Tubular and Glomerular Injury in Diabetesand the Impact of ACE InhibitionSTINE E. NIELSEN, MD

1

TAKESHI SUGAYA, MD, PHD2

LISE TARNOW, MD, DMSC1

MARIA LAJER, CANDSCI1

KATRINE J. SCHJOEDT, MD1

ANNE SOFIE ASTRUP, MD1

TSUNEHARU BABA, MD, PHD3

HANS-HENRIK PARVING, MD, DMSC4

PETER ROSSING, MD, DMSC1

OBJECTIVE — We studied tubular and glomerular damage in type 1 diabetic patients bymeasuring urinary–liver fatty acid binding protein (U-LFABP) and albuminuria. Subsequently,we evaluated the effect of ACE inhibition on U-LFABP in patients with diabetic nephropathy.

RESEARCHDESIGNANDMETHODS — We studied Caucasians with type 1 diabetes:58 with normoalbuminuria (urinary albumin �30 mg/24 h), 45 with persistent microalbumin-uria (30–300 mg/24 h), and 45 with persistent macroalbuminuria (�300 mg/24 h). A controlgroup consisted of 57 healthy individuals. The groups were matched by sex and duration ofdiabetes. In addition, U-LFABP was measured in 48 type 1 diabetic patients with diabeticnephropathy in a randomized crossover trial consisting of 2 months of treatment with 20, 40,and 60 mg lisinopril once daily in random order.

RESULTS — In the cross-sectional study, levels of U-LFABP were significantly higher innormoalbuminuric patients versus those in the control group (median 2.6 [interquartile range1.3–4.1] vs. 19 [0.8–3.0] �g/g creatinine, P � 0.02) and increased with increasing levels ofalbuminuria (microalbuminuric group 4.2 [1.8–8.3] �g/g creatinine and nephropathy group71.2 [8.1–123.4], P � 0.05 for all comparisons). U-LFABP correlates with the urinary albumin-to-creatinine ratio (R2 � 0.54, P � 0.001). In the intervention study, all doses of lisinoprilsignificantly reduced urinary albumin excretion rate and U-LFABP from baseline. The reductionsin U-LFABP were 43, 46, and 40% with increasing doses of lisinopril (NS).

CONCLUSIONS — An early and progressive increase in tubulointerstitial damage as re-flected by increased U-LFABP levels occurs in type 1 diabetic patients and is associated withalbuminuria. Furthermore, ACE inhibition reduces the tubular and glomerular damage anddysfunction.

Diabetes Care 32:1684–1688, 2009

D iabetic nephropathy is a serious andcommon complication in diabeticpatients. Although studies from se-

lected centers suggest a declining inci-dence of diabetic nephropathy (1), still30–40% of all patients with diabetes de-velop this complication (2). Diabetic ne-phropathy is associated with a higher riskof other complications such as cardiovas-cular disease, retinopathy, and neuropa-

thy. It is the most common cause of end-stage renal failure in Western countries.Therefore, it is of great interest to predictand prevent the development of diabeticnephropathy.

Persistent microalbuminuria is the es-tablished predictor of development of di-abetic nephropathy and progressive renalinsufficiency. However, use of the urinaryalbumin excretion rate (UAER), as an in-

dicator of renal damage, has some lim-i t a t i o n s . S e v e r a l p a t i e n t s w i t hmicroalbuminuria do not progress tomacroalbuminuria but continue havingmicroalbuminuria (30%) or even regressto normoalbuminuria (15–30% after 7.5years of follow-up) (3). As reviewed pre-viously, glomerular damage and tubulo-interstitial damage are important factorsin the pathology of diabetic nephropathy(2,4).

Liver fatty acid binding protein(LFABP) is an intracellular carrier proteinthat is expressed in the proximal tubulesin the human kidney and the liver (5). Byimmunohistochemical staining of renalbiopsy specimens, it has been shown thaturinary LFABP (U-LFABP) excretion ishighly associated with structural andfunctional tubular kidney damage. Thiswas confirmed in patients with chronickidney disease including minimal-changenephritic syndrome, nephrosclerosis, lu-pus nephritis, and diabetic nephropathy(6). A previous study in chronic kidneydisease has shown that serum LFABP lev-els do not affect the U-LFABP level, whichsuggests that there is no transglomerularpassage of LFABP in chronic kidney dis-ease and that the LFABP measured inurine originates primarily from tubularcells (7). It has been hypothesized thatLFABP is a protective protein; however,previous studies have, to our knowl-edge, not been able to confirm thishypothesis (8).

