Pathophysiology/Complications ORIGINAL ARTICLE

Polymorphisms of the C-␤ (PRKCB1) Accelerate Kidney Disease in Type 2 Diabetes Without Overt Proteinuria

1 1 SHIN-ICHI ARAKI, MD, PHD KEIJI ISSHIKI, MD, PHD methods, a case-control study and a family- 2 1 MASAKAZU HANEDA, MD, PHD ATSUNORI KASHIWAGI, MD, PHD based study (8). In our previous study (8), 1 1,3 TOSHIRO,SUGIMOTO, MD, PHD DAISUKE KOYA, MD, PHD 1 a T-G haplotype consisting of two single MOTOHIDE ISONO, MD, PHD nucleotide polymorphisms (Ϫ1504C/T and Ϫ546C/G) in the promoter region was associated with twofold increased OBJECTIVE — We investigated the contribution of PKC-␤ gene (PRKCB1) polymorphisms risk of diabetic nephropathy. Although to diabetic kidney disease in a prospective observational follow-up study. our report provided the first evidence that the PRKCB1 polymorphisms may contrib- RESEARCH DESIGN AND METHODS — A total of 364 Japanese subjects with type 2 ute to genetic susceptibility to diabetic ne- diabetes without overt proteinuria were enrolled during 1996–1998 and followed until 2004. phropathy (8), several questions remain Five single nucleotide polymorphisms (Ϫ1504C/T, Ϫ546C/G, Ϫ348A/G, Ϫ278C/T, and Ϫ238C/G) in the promoter region of PRKCB1 were genotyped. The end points were transition unclear: whether this relevance extrapo- from stage to stage of diabetic nephropathy as a time-to-event outcome and the annual decline lates to type 2 diabetic subjects, whether rate of estimated glomerular filtration rate (eGFR) as a slope-based outcome. the risk haplotype is associated with early diabetic nephropathy, and whether this RESULTS — During the study (median 6 years), 34 of 364 subjects (9.3%) progressed. risk haplotype of PRKCB1 influences kid- Kaplan-Meier estimation revealed that subjects with both T allele at Ϫ1054 C/T and G allele at ney function. Ϫ546 C/G polymorphisms frequently showed transition to advanced stages of diabetic nephrop- Therefore, in the present study, we athy (P ϭ 0.015). The annual change rate in eGFR in the subjects with both alleles was also investigated the effect of the risk haplo- significantly higher than in others (Ϫ2.96 Ϯ 0.62 vs. Ϫ1.63 Ϯ 0.15 ml/min per 1.73 m2/year, ϭ type of PRKCB1 on diabetic nephropathy P 0.02). The estimated frequency of this risk T-G haplotype was significantly higher in the by assessing two renal outcomes: transi- progressors who showed transition to advanced nephropathy stages (12%) than in the nonpro- gressors (5%) (odds ratio 2.3 [95% CI 1.0–5.2]), and it was also higher in those with accelerated tion from any given stage to the more ad- decline of the ⌬ eGFR (Ն3 ml/min per 1.73 m2/year) than in those without (2.1 [1.1–3.9]). vanced stage of diabetic nephropathy defined by albumin excretion rate (AER) CONCLUSIONS — Our study indicates that PRKCB1 is a predictor for worsening of kidney as a time-to-event outcome and kidney disease in Japanese subjects with type 2 diabetes. function defined by the annual decline rate of estimated glomerular filtration rate Diabetes Care 29:864–868, 2006 (eGFR) as a slope-based outcome. To this purpose, we carried out a prospective ob- iabetic nephropathy associated nase C (PKC)-␤ under diabetic condition servational follow-up study in Japanese with type 2 diabetes is a leading is proposed as a putative mechanism in subjects with type 2 diabetes without D cause of end-stage renal disease. the pathogenesis of diabetic nephropathy overt proteinuria. While clinical studies clearly show that (7). Thus, the gene (PRKCB1) encoding prolonged hyperglycemia is an important this protein is considered to be a candi- RESEARCH DESIGN AND risk factor for this microvascular compli- date gene for susceptibility to diabetic ne- METHODS — The subjects were re- cation (1,2), epidemiological and familial phropathy. Recently, we first reported the