clinical and cellular markers of diabetic nephropathy

Kidney International, Vol. 63 (2003), pp. 2319–2330

NEPHROLOGY FORUM

Clinical and cellular markers of diabetic nephropathy

Principal discussant: Daniel Batlle

Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA

initiated at that time because the patient was reluctant to startthis treatment in the event that she became pregnant.

When the patient was evaluated in our office at the age of21, we did not elicit a family history of renal disease. Her socialhistory was negative for tobacco, alcohol, and recreational druguse. Her medications included insulin, 10–15 units of NPH and20–25 units of regular insulin in the morning and 8 units ofNPH insulin and 10–15 units of regular insulin before dinner.Her blood pressure was 121/68 mm Hg in the seated positionand 120/68 mm Hg in the standing position. Her heart ratewas 70 beats/min and 75 beats/min in the seated and standingpositions, respectively. Her fundi showed mild proliferativechanges. No hemorrhages were seen. Her abdomen was soft,and no masses were felt. Her pulses in the lower extremitieswere 2� bilaterally.

At that time the BUN and serum creatinine were 15 mg/dLCASE PRESENTATIONand 1.1 mg/dL, respectively. Her blood glucose was 160 mg/dLA 21-year-old white woman was referred for evaluation ofand HbA1c was 9.9%. Her serum electrolytes were: sodium,microalbuminuria. She stated that at the age of 11 a diagnosis138 mmol/L; potassium, 4.1 mmol/L; calcium, 8.5 mg/dL (2.2of diabetes was made because of polyuria and polydipsia, andmmol/L); and chloride, 103 mmol/L. Total cholesterol was 190she was begun on a regimen of insulin. Two months later,mg/dL; fasting triglycerides, 140 mg/dL. Dipstick urinalysis wasshe was hospitalized with an episode of diabetic ketoacidosis.negative for protein; a 24-hour urine collection indicated aReview of her medical records revealed no further episodes ofglomerular filtration rate (GFR) (creatinine clearance) of 115ketoacidosis. During her adolescence, compliance with insulinmL/min, and an albumin excretion rate of 250 mg/24 hourstherapy was problematic despite frequent counseling and ad-(Fig. 1).justment of her insulin dose. On one evaluation at the age of 18,

A 24-hour ambulatory blood pressure monitoring was doneher blood glucose was 200 mg/dL, and her plasma electrolytes,at this time (Fig. 2). Mean systolic and diastolic blood pressuresblood urea nitrogen (BUN), and serum creatinine were in thewere 122 mm Hg and 70 mm Hg, respectively. Mean systolicnormal range. Her blood pressure while seated was 122/70and diastolic blood pressures during the sleep period were 121mm Hg, and her heart rate was 86 beats/min. A urinalysis wasmm Hg and 69 mm Hg, respectively. Mean systolic and diastolicnormal with a negative dipstick for protein; a 24-hour urineblood pressures during daily activities were 123 mm Hg andcollection revealed a creatinine clearance of 125 mL/min, and a71 mm Hg, respectively. At that time, renoprotective therapyurine albumin excretion (UAE) rate of 20 mg/24 hours (normal,with an ACE inhibitor, captopril at a dose of 25 mg orally twice0–30 mg/24 hours) (Fig. 1).daily, was initiated. She also was referred to the EndocrinologyOver the following years, her glycemic control was poor, as

revealed by glycosylated hemoglobin (HbA1c) ranging between Clinic to optimize her glycemic control.9% and 11%. When she was 20 years old, her pediatricianfound that the urine albumin excretion had increased to 200

DISCUSSIONmg/24 hours and referred the patient to us for nephrologicevaluation. Therapy with an angiotensin-converting enzyme Dr. Daniel Batlle (Chief, Division of Nephrology;(ACE) inhibitor was considered by her pediatrician but not

and Professor of Medicine, Northwestern University Fein-berg School of Medicine, Chicago, Illinois, USA): Dia-betic nephropathy is a microvascular complication of bothThe Nephrology Forum is funded in part by grants from Amgen,

Incorporated; Merck & Co., Incorporated; Dialysis Clinic, Incorpo- type 1 and type 2 diabetes, which is associated with end-rated; and Bristol-Myers Squibb Company. stage renal disease (ESRD) and premature death from

cardiovascular disease [1, 2]. The development of clinicalKey words: end-stage renal disease, microalbuminuria, NHE-1, Na�/H�

antiporter, nocturnal hypertension, cell cycle proteins, cellular markers. nephropathy is insidious, and macroalbuminuria [urinaryalbumin excretion rate �300 mg/24 hours], the hallmark 2003 by the International Society of Nephrology

2319

Nephrology Forum: Markers of diabetic nephropathy2320

Fig. 1. Three values of urinary albumin ex-cretion from the patient discussed today (�).The first value was obtained when the patientwas normoalbuminuric; the remaining twowere obtained years later when the patienthad developed microalbuminuria. The solidline depicts the progression of albumin excre-tion from normal to microalbuminuria andthen to overt proteinuria in type 1 diabeticswho ultimately will develop nephropathy.Years: duration from the diagnosis of diabetesat age 11 during the normoalbuminuric phase.