In patients with diabetic nephropathy,a reduction in albuminuria is a predictorof renoprotection (9). It is suggested thatthe combination of the two parameters,albuminuria and U-LFABP, would be use-ful in monitoring chronic kidney diseasemore than either alone.

The relationship between albumin-uria and U-LFABP has, to our knowledge,not been studied in type 1 diabetic pa-tients. Furthermore, whether U-LFABPcould be used to monitor the renoprotec-tive effect on the decline in glomerularfiltration rate (GFR) or the effect on uri-nary albumin excretion in short-termstudies in type 1 diabetic patients withdiabetic nephropathy has not beenstudied.

The aim of this study was to investi-gate the levels of U-LFABP in a cross-

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

From the 1Steno Diabetes Center, Copenhagen, Denmark; the 2Research Unit for Organ Regeneration, RikenKobe Institute, Hyogo, Japan; 3Third Department of Internal Medicine, School of Medicine, FukushimaPrefecture University, Fukushima, Japan; the 4Faculty of Health Sciences, University of Aarhus, Aarhus,Denmark, and Department of Medical Endocrinology, Rigshospitalet, University Hospital, Copenhagen,Denmark.

Corresponding author: Stine E. Nielsen, sene@steno.dk.Received 5 March 2009 and accepted 25 May 2009.Published ahead of print at http://care.diabetesjournals.org on 5 June 2009. DOI: 10.2337/dc09-0429.

Clinical trial reg. no. NCT00118976, clinicaltrials.gov.© 2009 by the American Diabetes Association. Readers may use this article as long as the work is properly

cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

P a t h o p h y s i o l o g y / C o m p l i c a t i o n sO R I G I N A L A R T I C L E

1684 DIABETES CARE, VOLUME 32, NUMBER 9, SEPTEMBER 2009

sectional study of type 1 diabetic patientswith different levels of albuminuria com-pared with those in a nondiabetic controlgroup. In addition, we wanted to explorethe short-term effect of increasing dosesof the ACE inhibitor lisinopril on U-LFABP levels in patients with type 1 dia-betes and diabetic nephropathy from arandomized, double-blind crossoverstudy.

RESEARCH DESIGN ANDMETHODS — The cross-sectionalstudy was based on data from a cohortused to identify biomarkers of diabeticnephropathy by proteomic analyses (10).The population consisted of Caucasianpatients with type 1 diabetes and differentlevels of albuminuria recruited from theoutpatient clinic at Steno Diabetes Centerin 2004. Based on albumin excretion in24-h urine samples that were collected aspart of the routine care of the patients be-fore the present study, patients were di-vided into three groups: 58 withnormoalbuminuria (UAER �30 mg/24h), 45 with persistent microalbuminuria(UAER between 30 and 300 mg/24 h in atleast two of three consecutive samples)and 45 with persistent macroalbuminuria(UAER �300 mg/24 h in at least two ofthree consecutive samples). The controlgroup consisted of 57 nondiabetichealthy individuals. The 24-h urine sam-ples were only used for assessing inclu-sion criteria. Groups were matched by sexand duration of diabetes.

Investigations were performed in themorning. Arterial blood pressure was mea-sured three times with an appropriate-sizedcuff after at least a 10-min rest. Urinary al-bumin concentration was measured by anenzyme immunoassay from early morn-ing spot urine samples and expressed asthe urinary albumin-to-creatinine ratio(UACR). Serum and urine creatinine con-centrations were assessed by a kineticJaffe method. Estimated GFR (eGFR) wascalculated using the reexpressed four-variable Modification of Diet in Renal Dis-ease study equation (eGFR � 175 �plasma creatinine�1.154 � age�0.203 �1.212 [if black] or � 0.742 [if female]) asdefined previously (11) where eGFR is ex-pressed as milliliters per minute per 1.73m2 and plasma creatinine is expressed asmilligrams per deciliter. U-LFABP wasmeasured in a two-step sandwich en-zyme-linked immunosorbent assay (12)and expressed as the U-LFABP-to-creatinine ratio. The inter- and intra-assayvariations were 6.8 and 8.2%, respec-

tively. Plasma samples were stored at�80°C, and urine samples were stored at�20°C until analysis.