cruited from among the participants at studies (3–6) suggest that genetic suscep- association between the PRKCB1 polymor- the outpatient clinic of the Department of tibility also plays a pivotal role. phisms and diabetic nephropathy in type 1 Medicine, Shiga University of Medical An abnormal activation of protein ki- diabetic patients by using two independent Science (9). During 1996–1998, patients ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● clinically diagnosed as having type 2 dia- betes in accordance with World Health From the 1Department of Medicine, Shiga University of Medical Science, Otsu, Shiga, Japan; the 2Second Department of Medicine, Asahikawa Medical College, Asahikawa, Hokkaido, Japan; and the 3Division of Organization criteria were examined with Endocrinology and Metabolism, Department of Medicine, Kanazawa Medical University, Kahoku-gun, Ish- multiple measurements of urinary AER ikawa, Japan. and estimated GFR in 24-h urine sample Address correspondence and reprint requests to Shin-ichi Araki, MD, PhD, Department of Medicine, collection in the initial 2 years (baseline Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan. E-mail: [email protected]. Received for publication 14 September 2005 and accepted in revised form 2 January 2006. period). On the basis of multiple mea- Abbreviations: AER, albumin excretion rate; eGFR, estimated glomerular filtration rate; MDRD, Modi- surements (average of 2.7 times [range fication of Diet in Renal Disease; PKC, . 2–9]), patients (n ϭ 518) were classified A table elsewhere in this issue shows conventional and Syste`me International (SI) units and conversion (regardless of diabetes duration) as hav- factors for many substances. ing normoalbuminuria, microalbumin- © 2006 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby uria, or overt proteinuria. Only patients marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. with normoalbuminuria or microalbu-

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Table 1—Clinical characteristics at baseline and allele frequencies at each polymorphic site of tion from any given stage to the more ad- PRKCB1 in subgroups according to the progression of diabetic nephropathy vanced stage of diabetic nephropathy. The progressors included both cases that Nonprogressors Progressors developed microalbuminuria and those that progressed from microalbuminuria n 330 34 to overt proteinuria. Subjects who Clinical characteristics showed intermittent transition to ad- Sex (male/female) 162/168 18/16 vanced stages were not counted among Age (years) 60 Ϯ 961Ϯ 10 the progressors. Duration of diabetes (years) 13 Ϯ 713Ϯ 8 The eGFR was estimated with one of Ϯ Ϯ HbA1c (%) 7.2 0.9 7.3 1.1 the equations developed in the Modifica- Total cholesterol (mg/dl) 213 Ϯ 30 210 Ϯ 32 tion of Diet in Renal Disease (MDRD) Triglycerides (mg/dl) 110 Ϯ 60 136 Ϯ 60* study (11). In the present study, the indi- Systolic blood pressure (mmHg) 133 Ϯ 16 137 Ϯ 15 vidual data for the daily urea excretion Diastolic blood pressure (mmHg) 76 Ϯ 977Ϯ 10 from a 24-h urine collection was avail- Hypertension (%) 48 65 able. Thus, we used the MDRD equation BMI (kg/m2) 23 Ϯ 324Ϯ 3* with demographic, serum, and urine vari- Retinopathy (%) 80 94 ables, which were reported to be the most AER (␮g/min) 20 Ϯ 24 38 Ϯ 43* precise in the original MDRD study (11): Ϫ Ϫ eGFR (min/ml per 1.73 m2) 105 Ϯ 20 100 Ϯ 20 GFR ϭ 198 ϫ (Pcr) 0.858 ϫ (Age) 0.167 Diabetes treatment (diet/oral agents/insulin) 64/191/75 9/13/12 ϫ (0.822 if patient is female) ϫ Ϫ ϩ Allele frequencies (%)† (SUN) 0.293 ϫ (UUN) 0.249, where Pcr is Ϫ1054 C/T 94.4/5.6 88.8/11.2* serum creatinine concentration (mg/dl), Ϫ546 C/G 94.9/6.1 88.8/11.2 SUN is serum urea nitrogen concentra- Ϫ348 A/G 66.7/33.3 61.8/38.2 tion (mg/dl), and UUN is urinary urea ni- Ϫ287 C/T 66.4/33.6 60.3/39.7 