signaling and gene regulation by urea in cells of the mammalian kidney medulla

Ž .Comparative Biochemistry and Physiology Part A 130 2001 429�436

Review

Signaling and gene regulation by urea in cells of themammalian kidney medulla�

Wei Tian, David M. Cohen�

Di�isions of Nephrology and Molecular Medicine, and Department of Cell and De�elopmental Biology, Oregon Health SciencesUni�ersity and the Portland Veterans Affairs Medical Center, Portland, OR 97201, USA

Received 24 November 2000; received in revised form 8 May 2001; accepted 11 May 2001

Abstract

Signaling by urea, although incompletely understood, is relevant both to cells of the mammalian kidney inner medullaand to all cells of the organism in the setting of advanced renal failure with its attendant accumulation of urea in thesystemic circulation. The molecular events initiated by urea stress are distinct from those occurring in response tohypertonic stress; urea activates a characteristic subset of signaling events, which are in large part specific to culturedrenal tubular epithelial cells. Interestingly, urea is protective of hypertonic NaCl-inducible apoptosis in this model.Details of this phenomenon are reviewed. The effect of urea has been likened to that of either hypertonicity or of apeptide mitogen. In preliminary expression array analyses, the profile of genes activated by urea stress in renalmedullary cells, however, was found to be unique. � 2001 Elsevier Science Inc. All rights reserved.

Keywords: Hypertonicity; Expression array; Signal transduction; NaCl; Urea; Kidney; Heat shock

1. Introduction

Urea functions as an organic osmolyte in elas-Žmobranchs sharks, rays and skates; Yancey et al.,

.1982 where it serves to offset the potential detri-mental effects of elevated extracellular levels ofNaCl, both in terms of preservation of cell volumeand in terms of limiting the intracellular accumu-

� This paper was originally presented at a symposium dedi-cated to the memory of Marcel Florkin, held within theESCPB 21st International Congress, Liege, Belgium, July`24�28, 2000.

� Corresponding author. Mailcode PP262, Oregon HealthSciences University, 3314 S.W. US Veterans Hospital Rd.,Portland, OR 97201, USA. Tel.: �1-503-220-8262, ext. 56654;fax: �1-503-721-7810.

Ž .E-mail address: [email protected] D.M. Cohen .

lation of potentially protein-denaturing concen-trations of ionic solutes. In mammals, both physi-ological and pathophysiological conditions mayresult in cell exposure to elevated urea concentra-tions. The renal medulla is unique in its perpetualexposure to urea concentrations in the hundredsof mM range; this level is subject to rapid fluctu-ations in response to changing diet and hydrationstatus of the organism. In chronic renal failure,characterized by an inability to adequately ex-crete nitrogenous protein catabolites, a substan-tial systemic accumulation of urea ensues whichmay achieve many tens of mM.

In contrast to the notion that urea is a passiveparticipant in renal medullary physiology, accu-mulating data indicate that changes in ambienturea concentration may influence gene expression

1095-6433�01�$ - see front matter � 2001 Elsevier Science Inc. All rights reserved.Ž .PII: S 1 0 9 5 - 6 4 3 3 0 1 0 0 4 4 1 - X

( )W. Tian, D.M. Cohen � Comparati�e Biochemistry and Physiology Part A 130 2001 429�436430

and intracellular signal transduction pathways.Through the use of a candidate gene approach,two genes have been shown to be regulated byurea in renal epithelial cells; neither is apprecia-

Ž .bly regulated by hypertonicity see below . Inaddition, a number of signaling events linked tothese transcriptional events have been defined.Most but not all of these signaling and transcrip-tional events appear to be restricted to cells ofrenal epithelial origin.

2. Signaling pathways activated by urea in renalepithelial cells

An overview of signaling events activated inresponse to urea treatment in renal medullaryŽ .mIMCD3 cells is depicted in Fig. 1. Aspects ofthese signaling events are reminiscent of thoseobserved in response to peptide mitogens; wehave, however, been unable to demonstrate anincrease in mitogenesis in response to urea treat-ment in this cell line. Indices of proliferation inresponse to urea treatment have variably beenobserved in other urea-responsive renal epithelialcell lines such as MDCK, LLC-PK , and NRK-1

Ž .52E Cohen and Gullans, 1993a,b; Logan, 1995 .

