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Continued Smoking Exacerbates but Cessation Ameliorates Progression of Early Type 2 Diabetic Nephropathy

      ABSTRACT

      Purpose

      We tested the hypothesis that continued cigarette smoking exacerbates and its cessation ameliorates progression of the early nephropathy of type 2 diabetes mellitus (DM2) from microalbuminuria to macroalbuminuria.

      Methods

      We recruited 91 DM2 subjects with microalbuminuria, 39 nonsmokers and 52 smokers. Smokers underwent smoking cessation intervention with 11 of the 52 smokers quitting, yielding 3 groups: nonsmokers (NS, n=39), continued smokers (S, n=41), and quitting smokers (Quit, n=11), all on angiotensin converting enzyme inhibition (ACEI), treated toward recommended BP and glycemic targets, and followed prospectively for 5 years. Subjects had yearly measurements of estimated glomerular filtration rate (eGFR) and albumin (mg)-to-creatinine (g) ratios (alb/cr) in spot morning urines. Comparison of changes in characteristics was done using analysis of variance, with all pair wise multiple comparison procedure at α=0.05.

      Results

      Although average urine alb/cr was not different among groups at recruitment, 7 of the 41 S (17%) but none of the 50 NS or Quit progressed to macroalbuminuria (P < 0.003). eGFR decline rate was faster in S (−1.79 ± 0.35 mL/min/yr) than in NS or Quit (−1.30 ± 0.43 and −1.54 ± 0.37 mL/min/yr, respectively, P < 0.001). Multivariate analysis revealed smoking to be the only measured baseline factor that influenced eGFR decline rate (P < 0.041).

      Conclusion

      Smoking exacerbates progression of early to advanced DM2 nephropathy and its cessation is an effective kidney-protective intervention in the early nephropathy of DM2.

      KEY INDEXING TERMS

      Type 2 diabetes mellitus (DM2) is the leading cause of stage 5 chronic kidney disease or CKD5 (formally called end-stage renal disease or ESRD) in the United States.
      • US Renal Data System
      Progression to CKD5 is preceded by a progressive increase in urine albumin excretion (UAE) from normal through microalbuminuria to macroalbuminuria.
      • Bennett P.H.
      • Haffner S.
      • Kasiske B.L.
      • et al.
      Screening and management of microalbuminuria in patients with diabetes mellitus. Recommendations to the Scientific Advisory Board of the National Kidney Foundation from an Ad Hoc Committee of the Council on Diabetes Mellitus of the National Kidney Foundation.
      Microalbuminuric DM2 subjects have kidney pathologic changes compared with those with normal UAE
      • Nakamura T.
      • Ushiyama C.
      • Suzuki S.
      • et al.
      Urinary excretion of podocytes in patients with diabetic nephropathy.
      • Adler S.G.
      • Kang S.W.
      • Feld S.
      • et al.
      Glomerular mRNAs in human type 1 diabetes: biochemical evidence for microalbuminuria as a manifestation of diabetic nephropathy.
      and these changes are even greater in those with macroalbuminuria, despite normal glomerular filtration rate (GFR).
      • Nakamura T.
      • Ushiyama C.
      • Suzuki S.
      • et al.
      Urinary excretion of podocytes in patients with diabetic nephropathy.
      Consequently, DM2 subjects with macroalbuminuria compared with microalbuminuria have more advanced nephropathy. Progression of macroalbuminuric DM2 nephropathy toward CKD5 is slowed but generally not stopped by improved blood pressure (BP) control and angiotensin converting-enzyme inhibition (ACEI)
      • Lebovitz H.E.
      • Wiegmann T.B.
      • Cnaan A.
      • et al.
      Renal protective effects of enalapril in hypertensive NIDDM: role of baseline albuminuria.
      • Yokoyama H.
      • Tomonaga O.
      • Hirayama M.
      • et al.
      Predictors of the progression of diabetic nephropathy and the beneficial effect of angiotensin converting enzyme inhibitors in NIDDM patients.
      • Chuahirun T.
      • Wesson D.E.
      Cigarette smoking predicts faster progression of type 2 established diabetic nephropathy despite angiotensin converting enzyme inhibition.
      or angiotensin II receptor antagonists.
      • Lewis E.J.
      • Hunsicker L.G.
      • Clarke W.R.
      • et al.
      Renoprotective effect of the angiotensin receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes.
      • Brenner B.M.
      • Cooper M.E.
      • de Zeeuw D.
      • et al.
      Effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy.
      By contrast, ACEI can stabilize UAE and GFR in microalbuminuric DM2 nephropathy.
      • Lebovitz H.E.
      • Wiegmann T.B.
      • Cnaan A.
      • et al.
      Renal protective effects of enalapril in hypertensive NIDDM: role of baseline albuminuria.
      • Ravid M.
      • Savin H.
      • Jutrin I.
      • et al.
      Long-term stabilizing effect of angiotensin converting enzyme inhibition on plasma creatinine and on proteinuria in normotensive type II diabetic patients.
      • Chan J.C.N.
      • Ko G.T.C.
      • Leung D.H.Y.
      • et al.
      Long-term effects of angiotensin-converting enzyme inhibition and metabolic control in hypertensive type 2 diabetic patients.
      Therefore, progression from microalbuminuric to macroalbuminuric DM2 nephropathy constitutes transition from a stage in which recommended kidney-protective interventions can halt further progression to one in which interventions slow but generally do not stop progression to CKD5.
      Cigarette smoking is associated with greater glomerular injury
      • Baggio B.
      • Budakovic A.
      • Vestra M.D.
      • et al.
      Effects of cigarette smoking on glomerular structure and function in type 2 diabetic patients.
      and UAE
      • Yokoyama H.
      • Tomonaga O.
      • Hirayama M.
      • et al.
      Predictors of the progression of diabetic nephropathy and the beneficial effect of angiotensin converting enzyme inhibitors in NIDDM patients.
      • Baggio B.
      • Budakovic A.
      • Vestra M.D.
      • et al.
      Effects of cigarette smoking on glomerular structure and function in type 2 diabetic patients.
      with faster GFR decline in macroalbuminuric DM2 nephropathy
      • Yokoyama H.
      • Tomonaga O.
      • Hirayama M.
      • et al.
      Predictors of the progression of diabetic nephropathy and the beneficial effect of angiotensin converting enzyme inhibitors in NIDDM patients.
      • Chuahirun T.
      • Wesson D.E.
      Cigarette smoking predicts faster progression of type 2 established diabetic nephropathy despite angiotensin converting enzyme inhibition.
      despite ACEI.
      • Chuahirun T.
      • Wesson D.E.
      Cigarette smoking predicts faster progression of type 2 established diabetic nephropathy despite angiotensin converting enzyme inhibition.
      Continued smoking was associated with a progressive UAE increase despite ACEI and improved BP control after 6 months in DM2 with microalbuminuric nephropathy but smoking cessation was associated with no UAE increase, similar to nonsmokers.
      • Chuahirun T.
      • Jan Simoni J.
      • Hudson C.
      • et al.
      Cigarette smoking exacerbates and its cessation ameliorates renal injury in type 2 diabetes.
      Consequently, cigarette smoking might abrogate the kidney protection of ACEI on microalbuminuric DM2 nephropathy. The present study tested the hypothesis that continued cigarette smoking exacerbates and its cessation ameliorates progression of microalbuminuric to macroalbuminuric DM2 nephropathy in subjects on ACEI.