trogen excretion (g/day). The rate of Ϫ238 C/G 74.5/25.5 79.6/20.6 annual decline in eGFR (⌬ eGFR) over the Data are means Ϯ SD. *P Ͻ 0.05 vs. nonprogressors. †Data are expressed as percent of for each course of the study was determined from allele. the slope of the plot of all measurements of eGFR for each individual (median 8 times [range 4–13]) calculated with lin- minuria in the baseline period were en- because they had undergone treatment ear regression analysis and was expressed rolled in this study. Patients with overt for cancer detected during the follow-up as ml/min per 1.73 m2/year. Also, we di- proteinuria (n ϭ 72) or with intermittent periods. Thus, data for 364 patients (261 chotomized the annual decline in eGFR as microalbuminuria (n ϭ 22) were ex- with normoalbuminuria and 103 with accelerated decline (⌬ eGFR Ն3 ml/min cluded. As other exclusive criteria, dia- microalbuminuria) were used in the anal- per 1.73 m2/year) on the basis of previous betic patients with complicating cancer, ysis. The study protocol and informed studies (12,13). liver disease, or nondiabetic kidney dis- consent procedure were approved by the ease confirmed by renal biopsy were ex- ethics committee of Shiga University of Genotyping of the PRKCB1 cluded (n ϭ 28). Each individual Medical Science. polymorphism provided a blood sample for biochemical Genomic DNA was extracted from pe- measurements and DNA extraction. The Definition of outcomes ripheral leukocytes with a DNA purifica- diagnosis of diabetic retinopathy was The diabetic nephropathy stage of each tion kit (QIAamp blood kit; Qiagen, made by ophthalmologists. Diabetic reti- patient was determined based on the de- Chatsworth, CA). Fragments containing the nopathy was defined as simple retinopa- gree of the AER measured by immunotur- five polymorphic sites in the promoter re- thy or more. At the beginning of the bidimetry assay (HITACHI 7070E; Hitachi gion of PRKCB1 (Ϫ1504C/T, Ϫ546C/G, study, 396 patients were eligible for en- High-Technologies, Tokyo, Japan) in Ϫ348A/G, Ϫ278C/T, and Ϫ238C/G) rollment in this study. The participants, 24-h urine samples after confirming the were amplified by the PCR method ac- during the follow-up periods, underwent absence of pyuria and hematuria and the cording to a previously described method the standardized physical examination, exclusion of other diseases that can in- (8). Genotyping for all polymorphisms biochemical measurements, and a mea- crease albuminunuria. Patients were des- was performed by hybridization with al- surement of AER in 24-h urine collection ignated as being in the stage of lele-specific oligonucleotide probes (14). at least once a year. The follow-up period normoalbuminuria if AER was Ͻ20 ␮g/ lasted at least 3 years, until the end of min, microalbuminuria if 20 Յ AER Statistical analysis 2004 or death. All participants received Ͻ200 ␮g/min, and overt proteinuria if Comparisons between frequencies in the treatment based on the standard strategies AER Ն200 ␮g/min and estimated GFR study groups were made by ␹2 tests with for diabetes, hypertension, and hyperlip- (eGFR) (ml/min per 1.73 m2) Ն60 (stage Fisher’s exact test. Comparisons between idemia during the follow-up periods. Of 1 or 2 according to the Kidney Disease groups were performed by using an un- 396 participants, 19 patients were ex- Outcomes Quality Initiative clinical prac- paired Student’s t test for normally dis- cluded from the analysis because the fol- tice guidelines [10]) in two consecutive tributed variables or a Mann-Whitney U low-up periods were Ͻ3 years. Thirteen measurements. The outcome of diabetic test for nonnormally distributed variables. patients were not included in the analysis nephropathy stage was defined as transi- For a time-to-event analysis, the association

