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BJ BANTAO Journal

Editor-in-Chief

Official Publication of the BANTAO Association Incorporating Proceedings of the BANTAO Association

Associate Editors Skopje

Goce Spasovski

Mustafa Arici Nada Dimkovic Dimitrios Goumenos Nikolina Basic-Jukic

Editores Emeriti Varna Skopje Belgrade Athens Izmir

Dimitr Nenov Momir Polenakovic Ljubica Djukanovic Charalambos Stathakis Ali Basci

Ankara Belgrade Patra Zagreb

Deputy Editors Veselin Nenov Adrian Covic

Varna Iasi

Editorial Board Aydin Turkmen Alketa Koroshi Amira Peco Antic Biljana Stojmirovic Boriana Kiperova Cengiz Utas Daniela Monova Dimitrios Memmos Dimitris Tsakiris Ekrem Erek Evgueniy Vazelov Fehmi Akcicek Fevzi Ersoy Georgios Vergoulas Gordana Peruncic-Pekovic Gultekin Suleymanlar Halima Resic Igor Mitic Jadranka Buturovic-Ponikvar Jelka Masin Spasovska John Boletis Kamil Serdengecti Kenan Ates

Istanbul Tirana Belgrade Belgrade Sofija Kayseri Sofia Thessaloniki Thessaloniki Istanbul Sofija Izmir Antalya Thessaloniki Belgrade Antalya Sarajevo Novi Sad Ljubljana Skopje Athens Istanbul Ankara

Katica Zafirovska Ladislava Grcevska Liliana Garneata Kostas Siamopoulos Marko Malovrh Milan Radovic Myftar Barbullushi Olivera Stojceva Taneva Paul Gusbeth-Tatomir Petar Kes Rade Naumovic Rafael Ponikvar Sanja Simic-Ogrizovic Sanjin Racki Serhan Tuglular Sevgi Mir Tekin Akpolat Velibor Tasic Vidosava Nesic Vidojko Djordjevic Visnja Lezaic Vladislav Stefanovic Mahmut Ilker Yilmaz

Skopje Skopje Bucharest Ioannina Ljubljana Belgrade Tirana Skopje Iasi Zagreb Belgrade Ljubljana Belgrade Rijeka Istanbul Izmir Samsun Skopje Belgrade Nis Belgrade Nis Ankara

International Advisory Board Andrzej Wiecek Claudio Ponticelli Carmine Zoccali David Goldsmith Dimitrios Oreopoulos Francesco Locatelli Horst Klinkmann John Feehally Jorg Vienken

Poland Italy Italy UK Canada Italy Germany UK Germany

Published by: Balkan Cities Association of Nephrology, Dialysis, Transplantation and Artificial Organs Printing: BANTAO, 2013

Jorge Cannata Jurgen Floege Marc De Broe Markus Ketteler Mohamed Daha Norbert Lameire Raymond Vanholder Rosanna Coppo Ziad Massy

Spain Germany Belgium Germany Netherlands Belgium Belgium Italy France

Contents I. Editorial Comments Improvement in Kidney Transplant Program in the Republic of Macedonia - What Might be the Clue? Goce Spasovski, Saso Dohcev, Oliver Stankov, Sotir Stavridis, Saso Josifov, Irena RambabovaBusletic and Jelka Masin-Spasovska ………………………………………..………………………

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II. Review Articles Vascular Access Options in Developing Countries Petar Dejanov ……………………………..…………………………………………………………..

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Consequences of Metabolic Acidosis in Chronic Kidney Disease Patients Lea Katalinic, Kristina Blaslov, Iva Pasini, Bojan Jelakovic, Petar Kes and Nikolina Basic- Jukic …

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III. Original Articles Association between Estrogen Receptor-α Gene Polymorphisms and Lupus Nephritis in Bulgarian Patients Zornitsa Kamenarska, Maria Hristova, Lyubomir Dourmishev, Silvia Andonova, Radka Kaneva, Vanio Mitev, Boris Bogov and Alexey Savov ………………………………..………………………

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Immunohistochemical and Morphometrical Study of the Tubulointerstitial Changes in Primary Glomerulopathies Slavica Kostadinova-Kunovska, Rubens Jovanovic, Ladislava Grcevska, Jelka Masin - Spasovska, Goce Spasovski, Momir Polenakovic and Gordana Petrushevska ……………………………………

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Pericardial Thickening and Cardiac Valvular Calcification in End-Stage Renal Disease Branimir Nikolov Kanazirev, Rossitsa Yordanova Zortcheva and Valentin Christoforov Ikonomov ……………...………………………………………………………………………………

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Association of Abdominal Adiposity, Inflammation Markers and Cardiac Valve Calcifications in Haemodialysis Patients Nereida Spahia Zeneli, Merita Rroji, Saimir Seferi, Alma Idrizi, Elvana Rista, Myftar Barbullushi, Elizina Petrela and Nestor Thereska …………………………………………………………………

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IV. Short Communication Breast Calcification in Women with End-Stage Renal Disease Undergoing Hemodialysis Rossitsa Zortcheva and Diana Gardevska ………………………………………………….…………

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V. Case Reports Listeria Monocytogenes Meningitis in a Renal Transplant Recipient-A Case Report Ines Mesar, Petar Kes, Ljubica Bubic-Filipi, Bruno Barsic, and Nikolina Basic-Jukic …..…………..

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Chylous Peritonitis in Patients on Continuous Ambulatory Peritoneal Dialysis Ina Georgieva, Vera Stamenova, Ivan Trendafilov, Mitko Georgiev, Velimir Papazov, Dimitar Terziev, Diana Yonova and Evgueniy Vazelov ……………………………………………………....

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Kidney Transplantation in a HIV Infected Patient: A Case Report Nikolina Basic-Jukic, Lea Katalinic, Ljubica Bubic-Filipi, Petar Kes, Josip Begovac, Eva Pasini and Josip Pasini ……………………………………………..………………………………………...