of the condition, is preceded by a phase of microalbumin- patients with nephropathy seem to have an increasedincidence of cardiovascular disease as compared withuria (UAE 30–300 mg/24 hours), which usually lasts 5

to 10 years. As albuminuria worsens and blood pressure parents of type 1 diabetic patients with normal albuminexcretion [10]. Albuminuria, cardiovascular disease, andincreases, there is a relentless decline in GFR and pro-

gression to ESRD [2]. Tight metabolic control can reduce hypertension might be linked by an inherited predisposi-tion to insulin resistance. Patients with nephropathy arethe incidence of nephropathy [3], but hyperglycemia

alone is not sufficient for the development of nephropa- more insulin-resistant than are non-nephropathic pa-tients [14]. Insulin resistance also has been noted in par-thy. This is apparent from the finding that nephropathy

develops in some patients with type 1 diabetes despite ents of type 1 diabetic patients with proteinuria [14].These clinical markers of increased risk for diabeticgood glycemic control and that a substantial proportion

of patients with poor control escape diabetic nephropa- nephropathy are useful in themselves, but we also needgenetic markers to define people at risk for nephropathythy [4–10]. Overall, only about one-third of type 1 diabe-

tes patients develop nephropathy, and the risk of ne- when the diagnosis of diabetes is made. Identification atan early age would not only allow for more intensivephropathy appears, at least in part, to be genetically

determined [4–11]. follow-up of such patients, but also would provide insightfor the design of therapeutic approaches based on the

Clinical markers particular genetic marker [11]. Genes encoding the vari-ous components of the renin-angiotensin-aldosteroneStrong evidence for genetic factors in nephropathysystem seem logical candidates for a genetic marker ofcame from studies of families with type 1 diabetes [6–9].

In families with two or more siblings with type 1 diabetes, susceptibility to nephropathy and its associated cardio-vascular disease. No significant genetic marker of dia-when one sibling has developed nephropathy, the other

has a fourfold risk of nephropathy compared with a sib- betic nephropathy has been conclusively identified amongthe components of the renin-angiotensin system or otherling of a patient without nephropathy [4, 6]. In a larger

study [7], if the proband had nephropathy, the cumula- potential candidates such as the aldose reductase geneand the apolipoprotein E gene [11]. Recent studies, how-tive risk of nephropathy to diabetic siblings was 71.5%,

and the risk fell to 25.4% when the proband did not ever, suggest that certain polymorphisms of the ACEgene might identify type 1 diabetic patients in whomhave nephropathy [2]. A recent analysis of the Diabetes

Control and Complications Trial (DCCT) cohort has diabetic nephropathy progresses at a faster rate (DDgenotype) and those who are responsive to renoprotec-confirmed these findings [8].

Other factors have been suggested as determinants of tive intervention using ACE inhibitors. These findingswere discussed by Dr. Hans-Henrich Parving in a recentrisk for diabetic nephropathy, including a family history

of hypertension [5, 12]. Parents of patients with type 1 Forum [2], and I will not review them here.A marker is not necessarily based on the genotypediabetes who develop nephropathy have higher arterial

blood pressures than do parents of patients who have no (that is, an alteration of DNA), but it can be based onphenotypic features, clinical or biochemical, that predictnephropathy [13]. Moreover, parents of type 1 diabetic

Nephrology Forum: Markers of diabetic nephropathy 2321

Fig. 2. Tracing of 24-hour ambulatory blood pressure in the patient being discussed taken at the age of 21 when she was microalbuminuric. (A )Systolic and diastolic blood pressure. (B ) Heart rate. Blood pressure throughout the 24-hour period, including the sleep period (0–6 hours), wasessentially the same.

Table 1. Markers of diabetic nephropathy in type 1 diabetes

Clinical Cellular phenotypes Genetic markers (potential)

Microalbuminuria Increased erythrocyte Na�/Li� countertransport Angiotensin-converting enzymeFamily history of nephropathy Na�/H� antiporter overactivity AngiotensinogenFamily history of HTN and/or insulin resistance Altered cell growth Angiotensin II, type 1 receptorIncreased GFR Cell cycle regulatory proteins (decreased p16?) Aldolase reductaseNocturnal HTN (non-dipping status) Apolipoprotein E

with reasonable sensitivity and specificity the susceptibil- a marker and a predictor of nephropathy. Later we willlook at the potential role of cellular markers of diabeticity to diabetic nephropathy before it becomes clinically

evident [15]. Table 1 lists markers of diabetic nephropa- nephropathy, such as overactivity of the Na�/H� antipor-ter and phenotypic features of altered cell growth.thy classified as clinical, cellular, or genetic. In this classi-

fication, the term marker is used loosely, in that it implies When today’s patient with type 1 diabetes was evalu-ated at the age of 21, she had developed microalbumin-a predictive value for the development of nephropathy.

In this context, the best available predictor of diabetic uria (Fig. 1). Her GFR, estimated by the creatinine clear-ance, was in the normal range, as is usually the casenephropathy to date is microalbuminuria. By the time

it develops, however, many patients have already ad- in microalbuminuric patients with type 1 diabetes. Herblood pressure was likewise in the normal range, whichvanced renal structural changes on renal biopsy [16–18].

This suggests that rather than being a predictor of dia- also is usual for type 1 diabetics with microalbuminuria[19, 20]. This finding is in sharp contrast with patientsbetic nephropathy, microalbuminuria is a marker of es-

tablished nephropathy. As part of my discussion of the with type 2 diabetes, in whom blood pressure is alwayselevated by the time microalbuminuria is documentedpatient presented here, I will try to distinguish between


[21]. In fact, type 2 diabetics are usually overtly hyperten- years of disease duration) is increasingly low. With adisease duration of less than 15 years, the rate of progres-sive (that is, systolic blood pressure �145 mm Hg) when

the diagnosis of diabetes is made. With this background sion from microalbuminuria to proteinuria over the next5 to 10 years has been estimated to be as much as 45%in mind, I would like to address two main issues: the

likelihood of progression to overt proteinuria from micro- [25]. This risk is still below the risk of nephropathy (about80%) estimated from earlier studies.albuminuria, and whether microalbuminuria could have

been predicted in this patient when she was first seen The impact of therapy with an ACE inhibitor and/orimproved glycemic control on progression from micro-years ago by her pediatrician.