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Clinical characteristics of the nonprogres- sors and the progressors at baseline are shown in Table 1. BMI, levels of triglycer- Figure 1—Kaplan-Meier curves for ides, and AER at the baseline were signif- transition from any given stage to icantly higher in the progressors than in the more advanced stage of diabetic the nonprogressors. The allele frequen- nephropathy for patients grouped cies at the Ϫ1054 C/T polymorphic site according to the genotypes at were significantly different in the progres- Ϫ1054 C/T and Ϫ546 C/G sites of sors and the nonprogressors (␹2 ϭ 4.03, the PRKCB1 polymorphisms. Log- ϭ ␹2 ϭ ϭ P 0.045). The G allele frequencies at rank test: 5.92, P 0.015. Ϫ546 C/G were weakly but not signifi- Solid line, subjects with both the T 2 Ϫ cantly different in the two groups (␹ ϭ allele at 1054 C/T (CT or TT) and ϭ the G allele at Ϫ546 C/G (CG or 3.26, P 0.071). The frequencies of the GG) (n ϭ 43). Broken line, subjects alleles at the other three polymorphic with other patterns (n ϭ 321). sites were similar in the two groups. For the time-to-event analysis, being a carrier of the T allele at the Ϫ1054 C/T between the PRKCB1 polymorphisms and drugs. Multiple logistic regression analy- polymorphic site (n ϭ 44) was signifi- the transition of stage of diabetic ne- sis was used to assess the prognostic effect ⌬ cantly associated with transition to ad- phropathy was estimated using Kaplan- for eGFR and to adjust the influence of vanced stages of diabetic nephropathy as Meier procedure and was compared by conventional risk factors. The linkage dis- examined with the Kaplan-Meier method the log-rank test. Follow-up time was equilibrium coefficients (the correlation ϭ ⌬ (log-rank test: P 0.021). The adjusted censored if the development or progres- coefficient ) and estimated haplotype risk of the T allele carrier was 2.9 (95% CI sion of microalbuminuria occurred or if frequencies were calculated by a statistical 1.3–6.5) after adjustment for conven- the patient was unavailable for follow-up. method described previously (15). All tional risk factors in the multiple Cox To adjust the influence of conventional data were analyzed using the SPSS soft- model. Also, being a carrier of the G allele risk factors for the transition, a multivar- ware package (Version 11; SPSS, Chicago, Ϫ ϭ Ͻ at 546 C/G (n 47) was associated with iate Cox proportional hazard regression IL). A value of P 0.05 was taken to be the transition to advanced stages (P ϭ model was applied. The independent statistically significant. 0.037), and the adjusted risk was 2.7 variables used as conventional risk factors (1.2–6.2). No association between the were sex, duration of diabetes, HbA1c,to- RESULTS — The mean follow-up pe- Ϯ other three polymorphic sites and transi- tal cholesterol, triglycerides, hyperten- riod after the baseline period was 6.0 tion to advanced stages was found. Inter- sion (present/absent), the use of renin- 0.9 years (median 6 years [range 3–7]). estingly, comparison for the frequency of angiotensin system blockade drugs (yes/ During the study period, 34 of 364 sub- transition to advanced stages in the sub- no), retinopathy (present/absent), BMI, jects showed transition to advanced jects carrying both risk alleles, which AER, and eGFR at baseline. Hypertension stages of diabetic nephropathy; they com- Ն were previously reported to be associated was defined as blood pressure 140/90 prised 18 of 261 with normoalbuminuria with diabetic nephropathy (8), revealed mmHg or on the use of antihypertensive and 16 of 103 with microalbuminuria. that 19% of 43 subjects with both the T allele (CT or TT) at Ϫ1054 C/T and the G allele (CG or GG) at Ϫ546 C/G polymor- phisms showed transition to advanced stages of diabetic nephropathy, in con- trast to 8% of 321 subjects with other pat- terns. For the time-to-event