Akin to an activator of receptor tyrosine ki-Ž . Žnases RTK , urea results in the activation tyro-

.sine phosphorylation of a variety of RTK effec-tors, presumably via interaction of effector-asso-ciated SH2 and�or PTB domains with newlyphosphorylated tyrosine residues on a putative

Ž .urea-responsive kinase Fig. 1 . Following ureatreatment, the adapter protein Shc becomes tyro-sine phosphorylated and recruits a second adapter

Ž .protein, Grb2 Zhang et al., 2000b . The Grb2interaction partner and guanine nucleotide ex-change factor, SOS, is also tyrosine phosphory-lated in the presence of urea and potentiallymediates the rapid activation of the small G-pro-

Žtein, Ras, that is observed in this context Tian et.al., 2000 . Although subsequent events including

activation of MEK, ERK, and the transcriptionfactor Elk-1, as well as the MEK-dependent tran-

Ž .scription of immediate�early genes see Fig. 1have all been detailed in this model of urea

Ž .signaling Cohen et al., 1994; Cohen, 1996, 1996b ,strict dependence upon prior activation of Ras isnot uniform. For example, Ras inhibition substan-tially blocks urea signaling to immediate�earlygene transcription, but only modestly inhibits ac-

Ž .tivation of ERK in this context Tian et al., 2000 .Other loci of signal integration distal to Ras

Ž .activation e.g. non-classical protein kinase C

Ž .Fig. 1. Overview of urea signaling pathways in renal epithelial cells. Based upon documented downstream signaling events see text ,Ž .urea appears to activate a cell surface-associated or cytosolic tyrosine kinase TK . Shaded boxes indicate confirmed involvement in

Ž . Ž .urea signaling; ? denotes uncertain involvement e.g. TK or conflicting data e.g. PKC . The nature of some of the interactions isdetailed in the key. Abbre�iations: ERK, extracellular signal-regulated kinase; MEK, MAPK and ERK kinase; PI3K, phosphatidylinosi-tol-3-kinase; PKC, protein kinase C; PTB, phosphotyrosine binding; TK, tyrosine kinase.

( )W. Tian, D.M. Cohen � Comparati�e Biochemistry and Physiology Part A 130 2001 429�436 431

may play a role in this process. The largely RTK-Ž .specific phospholipase C PLC isoform, PLC-�, is

also activated by urea treatment in renalmedullary cells and is potentially responsible forthe increase in inositol trisphosphate production

Ž .observed in this context Cohen et al., 1996a .Lastly, the lipid kinase, phosphatidylinositol-3-kinase, is activated by urea and indirectly results

Žin activation of Akt and p70S6 kinase Zhang et.al., 2000b . Not unexpectedly, this latter pathway

appears to be instrumental in conferring resis-tance to urea stress as is the case in other experi-mental contexts.

Urea may also signal via an oxidative stress-de-pendent pathway whose relation to RTK-media-ted signaling events remains unclear. The stress-inducible transcription factor, Gadd153, is tran-scriptionally upregulated by urea and this effect iscompletely inhibited by pretreatment with thiol-containing antioxidants. Consistent with theseobservations, urea treatment induces oxidativestress in renal medullary cells as determined via a

Ždecrease in reduced glutathione Zhang et al.,.1999 . Whether this oxidative stress is a conse-

quence of an adverse effect of urea or whether itrepresents an additional signaling event, as hasbeen observed in response to some cytokines and

Žpeptide growth factors reviewed in: Ha and Bahl.Lee, 2000 , is unknown.

3. Reciprocal relationship between urea and NaClstress

The adverse effects of urea upon conformationof biopolymers such as proteins and nucleic acidshas been appreciated for decades. Whereas adenaturing effect is also observed with high con-centrations of ionic solutes such as Na�, K� andCl�, increasing evidence supports a complex func-tional relationship between ionic solutes and ureain vivo. Specifically, their detrimental effects maynot be additive; in fact, they may even affordmutual protection.

The concept of a solute counteracting the po-tentially harmful effects of urea is not new; coun-teracting methylamine osmolytes such as betaineand glycerophosphocholine preserve structure and

Žfunction in urea-exposed proteins Yancey et al.,.1982 . In vitro data such as these are bolstered by

observations linking high concentrations of theseprotective compounds to cells and tissues exposed

to high concentrations of urea in vivo. The abilityof urea to protect from the detrimental effects ofhypertonicity, and conversely, the ability of NaClto protect from urea stress, has only recentlybeen appreciated.

