Increased Sodium-Lithium Countertransport Activity: A Cellular Dysfunction Common to Essential Hypertension and Diabetic Nephropathy
Gianpaolo Zerbini,
Daniela Gabellini,
Dora Ruggieri and
Anna Maestroni
Renal Pathophysiology Laboratory, Division of Medicine, San Raffaele Scientific Institute, Milan, Italy
Correspondence to Dr. Gianpaolo Zerbini, Divisione Medicina I, Istituto Scientifico San Raffaele, Via Olgettina, 60, I-20132 Milano, Italy. Phone: +39-02-26432419; Fax: +39-02-26433790; E-mail: g.zerbini{at}hsr.it
ABSTRACT. An increased activity of sodium-lithium countertransport(SLC) is a common finding in patients who have essential hypertension.The evidence that a similar dysfunction is shared also by patientswith type 1 diabetes and nephropathy has suggested the hypothesisthat a predisposition to essential hypertension may be the factorthat, along with hyperglycemia, underlies the development ofdiabetic nephropathy. Despite the initial enthusiasm surroundingthe potential use of SLC activity as a marker for the earlydetection and treatment of individuals who are predisposed tohypertension and diabetic nephropathy, its use has been so farrestricted to epidemiologic studies, as specificity and sensitivityof the test are still too low to justify any clinical use. Therecent finding, however, that the measurement of kinetic parametersof SLC can significantly increase the power to discriminateamong individuals with and without hypertension or diabeticnephropathy could be of help toward a future clinical use ofthe measurement of this membrane transport. A second major pointrelates to the possibility that SLC per se might be directlyinvolved in the pathogenesis of essential hypertension and diabeticnephropathy. This case has never been fully tested, as the generesponsible for this membrane transport has been, until recently,unknown. The recent identification of an alternative splicingof the first isoform of Na-H exchange that mediates SLC activityshould allow for a rapid comprehension of the role of this transportin the pathophysiology of essential hypertension and diabeticnephropathy.
The presence in the erythrocyte membrane of a transport mediatingthe exchange of intracellular lithium for extracellular sodiumwas first suggested after the finding that, in lithium-treatedpatients who had affective disorders, lithium concentrationwas systematically lower in the erythrocytes than in the plasma(1,2). Since the first identification, sodium-lithium countertransport(SLC)—so called because sodium, at opposite with the sodiumpump, is moved toward the intracellular compartment—wasconfirmed in the erythrocyte of humans and of a number of animalspecies (3,4).
SLC gained widespread reputation in 1980 after the demonstrationby Canessa et al. (5) that elevated activity rates of this transportare a consistent concomitant of essential hypertension. It isinteresting that although SLC activity rate can be affectedby several environmental factors (6,7), family and genetic epidemiologystudies have shown that the effect of polygenic inheritanceand/or of recessive major gene is nonetheless a major determinantin the interindividual variability of SLC (8–10).
Since the original finding, an increased activity of SLC hasalways been considered an established marker of essential hypertension.For this reason, after the demonstration that hyperglycemiaby itself is not sufficient to explain the development of diabeticnephropathy (11) and the discovery that arterial BP is higherin parents of patients with type 1 diabetes and proteinuria(12), it was consequential to evaluate SLC activity in patientswith type 1 diabetes with and without nephropathy.
As a result, increased SLC activity in diabetic nephropathywas demonstrated by two independent studies (13,14), suggestingthat an inherited predisposition to essential hypertension maybe related to the susceptibility to nephropathy in patientswith type 1 diabetes. Similar results were later confirmed byseveral (15–17) although not all studies (18,19). Thereason for these discrepancies has never been completely clarified,even though methodological differences among different studieshave been demonstrated (20,21). Finally, recent evidence indicatesthat an increased SLC activity predicts the development of bothessential hypertension (22) and diabetic nephropathy (23), suggestingthat this dysfunction cannot be considered simply a consequenceof the development of these human diseases.