The second study was a randomizeddouble-masked crossover trial performedin 2005 (13). Patients were treated in ran-dom order with 20, 40, and 60 mg lisin-opril, with each period lasting 2 months.A total of 56 patients with type 1 diabetes,hypertension (�135/85 mmHg), and di-abetic nephropathy were randomly as-signed in the study.

The primary end point was changesin UAER (micrograms per 24 h). Albu-minuria was determined in three consec-utive 24-h urine collections completedimmediately before the end of each treat-ment period. Among the secondary endpoints measured at the end of each treat-ment period were 24-h ambulatory bloodpressure and eGFR. U-LFABP was alsomeasured in 24-h urine collections (mi-crograms per 24 h). For safety reasons,blood pressure, plasma potassium,plasma sodium, and plasma creatininewere determined 3 weeks after the begin-ning of each treatment period. GFR (base-line) was measured at baseline after asingle intravenous injection of 3.7 MBq51Cr-EDTA at 8:30 A.M. by determiningthe radioactivity in venous blood samplestaken 180, 200, 220, and 240 min afterinjection. The results were standardizedfor 1.73 m2 body surface area. Blood pres-sure was measured by a 24-h ambulatoryblood pressure device (Takeda TM2421;A & D Medical, Tokyo, Japan).

The study was initiated by a 2-monthwashout period during which all antihy-pertensive drugs were withdrawn, exceptfor slow-release furosemide in individualdoses to prevent fluid retention and hy-perkalemia and control blood pressure.Thereafter, patients were treated in ran-dom order with 20, 40, and 60 mg lisino-pril, with each period lasting 2 months.Dietary intake of protein and salt was notrestricted.

Both studies were performed accord-ing to the principles of the Declaration ofHelsinki and the intervention study wasapproved by the ethics committee ofCopenhagen County. Written informedconsent was obtained from all patients.

Statistical analysisNormally distributed variables are ex-pressed as means � SD (baseline charac-teristics) and means � SEM. U-LFABPand UACR are given as medians (inter-quartile range [IQR]). All comparisonsbetween groups of normally or log-

normally distributed parameters wereperformed with a one-way ANOVA, andordinal data were compared using aKruskall-Wallis and a Mann-Whitney Utest. Changes in variables between visitsduring the intervention study are ex-pressed as mean differences (95% CI).Comparisons of log-U-LFABP betweeneach treatment period were performedusing linear mixed models. The adaptedmodel was one with fixed effects of treat-ment level, visit, and carryover (i.e., treat-ment level in the previous period) and arandom effect of person included to ac-count for the person independence indata. Linear regression analysis was usedto analyze for correlations between thechange from baseline in U-LFABP basedon differences in log-transformed valuesand changes in UAER, ambulatory bloodpressure, and eGFR, respectively. P �0.05 was considered significant (two-tailed test). Data were evaluated usingSPSS (version 14.0; SPSS, Chicago, IL).

RESULTS

Cross-sectional studyThe clinical characteristics of the patientsare shown in Table 1. The patients werewell matched regarding the duration ofdiabetes and sex. Patients with nor-moalbuminuria and microalbuminuriawere slightly older than control subjectsand patients with macroalbuminuria.

We found a significant difference insystolic blood pressure among the groups(P � 0.009) because of a difference be-tween control subjects and patients withmicroalbuminuria (P � 0.017) and mac-roalbuminuria (P � 0.001). Diastolicblood pressure was significantly higher incontrol subjects, whereas there was nodifference among diabetic groups. eGFRwas significantly lower in the macroalbu-minuric group than in all other groups.The significant difference in serum creat-inine was due to differences between themacroalbuminuric group and all othergroups.