Until recently, if we had asked whether the microalbu- albuminuria to proteinuria also needs to be considered.The DCCT demonstrated a positive effect of improvedminuria would have progressed to overt proteinuria, the

answer would have been a resounding “yes.” That is, it glycemic control on preventing microalbuminuria, butnot on the progression from microalbuminuria to pro-was only a matter of time before she progressed from

microalbuminuria to overt proteinuria, as depicted in teinuria [3]. Therapy with ACE inhibitors, by contrast,retards the progression from microalbuminuria to pro-the typical course of nephropathy in type 1 diabetics

(Fig. 1). Indeed, initial retrospective studies in type 1 teinuria (see [28] for review). As documented by a meta-regression analysis, ACE inhibitors reduce the progres-diabetics reported in the 1980s that the risk for progres-

sion from microalbuminuria to proteinuria over a period sion from microalbuminuria to overt proteinuria by 62%compared to placebo [28]. Importantly, the impact ofof 6 to 14 years was as high as 80% [22–24]. Such a high

predictive value led to the broad acceptance of microalbu- ACE inhibitors on preventing progression from micro-albuminuria to overt nephropathy lasts for many yearsminuria as a predictor of diabetic nephropathy. This

view, however, is no longer acceptable. Caramori, Fio- and is associated with preservation of GFR [29].Other predictors of progression from microalbumin-retto, and Mauer [25] have convincingly argued that micro-

albuminuria is likely a marker rather than a predictor uria to proteinuria that should be considered include afamily history of diabetes, an abnormal lipid profile, andof renal structural changes. This argument is based on

the finding that in some patients with microalbuminuria, smoking [2], none of which was present in our patient.Based on the foregoing considerations, I conclude thatrenal lesions are already quite advanced [11–13]. I know

of no prospective data regarding rates of progression while our patient is clearly at risk for progressing toovert nephropathy, her risk is less than 50%, far belowbased on results of renal biopsy. For individuals with

advanced renal lesions, microalbuminuria is not a pre- the 80% estimated from earlier studies. Indeed, persis-tent microalbuminuria does not predict clinical protein-dictor, but simply a sign of established nephropathy. In

others with only mild lesions on renal biopsy, microalbu- uria with more than 50% accuracy [25]. Clearly we needadditional markers or predictors of diabetic nephropa-minuria might not be a predictor of nephropathy. On

the other hand, regardless of findings on renal biopsy, thy. Moreover, with appropriate therapy, which includesoptimization of glycemic control and the use of an ACEmicroalbuminuria would be a powerful predictor of ne-

phropathy if most patients were to progress to overt inhibitor or perhaps an angiotensin receptor antagonist,the risk of progression to overt proteinuria in our patientproteinuria as initially reported. However, more recent

studies have clearly shown that the percentage of micro- could be further reduced. Both interventions seem inorder, and it is quite conceivable that they have an addi-albuminuric patients progressing to proteinuria over

more than 10 years is only 30% to 45% [25–27]. Of the tive or even a synergistic effect.I will now address the second issue, namely, whetherremaining patients in these more recent studies, 30%

became normoalbuminuric, and the rest remained micro- we could have predicted that this patient was going todevelop microalbuminuria when she was normoalbumi-albuminuric [25]. It should be noted that interpretation

of the data should take into account disease duration, nuric. At present, there is no simple way to predict thedevelopment of microalbuminuria among type 1 diabet-as most diabetic patients destined to develop proteinuria

will do so within 20 years. Duration of diabetes is often ics. Only about 25% to 30% of patients with type 1diabetes develop overt nephropathy, so one could infershorter in subjects with microalbuminuria progressing to

proteinuria (17 � 8 years) than in those remaining with that, in general, the risk for developing microalbumin-uria cannot be much higher than that. This assumes thatmicroalbuminuria (22 � 9 years, P � 0.005) [25].

In today’s 21-year-old patient, diabetes was diagnosed the development of proteinuria (macroalbuminuria) isalways preceded by a microalbuminuric phase that isat the age of 11 and microalbuminuria at the age of 20.

Accordingly, it is too early to predict, based on the crite- always progressive, but this might not be the case. Fors-blom et al [26] found that about 7% of normoalbumi-ria of microalbuminuria and disease duration, whether

she will progress to proteinuria. If she remains micro- nuric patients progressed to proteinuria and 14% tomicroalbuminuria over 10 years of follow-up. Thus, pro-albuminuric for another 10 years, her risk for progression

to proteinuria from that point on (that is, after about 20 gression took place in 21% of the patients when one


adds both groups. In a different study, Mathiesen et al[29] found a somewhat lower progression rate of micro-albuminuria (10%) over a mean observation also of 10years. Based on this and the review of other studies, Cara-mori, Fioretto, and Mauer [25] estimated that 5% ofnormoalbuminuric patients with at least 7 years of type1 diabetes will progress to proteinuria over the following5 to 10 years, whereas 17% will progress from normo-albuminuria to microalbuminuria. In a recent study, wefound that in adolescents and young adults with type 1diabetes, progression from normoalbuminuria to micro-albuminuria over a follow-up of approximately 5 yearsoccurred in 18% [30]. This finding concurs with datafrom other studies [25], although the follow-up periodwas shorter in our patients. Fig. 3. Kaplan-Meier curves showing the probability of microalbumin-