      Preliminary Studies

      To optimize study design, we conducted a preliminary, 5-year, prospective study to determine the course of nonsmoking DM2 getting recommended kidney protective intervention
      • Bennett P.H.
      • Haffner S.
      • Kasiske B.L.
      • et al.
      Screening and management of microalbuminuria in patients with diabetes mellitus. Recommendations to the Scientific Advisory Board of the National Kidney Foundation from an Ad Hoc Committee of the Council on Diabetes Mellitus of the National Kidney Foundation.
      and the following baseline characteristics: normal plasma creatinine (Pcr) by laboratory guidelines (<1.4 and <1.2 mg/dL for men and women, respectively), no evidence of cardiovascular disease, and no evidence of nephropathy except albuminuria. We excluded those with known secondary hypertension, pregnancy, or malignancy. Subjects were grouped by UAE determined by mean alb/cr of 3 weekly morning urines: normoalbuminuria (Normo, alb/cr <20 mg/g), microalbuminuria (Micro, alb/cr 20–200 mg/g), and macroalbuminuria (Macro, alb/cr >200 mg/g).
      • Bennett P.H.
      • Haffner S.
      • Kasiske B.L.
      • et al.
      Screening and management of microalbuminuria in patients with diabetes mellitus. Recommendations to the Scientific Advisory Board of the National Kidney Foundation from an Ad Hoc Committee of the Council on Diabetes Mellitus of the National Kidney Foundation.
      Subjects had yearly measurements of systolic BP (SBP), Pcr, hemoglobin A1c (HbA1c), and urine excretion of albumin and isoprostane 8-isoprostaglandin F (8-iso) (a measure of systemic oxidant stress
      • Devaraj S.
      • Hirany S.V.
      • Burk R.F.
      • et al.
      Divergence between LDL oxidative susceptibility and urinary F2-isoprostanes as measures of oxidative stress in type 2 diabetes.
      ), all factored for g creatinine excretion in a spot morning specimen. Normal GFR was initially determined by Pcr but we retrospectively estimated GFR (eGFR) with the modification of diet in renal disease (MDRDS)
      • Klahr S.
      • Levey A.S.
      • Beck G.J.
      • et al.
      The effects of dietary protein restriction and blood-pressure control on the progression of chronic renal disease. Modification of Diet in Renal Disease Study Group.
      formula. Subjects on ACEI at recruitment were maintained on ACEI. If not on ACEI and had either microalbuminuria or macroalbuminuria, they began ACEI as recommended
      • Bennett P.H.
      • Haffner S.
      • Kasiske B.L.
      • et al.
      Screening and management of microalbuminuria in patients with diabetes mellitus. Recommendations to the Scientific Advisory Board of the National Kidney Foundation from an Ad Hoc Committee of the Council on Diabetes Mellitus of the National Kidney Foundation.
      and enrolled. Only normoalbuminuric subjects with hypertension and on ACEI were enrolled. Goal BP was 130/85 as was recommended for diabetics with microalbuminuria.
      • Bennett P.H.
      • Haffner S.
      • Kasiske B.L.
      • et al.
      Screening and management of microalbuminuria in patients with diabetes mellitus. Recommendations to the Scientific Advisory Board of the National Kidney Foundation from an Ad Hoc Committee of the Council on Diabetes Mellitus of the National Kidney Foundation.
      • Joint National Commission on Detection, Evaluation, and Treatment of High Blood Pressure
      The fifth report of The Joint National Commission on Detection, Evaluation, and Treatment of High Blood Pressure (JNC V).
      Table 1 shows baseline data among groups. Macro urine 8-iso excretion was higher than Normo but not Micro. eGFR was similar among groups but Figure 1 shows that Macro eGFR at 5 years (72.8 ± 2.8 mL/min) was lower than Normo and Micro (87.2 ± 1.6 and 84.6 ± 1.3 mL/min, respectively, P < 0.001 versus Macro). Also, eGFR declined faster in Macro (−4.39 ± 1.86 mL/min/yr) than Normo and Micro (−1.45 ± 0.12 and −1.62 ± 0.27 mL/min/yr, respectively, P < 0.0001 versus Macro). Figure 2 shows that urine alb/cr did not increase in Normo, increased progressively in Macro, but increased in Micro compared with baseline at only year 5. Normo, Micro, and Macro had similar HbA1c (7.5 ± 0.6, 7.5 ± 0.9, and 7.6 ± 0.7%, respectively) and SBP (134.2 ± 4.9, 136.6 ± 6.8, and 135.5 ± 5.7 mm Hg, respectively) at 5 years.
      Table 1General Baseline Characteristics of the Preliminary Prospective Study Participants, All of Whom were Nonsmokers
      Normoalbuminuric (n=17)Microalbuminuric (n=23)Macroalbuminuric (n=20)
      % Males535250
      % Black/White/Hispanic12/53/3513/57/3010/60/30
      Age (yr)49.3 ± 2.449.7 ± 2.148.1 ± 2.2
      Diabetes duration (yr)4.4 ± 1.04.6 ± 0.85.1 ± 1.1
      Systolic BP (mm Hg)144.8 ± 8.5145.8 ± 9.7145.6 ± 2.3
      Hb Ale (%)7.7 ± 0.87.6 ± 0.87.5 ± 0.1
      Calculated GFR (ml/min)94.5 ± 6.792.7 ± 5.694.8 ± 1.4
      Urine albumin excretion (mg/g creatinine)8.4 ± 2.651.3 ± 26.8
      P < 0.05 vs. Normoalbuminuric
      435.1 ± 23.8
      P < 0.05 vs. Normoalbuminuric
      P < 0.05 vs. Microalbuminuria.
      Urine 8-iso excretion (μg/g creatinine)1.4 ± 0.11.7 ± 0.452.0 ± 0.1
      P < 0.05 vs. Normoalbuminuric
      Values are mean ± SE.
      BP indicates blood pressure; Hb A1c, hemoglobin A1c.
      a P < 0.05 vs. Normoalbuminuric
      b P < 0.05 vs. Microalbuminuria.
      Figure thumbnail gr1
      Figure 1Glomerular filtration rate estimated using the modification of diet in renal disease formula (eGFR) during 5-year follow-up of the first cohort of prospectively followed, nonsmoking subjects with type 2 diabetes mellitus (DM2) and either normoalbuminuria (Normo), microalbuminuria (Micro), or macroalbuminuria (Macro). *P < 0.05 versus Normo; +P < 0.05 versus Micro.
      Figure thumbnail gr2
      Figure 2Urine albumin excretion (UalbuminV) expressed as albumin (mg)-to-creatinine (g) ratio in a spot morning specimen of the first cohort of prospectively followed, nonsmoking Normo, Micro, or Macro DM2 subjects during the 5-year follow-up. *P < 0.05 versus respective baseline value.
      No Normo subject progressed to Micro but 3 Micro progressed to Macro level of urine alb/cr. Micro that did compared with Micro that did not progress had faster eGFR decline (−2.19 ± 0.31 versus −1.53 ± 0.13 mL/min/year, P < 0.0001), higher baseline urine alb/cr (102.3 ± 14.1 versus 51.6 ± 22.3 mg/g cr, P < 0.0002), urine 8-iso/cr (2.30 ± 0.08 versus 1.56 ± 0.4 μg/g cr, P < 0.006), and higher HbA1c (9.2 ± 0.3 versus 7.4 ± 0.5%, P < 0.0001). Baseline (153.3 ± 12.1 versus 144.7 ± 9.2 mm Hg, respectively, P=0.16) and 5-year SBP (135.7 ± 2.1 versus 136.8 ± 7.3 mm Hg, respectively, P=0.81) was similar in these two Micro groups.