U-LFABP and urinary albuminexcretionU-LFABP levels are shown in Table 1 andillustrated in Fig. 1. U-LFABP ratios weresignificantly higher in the normoalbu-minuric group versus the control group(median 2.6 [IQR 1.3–4.1] vs. 1.9 [0.8–3.0] �g/g creatinine, P � 0.02) and in-creased with increasing levels ofalbuminuria (microalbuminuric group4.2 [1.8–8.3] �g/g creatinine and mac-

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roalbuminuric group 71.2 [8.1–123.4]�g/g creatinine, P � 0.05 for allcomparisons.

U-LFABP correlated with UACR (R2 �0.54, P � 0.001) in the combined groupof all diabetic patients. In the normoalbu-minuric group, there was also a signifi-cant but weak association between UACRand U-LFABP (R2 � 0.07, P � 0.04).

In patients with macroalbuminuria,U-LFABP levels correlated with UACR(R2 � 0.50, P � 0.001) and eGFR (R2 �0.34, P � 0.001). When adjusted foreGFR, there was still a significant differ-ence in U-LFABP for comparisons be-tween patients with macroalbuminuriaand the other groups (P � 0.01).

There was a significant correlation be-tween U-LFABP and systolic blood pres-sure (R2 � 0.07, P � 0.001). However,

this may be explained by the associationbetween U-LFABP and renal function be-cause it disappeared after adjustment foreGFR and UACR. There were no signifi-cant associations between U-LFABP andsex, age, BMI, diastolic blood pressure,cholesterol, or A1C.

Effect of ACE inhibition onU-LFABP: intervention studyA total of 56 patients were randomly as-signed in the study, and 49 patients com-pleted the study. One patient did not haveU-LFABP measured at baseline; resultsare given for the remaining 48.

Age at baseline was (mean � SD)50 � 10 years and the duration of diabe-tes was 33 � 10 years. Baseline UAER was(geometric mean) 365 mg/24 h (95% CI

240 –554). Baseline GFR (51Cr-EDTA)was 73 � 28 ml/min per 1.73 m2.

All doses of lisinopril significantly re-duced UAER, U-LFABP, and arterialblood pressure compared with baseline(Table 2). At baseline, U-LFABP was me-dian 12.69 �g/24 h (IQR 3.88–49.82).Reductions from baseline in U-LFABPwere 43% (95% CI 15–62), 46% (19–64), and 40% (11–60) with increasingdoses of lisinopril (no significant differ-ence between doses). The reduction in U-LFABP was associated with the changes in24-h systolic ambulatory blood pressure(R2 � 0.22, P � 0.01) (similar for dia-stolic ambulatory blood pressure) andUAER (R2 � 0.38, P � 0.001) but notwith changes in eGFR. (Data are given forchange from baseline to 40 mg of lisino-pril, which gave the largest response, butthe same results were found for the otherdoses of lisinopril.) The decline in U-LFABP was still significant when adjustedfor the decline in UAER and for 24-h sys-tolic blood pressure (P � 0.011).

CONCLUSIONS — In our cross-sectional study, we have shown that themarker of tubulointerstitial damage, U-LFABP, is elevated in type 1 diabetic pa-tients compared with nondiabetic healthycontrol subjects. In addition, we haveshown that U-LFABP is further increasedin type 1 diabetic patients with micro-and macroalbuminuria, reflecting in-creased tubular damage with increasinglevels of albuminuria. There were no sig-nificant correlations between U-LFABPand sex, age, or A1C.

In our randomized, double-maskedcrossover study, ACE inhibition with lis-inopril reduced U-LFABP. There was nosignificant difference in effect betweendoses of lisinopril from 20 to 60 mg daily.

Figure 1—U-LFABP in control group and three diabetic groups with different levels ofalbuminuria.