3.1. NaCl protects from urea stress

A number of groups have observed the abilityof NaCl to protect from the adverse effects ofurea treatment. Santos et al. reported that bothurea and NaCl decreased cell viability in themIMCD3 cell line and that combining the two

Ž .solutes enhanced viability Santos et al., 1998 .Neuhofer similarly observed that pretreating re-nal epithelial MDCK cells with NaCl afforded

Ž .protection in terms of cell survival from subse-Ž .quent urea stress Neuhofer et al., 1998 , an ef-

fect attributed to heat shock protein inductionŽ .Neuhofer et al., 1999 . Whereas hypertonicity isassociated with upregulation of molecular chaper-

Ž .ones such as HSP70 Cohen et al., 1991 , mostinvestigators have failed to observe a correspond-ing increase in response to urea treatment.Therefore, the ability of this stress response toprotect from urea is unexpected. Interestingly,HSP72 has recently been shown to inhibit JNK

Ž .function Park et al., 2001 yet JNKs are cytopro-Žtective of hypertonic stress Wojtaszek et al.,

.1998 ; this apparent paradox may be a conse-quence of isoform specificity of the respectivephenomena.

3.2. Urea protects from NaCl stress

We have recently observed a relationship in-verse to the above: urea protects from the ad-

Ž .verse effects of hypertonicity Zhang et al., 2000a .For these studies, sensitive biochemical indices ofapoptosis were examined. Caspase-3 is a pivotalprotease instrumental in the execution phase ofapoptosis that participates in the final commonpathway shared by caspase-9-dependent mito-chondrially-mediated pathway and the caspase-8-dependent so-called ‘death domain’ receptor-mediated pathway. Therefore, it is an ideal candi-date for investigation of a relatively unexploredcontext of apoptosis. An additional downstreammolecular event in the apoptotic effector arm wasexamined in parallel. Membrane lipid polariza-tion is disrupted with apoptosis such that phos-phatidylserine, which is usually confined to the


inner leaflet of the cell membrane, translocates tothe outer leaflet where it is readily detected byvirtue of its in vitro affinity for the marker proteinannexin V.

In renal medullary mIMCD3 cells, hypertonic-ity activated caspase-3 in a dose- and time-depen-dent fashion; as little as 50 mM NaCl over 4 hwas sufficient for a statistically significant in-crease in activity and 100 mM NaCl produced a

Ž .four-fold upregulation Zhang et al., 2000a . Hy-pertonicity-inducible annexin V binding exhibited

Žsimilar temporal kinetics. Although urea 200.mM alone had no effect upon either caspase-3

activity or annexin V binding, it inhibited thehypertonic NaCl-inducible increment in caspase-3activity and annexin V binding by approximately

Ž .60% Fig. 2 . Dose�response studies indicatedthat at least 100 mM urea was required for theprotective effect. Urea protected from apoptosis

Žin response to other hypertonic stressors e.g..mannitol , although the degree of protection was

more modest. Importantly, another permeant so-lute, glycerol, failed to reproduce the protectiveeffect of urea implying a degree of specificity. Inaddition, in the non-renal 3T3 cell line, ureafailed to protect from hypertonicity-inducibleapoptosis. Furthermore, the ability of urea toprotect from apoptosis in the mIMCD3 cell linewas not universal. Ultraviolet irradiation-associ-ated apoptosis of a magnitude comparable to thatinduced by hypertonicity was markedly exacer-bated by urea pretreatment. The protective effect

of urea resembled the anti-apoptotic effect ofpeptide growth factors and was approximatelycomparable to the effect of either EGF or IGF.Interestingly, urea was additive with EGF in termsof protection from hypertonic stress implying in-

Ž .complete overlap or at least submaximal effectŽof the respective signaling events Zhang et al.,

.2000a .

4. Solute-inducible gene regulation

4.1. NaCl-inducible gene regulation

Hypertonicity induces expression of genes viaboth non-specific and specific mechanisms. In-hibitors of protein synthesis, such as cyclohex-imide, induce the remarkable accumulation, or‘superinduction’, of mRNAs encoded by so-calledimmediate�early genes. Immediate�early genesare inducible at the transcriptional level withoutthe necessity for a prior round of protein synthe-sis, i.e. they represent the first or earliest genesinduced in response to a given stimulus. Immedi-ate�early genes often encode transcription fac-tors, which serve to govern the transcription andexpression of downstream genes. Hypertonicstress inhibits translation in a cycloheximide-likefashion and, in so doing, super-induces many im-

Žmediate�early genes at the mRNA level Cohen.et al., 1991; and references therein . This incre-

ment in mRNA abundance, however, does not

Fig. 2. Effect of urea pretreatment upon indices of hypertonicity-inducible apoptosis in mIMCD3 cells. Confluent monolayers receivedŽ . Ž . Ž .pretreatment with urea 200 mM�30 min;�Urea or no pretreatment �Urea prior to 4-h exposure to NaCl �100 mM . Caspase-3

activity was determined via in vitro cleavage of a fluorogenic substrate and was normalized to Control in the absence of ureaŽ .pretreatment a . Annexin V binding was determined via cell-sorting using fluorescent-tagged annexin V and propidium iodide. Cells in

early apoptosis exhibiting high annexin V binding and low propidium iodide staining were used for gating; percent of cells wasnormalized to Control in the absence of urea pretreatment.


result in a corresponding increase in protein ex-pression as the translational block persists.

