Abstract
This study was conducted to see the effect of doxycycline on renal functions, especially proteinuria, in patients of diabetic nephropathy (DN). The study included 40 clinically proven adult patients of DN. All patients were on stable doses of angiotensin-converting enzyme inhibitors (ACEIs) and or angiotensin receptor blockers (ARBs) for 2 months before the study. The patients were divided into two groups of 20 patients each. Group A patients were maintained on stable dose of ACEIs and/or ARBs, whereas Group B patients received doxycycline (100 mg/day) for a period of 3 months in addition to ACEIs and or ARBs. Adequate glycemic control was achieved with insulin or oral hypoglycemic agents in all the patients. Renal parameters were assessed at the beginning of the study, at 1, 3, and 6 months (after a washout period of 3 months). All renal parameters remained unaltered during the study in both groups. However, proteinuria showed improvement in Group B (doxcycycline group).The mean basal value of proteinuria was 1.74 + 1.70 for Group A and 2.17 + 2.95 for Group B. At the end of 3 months, proteinuria was 1.22 + 2.11 in Group B whereas it was 1.50 + 1.50 in Group A (p < 0.05). However, the decrease in proteinuria at 6 months in the two groups did not show any statistically significant difference. No significant side effects of doxycycline were observed.
The study showed that doxycycline was effective in reducing proteinuria in patients of DN when used for the short duration of 3 months.
INTRODUCTION
Diabetic nephropathy (DN) is the leading cause of end-stage renal disease (ESRD) and the leading cause of diabetes mellitus (DM)-related morbidity and mortality in India and the rest of the world.Citation1 DN is characterized by specific renal morphologic and functional alterations. Features of early diabetic renal changes are glomerular hyperfiltration, glomerular and renal hypertrophy, increased urinary albumin excretion (UAE), increased basement membrane thickness; mesangial expansion with accumulation of extracellular matrix (ECM) proteins such as collagen, fibronectin, and laminin. Advanced DN is characterized by proteinuria, decreased renal function, decreased creatinine clearance, glomerular sclerosis, and interstitial fibrosis.Citation2
Proteinuria is one of the most characteristic functional changes in DN.Citation3 UAE tends to parallel glomerular basement membrane (GBM) thickening and/or mesangial expansion.Citation4,Citation5 The increase of mesangial expansion positively correlates with albumin excretion.Citation6 The progression to ESRD is accompanied by accumulation of ECM proteins. The degradation of ECM proteins occurs by action of proteases, notably matrix metalloproteinases (MMPs), which are zinc-dependent endopeptidases. Various types of MMPs such as interstitial collagenases, gelatinase, stromelysin, and membrane-type MMP are involved in cell proliferation and apoptosis.Citation7 An imbalance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) plays an important role in ECM remodeling that favors tissue fibrosis.Citation8 Diabetic environment including MMPs upregulates profibrotic transforming growth factor-β1 (TGF-β1) activity and expression in glomerular mesangial cells and proximal tubular epithelial cells.Citation9,Citation10 In combination, these pathways ultimately lead to increased renal albumin permeability and ECM accumulation, resulting in renal structural and functional changes causing increased proteinuria, glomerulosclerosis, and tubulointerstitial fibrosis.
Tetracycline and their analogues inhibit protein synthesis by binding to 30S ribosomes in susceptible organisms. They are potent inhibitors of MMPs, both collagenases and gelatinase. They have been investigated in the treatment of various disorders in which MMP activity is amplified, such as pyoderma gangrenosum, sarcoidosis, atherosclerotic aortic plaques, degenerating osteoarthritis, periodontitis, cancer metastasis, rheumatoid arthritis, scleroderma, aortic aneurysms, and amyloidosis.Citation11 Doxycycline is a potent nonselective MMP inhibitor. It can inhibit both mammalian collagenases and gelatinase as well as inhibit the synthesis of MMPs in human endothelial cells. The remodeling and excess deposition of ECM could be attenuated by doxycycline due to its property of MMP inhibition.Citation11,Citation12
The role of doxycycline in decreasing proteinuria in patients with DN is still largely experimental. Only a few human studies have shown preliminary short-term results. The present study therefore tries to find out if doxycycline has got any role in decreasing proteinuria in patients with DN.
MATERIAL AND METHODS
Forty clinically proven adult cases of DN, fulfilling the inclusion criteria, were taken up for this study. It was an open randomized trial conducted over a period of 6 months. The patients were enrolled in the study after obtaining a pre-informed consent. The study protocol duly got approved by Institutional Review Board.
Inclusion criteria
All adult patients (>18 years) with type 2 DM
Patients with overt proteinuria (>500 mg/24 h)
All patients had to be on stable doses of ACEIs and/or ARBs for at least 2 months before enrollment in the study.