Potential Clinical Significance of SLC Activity Measurement
Altogether, the evidence that an increased SLC activity precedesthe development of both essential hypertension and diabeticnephropathy and the reproducibility of the measurement overtime even in patients with diabetes (Figure 1) is in line withthe hypothesis that SLC activity might be of immediate clinicaluse. Unfortunately, as clearly shown in virtually every studyin which SLC has been measured (5,13,14,24) and also in Figure 2A,although SLC activity is significantly different among groups,a large overlap nonetheless exists between normotensive andhypertensive patients or between patient with diabetes withor without nephropathy, thus significantly reducing the clinicalusefulness of SLC activity measurement. In the past few years,prompted by the finding of Rutherford et al. (25) that SLC activityas usually evaluated (standard assay at 150 mM external Na;see Figure 1A) does not totally saturate the transport for externalNa, we investigated whether the measurement of kinetic parametersof SLC (Vmax and Km for external Na) could increase the sensitivityof the assay. Actually, this approach demonstrated that essentialhypertension is characterized by an increase of both Vmax andKm for external Na of SLC and that the measurement of theseparameters improves the sensitivity of the SLC in discriminatingbetween the two groups of individuals (24). We more recentlyobtained similar results in patients who have diabetic nephropathy.As shown in Figure 2, diabetic nephropathy is also characterizedby an increased Vmax (Figure 2B), and Km for external Na (Figure 2C)of SLC and differences among groups significantly increasewhen Vmax or Km for external Na are used instead of the standardassay at 150 mM external Na (Figure 2A). Altogether, these findingsnot only add one more clue to the hypothesis that essentialhypertension and diabetic nephropathy share a common backgroundbut probably also allow a step ahead toward a future clinicalapplication of SLC activity.
Figure 1. Correlation between sodium-lithium countertransport (SLC) activity measured in 54 patients with type 1 diabetes with a mean duration of diabetes of 5.8 ± 0.5 yr (mean ± SEM) and SLC activity measured in the same patients 6 yr later. All of the patients were studied in the outpatient clinic of the San Raffaele Scientific Institute, Milan, Italy.
Figure 2. (A) SLC activity measured with the standard assay (150 mM external Na) in 21 patients with type 1 diabetes and nephropathy and in 25 patients with type 1 diabetes and normoalbuminuria, matched for gender, age, and duration of diabetes. Medians are indicated by the bar. *P = 0.02. (B) Vmax of SLC measured in the same patients as described above. **P = 0.004. (C) Km for external Na of SLC measured in the same patients as described above. ***P = 0.0001. Methods used to measure SLC kinetic parameters are described in reference 24.
Is SLC a membrane transport present only in erythrocyte? Thisquestion is of obvious importance: if SLC activity were indeeddetectable only in erythroid cells, then its possible role inthe pathogenesis of essential hypertension and diabetic nephropathywould be virtually irrelevant, as erythrocytes are most likelynot involved in the pathogenesis of these diseases. AlthoughSLC activity has been for many years thought to be measurableonly in the mature erythrocyte, there nonetheless is evidencethat this transport is present also in other kind of cells.In particular, an SLC-like activity has been demonstrated inthe lymphocyte (26) and more recently in the human skin fibroblast(27). It is interesting that SLC activity in fibroblasts frompatients with essential hypertension is increased when comparedwith fibroblasts obtained from normotensive subjects (28), extendingthe association between SLC overactivity and essential hypertensionto nonerythroid cells and therefore, potentially, also to cellsactively involved in the pathogenesis of human diseases.
Increased SLC activity has been demonstrated in patients whohave IgA nephropathy and rapid progression of the disease (29,30)and, more recently, also in patients with type 1 diabetes andproliferative retinopathy (31). These findings are in line withthe hypothesis that a predisposition to essential hypertension(unmasked by an increased SLC activity) may modulate the rateof progression of several renal diseases and, possibly, of diabeticcomplications.