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VI. Letter to the Editor Art and Non-Governmental Organization Influence to Donor Pool Expansion – Possibilities for the Balkans Aleksandar Nesic ……………………………………………………………………...………………

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VII. In Memoriam In Memoriam - Prof. Dr. Gjorgji F. Masin (1930-2013) Momir Polenakovic and Goce Spasovski ……………………………………...……...………………

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BANTAO Journal 2012; 10 (2): 47-48

BJ BANTAO Journal Editorial

Improvement in Kidney Transplant Program in the Republic of Macedonia – What Might be the Clue? Goce Spasovski1, Saso Dohcev2, Oliver Stankov2, Sotir Stavridis2, Saso Josifov3, Irena RambabovaBusletic and Jelka Masin-Spasovska2 1

Department of Nephrology, 2Department of Urology, 3Department of Vascular Surgery, Medical Faculty, University "Ss. Cyril and Methodius" of Skopje, Republic of Macedonia

Key words: kidney transplantation, living donor, deceased donation

___________________________________________ It’s universally accepted that the best treatment option in patients with chronic kidney disease (CKD) in a need of renal replacement therapy is kidney transplantation [1]. Unfortunately, this is a multidisciplinary procedure that is not sufficiently developed in majority of the developing countries. The low expenditure on public health is surely one of the major disincentives for poor transplant activity, especially from a deceased donor. Here, the lack of an appropriate organizational infrastructure with national and hospital coordinators, public promotion of donation and education of well-trained and competent procurement teams are considered essential for a successful deceased donation and transplantation program [2]. Under such circumstances, at the question to the transplant professionals on how to increase the number of transplantations [3], the answer may be that an immediate improvement could be achieved increasing the living related transplant procedures. In this regard, a few committed surgical and transplant nephrological teams should be composed, given that transplant center facilities and therapeutic armamentarium is adequately provided. Macedonian kidney transplant program was initiated in 1977, but real continuum has been achieved in the period 1996-2011, with an average of around 13 living donor (LD) kidney transplantations per year, including 18 LD transplantations performed in patients from Kosovo. Unfortunately, such a small transplant program has been insufficient to equalize the number of incident dialysis patients [4], and to prevent opening of new dialysis centers and consequent health insurance fund expenditures. The small number of transplants in the presence of investigated and prepared LD pairs might be a result of at least two reasons. The first reason was the very low reimbursement per transplant procedure allocated according to the DRG code and the second one was the insufficient number of dedicated professionals (1 urologist and 1 nephrologist were in charge of the program).

The improvement can be achieved by acknowledging the great support from the newly created Regional Health Development Centre (RHDC) on Organ Donation and Transplant Medicine established in Croatia (Zagreb), under the umbrella of the South-eastern Europe Health Network (SEEHN) that is operating under the Regional Cooperation Council, as a successor to the Stability Pact for South-eastern Europe (SEE). In fact, the RHDC was intended to serve as a competent regional resource centre assisting SEEHN countries to create or improve their own donation and transplantation programs [5]. Thus, at the very first professional meeting (27-28 May 2011 in Skopje, Macedonia) of the national focal point (NFP) professionals from the SEEHN countries and the Task Force Group composed of professionals from The Transplantation Society and the European Society of Organ Transplantation in collaboration with the International Society of Organ Donation and Procurement and the European Transplant Coordinators Organization, the organ donation and transplantation needs of each country within the SEE geographical region were addressed. In order to accomplish the requirements for improvement of our transplantation program and to discuss the current situation we have held a couple of meetings with the Ministry of Health (MOH) and fund authorities. In addition, 3 urologists and 2 nephrologists were supported through RHDC and our MOH to spend a week in Zagreb, Croatia, at the Urology and Nephrology Department at the University Clinical Center, Rebro. During our stay we have been introduced with the system of the national coordination at the MOH in Croatia, a system of hospital coordinators, ICU brain death diagnostics and various SOPs for regular organisation of transplant program, also being a part of the Eurotransplant system. Secondly, in the outpatient clinics we have had a chance to see the regular work up for preparation of potential recipients from a nephrological and urological specific point of view. Thirdly, the urologists have participated in a few kidney transplantations as well as other procedures at the Urology Department. Furthermore, we have all had regular ward visits of patients at the urological ward on the first post-

________________________ Correspondence to: Goce Spasovski, University Department of Nephrology, "Vodnjanska" 17, 1000 Skopje, R. Macedonia; Mob.: +389 70 268 232; Tel/Fax: +389 2 3231 501 or +389 2 3220 935; E-mail: [email protected]

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operative transplant days when kidney transplant recipients were carefully evaluated with various therapeutic protocols for immunosuppression and prophylactic therapy. Finally, we have been familiarized with many standard operative procedures (SOPs) with regard to the surgical and nephrological requirements perioperatively. Finally, once the problems were recognized transplant professionals in R. Macedonia along with the health care authorities have tried to sort them out. Thus, the budget per transplant procedure DRG code was substantially increased from 3.500 to 10.000 Eur generating a positive budget at the Department of Urology. Secondly, the surgical team was composed of 3-4 urologists (donor nephrectomy and transplantation) and a vascular surgeon for the vessel anastomoses. Furthermore, the limited capacity of the transplant center (only 4 beds) was exceeded changing the peri/postoperative care of the transplant recipients, speeding up the turnover of patients whenever possible. In this regard, we initiated a modification of the anesthesiological treatment during the procedure with a special care to provide MAP of at least 85 mmHg trying to avoid hypoperfusion of the graft and related consequences of acute tubular necrosis, delayed graft function and a prolonged hospitalization. We also agreed to remove the urocatheter at 5-7 days. Finally, we agreed on hospital discharge as early as possible with a frequent outpatient visits in the following weeks. At the end of the year 2012, after 24 successfully performed LDKTx procedures over the period of only 7 months we are pleased to report that all these maneuvers have led towards the 4-fold improvement as compared to the year 2011. Apart from the enthusiasm of the team involved and a number of meetings held, we have been motivated to prove we can manage the program successfully.

Improvement in kidney transplantation in R. Macedonia

As for the future, we do hope that we could also be supportive for regional collaboration in preparation and transplantation of CKD patients from the neighboring countries. We are also willing to initiate the deceased donor program, once the national authorities have accomplished the preconditions and requirements in the organizational infrastructure [6]. There are many difficulties and obstacles to get successfully transplant patient, but the victory of the best and unique kidney transplantation treatment and the pleasure of a smile on the patient’s face at the discharge may be a substitute for all the efforts and time invested, so be it! Conflict of interest statement. None declared.

References 1.

2. 3.

4.

5.

6.

Wolfe RA, Ashby VB, Milford EL, et al. Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant. N Engl J Med 1999; 341(23): 1725-1730. Spasovski G. Can we decrease organ trafficking in the Balkans? Clin Transplant 2011; 25: E223-E224. Spasovski G, Stathakis Ch, Basci A. A struggle for kidney transplantation in a developing world? Bantao J 2007; 5: 2. http://www.bantao.org/5_2/5_2_2.pdf. Stel VS, Kramer A, Zoccali C, Jager KJ. The 2007 ERAEDTA Registry Annual Report-a Preґ cis. NDT Plus 2009; 2: 514. Spasovski G, Busic M, Raley L, et al. Current status of transplantation and organ donation in the Balkans-could it be improved through the South-Eastern European Health Network (SEEHN) initiative? Nephrol Dial Transplant 2012; 27(4): 1319-1323. Spasovski G, Vanholder R. Kidney transplantation in emerging countries: do we know all issues? Minerva Urol Nefrol 2012; 64(3): 183-189.