The following should be considered risk factors for uria according to daytime and nighttime systolic pressure. The probabil-ity of microalbuminuria differed significantly between the two groupsmicroalbuminuria: family history of nephropathy, poor(P � 0.01 by the log-rank test; chi-square � 6.217 with 1 df). The risk ofglycemic control, changes in UAE (even with the normal microalbuminuria was 70% lower in the subjects with a normal nocturnal

albuminuric range), and increased GFR. Since progres- pattern than in those with an abnormal nocturnal pattern. (Reprintedfrom the New England Journal of Medicine 347:797–805, 2002).sion from normoalbuminuria to proteinuria presumes

passing through a microalbuminuric phase, it seems rea-sonable to infer that family history of nephropathy, de-fined by dipstick proteinuria, also should predict the sure is already higher than in type 1 diabetics withoutdevelopment of microalbuminuria [29]. It also seems rea-

microalbuminuria or in age-matched control subjects [19,sonable to assume that poor glycemic control is a main

34–40]. A previous cross-sectional study led us to suggestdeterminant of progression because strict glycemic con-that in type 1 diabetics susceptible to nephropathy, antrol retarded the progression to microalbuminuria in theelevation in nocturnal blood pressure could antedate theDCCT study [3]. Indeed, normoalbuminuric patientsdevelopment of microalbuminuria [19].who progress to microalbuminuria have significantly

We examined the question of which comes first, micro-higher levels of HbA1c than do those who do not progressalbuminuria or a rise in blood pressure in patients with[3, 24, 27, 31]. There was some indication that today’stype 1 diabetes, using ABPM [30]. In this study by Lurbepatient was likely to progress to microalbuminuria,et al [30], we followed 75 healthy normoalbuminuric andnamely the high level of HbA1c. Her creatinine clearancenormotensive patients with type 1 diabetes prospectivelywas in the normal range when she was normoalbumi-over 5 years. Ambulatory blood pressure monitoring wasnuric, and we do not know whether her GFR had beenused to assess blood pressure changes during the awakeelevated earlier. While an increased GFR has been pro-and sleep periods at evaluation and approximately 2posed as an early marker of progression to microalbu-years later while all subjects had remained normoalbumi-minuria, controversy remains as to whether it is a pre-nuric. Of the 75 patients studied, 18% developed micro-dictor of microalbuminuria [24, 25, 27]. This patient hadalbuminuria and were classified as progressors. In theno family history of diabetes with renal disease, which, asremaining subjects, the UAE remained in the normalI mentioned, greatly increases the risk for nephropathy.range after a follow-up of about 5 years, and these pa-Measurements of UAE in the high rate of normal shouldtients were classified as non-progressors. At initial evalu-be considered a risk factor, especially if the level risesation, systolic and diastolic blood pressure, either at theover repeated measurements [32]. In our patient, we hadoffice or as assessed during the awake and sleep periods,only one data point on UAE in the middle of the normo-did not differ significantly between progressors and non-albuminuric range (Fig. 1). Therefore we cannot utilizeprogressors. After a mean follow-up of about 2 years,this as a predictor of progression to microalbuminuria.while all subjects were still normoalbuminuric, sleep sys-Now let’s look at the value of the blood pressure patterntolic blood pressure increased only in progressors. Byas a predictor of nephropathy in type 1 diabetics. Today’scontrast, in non-progressors, sleep systolic blood pres-patient had a striking “non-dipping status” (Fig. 2).sure remained unchanged. The risk of progression toIn type 1 diabetes with incipient nephropathy, definedmicroalbuminuria was further examined on the basis ofby the presence of microalbuminuria, hypertension is not“dipper” versus “non-dipper” status, defined by the ratiousually present [33]. Nevertheless, studies using ambula-of sleep/awake systolic blood pressure (Fig. 3). A 70.2%tory blood pressure monitoring (ABPM) have shown

that at the microalbuminuric stage, nocturnal blood pres- reduction of risk for developing microalbuminuria was


observed in “dippers” as compared with “non-dippers.” tein [5, 46]. Several isoforms of NHE (NHE-1 to 8) thatvary in molecular mass, level and type of glycosylation,A ratio of less than 0.9 had a negative predictive value

for the development of microalbuminuria of 90%. From sensitivity to inhibitors, and cell or tissue distributionhave been identified [47]. NHE-1, the first Na�/H� anti-these findings, we can conclude that in type 1 diabetics

susceptible to developing microalbuminuria, an increase porter cloned, is a 110 kD integral plasma membraneglycoprotein [47, 48]. NHE-1 activity is extremely sensi-in systolic blood pressure during the sleep period pre-

cedes the increase in UAE [30]. Moreover, microalbu- tive to intracellular pH (pHi) owing to the presence ofa proton activation site [49], and this pHi dependencyminuria is highly unlikely to develop in type 1 diabetics

in whom systolic blood pressure during sleep does not protects the cell against acute cytoplasmic acidification.NHE-1 activity, in concert with other pHi regulatoryincrease over about 5 years. The finding of a “dipping”

pattern of systolic blood pressure has a strong negative plasma membrane proteins such as the Na�-dependentand Na�-independent Cl�/HCO�

3 , determines the pHipredictive value for progression to microalbuminuria[30]. In other words, normal dipping is a strong predictor set point [50–52]. Erythrocyte Na�/Li� exchange activity

correlates with proximal tubular NHE-3 expression [53];of non-progression to microalbuminuria.The patient discussed here today had a 24-hour ambu- thus it may be used as a marker of the NHE-3 isoform,

which is located in the apical border of the proximallatory blood pressure pattern characterized by a flat linein both systolic and diastolic blood pressure (Fig. 2). In tubule and is involved with renal Na� reabsorption.