The nature of SLC and its role in human physiology have beeninvestigated since its first identification in the human erythrocyte.As lithium is barely detectable in the human body, SLC was suggestedto represent an in vitro mode of operation of some other sodiumtransport. Studies of Na flux across the membrane suggestedthat Na-H exchange—a membrane transport present in virtuallyevery animal cell involved in regulation of intracellular pH,cell volume, and cell proliferation (32)—exchanges alsoLi for Na, which has contributed to the hypothesis that SLCmay reflect an operational mode of this system (33,34).
A major argument against the common nature of SLC and Na-H exchange,however, is represented by their different response to inhibitors.Sensitivity to amiloride is an almost invariable feature ofNa-H exchange and of its isoforms (35), whereas SLC is totallyinsensitive to this drug (36). Moreover, the finding that theinterindividual variability of SLC activity cannot be explainedby polymorphisms of the gene encoding for the ubiquitous firstisoform of Na-H exchange favors the alternative view that SLCmay be mediated by a transmembrane carrier independent of amiloride-sensitivetransports (37).
In the past few years, we have searched for the presence inthe human erythrocyte of amiloride-insensitive Na-H exchangeisoforms that could, at least potentially, mediate SLC. To thisaim, we screened a cDNA library obtained from a pool of humanbone marrows using two probes specific for the high homologyregion shared by the Na-H exchange isoforms 1 to 7 (38–40).After restriction and sequencing analyses of the several clonesharboring partial transcripts of the Na-H exchange-isoform 1gene, only Na-H exchange-isoform 1 transcripts were finallydetected. Na-H exchange-isoform 1, because of its sensitivityto amiloride, already has been excluded as a possible mediatorof the amiloride-insensitive SLC (41).
Aimed to the identification of the gene responsible for SLCactivity, we recently succeeded in identifying in human erythrocytesand reticulocytes the presence of an alternative splicing ofthe above-described Na-H exchange-isoform 1, lacking the amiloridebinding site. This amiloride-insensitive isoform of Na-H exchange,once transfected in Na-H exchange-deficient cells, restoresSLC activity (42). This finding suggests that SLC is mediatedby a deleted variant of the Na-H exchange-isoform 1 that differsfrom the full-length isoform because of amiloride insensitivityand in some way re-candidate Na-H exchange-isoform 1 as a genepotentially involved in the pathogenesis of essential hypertension.
Finally, as both essential hypertension and diabetic nephropathyare characterized by an increased SLC activity, great attentionshould also be paid to the genes that modulate SLC activity.It is interesting that two studies have focused particularlyin this field. A very large study performed in baboons has suggestedthe baboon chromosome 5 (homologue of human chromosome 4) asa site potentially involved in the regulation of SLC activity(43), whereas a more recent genome-wide linkage study revealedthe chromosome 15q as a candidate for the presence of a quantitativetrait locus explaining SLC activity (44). Further studies noware necessary both in baboons and in humans to identify finallythe single genes involved in the regulation of SLC activity.
After the first demonstration by Dr. Canessa >20 yr ago thatSLC activity is increased in essential hypertension, a hugenumber of studies have followed, confirming the original finding,extending the identification of the same dysfunction also todiabetic nephropathy and to other diseases, and showing throughan epidemiologic approach that the activity of SLC is understrict genetic control. The only piece of the puzzle still missing,i.e., the gene responsible for this membrane transport, hasnow been identified as Na-H exchange-isoform-1 through an amiloride-insensitivealternative splicing. As the genes potentially involved in theincreased SLC activity seem to be close to the identification,it is possible to envisage for the near future the full comprehensionof the role of SLC in the pathophysiology of essential hypertensionand diabetic nephropathy.
Acknowledgments
The financial support of Telethon-Italy (grant no. E.816) isgratefully acknowledged.
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