analysis, the subjects having both the T allele at Ϫ1054 C/T and the G allele at Ϫ546 C/G showed more rapid transition to advanced stages than those without them (P ϭ 0.015) (Fig. 1). The adjusted risk of those sub- jects was 3.0 (1.3–6.7). Next, we examined the role of the risk T-G haplotype in affecting the kidney function evaluated by the rate of annual change in eGFR. In the subjects with both the T allele at Ϫ1054 C/T (CT or TT) and the G allele at Ϫ546 C/G (CG or GG), the rate of annual change in eGFR was signif- Figure 2—The rates of annual change in estimated GFR for patients grouped according to the icantly higher than in subjects with other Ϫ Ϫ patterns ([means Ϯ SE] Ϫ2.96 Ϯ 0.62 vs. genotypes at 1054 C/T and 546 C/G sites of the PRKCB1 polymorphisms. Data are expressed 2 as mean Ϯ SE. *P ϭ 0.02 by Mann-Whitney U test. f, subjects with both risk alleles, the T allele Ϫ1.63 Ϯ 0.15 ml/min per 1.73 m /year, at Ϫ1054 C/T (CT or TT) and the G allele at Ϫ546 C/G (CG or GG) (n ϭ 43). , subjects with P ϭ 0.02 by Mann-Whitney U test) (Fig. other patterns (n ϭ 321). 2). Eighteen (41.9%) of 43 subjects with

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Table 2—The estimated haplotype frequencies of ؊1054 C/T and ؊546 C/G polymorphisms of PRKCB1

Odds ratio Odds ratio Progressors Nonprogressors ␹2 P (95% CI) ⌬ GFR Ն3 ⌬ GFR Ͻ3 ␹2 P (95% CI) T-G 12% 5% 4.3 0.04 2.3 (1.03–5.2) 10% 5% 5.8 0.02 2.1 (1.1–3.9) C-C 88% 94% 3.0 0.08 0.5 (0.2–1.1) 90% 95% 3.4 0.07 0.6 (0.3–1.1) Data and P values for comparison of haplotype frequencies between case and control groups were generated by ␹2 test using a 2 ϫ 2 contingency table composed of the number for one haplotype and the sum for other haplotypes. both the T allele at Ϫ1054 C/T and the G an association between the PRKCB1 poly- (data not shown). This may have resulted allele at Ϫ546 C/G had an accelerated de- morphisms and diabetic nephropathy, from the difficulty of identifying the exact cline in eGFR (⌬ eGFR Ն3 ml/min per the results could not resolve the question diabetes duration in type 2 diabetes. 1.73 m2/year), in contrast to 81 (25.6%) of whether subjects with the risk haplo- A limitation of the present study is of 317 subjects with other patterns (␹2 ϭ type frequently develop diabetic ne- that we did not perform direct measure- 5.1, P ϭ 0.02). The risk for the ⌬ eGFR in phropathy and progress to more ment of GFR, such as inulin clearance. those with both risk alleles was 2.1 (95% advanced stages. In the present study, the Also, it is debatable whether the MDRD CI 1.1–4.0). Similarly, the risk for the ⌬ subjects with both risk alleles showed equation can be applied to Japanese pa- eGFR in the multiple logistic regression more rapid transition to advanced stages tients because ethnicity is a factor that analysis was twofold after adjustment for of diabetic nephropathy than those with- might influence results of the tested GFR- risk factors (adjusted odds ratio 2.2 [95% out them, and the risk was twice as great. estimating equations (11). We realize that CI 1.1–4.5]). This result from the time-to-event analysis more precise methods for the measure- Finally, we analyzed the structure and strongly supports the notion of the associa- ment of GFR will be needed to confirm estimated frequencies of haplotypes using tion between the PRKCB1 polymorphisms our results and that careful adjustment of the expectation maximization algorithm and diabetic nephropathy. MDRD equations is necessary in Japanese (Table 2). The degree of linkage disequi- One strength of the present study is populations with chronic kidney disease. librium of the G-T haplotype at Ϫ1054 its investigation of the influence of Ϫ However, the comparison of serum creat- C/T and 546 C/G was very strong, with PRKCB1 on the annual change in eGFR. It inine– and iothalamate-based