      Materials and Methods

      The objective of this prospective interventional study was to determine if continued smoking exacerbates and its cessation ameliorates progression of microalbuminuric to macroalbuminuric DM2 nephropathy treated toward recommended glycemic
      • Ismail N.
      • Becker B.
      • Strzelczyk P.
      • et al.
      Renal disease and hypertension in non-insulin-dependent diabetes mellitus.
      and BP
      • Bennett P.H.
      • Haffner S.
      • Kasiske B.L.
      • et al.
      Screening and management of microalbuminuria in patients with diabetes mellitus. Recommendations to the Scientific Advisory Board of the National Kidney Foundation from an Ad Hoc Committee of the Council on Diabetes Mellitus of the National Kidney Foundation.
      • Joint National Commission on Detection, Evaluation, and Treatment of High Blood Pressure
      The fifth report of The Joint National Commission on Detection, Evaluation, and Treatment of High Blood Pressure (JNC V).
      targets with regimens including ACEI. Primary outcome was development of macroalbuminuria (alb/cr >200).
      • Bennett P.H.
      • Haffner S.
      • Kasiske B.L.
      • et al.
      Screening and management of microalbuminuria in patients with diabetes mellitus. Recommendations to the Scientific Advisory Board of the National Kidney Foundation from an Ad Hoc Committee of the Council on Diabetes Mellitus of the National Kidney Foundation.
      UAE was the nephropathy indicator because its changes reliably indicate degree, course, and response to interventions in DM2 nephropathy.
      • Mogensen C.E.
      The kidney in diabetes: how to control renal and related cardiovascular complications.
      Secondary outcomes were eGFR change rate (mL/min/year) and urine 8-iso excretion because oxidative stress might mediate diabetic complications
      • Mezzetti A.
      • Cipollone F.
      • Cuccurullo F.
      Oxidative stress and cardiovascular complications in diabetes: isoprostanes as new markers on an old paradigm.
      and smoking increases oxidative stress.
      • Obata T.
      • Tomaru K.
      • Nagakura T.
      • et al.
      Smoking and oxidant stress: assay of isoprostane in human urine by gas chromatography-mass spectrometry.
      These latter and our preliminary studies led us to additionally explore if oxidative stress might mediate smoking-induced exacerbation of early DM2 nephropathy. Smoking cessation was determined by decrease in urine cotinine,
      • Chuahirun T.
      • Jan Simoni J.
      • Hudson C.
      • et al.
      Cigarette smoking exacerbates and its cessation ameliorates renal injury in type 2 diabetes.
      a nicotine metabolite.
      • Vartiainen E.
      • Seppala T.
      • Lillsunde P.
      • et al.
      Validation of self-reported smoking serum cotinine measurement in a community-base study.
      This method is among the more accurate ways to determine smoking cessation
      • Secker-Walker R.H.
      • Vacek P.M.
      • Flynn B.S.
      • et al.
      Exhaled carbon monoxide and urinary cotinine as measures of smoking in pregnancy.
      • Gariti P.
      • Alterman A.I.
      • Ehrman R.
      • et al.
      Detecting smoking following smoking cessation treatment.
      and is more accurate than self report.
      • Vartiainen E.
      • Seppala T.
      • Lillsunde P.
      • et al.
      Validation of self-reported smoking serum cotinine measurement in a community-base study.
      • Attenbring M.
      • Herlitz J.
      • Berndt A.K.
      • et al.
      Are patients truthful about their smoking habits? A validation of self-report about smoking cessation with biochemical markers of smoking activity amongst patients with ischaemic heart disease.
      The Institutional Review Board at Texas Tech University Health Sciences Center approved the protocol.