BANTAO Journal 2012; 10 (2): 49 -54

BJ BANTAO Journal Review

Vascular Access Options in Developing Countries Petar Dejanov Vascular Access Unit, University Clinic of Nephrology, Medical Faculty, University "Ss. Cyril and Methodius" of Skopje, Republic of Macedonia Abstract Vascular access (VA) creation for hemodialysis (HD) is important in health care system. The aim of this review is to clarify the most appropriate option for VA, which can be performed in the developing countries. It seems a difficult task to organize VA program in the developing countries. However, it is well known that 8-10% of all costs for HD are spent for VA creation and maintenance. Nephrologists in the developing countries have to make proper patient’s physical examination prior to surgery. In addition, they have to be skilled in surgical procedures during maintenance of VA. Therefore, good maintenance regarding optimal physical examination, Doppler sonography examination, good surgical technique for creating arteriovenous fistula and arteriovenous graft, VA catheters, are preferred. Strategies for optimal VA in the developing countries are important for patient’s quality of life as well as for properly used finances. Key words: vascular access; hemodialysis; developing countries

mination prior to surgery, as well as in surgical procedures during the maintenance of VA, for Doppler examination of arteries, veins, and anastomosis. Today, nephrologists have to coordinate the activities of the surgeons and radiologists, which is time consuming and healthcare finance wasting. In comparison to arteriovenous grafts (AVG) and catheters, arteriovenous fistula (AVF) is more preferred VA, due to its longer duration, less common infection and thrombosis complications. In addition, it leads in creation of optimal VA, especially for AVF, which is essential for patient’s quality of life with lower morbiddity and mortality, lower costs, all very important for healthcare system. Physical examination and prevention Physical examination of the patient is simple. The patient’s chest, breast, and upper arms should be evaluated for presence of scars, swelling, or collateral veins. The presence of a neck’s scar with arm swelling usually means previous placement of subclavian catheter associated with subclavian vein stenosis.

___________________________________________ Introduction Vascular access (VA) creation for hemodialysis (HD) is important for patient’s survival. It is time and money consuming procedure. During 2005, patients with end-stage renal disease in the USA represented only 1.2% of the Medicare population and 8.2% of the Medicare expenditures. Consequently, up to 17 billion US $ were associated with their care. This 6.8-fold cost ranked end-stage renal disease higher than other diseases: congestive heart failure (3-fold); chronic kidney disease (2.9-fold); and diabetes (1.7-fold). Expenditures for VA placement and access complications were over 1.5 billion US $. Moreover, these costs might rise out of proportion, since diabetes and hypertension affect the peripheral vasculature with potential detriment to long-term access options and also increased VA complication ratio [1]. It seems a difficult task to organize VA program in the developing countries, due to the fact that 8-10% of all costs for HD are spent for VA creation and maintenance. Nephrologists in the developping countries have to be skilled in patient’s physical exa-

b

a

Fig. 1. Central vein stenosis with arm swelling (a) and scars over subclavian site (b)

Subclavian vein stenosis might affect AVF creation (Figure 1) [2,3]. The arterial examination is performed by pulse examination, segmental blood pressure measurement and Allen

________________________ Correspondence to: Petar Dejanov, University Department of Nephrology, "Vodnjanska" 17, 1000 Skopje, R. Macedonia; Phone: +389 2 3147 193; E-mail: [email protected]

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Vascular access in developing countries

test. Pulse examination for axillary, brachial, radial and ulnar pulses has to be done in both upper extremities. The pulse quality is scored as either normal, diminished or absent. Differences in blood pressure between the two arms should not be higher than 20 mmHg. For good AVF, Allen test should be negative, and it means patent palmary arch [3].

Using physical examination, the nephrologist should be skilled to recognize early AVF stenosis during the first 3 months (Figure 2). Juxta-anastomotic venous stenosis and presence of cephalic vein side branches cause early AVF failure that could be easily diagnosed by physical examination (Figure 3).

a. radialis Fig. 2. Juxta-anastomotic stenosis. A-radial artery. A strong pulse and thrill are present at anastomosis (B), and it disappears as one moves up the fistula to the level of the lesion (C)

a. radialis Fig. 3. Fistula occluded at point A, the thrill will disappear at the anastomosis. As the occlusion is moved to point B, the thrill will continue when the fistula is occluded

Thrill / bruit

Dilated / pulsatile

Collapsed / Dilated

a.radialis

Elevation test Fig. 4. Venous stenosis. Elevation test, distal part of the vein is dilated and pulsatile, and proximal part of the vein is collapsed

Late fistula stenosis is detected by physical examination. Venous stenosis is detected by elevation test (Figure 4) [3]. Thrill, pulse and bruit characteristics are checked on daily

basis. These parameters can be checked either by medical staff or patients. Therefore, this will result in a lower complication rate and a prolonged fistula survival (Table 1).

Table 1. Physical findings of venous stenosis Parameter Normal Thrill At the arterial anastomosis only Pulse Soft, easily compressible Bruit Low pitch, continuous, diastolic and systolic

Venous stenosis is related to the presence of: aneurysm formation, 3 HD sessions with blood flow rate less than 200 ml/min, increased venous blood pressure, bleeding

Stenosis At stenotic lesion site Water hammer High pitched, systolic only

more than 10 minutes after withdrawing the needles, Kt/V (a number used to quantify adequacy of HD, according to National Kidney Foundation) less than 1.2.

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Dejanov P.

Aneurysm of AVF is recognized as a vein ballooning. Aneurysms are developing upstream from a venous ste-

nosis, at sites of repetitive needle insertions (Figure 5).

Fig. 5. AVF with aneurysm (a), angiogram of fistula with aneurysm (arrows) (b)

Their progress can be followed up by skin changes. Surgical intervention is indicated when the skin shows signs of possible rupture due to infection and thinning (Figure 6).

Important issues related to the cannulation procedures are: needle size, route of needle placement and needle design. Doppler sonography examination

Fig. 6. Aneurysm associated with skin thinning

To avoid serious complications, prevention of aneurysm appearance is advised, using two cannulation procedures: rope ladder and button hole technique. Rope ladder technique involves needle placement in different sites, while button hole technique requires insertion of a needle in the same spot, at the same angle and depth (Figure 7).

Doppler sonography examination is a valuable tool for vasculature evaluation regarding access creation. Optimal preoperative radial artery diameter is found to be 2 mm or greater. Venous anatomy is important for access creation, for both AVF and AVG. Optimal features for the procedure are: luminal diameter at the point of anastomosis of 2.5 mm or greater, a straight segment of vein, continuity with the proximal central veins without obstruction. Doppler measurement of brachial artery flow more than 80 ml/ min prior to access construction is predictive of access maturation [4]. Graft-venous anastomotic stenosis can be seen with Doppler examination. The use of ultrasound guidance has been promoted for reducing the risk of complications during central venous catheterisation. Ultrasound probe is used to localize the vein and to measure its depth beneath the skin. Under Doppler ultrasound visualisation, the introducer needle is guided through the skin and into the vessel. During internal jugular venous catheterization, Doppler guidance reduces the number of complications, and the time required for insertion (Figure 8) [5]. Surgical technique

Fig. 7. Button hole and rope ladder techniques for cannulating AVF

A good surgical technique is important for creating AVF and AVG. Nephrologists are familiar with these techniques in several countries, including Macedonia, Italy, Slovenia and The Netherlands. Local anesthesia with 2% lidocaine in the forearm is used. After few minutes a longitudinal incision of 4-5 cm has to be performed. Using 3.5 x loupes, the cephalic vein is gently dissected to the length to reach the arterial anastomotic site. Upper aspect of the vein is marked in order to prevent rotation, and future post-anastomotic stenosis. During anastomo-

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Vascular access in developing countries

sis creation, 0.8-1.0 mg/kg heparin is given intravenously. A 5-6 mm arteriotomy is performed. The vein is cut and end to side anastomosis is made with two running su-

tures with 6/0 Prolene. The wound is closed in two layers using 3/0 absorbable sutures for the subcutaneous layer.

