protocols-a guide to methods and

applications. New York: Academic Press 1990:3-12.

Latge J-P, Kobayashi H, Debeaupuis J-P, Diaquin M, Sarfati J,

Wieruszeski JM, et al. Chemical and immunological characterization

of the extracellular galactomannan of Aspergillus fumigatus. Infect

Immun 1994;62:5424-33.

Notermans S, Dufrenne J, Wijnands L, Engel H. Human serum antibodies

to extracellular polysac-charide (EPS) of moulds. J Med Vet Mycol

1988;26:41-8.

Garner R, Hudson J. Intravenous injection of Candida-derived mannan

results in elevated TNFa levels in serum. Infect Immun 1996;4561-66.

Roby R, Sneller M. Incidence of fungal spores at the homes of

allergic patients in an agricultural community. II. Correlations of

skin test with mold frequency. Ann Allergy Asthma Immunol

1979;43:286-8.

De Nus M, Rombouts F, Notermans S. Fusarium molds and their

mycotoxins. Journal of Food Safety 1996;16:15-58.

Badria FA, Li S, Shier WT. Fumonisins as potential causes of kidney

disease. Journal of Toxicology-Toxin Reviews 1996;15:273-92.

Scheff R, Zuckerman G, Harter H, Delmez J, Koehler R. Diverticular

disease in individuals with chronic renal failure due to polycystic

kidney disease. Ann Intern Med 1980;92:202-4.

Gans H, Matsumoto K. The escape of endotoxin from the intestine.

Surgery, Gynecology and Obstetrics 1974;139:395-402.

Grantham JJ. Acquired cystic kidney disease. Kidney Int 1991;40:143-

52.

Gardner KD Jr, Glew RH, Evan AP, McAteer JA, Bernstein J. Why renal

cysts grow. Am J Physiol 1994;266:F353-9.

Cook J, Dougherty W, Holt T. Enhanced sensitivity to endotoxin

induced by the R-E stimulant, glucan. Circulatory Shock 1980;7:225-

38.

Rylander R. Endotoxin in the environment. In: Levin J, Alving C,

Munford R, Redl H, editors. Bacterial endotoxin: lipopolysaccharides

from genes to therapy. New York: Wiley-Liss, Inc. 1995;392:79-90.

J. Mycotoxins. In: Rylander R, s R, editors. Organic

dusts: exposure, effects and prevention. Boca Raton (FL):

Pubishers, 1994;87-92.

O\'Donnell K. Progress towards a phylogenetic classificaton of

Fusarium. Sydowia 1996;48:57-70.

Spiegel S, Merrill AH Jr. Sphingolipid metabolism and cell growth

regulation. FASEB J 1996;10:1388-97.

Deshmukh GD, Radin NS, Gattone V, Shayman JA. Abnormalities of

glycosphingolipid, sulfatide, and ceramide in the polycystic

(cpk/cpk) mouse. J Lipid Res 1994;35:1611-21.

Hjelle JT, Dombrink-Kurtzman M, Nowak DM, -Hjelle MA, Darras

F, Dobbie JW. Ceramide pathways in human polycystic kidney disease.

Peritoneal Dialysis International 1996;16:S94.

Riley RT, Wang E, Schroeder JJ, ER, Plattner RD, Abbas H.

Evidence for disruption of sphingolipid metabolism as a contributing

factor in the toxicity and carcinogenicity of fumonisins. Nat Toxins

1996;4:3-15.

Merrill AH Jr, Liotta DC, Riley RT. Fumonisins: fungal toxins that

shed light on sphingolipid function. Trends in Cell Biology

1996;6:218-23.

Merrill AH Jr, Grant AM, Wang E, Bacon CW. Lipids and lipid-like

compounds of Fusarium. In: Prasad R, Ghannoun MA, editors. Lipids of

pathogenic fungi. New York: CRC 1996;199-217.

Rutledge G, Wilkinson SG, editors. Microbial lipids, Vol. 1, New

York: Academic Press, 1988.

SD, Kolesnick RN. Does endotoxin stimulate cells by mimicking

ceramide? Immunol Today 1995;16:294-302.

Barber SA, Detore G, McNally R, Vogel SN. Stimulation of the

ceramide pathway partially mimics lipopolysaccharide-induced

responses in murine peritoneal macrophages. Infect Immun

1996;64:3397-400.

Ho AK, Peng R, Ho AA, Duffield R, Dombrink-Kurtzman MA. Interactions

of fumonisins and sphing-oid bases with GTP-binding proteins.

Biochemical Archives. In Press.

Portner A, -Katalinic J, Brade H, Unland F, Buntemeyer H,

Muthing J. Structural characterization of gangliosides from resting

and endotoxin-stimulated murine B lymphocytes. Biochemistry

1993;32:12685-93.

Ballou LR, Laulederkind SJF, Rosloniec EF, Raghow R. Ceramide

signalling and the immune response. Biochim Biophys Acta

1996;1301:273-87.

Hannun Y. Functions of ceramide in coordinating cellular responses

to stress. Science 1996;274:1855-9.

Shayman JA. Sphingolipids: their role in intracellular signaling and

renal growth. J Am Soc Nephrol 1996;7:171-82.

Calvet JP. Injury and development in polycystic kidney disease. Curr

Opin Nephrol Hyperten 1994;3:340-8.

Shayman JA, Radin NS. Structure and function of renal

glycosphingolipids. Am J Physiol 1991;260:F291-302.

Bibel DJ, Aly R, Shah S, Shinefield HR. Sphingosines: antimicrobial

barriers of the skin. Acta Derm Venereol Suppl (Stockh) 1993;73:407-

11.

Iwata M, Herrington J, Zager RA. Sphingosine: a mediator of acute

renal tubular injury and subsequent cytoresistance. Proc Natl Acad

Sci USA 1995;92:8970-4.

Woolf AS, Neuhaus TJ, Kolatsi M, Winyard PJ, Klein NJ. Nephron

formation is inhibited by lipopolysaccaride and by tumor necrosis

factor-a. J Am Soc Nephrol 1994;5:641.

Counts RS, Nowak G, Wyatt RD, Schnellmann RG. Nephrotoxicant

inhibition of renal proximal tubule cell regeneration. Am J Physiol

1995;269:F274-81.

Lim CW, HM, Vesonder RF, Haschek WM. Intravenous fumonisin B1

induces cell proliferation and apoptosis in the rat. Nat Toxins

1996;4:34-41.

Wang W, C, Ciacci-Zanella J, Holt T, Gilchrist DG, Dickman MB.

Fumonisins and Alternaria alternata lycopersici toxins: sphinganine

analog mycotoxins induce apoptosis in monkey kidney cells. Proc Natl

Acad Sci USA 1996;93:3461-5.

Gelderblom WCA, Kriek NPJ, Marasas WFO, Thiel PG. Toxicity and

carcinogenicity of the Fusarium moniliforme metabolite, fumonisin

B1. Carcinogenesis 1991;12:1247-51.

Woo D. Apoptosis and loss of renal tissue in polycystic kidney

disease. New Eng J Med 1995;333:18-25.

Winyard PJD, Nauta J, Lerienman DS, Hardman P, Sams VR, Risdon RA,

et al. Deregulation of cell survival in cystic and dysplastic renal

development. Kidney Int 1996;49:135-46.

Mays RW, Siemers KA, Fritz BA, Lowe AW, van Meer G, WJ.

Hierarchy of mechanisms involved in generating Na/K-ATPa