      Study Design

      Inclusion criteria for this 5-year prospective study were: DM2 as defined,
      • Borg W.P.
      • Sherwin R.S.
      Classification of diabetes mellitus.
      eGFR >90 mL/min, and alb/cr (mean of 3 spot morning urines ≥1 week apart) 20–200 mg/g. Smokers consumed ≥10 cigarettes/d for ≥1 year. Recruited subjects received ACEI if not already on it and enrolled 4 weeks later. If on ACEI, they enrolled 4 weeks later. At the time that the protocol began, no published studies examined effects of angiotensin receptor blockers (ARB) on DM2 nephropathy progression but studies subsequently showed this drug class to be effective in slowing progression of macroalbuminuric DM2 nephropathy.
      • Lewis E.J.
      • Hunsicker L.G.
      • Clarke W.R.
      • et al.
      Renoprotective effect of the angiotensin receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes.
      • Brenner B.M.
      • Cooper M.E.
      • de Zeeuw D.
      • et al.
      Effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy.
      Importantly, studies available at initiation of this protocol showed no kidney protection difference between ACEI and ARB in DM2.
      • Lacourciere Y.
      • Belanger A.
      • Godin C.
      • et al.
      Long-term comparison of losartan and enalapril on kidney function in hypertensive type 2 diabetics with early nephropathy.
      Consequently, subjects taking ARBs were excluded to simplify study design. Nevertheless, PCP’s could add ARB during follow-up. Exclusion criteria were: smokers who consumed <10 cigarettes/d or who had discontinued smoking <1 year, nonsmokers who last smoked <1 year before recruitment, and unable to tolerate ACEI. Because poor glycemic
      • UK Prospective Diabetes Study (UKPDS) Group
      Intensive blood glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPKS 33).
      and BP
      • UK Prospective Diabetes Study (UKPDS) Group
      Tight blood pressure control and risk of macrovascular and microvascular complications in type 2 diabetes (UKPKS 38).
      control increases progression risk for microalbuminuric DM2 nephropathy, subjects with hemoglobin A1c ≥8.0% and SBP ≥140 mm Hg were excluded. Goal SBP was 130/85 mm Hg as recommended.
      • Bennett P.H.
      • Haffner S.
      • Kasiske B.L.
      • et al.
      Screening and management of microalbuminuria in patients with diabetes mellitus. Recommendations to the Scientific Advisory Board of the National Kidney Foundation from an Ad Hoc Committee of the Council on Diabetes Mellitus of the National Kidney Foundation.
      • Joint National Commission on Detection, Evaluation, and Treatment of High Blood Pressure
      The fifth report of The Joint National Commission on Detection, Evaluation, and Treatment of High Blood Pressure (JNC V).
      Smokers received 8 weeks of daily transdermal nicotine (14 to 21 mg as needed for cessation) and twice-daily bupropion (150 mg tablets) and 12 weekly outpatient sessions of substance abuse counseling, similar to a previous protocol
      • Jorenby D.E.
      • Leischow S.J.
      • Nides M.A.
      • et al.
      A controlled trial of sustained-release bupropion, a nicotine patch, or both for smoking cessation.
      but we used 12 instead of 9 wks of counseling. Seven of the 52 smokers reported side effects from the nicotine patch and 5 continued it after reducing the daily dose to 14 or 7 mg. The 2 remaining subjects could not continue the nicotine patch despite reduced dosage but maintained bupropion and counseling. None reported stopping bupropion.
      All subjects submitted 3 spot morning urines, 1 week apart, for alb/cr, cotinine, 8-iso, and serum for cotinine and creatinine at study entry, 12 weeks (ie, after completing smoking cessation intervention), and 24 weeks (when determination was made if they were quitting smokers
      • Jorenby D.E.
      • Leischow S.J.
      • Nides M.A.
      • et al.
      A controlled trial of sustained-release bupropion, a nicotine patch, or both for smoking cessation.
      ). To be labeled quitting smokers, urine and plasma cotinine of smokers at 12 and 24 weeks had to be ≤10% of the respective 1-week value for subsequent determinations.
      • Chuahirun T.
      • Jan Simoni J.
      • Hudson C.
      • et al.
      Cigarette smoking exacerbates and its cessation ameliorates renal injury in type 2 diabetes.
      All other smokers were labeled continued smokers. Thereafter, each subject provided urine and serum specimens at 6 months (1 year after study initiation) and at years 2, 3, 4, and 5. No dietary instructions were given beyond standard diabetic recommendations to take 20% of caloric intake as protein. Smokers used only cigarettes and none admitted to chewing tobacco.