Fig. 8. Use of Doppler sonography in cannulation of right internal jugular vein (RIJV)

The skin is closed with 3/0 silk sutures. Control of the thrill, pulse, bruit, and inspection for eventual hematoma or infection, is important for the AVF function the first week after the surgery [6,7]. Vascular access with catheters Vascular access catheters are more often used due to their easier way of placement at bedside. They are used in emergency, in acute renal failure, in plasmapheresis, in intoxications, and when AVF or AVG are thrombosed. Catheters are used for permanent access when patients have exhausted blood access sites or when they have short life expectancy due to cancer, advanced age, or myocardiopathy with heart failure. Different types of catheters, different materials, impregnated with anti-microbial substances are produced. Tunneled cuff catheters (TCC) have been found to be associated with three-fold increased mortality compared with AVF, because of their high infections ratio. Poor quality of life, reduced dialysis adequacy, and central venous stenosis are additional complications associated with TCC [8-10].

Table 2. Duration of femoral and subclavian catheters Femoral catheters Subclavian catheters Days % of total Days % of total 1-7 16 1-7 7.0 8-14 25.4 8-14 16.3 15-30 44.3 15-30 50.6 31-60 12.3 31-60 24.1 > 60 2.0 > 60 2.0 Table 3. Complication rates in femoral catheters with Medcomp and Gam Cath catheters Femoral 20-24 cm Medcomp GamCath N=1200 N=800 Catheter infection 93 (7.75%) 50 (6.25%) Sepsis 36 (3.0%) 16 (2%) Thrombosis 66 (5.5%) 32 (4%) Arterial puncture 90 (7.5%) 88 (11%) Letal outcome 0 0 Pneumothorax 0 0 Total 23.75% 23.25%

Table 4. Complication rates in subclavian and jugular catheters with Medcomp and Gam Cath catheters Subclavian 20 cm Jugular 20 cm Medcomp GamCath Medcomp GamCath N=30 N = 40 N=30 N=30 Catheter infection 2 (6.6%) 4 (10%) 1 (3.3%) 1 (3.3%) Sepsis 0 0 1 (3.3%) 1 (3.3%) Thrombosis 2 (6.6%) 4 (10%) 1 (3.3%) 1 (3.3%) Arterial puncture 3 (10%) 3 (7.5 %) 3 (10%) 3 (10%) Letal outcome 0 0 1 (3.3%) 0 Pneumothorax 2 (6.6) 2 (5%) 2 (6.6%) 1 (3.3%) Total 29.8% 32.5% 29.3% 23.2%

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Dejanov P.

Experience with HD catheters in Macedonia In our VA Unit, AVF is the first choice for VA. It is used in app. 90% of the cases. Catheters are used in app. 9%, when there is no place for AVF creation. AVG is used in not more than 1%. We prefer femoral route for VA, as opposed to literature data or DOQI guidelines. Duration of femoral and subclavian catheters in our group of patients is presented in Table 2. Complication ratio resulting from the use of catheters, produced by Medcomp and Gam Cath is presented in Table 3 and Table 4. We have been using antibiotic lock technique for the last two decades. After catheter placement we observe if clinical signs of infection occur. In case of infection, half

dose of the antibiotics is flushed into circulation. The other half remains locked into the catheter lines, between HD sessions. Antibiotics (usually cephalosporins) show antimicrobial effects, antithrombotic, as well as coumarin-like effect which reduces the rate of stenosis and thrombosis. Cephalosporins might cause bleeding and other hemostatic disturbances. When locked in the catheter lumen, they also achieve a favourable concentration against a large number of bacteria. This technique can prevent occurrence of the central veins stenosis and thrombosis [11,12]. Distribution of bacterial infections from routinely analyzed catheter tips in 102 patients with 105 cannulations (84 femoral and 21 subclavian) are presented in Table 5.

Table 5. Distribution of bacteria infections from routinely analyzed catheter tips % of infected % of infected Bacteria femoral tips subclavian tips Staphilococcus coagulase 58.0 56.4 negative Staphilococcus aureus 18.1 20.1 Enterococcus 16.1 20.5 Eschericia colli 4.6 1.4 Streptococcus viridans 1.6 0 Pseudomonas aeruginosa 1.6 1.6

During 30-year-period of cannulation, using numerous catheters of different material, form, length and impregnation, no significant difference among catheters has been noticed [13,14]. However, staff education, rigorous sterile conditions during placement and maintenance of the catheters are essential for their longer duration. Vascular access in developing countries-conclusions VA data from literature and our 3 decades of experience are a solid ground that we may suggest certain strategies for optimal VA in the developing countries, which are important for patients’ quality of life and properly used finances. According to our experience, AVF is preferred in approximately 90% of total VA, while TCC are used in less than 10% and AVG are used in less than 1% of the cases. Nephrologists have to coordinate the activities of surgeons and radiologists. In addition, they have to be skilled in creation of AVF, AVG, TCC, and have to be educated for Doppler examination. They should also increase a number of preventive AVF, which means to create AVF in the IV stage of chronic kidney disease. Antibiotic locking technique may contribute in achieving long duration catheters. Subclavian vein cannulation should be avoided, due to possible vein stenosis. The increased use of citrate as a locking solution, and the limited use of heparin are important for optimal VA. Moreover, permanent education of nephrologists, medical staff and patients, and implementing updated VA strategies are essential for optimized VA in the developing countries. Conflict of interest statement. None declared.

References 1.