Exclusion criteria
Any history of systemic long-term antimicrobial therapy over the past 6 months
History of hypersensitivity to tetracycline derivatives
Chronic renal insufficiency (serum creatinine levels >1.4 mg/dL)
Hepatic dysfunction (transaminase levels greater than twice the upper limit of normal)
Incompatibility of doxycycline with the patient's concurrent medications.
The patients were divided into two groups of 20 patients each.
Group A (Control) (n = 20) – subjects with DN on ACEIs and/or ARBs.
Group B (Study) (n = 20) – subjects with DN on ACEIs and/or ARBs and doxycycline.
The control group received only ACEIs and/or ARBs while the study group was given doxycycline in a dose of 100 mg per day in addition to ACEIs and/or ARBs for a period of 3 months. Investigations to assess the renal parameters including proteinuria were carried out at the beginning of the study, and after 1, 3, and 6 months (washout period of 3 months). Adequate glycemic control was achieved with insulin and/or oral hypoglycemic agents.
After screening the patients for inclusion and exclusion criteria and before starting therapy, all were subjected to detailed history, clinical examination, and a battery of investigations with special emphasis on renal parameters. All the patients were observed for the side effects of doxycycline such as gastrointestinal intolerance and photosensitivity.
Statistical application
The data obtained were subjected to appropriate statistical analysis. The continuous variables were recorded as mean and standard deviations. For comparison, paired t-test was applied. p-Values were calculated and p < 0.05 was considered to be statistically significant.
RESULTS
The mean age of the patients in Group A was 52.8 ± 8.74 years and in Group B 56.7 ± 10.3 years. Majority of the patients were above 50 years of age. There were 9 males and 11 females in Group A, whereas in Group B there were 13 males and 7 females ( and ). All patients had type 2 DM and overt proteinuria. Most of the patients had nonproliferative diabetic retinopathy (80% in Group A and 85% in Group B), whereas the remaining patients had proliferative retinopathy. The electrocardiogram and chest X-ray were largely normal. All the patients had normal kidney size and echo texture as per ultrasonographic examination. The baseline renal parameters were within normal limits ().
TABLE 1. (a) Age distribution of the patients
TABLE 1. (b) Sex distribution of the patients
TABLE 2. Basal values of renal function tests
The mean basal hemoglobin level was 9.92 ± 1.85 g/dL in Group A and 10.2 ± 1.58 g/dL in Group B. No significant difference in the levels of hemoglobin between the two groups was observed after 3 and 6 months (p > 0.05). Both systolic and diastolic blood pressure showed no statistical difference between the two groups throughout the study ( and ). The basal values for the various renal parameters were largely within normal limits and did not vary significantly throughout the study ( and ). Doxycycline was seen to exert no significant deleterious or beneficial effects on the renal parameters, despite continued low-dose therapy for 3 months.
TABLE 3. (a) Blood pressure (systolic)
TABLE 3. (b) Blood pressure (diastolic)
TABLE 4. Blood urea levels
TABLE 5. Serum creatinine levels
The mean basal levels of proteinuria was 1.74 ± 1.70 g/day (range 0.56–5.4 g/day) for Group A and 2.17 ± 2.95 g/day (range 0.54–6.48 g/day) for Group B. It reduced to 1.56 ± 1.52 for Group A and 1.89 ± 2.72 for Group B, at the end of 1 month. At the end of 3 months, a significant decline of proteinuria was observed in both the groups. In Group B it had a mean of 1.22 ± 2.01, whereas it was 1.50 ± 1.50 in Group A. A statistically significant difference existed between the control and study groups (p < 0.05), at 3 months. The levels of proteinuria increased nearing the pre-treatment levels (1.98 ± 2.65) after a washout period of 3 months, that is, at 6 months in Group B (study group) ( ).
TABLE 6. Level of proteinuria (g/day)
FIGURE 1. The level of proteinuria.
The mean change in the level of proteinuria was 0.956 g/day in the study group (Group B) after 3 months of doxycycline therapy, whereas it was 0.253 g/day in the control group (Group A), indicating the beneficial effect of doxycycline in decreasing proteinuria. Mean change in the values of proteinuria tended to decrease to 0.193 g/day at the end of the study period at 6 months. There was no significant difference in the mean change of proteinuria at the baseline from that at 6 months, indicating that there was no carryforward effect of doxycycline, and the increase of proteinuria seen was due to withdrawal of the additive effect of the drug ().
TABLE 7. Mean change in levels of proteinuria (g/day)
There were no major adverse effects due to doxycycline. Only three patients complained of mild gastrointestinal disturbance.