      Analytical Methods

      Serum and urine creatinine were measured with the Sigma Diagnostics Creatinine Kit (Procedure No. 555, Sigma Diagnostics) and urine albumin (mg/g creatinine) as done previously.
      • Chuahirun T.
      • Wesson D.E.
      Cigarette smoking predicts faster progression of type 2 established diabetic nephropathy despite angiotensin converting enzyme inhibition.
      Urine cotinine was measured with the double antibody Nicotine Metabolite radioimmunoassay kit (Diagnostic Products, Los Angeles, CA) after C18 column solid phase extraction
      • Perkins S.L.
      • Livesey J.F.
      • Escares E.A.
      • et al.
      High performance liquid-chromatographic method compared with a modified radioimmunoassay of cotinine in plasma.
      as done previously
      • Chuahirun T.
      • Jan Simoni J.
      • Hudson C.
      • et al.
      Cigarette smoking exacerbates and its cessation ameliorates renal injury in type 2 diabetes.
      was expressed as μg/g creatinine. Urine 8-iso was measured using direct ELISA after NaOH hydrolysis and ethyl acetate phase extraction with a kit (Cayman Chemical, 8-iso-prostane EIA kit Cat. No. 516351, Cayman Chemical, Ann Arbor, MI)
      • Obata T.
      • Tomaru K.
      • Nagakura T.
      • et al.
      Smoking and oxidant stress: assay of isoprostane in human urine by gas chromatography-mass spectrometry.
      as previously
      • Simoni J.
      • Simoni G.
      • Wesson D.E.
      • et al.
      A novel hemoglobin-adenosine-glutathione based blood substitute: evaluation of its effects on human blood ex vivo.
      and expressed as μg/g creatinine.

      Statistical Analysis

      Values were expressed as means ± SD and range. Continuous variables were first examined for normality and then compared using paired t test or the Signed-Rank test, as appropriate. We used the χ2 test for proportional changes. Comparison of changes in clinical characteristics over the 5-year period among the 3 groups (nonsmokers, continued smokers, and quitters) was done using one way Analysis of Variance (ANOVA), with all pairwise multiple comparison procedure at α=0.05. Data management and statistical analysis, including multivariate analysis, were performed using SAS software, version 9.1.2 (SAS Institute, Cary, NC). The distinguished level of significance was 0.05.