Table 1—Clinical data of control group and type 1 diabetic patients differentiated according to the level of albuminuria in cross-sectional study

Controlgroup Normoalbuminuria Microalbuminuria Macroalbuminuria P

n (male/female) 57 (37/20) 58 (30/28) 45 (24/21) 45 (27/18) 0.273Age (years) 51 � 11.0 56 � 10.8 54 � 11.1 49 � 9.3 0.004Diabetes duration (years) — 37 � 11 35 � 11 34 � 11 0.411Systolic blood pressure (mmHg) 132 � 16 138 � 21 142 � 23 145 � 19 0.009Diastolic blood pressure (mmHg) 81 � 11 75 � 11 74 � 12 78 � 10 0.002A1C (%) 5.5 � 0.3 8.2 � 1.1 8.8 � 1.2 8.8 � 1.1 �0.001eGFR (ml/min per 1.73m2) 70.5 � 9.6 70.3 � 10.3 71.0 � 13.3 48.7 � 19.2 �0.001Creatinine (�mol/l)* 95 (73–146) 91 (69–121) 89 (70–143) 127 (84–144) �0.001UACR (mg/g)† 2 (1–5) 5 (3–9) 27 (11–68) 461 (173–1,172) —U-LFABP (�g/g creatinine)† 1.9 (0.8–3.0) 2.6 (1.3–4.1) 4.2 (1.8–8.3) 71.2 (8.1–123.4) �0.05

Data are means � SD, *median (range), or †median (IQR). P value refers to allover difference between groups (ANOVA).

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1686 DIABETES CARE, VOLUME 32, NUMBER 9, SEPTEMBER 2009

Previous studies of U-LFABP in diabe-tes were cross-sectional studies on U-LFABP in type 2 diabetic patients. Suzuki etal. (14) performed a cross-sectional study in356 adult type 2 diabetic patients. They di-vided the patients into four groups: nor-moalbuminuric, microalbuminuric,macroalbuminuric, and renal failure, but nocontrol group was included. They reporteda significant association between the stageof diabetic nephropathy and U-LFABP, al-though no significant difference betweenthe normoalbuminuric and microalbumin-uric groups was seen.

The results from our cross-sectionalstudy show that patients with normoalbu-minuria and type 1 diabetes had higherU-LFABP than the healthy control sub-jects (Table 1). The normoalbuminuricgroup had a significantly higher level ofalbuminuria than the healthy control sub-jects, but even with adjustment for thisresult, there was still a significant differ-ence in U-LFABP between the two groups(P � 0.014). One possible explanation isthat having diabetes elevates U-LFABP.However, this possibility is not likely be-cause U-LFABP is not correlated withA1C. Another hypothesis is that some ofthe patients in the normoalbuminuricgroup had higher levels of U-LFABP as apredictor of future development of mi-croalbuminuria and diabetic nephropa-thy. The significant association betweenUACR and U-LFABP in the normoalbu-minuric group also indicated this. How-ever, to test this hypothesis we needprospective follow-up studies in nor-moalbuminuric type 1 diabetic patients.

We also saw that with increasing lev-els of albuminuria, from normoalbumin-

ur i a to mic roa lbuminur i a andmacroalbuminuria, U-LFABP is increas-ing. Part of this association can be ex-plained by the transport with albumin offatty acids to the proximal tubules. Herethe fatty acids are absorbed into the prox-imal tubular cells, where the role ofLFABP is to transport the fatty acids to themitochondria. Therefore, when albumin-uria increases, the LFABP gene is upregu-lated and more LFABP is excreted intothe urine (15). U-LFABP is also elevatedindependently of albuminuria because ofincreased tubular production due to tu-bular hypoxia and oxidative stress (16),which is seen in diabetes (17). Our studyis cross-sectional, and therefore we can-not draw conclusions on the time per-spective between elevation in U-LFABPand development of nephropathy. How-ever, from earlier studies in nondiabeticchronic renal disease, the potential of U-LFABP as an early predictor of nephropa-thy is supported. Kamijo et al. (18)performed a multicenter observationaltrial in 48 patients with nondiabeticchronic kidney disease. Retrospectively,they divided the patients into progressorsand nonprogressors in their chronic kid-ney disease based on changes in creati-nine clearance during 1 year of follow-up.They found that U-LFABP had a highersensitivity (94%) than urinary albumin(69%) but a lower specificity (63%) thanurinary albumin (94%) in predicting pro-gression in chronic kidney disease.