DISCUSSION
Diabetes and its devastating complications greatly reduce life expectancy and target nearly every organ system in the body. Despite advances in clinical care, the incidence of type 2 diabetes-related cases of ESRD show an increasing trend over the years.Citation13
Data from the Diabetes Control and Complications trial have established that glycemic control plays an important role in the prevention and treatment of DN.Citation14 Major landmarks including the irbesartan DN trialCitation15 and the Reduction of Endpoints in NIDDM with the Angiotensin II Antagonist Losartan (RENAAL) studyCitation16 have provided compelling evidence to support the renoprotective effect of angiotensin II receptor antagonists in DN. In addition, a low-protein diet prevents the decline of glomerular filtration rate (GFR) and reduces the risk of ESRD.Citation17 Lipids may be important in the pathogenesis of progressive renal injury, thus advocating the role of anti-lipemic agents in the prevention of development of macroalbuminuria.Citation18,Citation19 Correction of anemia is vital to prevent the decline of renal functions in diabetes.Citation20 The use of low-molecular-weight heparin has been reported to prevent diabetes-induced albuminuria and glomerulosclerosis.Citation21 Vitamin E prevents oxidant injury by quenching reactive oxygen species.Citation22 However, despite receiving the best of therapy, even in a controlled trial setting, a considerable proportion of patients progress to develop irreversible ESRD. Thus, it is urgently necessary to elucidate the natural history of DN and the clinical course of renal structural-functional relationships.
Increased ECM is a characteristic feature of DN that is directly linked to declining renal functions. However, rather than being a static phenomenon, ECM is in dynamic flux, with both synthetic and degradative components contributing to its turnover. The MMPs are zinc-dependent endopeptidases that play an important role in the remodeling of connective tissue and are involved in the degradation of all components of the ECM. Remodeling is attributed to interaction between the proteases and their inhibitors. Changes in the balance between ECM synthesis and degradation may lead to expansion of the glomerular ECM, that is, glomerulosclerosis and decline in renal function.Citation7
Zua et al.Citation23 and Nakamura et al.Citation24 have shown an increased level of MMP-9 in DN, which may precede the onset of microalbuminuria in type 2 diabetes patients. Moreover, diabetic environment including MMPs upregulates TGF-β activity and expression in glomerular mesangial cells and proximal tubular cells. TGF-β is accepted as the main profibrotic factor in DN. Thus, treatment aimed at modifying MMP expression may prevent activation of TGF-β and decrease progression.Citation9,Citation10
The tetracyclines are potent inhibitors of the MMP enzyme family. Their role has been investigated in several disorders in which MMP activity is amplified. Doxycycline is a potent, broad-spectrum, nonselective MMP inhibitor, acting on both mammalian collagenases and gelatinase and inhibiting the synthesis of MMPs in vivo. This is achieved by binding of the drug to zinc or calcium associated with the enzyme and blocking the active site, resulting in decreased enzyme activity leading to decreased expression of TGF-β, therefore attenuating mesangial cell proliferation, ECM deposition, glomerular hypertrophy, and proteinuria. Also, doxycycline can reduce the steady-state level of mRNA for several MMPs. Tension in floating collagen gels is dependent on the presence of serum or growth factors sequestered in the matrix such as platelet-derived growth factor or TGF-β. Thus doxycycline may inhibit collagen gel remodeling by preventing the release or activation of growth factors sequestered in the ECM. Moreover, CMT-3, a derivative of doxycycline that lacks antibiotic activity but retains anti-MMP activity, inhibited intimal thickening, demonstrating that these effects were independent of the antibiotic activity of the drug.Citation11
In the present study, diabetic proteinuria was seen to decrease significantly after 3 months of doxycycline therapy (p < 0.001), though the other renal parameters, which were normal at baseline, remained unaltered. When interpreted in terms of mean change in the levels of proteinuria, the mean change in the levels of proteinuria in Group B at 3 months was high as compared to the basal values and this mean change decreased significantly on withdrawal of doxycycline. Naini demonstrated the favorable effects on diabetic proteinuria by using low-dose doxycycline in a similar study performed over a period of 2 months on 35 patients.Citation12 A similar dose was used by Ahuja who reported a 70% decline in proteinuria in a patient with glomerulonephritis.Citation25 A study by McLennan postulates that MMP-7, responsible for degradation of non-collagenous glycoproteins, is reduced in diabetic kidney and that advanced glycation end products (AGEs), acting via TGF-β-dependent mechanisms, contribute to this process.Citation26
The present study showed that the level of proteinuria in patients with overt DN can be decreased with a low-dose doxycycline therapy. The delayed response seen after 3 months and not immediately after 1 month may be due to altered expression of MMPs and degradation of ECM proteins in the presence of the drug. This was achieved with a high degree of compliance and no apparent serious adverse effects.
Although promising, the studies published so far including our study have been too short term to clarify whether doxycycline treatment in diabetic patients is capable of preventing progression of DN, instead of simply influencing one of its surrogate end points, albuminuria. Further long-term multicentric trials are required to determine the benefits of doxycycline in patients of DN.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
Related Research Data
REFERENCES
- Barit D, Cooper ME. Diabetic patients and kidney protection: An attainable target. J Hypertens. 2008;26:53–57.