measure- a value of 0.94 for the correlation coeffi- is now well-appreciated that the majority ⌬ ments of GFR was reported to give similar cient (15). The estimated frequency of of the subjects who progress to end-stage time-to-event and slope-based renal out- the T-G haplotype was significantly renal disease have a chronic and progres- come results in the African-American higher in the progressors (12%) than in sive decline of renal function over the ␹2 ϭ ϭ Study of Kidney Disease and Hyperten- the nonprogressors (5%) ( 4.32, P years. This process may continue even sion (20). Therefore, in serial measure- 0.038). The odds ratio of the T-G haplo- when no initial renal damage is present. ments of eGFR, the difference between type was 2.3 (95% CI 1.0–5.2). Regard- In the diabetic patients, increased urinary the estimation and the real measurement ing the ⌬ eGFR, the estimated frequencies albumin excretion has been considered to of GFR is unlikely to change the main of the T-G haplotype were also different in reflect the initial renal damage and to be a 2 conclusion. Also, the mechanism by the two groups (␹ ϭ 5.8, P ϭ 0.02). The strong predictor of the progression (16). risk associated with the T-G haplotype for However, albuminuria does not always which the risk haplotype of PRKCB1 in- the ⌬ eGFR was 2.1 (95% CI 1.1–3.9). reflect the change of the renal function. In fluences renal function remains unclear. We previously demonstrated that fact, several studies (17,18) showed that ␤ ␤ CONCLUSIONS — The present some diabetic subjects with normoalbu- PKC- 1 and - 2 mRNA levels in lympho- study provides evidence that PRKCB1 as- minuria have low GFR. Also, Rudberg blasts cultured under high-glucose condi- sociates with susceptibility to the worsen- and Osterby (19) reported that the de- tions were influenced by the presence of ing of diabetic kidney disease in Japanese cline rate in GFR, although still within the the T-G haplotype (8). However, the dif- ␤ subjects with type 2 diabetes. In this normal range, was positively correlated ferent expression of PKC- or the func- study, we used both time-to-event and with glomerular structural changes. Here, tional significance according to the slope-based renal outcomes to evaluate we provide evidence that the subjects PRKCB1 polymorphisms has not been the effect of PRKCB1 on diabetic kidney with the T-G haplotype of the PRKCB1 clarified. Further study regarding the disease. The subjects having the T allele at polymorphisms show accelerated annual functional role is required to verify this Ϫ1054 and the G allele at Ϫ546 polymor- decline in eGFR. Thus, the T-G haplotype effect. phisms of PRKCB1 showed transition of PRKCB1 may be one of the valuable In conclusion, our study indicates from any given stage to the more ad- predictors for the rapid deterioration of that the T-G haplotype of the PRKCB1 vanced stage of diabetic nephropathy de- renal function in type 2 diabetic patients. polymorphisms is a predictor for worsen- fined by AER. Furthermore, those In the previous study, the association ing of kidney disease in Japanese patients subjects at risk showed an accelerated rate between the risk haplotype of PRKCB1 with type 2 diabetes. Furthermore, this of annual decline in eGFR. and diabetic nephropathy was particu- risk haplotype may also be a risk factor for Our finding is consistent with the larly strong in subjects with a short dura- cardiovascular disease because an ele- previous results (8) in Caucasian subjects tion of diabetes (8). However, in the vated AER and a reduced GFR were iden- with type 1 diabetes. Although the previ- present study, we observed no such effect tified as independent risk factors for it ous results provided the first evidence of of diabetes duration on our outcomes (21,22).

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