In type 1 diabetic patients with nephropathy, an in-fact, this pattern is unusual because there is no trace of“dipping” whatsoever during sleep; she thus is an ex- crease in the activity of the Na�/H� antiporter has been

reported in a variety of cell types: cultured skin fibro-treme case of non-dipping status reflecting nocturnal hy-pertension. If this pattern had already been present when blasts [54–58], immortalized lymphoblasts [58–61], plate-

lets [62], and leukocytes [63]. We evaluated Na�/H� anti-she was normoalbuminuric, as in the subjects in the pro-spective study that I just described, it could be inferred porter activity and its relationship to DNA and protein

synthesis in cultured skin fibroblasts from patients withthat she had a high risk for microalbuminuria. Stateddifferently, her lack of a normal dipping status—a strong type 1 diabetes classified as having either overt nephrop-

athy or the absence of nephropathy on the basis of urinarynegative predictor of microalbuminuria—puts her athigh risk of progression. Moreover, we know from previ- albumin excretion and renal biopsy findings [54]. In type

1 diabetic patients with overt nephropathy (mean urinaryous cross-sectional studies that most microalbuminuricpatients with type 1 diabetes are non-dippers [19, 34–40]. albumin excretion, 979 � 292 �g/min), Na�/H� antipor-

ter activity in serum-stimulated cells was increased asIn our initial cross-sectional study, 9 of 11 microalbumi-nuric patients (82%) had an abnormal night-day ratio compared to cells from control subjects and cells from

patients without nephropathy (UAE, 6.7 � 7.9 �g/min).consistent with a non-dipping status [19]. Altogether, thelack of “dipping” is a sign of progression to microalbu- By contrast, in cells made quiescent by serum depriva-

tion, we found no significant differences in Na�/H� ex-minuria, and the mere presence of microalbuminuriasuggests a non-dipping status. Since a non-dipping status change activity among the three groups [54]. Intracellular

pH in serum-stimulated fibroblasts from patients withprecedes the development of microalbuminuria, it fol-lows that non-dipping might be an earlier and thus a type 1 diabetes and nephropathy was higher than that of

patients without nephropathy (7.34 � 0.04 and 7.24 �more sensitive predictor of nephropathy. Also, rapidprogression from microalbuminuria to overt nephropa- 0.03, respectively), but this difference did not achieve

statistical significance [54].thy was described in a small group of “non-dippers” [41].A blunted nocturnal decline in arterial blood pressure Several other studies have used the skin fibroblast

model to study Na�/H� antiporter activity in diabeticsis also seen in type 2 diabetes with nephropathy [42] andis increasingly recognized as a risk factor for cardiovascu- with and without nephropathy [55–58]. All these studies

have consistently reported increased activity in cells fromlar disease in diabetic and non-diabetic subjects [43, 44].Non-dipping therefore can be viewed as both a predictor diabetics with nephropathy [54–58]. An important fea-

ture in all these studies is that blood pressure was higherof microalbuminuria and a marker of progression to ne-phropathy and its associated cardiovascular complications. in diabetics with nephropathy than in those without ne-

phropathy (or was similar, but the diabetics with nephrop-Cellular markers athy were hypertensives treated with antihypertensive

medications). Thus, hypertension cannot be excluded asA list of possible cellular markers of nephropathy isgiven in Table 1. Na�/Li� exchange activity [5, 45] and the link between the Na�/H� antiporter overactivity and

nephropathy in type 1 diabetics.Na�/H� (NHE) exchange activity [46] are both increasedin type 1 diabetics with nephropathy. In fact, Na�/Li� ex- Our group and others found a difference in Na�/H�

antiporter activity in studies in which HbA1c was higherchange might represent a functional mode of the plasmamembrane Na�/H� exchanger or a closely related pro- in diabetics with nephropathy but also in others in which


differences in this parameter were not apparent [54–58].That the difference in Na�/H� antiporter activity cannotbe explained by differences in metabolic control is fur-ther suggested by the findings that, in vitro, such differ-ence in activity has been reported in studies in which theglucose concentration was high (17.9 mmol/L) [54–57] ornormal (5 mmol/L) [58]. In fact, one of the great advan-tages of the in vitro model is that the external environmentcan be controlled at will, including the glucose concentra-tion and the presence of insulin and other hormones inthe media. Familial concordance for Na�/H� antiporteractivity in long-term cultured skin fibroblasts from type1 diabetic siblings suggests that at least some of the in

Fig. 4. Thymidine incorporation in cultured skin fibroblasts from threevitro phenotypic characteristics of these cells are likelygroups of patients—controls, type 1 diabetics without nephropathy, andto be genetically determined and, at least in part, inde- those with nephropathy—in the absence and presence of ethyl-isopro-

pendent of in vivo metabolic control [64]. The fact that pyl-amiloride (EIPA). [3H] thymdine incorporation was significantlyincreased in cells from diabetic patients with nephropathy as compareddifferences in Na�/H� antiporter activity were seen onlyto controls, whereas cells from diabetic subjects without nephropathyin serum-stimulated fibroblasts suggested to us that these had an intermediate level. The percent reduction of [3H] thymidine by

differences were related to cell growth. To address this EIPA was similar in all three groups, so that the difference persisted.(Figure modified from data published in Kidney International 50:1684–issue, our studies also measured DNA synthesis and cell1693, 1996).size concurrently with Na�/H� antiporter activity. The

activity of the Na�/H� antiporter (NHE-1) has long beensuspected of playing a role in cell growth.