USRDS. The 2007 USRDS annual data report. http://www. usrds.org/adr.htm. Accessed September 30, 2007. 2. Beathard GA. Physical examination: The forgotten tool, in A Multidisciplinary Approach for Hemodialysis Access, edited by Gray R and Sands J, New York New York, Lippincott Williams & Wilkins, 2002, pp 111-118. 3. Beathard G. Complications of vascular access, in Complications of Dialysis-Recognition and Management, edited by Lameire N, Mehta R, New York, Marcel Dekker, 2000; pp 1-27. 4. Malovrh M. Native arterio-venous fistula: Preoperative evaluation. Am J Kidney Dis 2002; 39: 1218-1223. 5. Lenng J, Duffy M. Real time ultasonographically guided internal jugular vein catheterization in the emergency department increases success rates and reduces complications: A randomized prospective study. Annals of emergency medicine 2006; 48; 4-9. 6. Shmesh D, Zigelman O. Primary forearm arterio-venous fistula for hemodialysis access an integrated approach to improve outcomes. Cardiovascular surgery 2003; 11: 35-41. 7. Robbin ML et al. Hemodialysis arterio-venous fistula maturity: US evaluation: Radiology 2002; 225: 59-64. 8. Di Iorio et al. Vascular access for hemodialysis: the impact on morbidity and mortality. J Nephrol 2004; 17(1): 19-25. 9. Schwab SJ. The hemodialysis catheter conundurum: Hate living with them, but can’t live without them. Kidney Int 1999; 56: 1-17. 10. Katneni R. Hemodialysis central venous catheter related bacteriemia in chronic hemodialysis patients; Epidemiology and evidence-based management. Nat Clin Pract Nephrol 2007; 3: 256-266. 11. Dejanov P, Oncevski A, Gerasimovska V. Locking of hemodialysis catheters with proper antibiotics in catheter related infections. Paper presented at: Angioaccess for

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hemodialysis, second international multidisciplinary symposium, France 1999. 12. Gerasimovska V, Oncevski A, Dejanov P, Polenakovic M. Are ambulatory femoral catheters for hemodialysis a safe vascular access? J Vasc Access 2002; 3(1): 14-20. 13. Oncevski A, Dejanov P, Gerasimovska V, Polenakovic MH. Approach to vascular access for hemodialysis: experien-

Vascular access in developing countries

ces from the Republic of Macedonia. Int J Artif Organs 2002; 25(5): 354-364. 14. Dejanov P, Oncevski A, Gerasimovaska V. The advantages of femoral catheters for hemodialysis. In: Vascular access for hemodialysis-VII. Precept Press 2001, Gore & associates.

BANTAO Journal 2012; 10 (2): 55-58

BJ BANTAO Journal Review Articles

Consequences of Metabolic Acidosis in Chronic Kidney Disease Patients Lea Katalinic, Kristina Blaslov, Iva Pasini, Bojan Jelakovic, Petar Kes and Nikolina Basic- Jukic Department of Nephrology, Arterial Hypertension, Dialysis and Transplantation, University Hospital Centre Zagreb and School of Medicine, University of Zagreb Abstract Metabolic acidosis is an inevitable complication associated with progressive loss of kidney function. It appears when glomerular filtration rate (GFR) falls below 25 ml/min/ 1.73 m2. Metabolic acidosis arises from the difference between the excretion of hydrogen and the synthesis of ammonia ions. Damaged renal tubules cannot contribute in maintaining the acid base balance by reabsorbing the daily filtrated HCO3− and synthesizing new HCO3− ions. Although usually mild to moderate, it may lead to several metabolic complications. Metabolic acidosis is one of the important causes of protein energy wasting (PEW) and can trigger muscle loss in patients dealing with chronic kidney disease (CKD). Furthermore, it contributes to the development of chronic kidney disease-metabolic bone disease by increasing the bone resorption and inhibiting the bone formation. It has been shown that the acidic environment could play an important role in regulation of the hepcidin homeostasis and thus, be one of the factors contributing to the etiology of the anemia of chronic disease. It is also known that metabolic acidosis can severely affect kidney function by causing progressive tubulointerstitial injury and decline in glomerular filtration rate. All these metabolic effects are complex but can be successfully managed. Series of studies provide evidence that bicarbonate therapy is beneficial in these patients. It helps in achieving the balance between calcium and phosphate. Furthermore, it improves dietary protein intake and lean body mass and slows down the decline in glomerular filtration rate. Thus, metabolic acidosis needs to be monitored and carefully corrected. Keywords: metabolic acidosis, chronic kidney disease, kidney transplantation, bone metabolism, anemia

___________________________________________ Introduction

fully protected. If an increase in acidity or alkalinity occurs, three lines of defence activate-the blood buffers, the respiratory system's control of CO2 and the renal excretion of the excess acid or base [2]. The third defence line, kidney, is the most important in preserving this balance [3]. Metabolic acidosis is an inevitable complication associated with progressive loss of kidney function [4]. Its incidence and prevalence increase with declining kidney function [5]. When glomerular filtration rate (GFR) falls below 25 ml/min/1.73 m2 kidney's capacity to synthesize new ammonia and excrete excess hydrogen ions significantly reduces [5-7]. Furthermore, damaged renal tubules cannot contribute in maintaining the acid base balance by reabsorbing the daily filtrated HCO3− and synthesizing new HCO3− ions [5,6]. Although metabolic acidosis is usually mild to moderate, it can have a major impact on bone and muscle metabolism, nutritional status and anemia [5,6,8]. It may negatively affect kidney allograft function and is also associated with an increased morbidity and mortality of these patients [4,6,9,10]. These metabolic effects are very complex but can be successfully managed [8]. We present an overview of clinical consequences of metabolic acidosis in patients with chronic kidney disease. Metabolic acidosis and bone disease Disorders of mineral and bone metabolism are common in patients with chronic kidney disease (CKD). Serum phosphate levels rise due to decreased renal phosphate excretion [11]. Moreover, the conversion of vitamin D to its active form is decreased leading to decreased serum calcium levels and reduced intestinal calcium absorption. The disturbed ion balance provokes the synthesis and secretion of parathyroid hormone (PTH) in order to normalize the disturbed values [6,11]. Series of studies confirmed that metabolic acidosis contributed to the development of chronic kidney disease-metabolic bone disease [6]. Bone is considered to be an important buffering component since it is a reservoir of labile base in the form of alkaline salts of calcium [6,12]. Many studies suggested that a decrease of pH level and plasma bicarbonate concentration stimulate bone resorption and inhibit bone for-

Maintaining the acid base homeostasis is one of the most important factors which contribute to normal protein function [1]. The extracellular fluid pH value of 7.4 is care________________________ Correspondence to: Lea Katalinic, Department of nephrology, arterial hypertension, dialysis and transplantation, University hospital centre Zagreb, Kispaticeva 12, Zagreb, Croatia; E-mail: [email protected]