      Results

      Thirty-six of the 52 smokers (69%) and 36 of the 39 (93%) nonsmokers completed all protocol visits and provided all required urine and serum samples at the indicated times. Smokers attended 562 of 624 (90%) of scheduled counseling sessions. All subjects completed at least 80% of protocol visits. Eleven (21%) of the 52 smokers met criteria for quitters, yielding 3 comparison groups: nonsmokers (NS, n=39), continued smokers (S, n=41), and quitting smokers (Quit, n=11). Urine cotinine excretion decreased in Quit (2894 ± 1287 to 162.0 ± 85.1 μg/g cr, P < 0.001 paired t) 6 months after smoking cessation intervention as shown in Figure 3. By contrast, urine cotinine excretion did not decrease in S (4211 ± 1705 to 3882 ± 1521 μg/g cr, P=0.56, paired t). Twelve of the 39 NS (31%) and 19 of the 52 smokers (37%) were on ACEI before recruitment. One NS, 3 S, and no Quit had ARB added to ACEI during follow-up. Thirty NS (51%), 22 S (54%), and 6 Quit (55%) were also on calcium channel blockers during follow-up. Pharmacy records show that 36 of the 39 (92%) NS, 10 of the 11 (91%) Quit, and 38 of the 41 (93%) S refilled 100% of scheduled prescriptions for ACEI or ARB medications.
      Figure thumbnail gr3
      Figure 3Urine cotinine excretion (UcotinineV) during 5-year follow-up in the second cohort of prospectively followed DM2 subjects with early nephropathy (microalbuminuria) who were nonsmokers, continued smokers, and quitting smokers. *P < 0.05 versus nonsmokers.
      Table 2 shows general baseline characteristics. Recruitment values where when subjects were identified as having met study criteria and enrollment values are 4 weeks later, either continuing or after starting ACEI (see Methods). Both S and Quit had higher urine excretion of cotinine and 8-iso than NS. Recruitment urine alb/cr was similar among groups but enrollment alb/cr was higher in S and Quit than NS due to a greater urine alb/cr fall in NS than S and Quit (P < 0.05) after those not on ACEI at recruitment were started on ACEI and then enrolled (see Methods). Urine alb/cr decreased 37.1 ± 28.6 to 18.7 ± 25.0 mg/g cr, respectively, (P < 0.01, paired t) in the 27 NS not taking ACEI at recruitment but 38.8 ± 30.5 to 33.4 ± 27.8 mg/g cr, respectively, (P < 0.05, paired t) in the 33 smokers not on ACEI at recruitment. Enrollment HbA1c was higher than NS in S but not in Quit. Enrollment LDL, HDL, and eGFR were similar among groups. Although all subjects met recruitment criteria for having eGFR >90 mL/min (see Methods), eGFR of many subjects from all 3 groups was lower at enrollment, some to <90 mL/min. The absolute (−5.8 mL/min) and proportional (−5.9%) decrease was similar for all 3 groups, likely reflective of the well-described effects of ACEI to initially lower GFR in some subjects.
      • Klahr S.
      • Levey A.S.
      • Beck G.J.
      • et al.
      The effects of dietary protein restriction and blood-pressure control on the progression of chronic renal disease. Modification of Diet in Renal Disease Study Group.
      Table 2General Baseline Characteristics of Microalbuminuria Subjects by Smoking Group (Mean ± SD)
      VariableNon-Smokers (n=39)Continued Smokers (n=41)Quitters (n=11)P
      % Males464551
      % Black/White/Hispanic10/77/139/73/189/78/13
      Age (yr), (min, max)53.4 ± 9.9, (29, 67)49.1 ± 9.9, (29, 65)48.1 ± 9.3, (36, 61)0.09
      Diabetes duration (yr), (min, max)7.2 ± 23,(4, 12)4.9 ± 1.7
      Difference from nonsmokers.
      , (2, 10)
      4.7 ± 1.4
      Difference from nonsmokers.
      , (3, 7)
      <0.0001
      Enrollment urine cotinine (μg/g creatinine), (min, max)76.8 ± 49.1, (31.2, 99.3)4773 ± 1985.0
      Difference from nonsmokers.
      , (3160, 6831)
      3178 ± 1446.0
      Difference from nonsmokers.
      , (2169, 4914)
      <0.0001
      Smoking intensity (packs/d), (min, max)1.29 ± 0.70, (0.9, 1.9)0.91 ± 0.43, (0.6, 1.5)0.14
      Smoking load, (packs/d × yr), (max, min)33.2 ± 19.2,(22, 47)24.9 ± 11.9,(16, 38)0.18
      Enrollment SBP (mm Hg), (min, max)135.5 ± 4.7,(126, 147)132.4 ± 5.4
      Difference from nonsmokers.
      , (120, 146)
      133.0 ± 4.6,(125, 139)0.02
      Recruitment urine albumin (mg/g creatinine), (min, max)35.5 ± 19.9, (21.7,101.3)45.6 ± 30.2,(21.3, 155.2)42.9 ± 26.3,(21.8, 95.2)0.329
      Enrollment urine albumin (mg/g creatinine), (min, max)18.7 ± 24.8, (3.6,103.2)34.0 ± 30
      Difference from nonsmokers.
      , (3.1, 148)
      29.7 ± 15.7
      Difference from nonsmokers.
      , (14.8, 65.2)
      0.038
      Enrollment urine 8-iso (μg/g creatinine), (min, max)1.9 ± 0.3, (1.5, 2.3)4.3 ± 0.5
      Difference from nonsmokers.
      , (3.0, 7.1)
      3.8 ± 0.5
      Difference from nonsmokers.
      , (2.8, 6.4)
      0.012
      Enrollment HbAlc (%), (min, max)7.04 ± 0.4, (6.2, 7.9)7.26 ± 0.3, (6.5,7.9)7.00 ± 0.24
      Difference from continued smokers.
      , (6.6, 7.3)