Pharmacologic NHE-1 inhibitors block vascular smooth fibroblasts from type 1 diabetics with nephropathy displayan abnormal growth phenotype characterized by cell hy-muscle cell (VSMC) proliferation in vitro [65] and in

vivo [66]. Moreover, overexpression of NHE-1 increased perplasia but not hypertrophy and that this phenotypeis associated with overactivity of the Na�/H� antiporterproliferation of VSMC [67], and specific mitogens have

an effect on the NHE-1 promoter [67, 68]. Glucose is during stimulation of cell growth by serum [54]. Ourstudy was unique in that the type 1 diabetics withoutalso thought to act through protein kinase C (PKC) to

increase message levels in certain cell types [69]. During microalbuminuria were selected on the basis of long dis-ease duration and nearly normal mesangial cell structurecell proliferation, NHE-1 is responsible for an elevation

of pHi, which seems to play a permissive role in the by morphometric analysis [54]. Therefore the risk ofprogression to nephropathy under these conditions shouldgrowth of some cell types. Also, actively proliferating

tumor cells often are more alkaline than are normal cells, be very low. The findings of intermediate values of bothNa�/H� antiporter activity and DNA synthesis in fibro-and inhibitors of the exchanger help reduce their rate

of proliferation. It remains to be proven, however, that blasts from such patients, as compared to those fromnormal subjects and type 1 diabetics with overt nephrop-this activation is essential for the cells to progress through

the cell cycle. athy, might reflect either an effect of diabetes per se(hyperglycemia) or attenuated phenotypic features re-Earlier studies using skin fibroblasts from patients with

type 1 diabetes showed a decreased replicative lifespan, flecting individuals less likely to progress in terms ofnephropathy and/or microvascular complications.consistent with premature senescence, although other

studies have not confirmed this finding [70–72]. An in- If overactivity of the antiporter were the primary rea-son for increased DNA synthesis, then its inhibitioncreased proliferation rate and increased DNA synthesis

in type 1 diabetics with nephropathy have been found in should obliterate any differences in cell growth betweendiabetics with and without nephropathy. A pharmaco-studies by Viberti and his associates [55, 56]. Interestingly,

some of these phenotypic features have been reported logic approach consisting of pre-incubating cultured skinfibroblasts with a relatively specific inhibitor of the anti-in cells from hypertensive patients as well [73]. In our

study, DNA synthesis, assessed by [3H] thymidine incor- porter, ethyl-isopropyl-amiloride (EIPA), was used totest this hypothesis [54]. After chronic inhibition of theporation, was increased in a group of type 1 diabetics

with nephropathy as compared to a group without ne- Na�/H� antiporter using EIPA (25 �mol/L), [3H] thymi-dine incorporation was reduced by about 20% in cellsphropathy and to control subjects (Fig. 4). By contrast,

protein synthesis assessed by [14C] L-leucine incorpora- from diabetic patients and from controls. [3H] thymidineincorporation therefore remained higher in cells fromtion was not increased in fibroblasts from type 1 diabetics

with nephropathy; this finding suggested a lack of cellular type 1 diabetics with nephropathy than in those fromcontrols, whereas cells from type 1 diabetics withouthypertrophy [54]. We thus concluded that cultured skin


nephropathy displayed intermediate values (Fig. 4). We is a predictor of nephropathy or simply an alterationconcluded that Na�/H� exchange activity is required for associated with nephropathy.optimal cell growth, but that in itself it cannot account Finally, hyperglycemia itself increases VSMC Na�/H�

for differences in DNA synthesis in diabetics with ne- exchange activity and growth [69]. It is not completelyphropathy as compared to those without nephropathy possible to separate the effect of metabolic control inand to controls [54]. vivo from its long-term effect in vitro, even after several

Further insight into the risk of nephropathy might passages in culture. Genes might be turned on in vivocome from studies at the cellular level focusing on alter- during poor hyperglycemic control that could remainations in cell-cycle regulatory proteins, which control the overexpressed after several passages in culture. The issuerate of DNA synthesis. Such alterations could explain of hyperglycemic memory is unraveling quickly, particu-the abnormal growth phenotype of diabetics, particularly larly because its long-term effect in vivo is becomingthose with nephropathy. Progression through the G1 evident [78, 79]. For instance, the effects of intensivephase of the cell cycle is not only controlled by positive versus conventional insulin therapy on the occurrenceregulators, the G1 cyclins, but is also under negative and severity of post-study retinopathy and nephropathycontrol of a number of different cyclin-dependent kinase were shown to persist for 4 years after the DCCT, despiteinhibitor proteins (CDK1). My laboratory has been ex-

nearly identical glycosylated hemoglobin values duringamining the hypothesis that cultured skin fibroblasts

the 4-year follow-up [79]. Hence, the so-called phenome-from diabetics with nephropathy have a hyperplasticnon of hyperglycemic memory, which refers to the occur-growth phenotype either because of overexpression ofrence of persistent renal damage during subsequent peri-one or several of the cell cycle positive regulators, orods of normal glucose control. This phenomenon mightunderexpression of one or more of the negative regula-translate into phenotypic features in vitro and partlytors [74, 75]. One of the negative cell cycle regulatoryexplain why alterations in cell growth and Na�/H� anti-proteins, p16, a member of the INK4 family, inhibits theporter activity observed in cultured skin fibroblasts arekinase activity associated with CDK4 and CDK6 withoutusually described in patients with poor metabolic control.affecting other CDKs and appears to act by blocking the

association of CDK4,6 with D-type cyclins, thus pre-venting the activation of the kinases [76, 77]. We have

CONCLUSIONshown that the levels of p16 protein are suppressed inProspective studies are needed to establish the pre-skin fibroblasts from type 1 diabetics with overt nephrop-

athy as compared to those without nephropathy (ab- dictive value of clinical and cellular markers for earlystract; Danesh et al, J Am Soc Nephrol 11:638A, 2000); identification of diabetic patients at risk for the develop-this finding suggests the possibility that low levels of p16 ment of nephropathy. Such patients at risk could benefitare a marker for nephropathy in type 1 diabetics. from early intervention, such as the use of ACE inhibi-