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mation [6]. This happens through activation of osteoclast's and inhibition of osteoblast's activity. In vitro studies provided evidence that a bone mineral base was released into the circulation when administering exogenus acid [12]. Furthermore, these studies provided evidence that metabolic acidosis could reduce vitamin D levels and stimulate PTH secretion. Not only does chronic metabolic acidosis stimulate PTH secretion but in the same time attenuates the cellular response to it [6]. If the acidification lasts long enough bone mineral content and bone mass progressively decline and osteoporosis occurs [12]. In children, uncontroled metabolic acidosis can lead to stunted growth. This is not only the outcome of inhibited bone formation but happens also as the consequence of decreased cartilage production. The assumption is that metabolic acidosis could decrease growth hormone and insulin-like growth factor 1 (IGF-1) secretion and attenuate their actions on targeted tissues [6]. Metabolic acidosis and malnutrition Malnutrition is a very frequent condition among patients with functioning graft. Chrusciel et al. showed in their study that malnutrition could be found in more than 20% of transplant recipients [13]. Both inadequate nutrient intake and ineffective nutrient utilization can contribute to nutritional disorders in CKD patients [14]. Protein energy wasting (PEW) is a disorder characterized by progressive loss of muscle and visceral protein stores, including albumin, which becomes noticeable in the advanced stage of kidney disease [15,16]. It is very common in patients with end-stage renal disease (ESRD) and can lead to increased debility and mortality, especially because PEW is strongly linked with an increased cardiovascular risk. Mechanisms responsible for muscle protein breakdown are complex and cannot be associated only with lower protein intake [16]. Uremic milieu leads to loss of appetite but uremia also inhibits the regenerative potential of skeletal muscle by acting on muscle stem cells [6,16]. Several abnormalities such as increased levels of circulating cytokines due to the presence of chronic inflammation, oxidative stress and endothelial damage, metabolic acidosis and disturbed insulin signaling also stimulate protein degradation and inhibit protein synthesis [16,17]. Inflammation markers such as CRP, IL-1 and IL-6 are often elevated in CKD patients. This happens due to the decreased excretory function of the kidneys but also during hemodialysis treatment which can activate microinflammation cascade through the exposure of the blood to dialysis membranes. Furthermore, oxidative stress arises from the difference between increased oxidant generation and insufficient anti-oxidant defence mechanisms. The presence of inflammation and generated free radicals results with tissue injury. It is a well-known fact that insulin is one of the most important factors in preserving lean body mass. However, it is not confirmed that disturbed insulin signaling, which can occur in these patients, could lead to muscle degradation [16]. Series of studies have suggested that metabolic acidosis was also one of the most important contributing factors

Metabolic acidosis in CKD patients

of PEW [17]. Acidosis increases muscle catabolism through an upregulation of the ATP-dependent ubiquitin-requiring pathway [6,18,20]. It also reduces protein synthesis, especially of albumin, and enhances amino acid oxidation, especially degradation of valine [6,16,17]. Studies in rats and humans indicated that the correction of metabolic acidosis raised both plasma and muscle protein levels by decreasing the transamination and decarboxylation in muscle [6]. Metabolic acidosis and anemia A normochromic, normocytic anemia is very common in the advanced stages of CKD. About 50% of all ESRD patients and 30-40% of kidney transplant recipients deal with its consequences [14]. Not only does anemia increase the overall cardiovascular risk, but it also leads to progression of renal impairment due to tissue hypoxia [10,14]. Deficient erythropoietin synthesis, shortened erythrocyte half-life and iron deficiency are the most important causes of anemia associated with CKD [1]. Hepcidin is a recently discovered but very important central regulator of body iron homeostasis [18]. This 25-amino acid peptide is synthesized predominantly in the liver but it is also expressed in other cells, including microphages. If overly expressed, hepcidin is associated with anemia of inflammation, chronic kidney disease and iron-refractory iron deficiency anemia [19]. In their study Rivera et al. Showed the potent role of hepcidin. When injected into mice it resulted in a dramatic drop of serum iron levels within an hour while the effect of a single dose lasted for 3 days even though hepcidin alone was rapidly cleared from plasma [20]. Hepcidin regulates cellular iron metabolism by binding to ferroportin, its receptor and the only known cellular exporter in mammals. This complex is degraded in lysosomes and iron cannot be released in the plasma [19]. Hepcidin is homeostatically regulated by iron and erythropoietic activity but interestingly enough, its transcription is also regulated by acid base status [19,21]. A recent study of Mizumoto and al. has shown that hepcidin levels were high in patients with ESKD and that acidic environment induced hepcidin transcription in the hepatoma cells. Thus, serum hepcidin levels are increased and could play a very important role in the etiology of the anaemia of chronic disease [21]. Metabolic acidosis, progression of CKD and effect on the kidney allograft Even though CKD inevitably progresses to ESRD, this process might be attenuated. Series of factors contribute to this such as cardiovascular disease, anemia, microinflammatory state, oxidative stress [10]. Metabolic acidosis is also among the factors which affect the kidney. Furthermore, even mild metabolic acidosis may affect the function of kidney allograft. This happens possibly through the stimulation of adaptive mechanisms aimed at maintaining acid base homeostasis [4]. Increased ammonia and endothelin production may cause progressive tubulointerstitial injury and decline in glomerular

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filtration rate while the newly synthesized bicarbonate alkalinizes the interstitium and encourages precipitation of calcium in the kidney [6,8,9]. Finally, studies performed on rats using the remnant kidney model of CKD indicated that GFR decline was mediated in part by the actions of excess aldosterone and endothelin stimulated through acid retention [8]. A limited number of studies performed on humans have also supported the potential role of metabolic acidosis in the progression of CKD [6]. Administration of bicarbonates to individuals with CKD of diverse etiology and metabolic acidosis not only slowed progression of CKD (GFR decline was less than half of the control group who received sodium chloride), but the number of individuals developing ESRD was significantly reduced [6]. Studies performed on rats provided evidence that bicarbonate therapy could decrease the severity of tubulointerstitial disease by preventing a development of interstitial inflammation and chronic fibrosis [8]. As previously mentioned, metabolic acidosis may also seriously damage the kidney allograft [4]. This is strongly associated with the use of immunosuppressive medications. These agents also contribute to the development of hyperkalemia, hyperchloremia and metabolic acidosis [22-24]. Immunosuppressive therapy is one of the cornerstones of successful kidney transplantation but we often neglect its negative effects on graft function [22,25]. Chronic allograft nephropathy (CAN) is the leading cause of graft loss one year after transplantation and is associated with a significant rise in morbidity and mortality. It is manifested with a decrease in allograft function at least 3 months after transplantation. Calcineurin inhibitors (CNIs), cyclosporine A (CsA) and tacrolimus contribute to CAN and are strong profibrotic agents. Most allografts show histopathologic signs of CNI toxicity 10 years after transplantation. Initiated fibrogenesis can in the end lead to organ failure thus making it diffucult to achieve a successful long-term allograft outcome [26]. Correction of metabolic acidosis with bicarbonate supplementation Metabolic acidosis is a very important clinical issue that needs to be monitored in patients suffering from CKD as well as in kidney transplant recipients. Its effects on kidney function, nutritional status, anemia and bone mass are complex and sometimes severe but can be successfully managed. There have been many studies showing benefits of the base therapy. It has been previously mentioned that metabolic acidosis supports the presence of mineral disturbance and thus aggravates bone disease. It provokes PTH secretion but attenuates the cellular response to it, reduces vitamin D plasma level, stimulates bone absorption and inhibits bone formation. Acidification encourages osteoclast activity while prohibiting the activation of osteoblasts. If this process lasts long enough, bone mass will be lost and osteoporosis will occur [12]. Sebastian at al. examined the influence of administering potassium bicarbonate on bone metabolism in postmenopausal women in their study in 1994. The study showed that supplementation with potassium bicarbonate had lowered urinary