Specific gene regulation by hypertonicity is ex-emplified by the coordinate regulation of genescoding for proteins essential for the adaptive ac-cumulation of organic osmolytes in response to

Ž .hypertonic stress reviewed in Burg et al., 1997 .Examples of target genes include the enzymealdose reductase, essential for the synthesis of theprotective osmolyte, sorbitol, and the co-transporters essential for importation of the os-molytes myoinositol and betaine. Upstreamflanking regions of the genes coding for these

Žproteins contain osmotic response elements alsoknown as tonicity enhancer elements; see accom-

.panying review of Kwon which confer osmoticinducibility at the transcriptional level. Recently,several groups have identified a putativeosmotic-responsive transcription factor which di-rectly interacts with this DNA consensus se-

Žquence Miyakawa et al., 1999; Lopez-Rodriguez.et al., 1999; Ko et al., 2000 . Signaling events

leading to activation of this nuclear phosphopro-tein are incompletely understood with some butnot all groups reporting a role for the MAPK, p38Ž .Kultz et al., 1997; Sheikh-Hamad et al., 1998 .Additional mechanisms of tonicity-inducible tran-scription have been observed in other contexts.For example, the serum and glucocorticoid re-sponsive kinase is activated at the transcriptionallevel in an apparently p38-dependent fashion in-volving the ubiquitous transcription factor, Sp1Ž .Bell et al., 2000 . Hypertonic regulation of aqua-porin 5 expression likely requires a different

Ž .MAPK, ERK Hoffert et al., 2000 . Other tran-scription factors including members of the STAT

Žfamily are also activated by hypertonicity Bode et.al., 1999 but downstream targets in this context

remain unknown.

4.2. Urea-inducible gene regulation

In contrast to hypertonic shock where a rela-tively large number of genes have been identifiedthat are transcriptionally regulated, much less isknown about the molecular consequences of ureastress. Genes encoding two stress-responsive tran-scription factors are specifically transcriptionallyactivated in response to urea treatment in renalmedullary cells: the zinc finger containing Egr-1Ž .Cohen et al., 1994 ; and the DNA damage-in-

Ž .ducible GADD153 Zhang et al., 1999 . Synthesis

of Egr-1 and GADD153 protein is correspond-ingly upregulated. Signaling events leading to ac-tivation of the Egr-1 gene have been explored in

Ž .detail and were described above Fig. 1 . Less isknown about regulatory events leading toGADD153 transcription in response to urea, al-though interestingly, the phenomenon is depen-

Ž .dent upon oxidative stress Zhang et al., 1999 . Incontrast to the effect of urea, neither of thesegenes is activated to an appreciable degree byhypertonicity or by any other permeant or im-permeant solute examined. Although hypertonic-ity activates the heat shock response in renal

Žepithelial cells, urea produces no effect Cohen et.al., 1991 . The urea transport protein, UT-A2 was

upregulated in a synergistic fashion in vitro by thecombination of urea and NaCl, whereas neither

Žsolute alone exerted a substantial effect Leroy et.al., 2000 . In this latter model, however, upregula-

tion of UT-A2 protein was not demonstrated.

4.3. Expression profiling in response to ureatreatment: a comparison

Because urea signaling in renal epithelial cellsbears hallmarks of mitogenic as well as hyper-tonic signaling, a preliminary effort was made todirectly compare signaling events activated byeach of these stimuli in a large-scale fashion.Because gene regulation is a principal down-stream locus of signal integration, the effect ofurea treatment, and of treatment with the bonafide osmotic stressor, mannitol, as well as thepeptide mitogen, EGF, was explored. It was rea-soned that if the gene expression profile observedin response to urea treatment more closely re-sembled that seen in the setting of hypertonicityor in response to mitogen treatment, then the neteffect of urea treatment upon whole-cell pheno-type would be more akin to the response to thatstimulus.