As I said, most type 1 diabetics with nephropathy tors or angiotensin II blockers, which might not beinvolved in studies of Na�/H� antiporter activity were needed in patients at reduced risk for nephropathy. Thehypertensive. Importantly, an increased turnover number best clinical marker available to date, microalbuminuria,of NHE-1 also has been noted in immortalized lympho- predicts clinical nephropathy to a lesser extent than origi-blasts from hypertensive subjects [73]. It is therefore pos- nally described. Thus, the need exists for additionalsible that the NHE-1 alteration reflects a hypertensive markers that either alone or in combination with micro-rather than a diabetic phenotype. Additional studies us- albuminuria will identify early the individuals susceptibleing patients with diabetic nephropathy who have not to clinical nephropathy. We have suggested that a “non-developed hypertension would be critically important to

dipping” status in type 1 diabetics without either micro-see whether a diabetic “nephropathic” phenotype exists

albuminuria or hypertension predicts the developmentindependently of hypertension. Cells from nondiabeticof microalbuminuria [30]. Further studies are needed tohypertensive subjects should be used as controls to fur-see whether it also predicts the development of clinicalther elucidate whether the phenotype is “hypertensive”nephropathy. This clinical marker, used in conjunctionor “nephropathic.” Ideally, diabetic patients who havewith other known risk factors, such as high level of HbA1cnot yet developed hypertension, not even nocturnal hy-and family history of nephropathy, translates into a pro-pertension, should be followed longitudinally until thefile of high risk for diabetic nephropathy. Potential cellu-development of microalbuminuria. Such studies alsolar markers such as overactivity of the Na�/H� antiporterwould be the first to examine whether overactivity of(NHE-1 isoform) and phenotypic changes in cell growthNHE-1 has a predictive value for the development ofand cell cycle proteins expressed in vitro by cells ofnephropathy. To my knowledge, all studies to date havediabetics with nephropathy are also worthy of furtherbeen cross-sectional and therefore have not established

whether the overactive Na�/H� antiporter phenotype study.


QUESTIONS AND ANSWERS Dr. Batlle: We made this observation in type 1 dia-betics with overt nephropathy [54]. There are no studiesDr. John T. Harrington (Dean, Tufts Universityshowing that this alteration is already present prior to theSchool of Medicine, Boston, Massachusetts): Do we knowdevelopment of nephropathy. Accordingly, we cannotwhat factors are the root cause of the “non-dipping”claim that it has a predictive value at this time. Perhapsphenomenon? If so, can we block some of those factors?certain cell cycle regulatory proteins are abnormally ex-Dr. Batlle: This question can be addressed by consid-pressed or regulated in patients with nephropathy andering the normal regulation of nocturnal blood pressure.this is the cause of this abnormal growth pattern.During sleep the blood pressure falls as a result of a

Dr. Andrew S. Levey (Division of Nephrology, Tufts-decrease in both cardiac output and peripheral resistance.New England Medical Center, Boston, Massachusetts):This physiologic fall in cardiac output is associated withLet me ask about the cyclin-dependent kinase inhibitor,a decrease in heart rate, an increase in vagal tone, andp16, which you showed to be abnormal. Is this a genetica decrease in sympathetic tone. By extrapolation, it isfactor? Is this a susceptibility factor? Are the peoplereasonable to postulate that diabetics in whom blood pres-more prone to increased cell growth the ones who de-sure does not fall normally during sleep have an overactivevelop nephropathy, or is this a consequence of havingsympathetic system, which is consistent with the auto-diabetes?nomic dysfunction that is so prevalent in diabetics.

Dr. Batlle: The hyperplastic phenotype observed inDr. Harrington: Do we have evidence for that, or iscultured skin fibroblasts from patients with type 1 diabe-it speculation?tes seems to be limited to those who have establishedDr. Batlle: Studies suggest that autonomic dysfunc-nephropathy [54]. Our working hypothesis has been thattion is prevalent in diabetics who are “non-dippers”a high level of cyclin D1 or a low level of a cyclin-[40, 80]. In type 1 diabetics with intermittent microalbu-dependent kinase inhibitor, such as p16, could be aminuria, early signs of subclinical autonomic neuropathy,marker of susceptibility to nephropathy and not the con-such as reduced papillary reaction to darkness, are al-sequence of diabetes per se [74]. I know of no dataready present [81]. We have shown that most microal-showing that diabetes (that is, a hyperglycemic environ-buminuric patients with type 1 diabetes have an abnor-ment) regulates the p16 gene. We know that the p16mal nocturnal blood pressure pattern [19]. It seems safegene is involved in the regulation of cell growth by re-to extrapolate that autonomic dysfunction is usuallytarding G1 transition and entry into the S phase of thepresent during the microalbuminuric phase of diabeticcell cycle. It makes sense that a low level of an inhibitornephropathy. Type 2 diabetics also manifest evidence ofof cell cycle progression would facilitate progressionautonomic dysfunction. Moreover, in type 2 diabeticsthrough G1 phase and potentially lead to increased cellwith nephropathy, the “non-dipping” phenomenon hasreplication and exaggerated cell growth [76, 77].been associated with orthostatic hypotension [80]. Thus,

Dr. Levey: I would like to challenge the notion thatin both type 1 and type 2 diabetics, autonomic dysfunc-microalbuminuria predicts the development of diseasetion, non-dipping, and nephropathy often co-exist andas opposed to being a marker for the presence of disease.might be intimately related. That is not to say, however,Would you at least acknowledge that the relatively shortthat autonomic dysfunction is the sole explanation forfollow-up in some studies, 10 years, might be related tothe “non-dipping” phenomenon often seen in diabeticsthe lower incidence of clinical nephropathy? It mightwith nephropathy.be that another 5 to 10 years of follow-up would haveDr. Harrington: What diseases other than diabetesdemonstrated the development of clinical proteinuria,also have a “non-dipping” phenomenon?thereby improving the predictive value of microalbumin-Dr. Batlle: The “non-dipping” phenomenon was firsturia.described, to my knowledge, in subjects with secondary