excretion of calcium and phosphorus thus inducing the increase in the balance of these minerals. Secondly, the serum osteocalcin concentration had also risen. In conclusion, the administration of potassium bicarbonate reduced the bone absorption and increased the bone formation. Studies performed in adult patients on chronic maintenance hemodialysis showed similar results-correction of acidosis by raising the dialysate base concentration atenuated the rise in PTH, reduced bone absorption and improved bone formation [7,27]. Metabolic acidosis is one of the major factors in the development of PEW disorder. This disorder is characterized by the increase in protein catabolism due to the upregulation of the ubiquitin-proteasome system, increased amino acid oxidation and decreased visceral protein synthesis. The presence of uremia is also a contributing factor, which leads to loss of appetite and blunts muscle regenerative potential. Ione de Brito-Ashurst et al. showed how bicarbonate therapy could be beneficial in fighting this specific disorder. They observed bicarbonate therapy action by comparing the difference between two groups of patients-one group received bicarbonate treatment while the other did not. First of all, the group which was given bicarbonate supplementation at a dosage of 1.82 ± 0.8 g/ day showed a significant increase of serum HCO3-levels. Secondly, a significant improvement in dietary protein intake, lean body mass and an increase of albumin plasma levels was apparent in this group of patients [15]. Series of studies performed on animals and humans confirmed that metabolic acidosis is one of the risk factors for CKD progression [8]. It activates the alternative complement pathway and causes tubulointerstitial injury which leads to increased proteinuria, fibrosis and faster rate of decline in renal function [6,15]. There is also substantial evidence that bicarbonate therapy could slow the GFR decline. Administration of citrate or bicarbonate to rats with CKD slowed a decline in GFR and prevented tubulointerstitial inflammation and fibrosis. The results of the previously mentioned study of Ione de Brito-Ashurst and colleagues showed that the rate of loss of creatinine clearance was significantly lower in the treated group in comparison to control subjects. The observed decline in creatinine clearance was 5.93 ml/min/1.73 m2 in the control group compared with 1.88 ml/min/1.73 m2 in the treated group [15]. It needs to be emphasized that base therapy is not without risks. Possible adverse effects are volume overload, congestive heart failure and hypertension, all due to sodium retention. Thus, all of the patients receiving this therapy should be carefully monitored. It is recommended to give enough alkaline therapy in order to achieve normal serum HCO3− values [6]. Conclusions In conclusion, metabolic acidosis is a complex but inevitable condition of CKD. It has a major impact on bone and muscle metabolism, nutritional status, anemia and kidney function and is also associated with an increased morbidity and mortality of these patients. All these complications clearly respond to base therapy. Therefore, all

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patients in the state of metabolic acidosis should be monitored and treated in order to prevent complications and improve their health and quality of life. Conflict of interest statement. None declared.

References 1.

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Adrogue HJ, Madias NE. Management of life-threatening acid-base disorders. First of two parts. N Engl J Med 1998; 338: 26-34. Chan JC. Acid-base disorders and the kidney. Adv Pediatr 1983; 30: 401-471. Alcazar Arroyo R. Electrolyte and acid-base balance disorders in advanced chronic kidney disease. Nefrologia 2008; 28 Suppl 3: 87-93. Kovesdy CP. Metabolic acidosis and kidney disease: does bicarbonate therapy slow the progression of CKD? Nephrol Dial Transplant 2012; 27: 3056-3062. Navaneethan SD, Schold JD, Arrigain S, et al. Serum bicarbonate and mortality in stage 3 and stage 4 chronic kidney disease. Clin J Am Soc Nephrol 2011; 6: 2395-2402. Jeffrey A. Kraut, Nicolaos E. Madias. Consequences and therapy of the metabolic acidosis of chronic kidney disease. Pediatr Nephrol 2011; 26: 19-28. Roderick P, Willis NS, Blakeley S, et al. Correction of chronic metabolic acidosis for chronic kidney disease patients. Cochrane Database Syst Rev 2007; (1): CD001890. Kraut JA. Effect of metabolic acidosis on progression of chronic kidney disease. Am J Physiol Renal Physiol 2011; 300: F828-F829. Klaboch J, Opatrna S, Matousovic K, Schuck O. End stage of chronic kidney disease and metabolic acidosis. Vnitr Lek 2012; 58: 519-524. Schaefer B, Wuhl E. Educational paper: Progression in chronic kidney disease and prevention strategies. Eur J Pediatr 2012; 171: 1579-88. Abboud H, Henrich WL. Clinical practice. Stage IV chronic kidney disease. N Engl J Med 2010; 362:56-65. Sebastian A, Harris ST, Ottaway JH, et al. Improved mineral balance and skeletal metabolism in postmenopausal women treated with potassium bicarbonate. N Engl J Med 1994; 330: 1776-1781. Chrusciel B, Stompor T, Sułowicz W. Nutritional status of patients with functioning graft assessed by clinical examination, anthropometry and bioimpedance. Przegl Lek 2001; 58(9): 828-832.

14. Robert Thomas, Abbas Kanso, John R. Sedor. Chronic Kidney Disease and Its Complications. Prim Care 2008; 35: 329-vii. 15. Ione de Brito-Ashurst, Mira Varagunam, Martin J. Raftery, Muhammad M. Yaqoob. Bicarbonate Supplementation Slows Progression of CKD and Improves Nutritional Status. J Am Soc Nephrol 2009; 20: 2075-84. 16. Bonanni A, Mannucci I, Verzola D, Sofia A, et al. Protein-Energy Wasting and Mortality in Chronic Kidney Disease. Int J Environ Res Public Health 2011; 8(5): 163154. 17. Chiu YW, Kopple JD, Mehrotra R. Correction of metabolic acidosis to ameliorate wasting in chronic kidney disease: goals and strategies. Semin Nephrol 2009; 29: 67-74. 18. Hugman A. Hepcidin: an important new regulator of iron homeostasis. Clin Lab Haematol 2006; 28: 75-83. 19. Elizabeta Nemeth, Tomas Ganz. The Role of Hepcidin in Iron Metabolism. Acta Haematol 2009; 122: 78-86. 20. Rivera S, Nemeth E, Gabayan V, Lopez MA, Farshidi D, Ganz T. Synthetic hepcidin causes rapid dose-dependent hypoferremia and is concentrated in ferroportin-containing organs. Blood 2005; 106: 2196-9. 21. Mizumoto C, Kawabata H, Uchiyama T, et al. Acidic milieu augments the expression of hepcidin, the central regulator of iron homeostasis. Int J Hematol 2012; 96: 701-709. 22. Djamali A, Samaniego M. Fibrogenesis in kidney transplantation: potential targets for prevention and therapy. Transplantation 2009; 88: 1149-1156. 23. Kamel KS, Ethier JH, Quaggin S, et al. Studies to determine the basis for hyperkalemia in recipients of a renal transplant who are treated with cyclosporine. J Am Soc Nephrol 1992; 2: 1279-1284. 24. Mohebbi N, Mihailova M, Wagner CA. The calcineurin inhibitor FK506 (tacrolimus) is associated with transient metabolic acidosis and altered expression of renal acid-base transport proteins. Am J Physiol Renal Physiol 2009; 297: F499-509. 25. Marcen R, Morales JM, Fernandez-Rodriguez A, et al. Long-term graft function changes in kidney transplant recipients. NDT Plus 2010; 3(Suppl_2): ii2-ii8. 26. Schaefer HM. Long-term management of the kidney transplant recipient. Blood Purif 2012; 33(1-3): 205-211. 27. Mathur RP, Dash SC, Gupta N, et al. Effects of correction of metabolic acidosis on blood urea and bone metabolism in patients with mild to moderate chronic kidney disease: a prospective randomized single blind controlled trial. Ren Fail 2006; 28: 1-5.