U-LFABP is different from other sug-gested biomarkers, e.g., �1- and 2-microglobulins: U-LFABP is produced inthe tubular cells, whereas �1- and 2-microglobulins are freely filtered through

the glomerular basement membrane.During poor glycemic control, urinary ex-cretion of 2-microglobulin is increased(19); this is a result of a lack of reabsorp-tion in the damaged tubular cell (20,21).2-Microglobulin is unstable at low pH,causing an underestimate of the tubulardamage (22).

In our randomized, crossover study,we saw that 2 months of ACE inhibitionreduces U-LFABP by 40%. There wasno significant difference in the decline inU-LFABP among doses of lisinopril, sug-gesting an optimal effect with 20 mg lis-inopril daily, which was in contrast to theincreased decline in UAER when dosesof lisinopril were increased from 20 to40 mg.

We observed that the decrease in U-LFABP is associated with a decrease in al-buminuria, but the relatively weakassociation (R2 � 0.38, P � 0.001) sup-ports the fact that the decline in U-LFABPis not only explained by reduced albu-minuria but also suggests, as mentionedearlier, that the reduction also reflects re-duced tubular damage. The decrease inU-LFABP indicates that the tubular dam-age and upregulation of the U-LFABPgene are reversible.

Our finding is in accordance with ear-lier studies in type 2 diabetic patients(23), which showed a significant decreasein U-LFABP when these patients weretreated with an angiotensin II receptor an-tagonist. Experimental studies in diabeticrats have shown that renin-angiotensin-aldosterone system blockade reduces an-tiapoptotic factors (24) and that oxidativestress in tubular cells is reduced andchronic hypoxia is corrected independentof the blood pressure–lowering effect (25)and thereby preserves tubular function.

In summary, an early and progressiverise in tubulointerstitial damage as re-flected by increased U-LFABP levels oc-curs in type 1 diabetic patients and isassociated with albuminuria. Further-more, ACE inhibition reduces the tubularand glomerular damage and dysfunction.Our studies indicate that U-LFABP is anew marker of tubular damage and a po-tential supplement to the glomerulardamage marker albuminuria for progno-sis, diagnosis, and treatment of kidney in-jury, although further longitudinalstudies are needed.

Acknowledgments— The studies were fi-nanced out of local funds and were not sup-ported by the pharmaceutical industry. T.S. is

Table 2—Laboratory data during treatment with 20, 40, and 60 mg lisinopril in random ordercompared with baseline in 48 type 1 diabetic patients with diabetic nephropathy

Baseline

Lisinopril

20 mg 40 mg 60 mg

24-h systolic blood pressure (mmHg),reduction from baseline — 10 (6–14)* 13 (8–18)*† 12 (8–17)*†

24-h diastolic blood pressure(mmHg), reduction from baseline — 5 (3–7)* 7 (5–10)*† 7 (5–10)*†

Reduction in UAER compared withbaseline — 63 (55–69)* 71 (66–76)* 70 (64–75)*

eGFR (ml/min per 1.73 m2) 75 � 4 69 � 4* 68 � 4* 67 � 4*A1C (%) 8.6 � 0.1 8.7 � 0.2 8.8 � 0.2 8.9 � 0.1*Plasma potassium (mmol/l) 3.9 � 0.1 4.3 � 0.1* 4.4 � 0.1* 4.4 � 0.1*U-LFABP reduction compared with

baseline — 43 (15–62)* 46 (19–64)* 40 (11–60)*

Data are means � SEM or mean difference (95% CI). The Friedman test for several related samples was usedfollowed by a paired-samples t test if significant. *P � 0.05 vs. baseline; †P � 0.05 vs. 20 mg.

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the director and senior scientist of CMIC, To-kyo, Japan, the company that produced thekits for LFABP analysis. No other potentialconflicts of interest relevant to this article werereported.

Parts of this study were presented in ab-stract form at the 44th annual meeting of theEuropean Association for the Study of Diabe-tes, Rome, Italy, 11 September 2008.

We thank B.R. Jensen, T.R. Juhl, B.V. Han-sen, U.M. Smidt, and L. Pietraszek for theirhelp with collecting the data.

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