Dr. Batlle: Your question really brings in the pointcauses of hypertension. “Non-dipping” is also more prev-of why the more recent studies have shown a much loweralent in certain populations with essential hypertensionrate of transition of microalbuminuria to overt protein-such as African-Americans. I believe the percentage ofuria than the initial ones. The excellent review by Cara-“non-dippers” in African-Americans with hypertensionmori, Fioreto, and Mauer [25] could not find a singleis close to 30%. So, it is not a unique phenomenon ofexplanation for this. Maybe the natural history of thetype 1 diabetics but, in the context of an individual withdisease has changed, and it seems clear that glycemicdiabetes who is not yet hypertensive in the office, “non-control has improved over the last decade. I cannot saydipping” should be viewed as a potential index of pro-that the disease duration was a main factor. In the reviewgression to microalbuminuria.by Caramori, Fioreto, and Mauer, only data that metDr. Harrington: You showed increased thymidine in-the criterion of an interval of about 10 years of knowncorporation in cells from diabetic patients who are likelydiabetes (disease duration) were included for analysisto progress to nephropathy. What is the cause of this

phenomenon? [25]. So disease duration was taken into consideration


already. Despite similar disease duration, the conversion not always taken into account has led to conflicting re-sults. Another limitation probably comes from the diffi-rate to overt proteinuria seems to be much lower than

originally stated. Having said that, I agree that in some culty in measuring GFR accurately and frequently. Eventhe better methods of measuring GFR have been difficultpatients conversion from microalbuminuria to protein-

uria takes place after more than 10 years of microalbu- to standardize. I am fond of the MDRD formula thatDr. Levey has popularized. I believe this formula is goodminuria. After more than 20 years of diabetes, however,

the development of nephropathy is very unlikely. for estimating the GFR, and it allows us to have frequentand serial values of GFR.Dr. Bertrand L. Jaber (Division of Nephrology, Tufts-

New England Medical Center): Since the absence of noc- Dr. Levey: I had the opportunity to review renal func-tion data from the DCCT, in which GFR was measuredturnal blood pressure “dipping” is associated with pro-

gression of diabetic nephropathy, do ACE inhibitors in a large subgroup of patients from the beginning of thestudy. Creatinine clearance was measured in all patients,re-establish nocturnal blood pressure “dipping” patterns?

Such an effect could account for some of the beneficial and baseline GFR was measured in a large subgroup ofthe patients. We found no association between hyperfil-properties of this class of medications.

Dr. Batlle: I am not aware of any studies on this tration and subsequent development of microalbumin-uria in that study. Hyperfiltration was not a predictor,area but it is something we are studying now. In general,

most antihypertensives lower the blood pressure through- nor did it interact with the treatment effect of intensiveglycemic control.out a 24-hour period. Some clinical trials of antihyperten-

sive drugs, including those using ACE inhibitors, have Dr. Harrington: Do you think the prediction equa-tion would be the standard equation for GFR in thisprovided us with 24-hour ambulatory blood pressure

data before and after treatment. Usually, blood pressure early stage of nephropathy?Dr. Levey: I would have some reservations about us-falls throughout the day and night during treatment, and

striking effects limited to the nocturnal pattern have not ing GFR prediction equations in this early stage of ne-phropathy because they have not been shown to be accu-been reported. Whether ACE inhibitors have a special

effect on nocturnal hypertension needs to be investi- rate in individuals with very low serum creatinine levels.gated. My collaborators in Spain have observed near The problem is using non-systemic calibration of creati-normalization of nocturnal hypertension in a few mi- nine assays to account for errors due to interferencecroalbuminuric patients treated with captopril (Lurbe by non-creatinine chromogens. In patients with serumet al, unpublished data). There might be interactions creatinine below 1.0 mg/dL, systemic differences in cali-between the renin-angiotensin and sympathetic nervous bration among laboratories could lead to large errors insystems that ACE inhibitors affect, returning to normal estimated GFR. The formulae were very accurate in thethe abnormal nocturnal blood pressure pattern. MDRD study in patients with GFR as high as 90 mL/min/

Dr. Harrington: How would you incorporate those 1.73 m2 and in the African-American Study of Kidneyobservations on the “non-dipping” phenomenon with Disease and Hypertension (AASK) with GFRs up towhat we know about the adverse prognostic effect of 100 to 110 mL/min/1.73 m2. However, young patientshaving the “DD” ACE alleles? with diabetes can have GFRs �140 mL/min/1.73 m2 with

Dr. Batlle: Only by way of a possible association. serum creatinine levels below 1.0 mg/dL.What has been suggested so far is that diabetics with the

Reprint requests to Dr. D. Batlle, Chief, Division of Nephrology,DD genotype of the ACE gene have an accelerated Northwestern University Feinberg School of Medicine, 303 East Chicagoprogression to ESRD [82]. I also can cite a study showing Avenue, Chicago, IL 60611-3008, USA.

E-mail: [email protected] proteinuric patients with a “non-dipping” status alsoprogress at a faster rate [37]. Perhaps those individuals

ACKNOWLEDGMENThave unique features, and it would be a good idea tocombine the assessment of ACE polymorphisms with The work described in this Forum was supported by the National

Institutes of Health (DK 53446) and a Merit Review from the Veteransthe patterns of “dipping” and “non-dipping.”Administration.Dr. Harrington: I was taught years ago that the in-

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