- Schrijvers B, De Urieso AS, Flyobjerg A. From hyperglycemia to diabetic kidney disease. Endocr Rev. 1997;25:971–1010.
- Toshio D. The current clinical problems for early phase of diabetic nephropathy and progression. Kidney Int. 1995;48:1929–1935.
- Fioretto P, Steffes MW, Mauer SM. Glomerular structure in non proteinuric IDDM patients with various levels of albuminuria. Diabetes. 1994;43:1358–1364.
- Chavers BM, Bilous RW, Elis EN, Steffes MW, Mauer SM. Glomerular lesions and urinary albumin excretion in type I diabetes without overt proteinuria. N Engl J Med. 1989;320:966–970.
- Fioretto P, Steffes MW, Sutherland DE, Mauery M. Sequential renal biopsies in IDDM patients; structural factors associated with clinical progression. Kidney Int. 1995;48:1929–1935.
- Zaoui P, Cantin JF, Alimardani Bassette M, Role of MMPs and inhibitors in the occurrence and progression of diabetic renal lesions. Diabetes Metab. 2000;26:25–29.
- Han SY, Jee YH, Han KH, An imbalance between matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 contributes to the development of early diabetic nephropathy. Nephrol Dial Transplant. 2006;21: 2406–2416.
- Wahab NA, Harper K, Manson RM. Expression of extracellular matrix molecules in human, mesangial cells in response to prolonged hyperglycemia. Biochem J. 1996;316:985–992.
- Sharma K, Zi Yodeah FN. Hyperglycemia and diabetic kidney disease: The care for TGF β as a key mediator. Diabetes. 1995;44:1139–1146.
- Franco C, Bernard HO, Mullholland D, Hou G, Guignabert C. Doxycycline alters vascular smooth muscle cell adhesion, migration and reorganization of fibrillar collagen matrices. Am J Pathol. 2006;168:1697–1709.
- Naini AE, Harandi AA, Moghtaderi J, Bartani B, Amiran A. Doxycycline: A pilot study to reduce diabetic proteinuria. Am J Nephrol. 2007;27:269–273.
- Pradeepa R, Deepa R, Mohan V. Epidemiology of diabetes in India – Current perspective and future projections. J Indian Med Assoc. 2002;100:144–148.
- The Diabetes Control and Complications trial research group. The effect of intensive treatment of diabetes on the development and progression of long term complications in IDDM. N Engl J Med. 1993;329:977–986.
- Lewis EJ, Hunsicker LG, Clarke WR, Renoprotective effect of angiotensin receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes. N Eng J Med. 2001;345:851–860.
- Brenner BM, Cooper ME, Dezeeuw D, Effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy. N Eng J Med. 2001;345:861–869.
- Pedrini MT, Levey AS, Lau J, Chalmers TC, Wang PH. The effect of dietary protein restriction on the progression of diabetic and non diabetic renal disease: A meta-analysis. Ann Intern Med. 1996;124:627–632.
- Wilens SL, Elster SK. The role of lipid deposition in renal arteriolar sclerosis. Am J Med Sci. 1951;219:183–196.
- Grundy SM, Cleeman JI, Merz CN, Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III guidelines. Circulation. 2004;110:227–239.
- Laville M. New strategies in anaemia management; Anaemia Corrections in Diabetes (ACORD) trial. Acta Diabetol. 2004;41:18–22.
- Kelly DJ, Zhang Y, Hepper C, Protein kinase C β inhibition attenuates the progression of experimental diabetic nephropathy in the presence of continued hypertension. Diabetes. 2003;52:512–518.
- Trachtman H, Schwob N, Maesaka J, Valderama E. Vitamin E supplementation ameliorates renal injury in chronic puromycin aminoglycoside nephropathy. J Am Soc Nephrol. 1995;5:1811–1819.
- Zaoui D, Kim Y, Steffes MW, Groppoli TJ, Butkowski RJ, Mauer SM. Glomerular distribution of type IV collagen in diabetes by high resolution quantitative immunochemistry. Kidney Int. 1994;45:425–4233.
- Nakamura T, Ushiyama C, Suzuki S, Urinary excretion of podocytes in patients with diabetic nephropathy. Nephrol Dial Transplant. 2000;15:1379–1383.
- Ahuja TS. Doxycycline decreases proteinuria in glomerulonephritis. Am J Kidney Dis. 2003;42:376–380.
- McLennan SV, Kelly DJ, Schache M, Advanced glycation end products decrease mesangial cell MMP-7: A role in matrix accumulation in diabetic nephropathy. Kidney Int. 2007;72:481–488.