      0.015
      Enrollment LDL, (min, max)149.4 ± 21.9,(111, 201)161.3 ± 33,(125,254)154.2 ± 15.8,(125, 182)0.152
      Enrollment HDL, (min, max)45.9 ± 11.9, (26, 65)44.4 ± 10, (25, 69)47.6 ± 9.2, (36, 64)0.63
      Enrollment GFR, (min, max)92.8 ± 5.5, (84.2, 104.5)92.9 ± 6.2, (83.2, 104.3)92.6 ± 7.2, (84.2, 105.3)0.92
      BP indicates blood pressure; HbA1c, hemoglobin A1c.
      a Difference from nonsmokers.
      b Difference from continued smokers.
      Table 3 shows 5-year values for selected parameters. Urine excretion of cotinine, albumin, and 8-iso were each higher than NS in S but were not different between Quit and NS. HbA1c was higher in S than NS but was not different between Quit and NS. Despite similar follow-up eGFR, eGFR decline rate was faster in S than NS and Quit. Multivariate analysis showed smoking status was the only baseline parameter that influenced eGFR decline rate (smoking predicted faster eGFR decline, P < 0.041).
      Table 3Five-Year Follow-Up Values in Microalbuminuria Subjects by Smoking Group (Means ± SD)
      VariableNonsmokers (n=39)Continued Smokers (n=41)Quitters (n=11)P
      SBP (mm Hg)133.4 ± 4132.2 ± 4131.0 ± 4.60.33
      Urine cotinine (μg/g creatinine)73.1 ± 69.94321 ± 1269
      Difference from nonsmokers.
      89.2 ± 16.0
      Difference from continued smokers.
      <0.0001
      Urine albumin (mg/g creatinine)21.24 ± 1199.2 ± 85
      Difference from nonsmokers.
      23.9 ± 7.6
      Difference from continued smokers.
      <0.0001
      Urine 8-iso (μg/g creatinine)1.7 ± 0.24.4 ± 0.5
      Difference from nonsmokers.
      1.7 ± 0.3
      Difference from continued smokers.
      <0.0001
      HbAlc (%)6.99 ± 0.47.17 ± 0.3
      Difference from nonsmokers.
      6.95 ± 0.3
      Difference from continued smokers.
      0.04
      LDL (mg/dL)132.6 ± 14.1138.1 ± 19.1134.3 ± 8.00.33
      HDL (mg/dL)48.8 ± 12.148.4 ± 11.050.7 ± 8.00.83
      eGFR (mL/min)86.4 ± 6.084.0 ± 7.085.2 ± 7.10.24
      eGFR change (mL/min/mo)–1.30 ± 0.43 (0.64, 2.22)–1.79 ± 0.35
      Difference from nonsmokers.
      (0.86, 2.80)
      –1.54 ± 0.37
      Difference from nonsmokers.
      Difference from continued smokers.
      (0.62, 1.96)
      <0.001
      BP indicates blood pressure; HbA1c, hemoglobin A1c.
      a Difference from nonsmokers.
      b Difference from continued smokers.
      Urine alb/cr increased after enrollment in S but not in Quit compared with NS as shown in Figure 4. Urine alb/cr declined in year 5 compared with year 0 (enrollment) in 18% of NS, 73% of Quit, and in 0% of S. Seven of the 41 (17%) S but none of the 50 Quit or NS progressed to macroalbuminuria (P < 0.003) after an average of 3.0 ± 1.4 yr (range 1–5 yr). When the 7 S who progressed to macroalbuminuria were excluded, urine alb/cr of the remaining S (66.9 ± 48.1 mg/g cr) was higher than NS (21.2 ± 11.0 mg/g cr) and Quit (23.9 ± 7.6 mg/g cr) at 5 yr (P < 0.02). The 7 S who progressed to macroalbuminuria had higher urine alb/cr at recruitment (67.2 ± 30.3 versus 41.2 ± 28.7 mg/g cr, P < 0.04) and at enrollment (62.4 ± 33.9 versus 28.2 ± 26.1 mg/g cr, P < 0.005). Smoking intensity (1.42 ± 1.22 versus 1.24 ± 0.73 packs/d, P=0.60) and smoking load (37.7 ± 31.8 versus 31.6 ± 22.5 pack-years, P=0.55) were not different in the 7 S who did compared with S who did not progress to macroalbuminuria. Multivariate analysis showed that smoking status (smoking predicted greater alb/cr increase, P < 0.0001) and HDL (HDL decrease predicted greater alb/cr increase, P < 0.037) were the only measured parameters that influenced urine alb/cr change.
      Figure thumbnail gr4
      Figure 4Urine albumin excretion (UalbuminV) expressed as albumin (mg)-to-creatinine (g) ratio in a spot morning specimen during the 5-year follow-up in the second cohort of prospectively followed DM2 subjects with early nephropathy (microalbuminuria) who were nonsmokers, continued smokers, and quitting smokers. *P < 0.05 versus nonsmokers.
      Figure 5 shows that after smoking cessation intervention, urine 8-iso decreased in Quit (3.8 ± 1.7 to 2.0 ± 1.0 μg/g cr, P < 0.001, paired t) at 6 months but did not change at this time point in S (4.3 ± 1.7 to 4.2 ± 1.5 μg/g cr, P=0.71, paired t) or NS (1.9 ± 1.9 to 1.8 ± 1.9 μg/g cr, P=0.86, paired t). Figure 5 also shows that urine 8-iso/cr decreased in Quit to a level comparable to NS and remained so throughout. By contrast, urine 8-iso/cr did not decrease in S but remained higher than NS and Quit after the smoking cessation intervention.
      Figure thumbnail gr5
      Figure 5Urine excretion of isoprostane 8-prostaglandin F (U8-isoV), expressed as (μg)-to-creatinine (g) ratio in a spot morning specimen during 5-year follow-up in the second cohort of prospectively followed DM2 subjects with early nephropathy (microalbuminuria) who were nonsmokers, continued smokers, and quitting smokers. *P < 0.05 versus nonsmokers.