Of 1200 genes observed under four separateŽconditions at a single time-point 24 h of treat-

.ment , almost 400 genes were detectable under atleast one of the conditions. In response to urea

Ž .treatment vs. control treatment , 42 genes wereupregulated whereas 128 genes were downregu-lated. EGF treatment upregulated far more genesthan urea and resulted in downregulation of fewergenes. Mannitol, like EGF, upregulated a fargreater number of gene products, but also down-regulated nearly as many genes as urea. Perusal


Fig. 3. Relationship between the genetic program activated by urea and by related stimuli in mIMCD3 cells . Total number of genesup- or downregulated by EGF or mannitol is indicated by overall height of bars. Fraction of these regulated genes that is shared by allstimuli, or by only urea, is indicated by the dark and intermediate shading, respectively. Lighter shading and no shading indicatefraction common to only a non-urea stimulus and fraction that is unique to the indicated stimulus, respectively. Therefore, for example,

Žin the first column of panel A, of the 187 genes upregulated by EGF, 13 are upregulated by all stimuli examined EGF, urea, and. Ž .mannitol; dark shading , 3 are upregulated by both EGF and urea medium shading , 87 are upregulated by both EGF and mannitol

Ž . Ž .light shading , and 84 are upregulated by only EGF no shading . See text for interpretation.

of genes upregulated in response to urea treat-ment underscored the specificity of this approach;although all cDNAs were examined in a blindedŽ .coded fashion, two of the 42 upregulated genesŽ .the transcription factors, Egr-1 and Gadd153were previously shown to be transcriptionally reg-

Žulated by urea Cohen et al., 1994; Zhang et al.,.1999 . We believe these findings validate the util-

ity of this approach and support future moredetailed studies.

One focus concerned genes uniquely regulatedŽ .either up or down by individual treatments. EGF

Ž .uniquely upregulated the most genes 85 genes ;the effects of mannitol and urea were more mod-est. A total of 74 genes were uniquely downregu-lated by the stimuli, nearly all in response to ureaor mannitol. A related analysis was performed toestablish the degree to which EGF and mannitoltreatment resembled urea treatment. Total up-

Žregulated genes not merely uniquely upregulated.genes were assigned to one of several categories:

those upregulatory events that were common toall stimuli; those that were common to only urea;

Žthose that were common to other non-urea,.non-self stimuli; and those that were unique to

the stimulus in question. Genetic programs acti-vated by EGF and mannitol treatment bear littleresemblance to that initiated in response to ureaŽ .Fig. 3; see below . Applying this analysis todownregulated genes led to a different conclusion

Ž� of the transcripts downregulated by EGF but.not mannitol , a substantial percentage were

downregulated by either urea or by all stimuliexamined.

An approach using Venn diagrams was adoptedto visually unify these preliminary observations.Each circle in Fig. 4 represents the number ofgenes subject to regulation by a given stimulus;the area of the circle is proportional to the num-ber of regulated genes. Overlap between adjacentcircles indicates the percentage of regulated genesthat are ‘shared’ by two stimuli. Highly relatedgenetic programs would, therefore, be repre-sented as nearly superimposable circles of approx-imately equal diameter. The most striking findingwhen data are visualized in this fashion is thatwhereas neither EGF treatment nor mannitoltreatment closely resembles urea treatment interms of upregulated genes, they exhibit an im-pressive degree of similarity to each other. Thisfinding is perhaps consistent with earlier observa-tions that hypertonic stress may result inoligomerization and activation of cell surface-as-sociated receptors for peptide growth factors such

Ž .as EGF e.g. Rosette and Karin, 1996 . In termsof downregulated genes, the picture is less clear.Urea appears to downregulate far more genesthan does EGF, however, there is substantialoverlap among them. The preponderance of genesdownregulated by urea underscores the functio-nal specificity of those genes previously shown tobe upregulated in this context.

In aggregate, these expression array data serveto highlight the unique nature of urea signaling in


Fig. 4. Venn diagram depicting the relationship of the urea-Ž . Ž .inducible genetic program U to that of EGF E and manni-

Ž .tol M in mIMCD3. Genetic program is depicted as circle ofa diameter proportional to the number of regulated genes;percentage of genes ‘shared’ by two genetic programs is pro-portional to the area of intersection of two circles. Upregu-

Ž . Ž .lated left panel and downregulated right panel genes areconsidered independently. In this analysis, closely related ge-netic programs would appear as nearly superimposable circles

Ž .of approximately equal diameter see text . For scale, a circleof area equal to 10 genes is included at lower right. Pro-grams�treatments: U, urea 200 mM�24 h; E, EGF 100nM�24 h; M, mannitol 200 mM�24 h.

cells of the renal medulla. Further studies areunderway to examine and quantify this pheno-menon in both renal and non-renal cell models atvarious time-points using much more comprehen-

Ž .sive gene coverage �7000 . It is anticipated thatstudies such as these will provide a more globalpicture of both physiological and pathological re-sponses to urea stress.

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signaling and gene regulation by urea in cells of the mammalian kidney medulla

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