      Discussion

      The present studies tested the hypothesis that cigarette smoking enhances and its cessation ameliorates progression of microalbuminuric to macroalbuminuric DM2 nephropathy in subjects treated toward recommended glycemic and BP targets with regimens that include ACEI. Shorter term studies showed that continued smoking in microalbuminuric DM2 nephropathy was associated with a progressive increase in albuminuria over 6 months and its cessation was associated with no further increase in a pattern similar to that of nonsmokers.
      • Chuahirun T.
      • Jan Simoni J.
      • Hudson C.
      • et al.
      Cigarette smoking exacerbates and its cessation ameliorates renal injury in type 2 diabetes.
      These shorter-term studies did not determine if the apparent deleterious effects of continued smoking or if the apparent beneficial effects of smoking cessation were sustained over a longer follow-up. The follow-up period for these earlier studies was also not sufficient to determine if continued smoking or its cessation influenced the risk for progression to macroalbuminuria. This microalbuminuria to macroalbuminuria transition is important because macroalbuminuric compared with microalbuminuric DM2 nephropathy have faster eGFR decline despite ACEI, shown in our preliminary study. The present study shows that continued smoking exacerbates and its cessation ameliorates progression of DM2 microalbuminuric to macroalbuminuric nephropathy with its greater risk for CKD5, again, despite ACEI. The data support that smoking cessation is kidney-protective in microalbuminuric DM2 nephropathy for at least 5 years.
      Previous studies support that smoking enhances progression of DM2 nephropathy
      • Yokoyama H.
      • Tomonaga O.
      • Hirayama M.
      • et al.
      Predictors of the progression of diabetic nephropathy and the beneficial effect of angiotensin converting enzyme inhibitors in NIDDM patients.
      • Chuahirun T.
      • Wesson D.E.
      Cigarette smoking predicts faster progression of type 2 established diabetic nephropathy despite angiotensin converting enzyme inhibition.
      • Chuahirun T.
      • Jan Simoni J.
      • Hudson C.
      • et al.
      Cigarette smoking exacerbates and its cessation ameliorates renal injury in type 2 diabetes.
      despite ACEI
      • Chuahirun T.
      • Wesson D.E.
      Cigarette smoking predicts faster progression of type 2 established diabetic nephropathy despite angiotensin converting enzyme inhibition.
      • Chuahirun T.
      • Jan Simoni J.
      • Hudson C.
      • et al.
      Cigarette smoking exacerbates and its cessation ameliorates renal injury in type 2 diabetes.
      but the present studies additionally support that cigarette smoking abrogates kidney protection by ACEI. Specifically, we showed that the typical ACEI-induced reduction in urine albumin excretion by ACEI in the microalbuminuric DM2 nephropathy
      • Ravid M.
      • Savin H.
      • Jutrin I.
      • et al.
      Long-term stabilizing effect of angiotensin converting enzyme inhibition on plasma creatinine and on proteinuria in normotensive type II diabetic patients.
      was less in smokers than nonsmokers. In addition, none of the continued smokers but most of the quitting smokers experienced a decrease in urine alb/cr during follow-up, the latter occurring chronologically with smoking cessation. Because change in urine albumin excretion is a surrogate for the course of kidney injury in DM2 nephropathy,
      • Mogensen C.E.
      The kidney in diabetes: how to control renal and related cardiovascular complications.
      these data support that smoking abrogates kidney protective effects of ACEI in microalbuminuric DM2 nephropathy. Because only a very small number of subjects received ARB in addition to ACEI, we cannot comment as to whether smoking might have this apparent effect in subjects on ACEI and ARB in combination.
      Only a minority of continued smokers (17%) progressed to macroalbuminuric nephropathy although the 5-year alb/cr for the remaining continued smokers was still higher than quitting smokers and nonsmokers. These remaining continued smokers might indeed have progressed to macroalbuminuric nephropathy with longer follow-up. Nevertheless, the fact that this minority of smokers with microalbuminuric DM2 nephropathy progressed faster than did their counterparts suggests that they are somehow distinct. These faster progressors had higher urine alb/cr at recruitment and enrollment than did their counterpart continued smokers, an association that has already been shown in previous studies.
      • Mogensen C.E.
      The kidney in diabetes: how to control renal and related cardiovascular complications.
      • Chuahirun T.
      • Khanna A.
      • Kimball K.
      • et al.
      Cigarette smoking and increased urine albumin excretion are interrelated predictors of nephropathy progression in type 2 diabetes.
      Because there were no identifiable differences in smoking history, we could not establish heavier smoking as the distinguishing factor. Identifying these distinguishing features will require further study.
      The mechanism(s) by which cigarette smoking exacerbates progression of DM2 nephropathy was not determined. Nevertheless, oxidative stress is a phenomenon common to both cigarette smoking
      • Obata T.
      • Tomaru K.
      • Nagakura T.
      • et al.
      Smoking and oxidant stress: assay of isoprostane in human urine by gas chromatography-mass spectrometry.
      and diabetes
      • Devaraj S.
      • Hirany S.V.
      • Burk R.F.
      • et al.
      Divergence between LDL oxidative susceptibility and urinary F2-isoprostanes as measures of oxidative stress in type 2 diabetes.
      that mediates progression of experimental diabetic nephropathy.
      • Melhem M.F.
      • Craven P.A.
      • Derubertis F.R.
      Effects of dietary supplementation of α-Lipoic acid on early glomerular injury in diabetes mellitus.
      The present studies showed that smokers had higher urine excretion of 8-iso, an indicator of systemic oxidant stress in DM2
      • Mezzetti A.
      • Cipollone F.
      • Cuccurullo F.
      Oxidative stress and cardiovascular complications in diabetes: isoprostanes as new markers on an old paradigm.
      and continued smokers maintained these higher levels. By contrast, urine 8-iso excretion of quitting smokers decreased compared with continued smokers to a level comparable to nonsmokers. Consequently, cigarette smoking might exacerbate kidney injury in microalbuminuric DM2 nephropathy by enhancing oxidant stress. In addition, because cigarette smoking damages arterioles,
      • Black H.R.
      • Zeevi G.R.
      • Silten R.M.
      • et al.
      Effect of heavy cigarette smoking on renal and myocardial arterioles.
      smoking might exacerbate vasculopathy in diabetic nephropathy
      • Harris R.D.
      • Steffes M.W.
      • Bilous R.W.
      • et al.
      Global glomerular sclerosis and glomerular arteriolar hyalinosis in insulin-dependent diabetes mellitus.
      by mechanisms similar to those by which it exacerbates extra-kidney vascular disease.
      Although the present studies focused on the effects of cigarette smoking on progression of microalbuminuric DM2 nephropathy, it is quite likely that smoking interacts with other factors such as chronically elevated SBP or glucose to exacerbate progression of DM2 nephropathy. Intensive compared with less intensive blood pressure control (mean blood pressure 118 versus 124 mm Hg) better protected against a subsequent increase in urine albumin excretion in subjects with microalbuminuric DM2 nephropathy.
      • Estacio R.O.
      • Coll J.R.
      • Tran Z.V.
      • et al.
      Effect of intensive blood pressure control with valsartan on urinary albumin excretion in normotensive patients with type 2 diabetes.
      Other studies suggest that achieved systolic BP (SBP) is more important than baseline SBP in predicting kidney outcomes in DM2 nephropathy.
      • Pohl M.A.
      • Blumenthal S.
      • Cordonnier D.J.
      • et al.
      Independent and additive impact of blood pressure control and angiotensin 2 receptor blockade on renal outcomes in the Irbesartan Diabetic Nephropathy Trial: clinical implications and limitations.
      Specifically, subjects with achieved SBP less than 134 mm Hg had the best kidney outcome but only 17% of subjects studied achieved this SBP.
      • Pohl M.A.
      • Blumenthal S.
      • Cordonnier D.J.
      • et al.
      Independent and additive impact of blood pressure control and angiotensin 2 receptor blockade on renal outcomes in the Irbesartan Diabetic Nephropathy Trial: clinical implications and limitations.
      These data suggest that SBP targets less than the recommended 130 mm Hg
      • Chobanion A.V.
      • Bakris G.L.
      • Black H.R.
      • et al.
      The seventh report of the Joint National Commission on Detection, Evaluation, and treatment of high blood pressure. The JNC 7 Report.
      might be necessary to achieve this 134 mm Hg target in a greater proportion of treated subjects. In addition, more stringent glycemic control than is currently recommended
      • Ismail N.
      • Becker B.
      • Strzelczyk P.
      • et al.
      Renal disease and hypertension in non-insulin-dependent diabetes mellitus.
      might be necessary to reduce risk for the appearance or progression of early nephropathy as suggested by some investigators.
      • Maeda Y.
      • Shiigai T.
      Diet therapy in diabetic nephropathy.
      In summary, the present studies show that continued cigarette smoking exacerbates and its cessation ameliorates progression from microalbuminuric to macroalbuminuric DM2 nephropathy. This detrimental effect of cigarette smoking occurred despite reasonable SBP and glycemic control and, most importantly, despite ACEI. These studies support that smoking cessation is an effective kidney-protective intervention in DM2 subjects with microalbuminuric nephropathy without poor glycemic control and who are being treated toward recommended BP targets with medical regimens that include ACEI.

      Acknowledgments

      We are thankful to the Inside Out Community Outreach Program of Lubbock, Texas for providing the substance abuse counseling and to the nursing and clerical staff of the Internal Medicine Clinic of the Department of Internal Medicine at Texas Tech University Health Sciences Center for their assistance.

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