BANTAO Journal 2012; 10 (2): 59-62

BJ BANTAO Journal Original article

Association between Estrogen Receptor-α Gene Polymorphisms and Lupus Nephritis in Bulgarian Patients Zornitsa Kamenarska1, Maria Hristova2,4, Lyubomir Dourmishev3, Silvia Andonova5, Radka Kaneva1, Vanio Mitev1, Boris Bogov4 and Alexey Savov5 1

Molecular Medicine Center, Medical University-Sofia and Department of Medical Chemistry and Biochemistry, Medical University-Sofia, 2Department of Clinical Laboratory and Clinical Immunology, Medical University-Sofia, 3Department of Dermatology and Venerology, Medical University-Sofia, 4Clinic of Nephrology at University Hospital "Alexandrovska", Medical University-Sofia, 5National Genetic Laboratory, Majchin Dom Hospital, Sofia, Bulgaria Abstract Introduction. Systemic Lupus Erythematosus (SLE) is an autoimmune disease with a complex etiology. Genetic and hormonal factors as well as signal transduction changes contribute to its pathogenesis. There is compelling evidence that autoimmunity is under genetic control and estrogens and their receptors can play an important role in its pathogenesis. Our stady aimed to investigate the association of estrogen receptor - ESR1 XbaI x/X and PvuII p/P polymorphisms with SLE and lupus nephritis and their variation across different populations. Methods. Forty-nine patients with lupus nephritis and sixty-nine healthy controls were examined in this pilot study. Two single nucleotide polymorphisms (SNPs) in the ESR1 gene XbaI x/X (-351A/G, rs9340799) and PvuII p/P (397T/C, rs2234693), previously associated with altered receptor expression, were selected for examination. Results. No significant differences in the genotype and allele distributions between patients and control groups were found. The XbaI X allele was associated with malar rash in SLE patients. The XX+Xx genotypes and the P allele and PP+Pp genotypes were strongly associated with the hematological disease in SLE patients. Conclusions. The results of this pilot study suggest that polymorphisms of the ESR1 gene are possible disease modifying factors for SLE but they are not associated with lupus nephritis.

Environmental and hormonal factors are known to contribute to the expression of the disease. Among the risk factors, female gender is considered to be of greatest importance [1]. Since women suffer about nine times more often from SLE, estrogens have been proposed as obvious candidates to explain this sexual dimorphism [2]. The renal glomerulus has been reported to be a direct-target tissue for estrogens [3,4] and it is well-known that estrogens aggravate lupus and nephritis [5]. Estrogens act through two nuclear receptors-estrogen receptor-α (ER-α) coded by ESR1 and estrogen receptor-β (ER-β) coded by ESR2. The PvuII P (C) and XbaI X (G) variants were found to lead to enhanced ER-α activity [6,7]. A recent study has proved relationship between estrogen receptor Pvu II and XbaI polymorphisms as well as the Th1 and Th2 cytokine expression in patients with SLE. According to the results of this study, the polymorphisms are associated with an alteration in the Th-1/ Th-2 balance in favor of Th-2, increasing the susceptibility to SLE. In addition, ER-α gene polymorphisms could influence the expression of IL-10, IL-4, IL-2 and IFN-γ in SLE, with the Th-2 cell being predominant in patients with PpXx and Ppxx genotypes [8]. Up-to-now a few studies have investigated the association of ESR1 XbaI x/X and PvuII p/P polymorphisms with SLE and lupus nephritis showing variation across different populations which was the aim of our study. Materials and methods

Key words: Systemic Lupus Erythematosus, Estrogen Receptor-α Gene Polymorphisms

Patients

___________________________________________ Introduction The etiology of SLE is not clearly understood but strong genetic predisposition has been recognized.

A total of forty-nine patients with SLE met the American College of Rheumatology (ACR) criteria. All of them had lupus nephritis varying from class II to class VI. There were forty females and nine males. The mean age was 40 years (range between 18 and 78 years old). The patients

________________________ Correspondence to: Zornitsa Kamenarska, Molecular Medicine Center, Medical University-Sofia and Department of Medical Chemistry and Biochemistry, Medical University-Sofia, 2 Zdrave Street, 1431 Sofia, Bulgaria E-mail: [email protected], Tel: 359 2 9172268

60 had been followed up for a mean period of 10 years at the Department of Nephrology, Medical University-Sofia and at the Department of Nephrology, Ministry of Interior Hospital-Sofia. Sixty-nine anonymous non-related healthy individuals, matched for sex, age and ethnicity with SLE patients were included for genetic analysis. They were selected from the BioBank of the Molecular Medicine Center and National Genetic Laboratory. Genetic analysis The study has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. The study was approved by the local ethics committee at the Medical University, Sofia. All participants signed an informed consent and venous blood was drawn for DNA isolation. Genomic DNA was extracted from the peripheral blood with the Chemagen DNA purification kit, using Chemagic Magnetic Separation Module I (Chemagen AG). The PvuII and XbaI polymorphisms were analyzed by polymerase chain reaction restriction fragment lengths polymorphism (PCR-RFLP). A 1.372 kb DNA fragment that contains the 2 polymorphic site was amplified using forward and reverse primers 5'CTG CCA CCC TAT CTG TAT CTT TTC CTA TTC TCC 3' and 5' TCT TTC TCT GCC ACC CTG GCG TCG ATT ATC TGA 3'. PCR was performed through 30 cycles including the following steps: denaturation at 95°C for 30s; annealing at 50°C for 1 min 30s; and extension at 72°C for 2min 30s. PCR products were digested with the restriction endonucleases PvuII and XbaI (Promega, Madison, WI, USA). The digested products were analyzed on 3% agarose gel stained with ethidium bromide. Heterozygous Pp genotype exhibited fragments 1372, 936 and 436 bp lengths and heterozygous Xx genotype exhibited fragments 1372, 982 and 390 bp lengths. Capital P or X represent the absence of restriction site while lower-case p or x indicate the presence of restriction site.

ESR polymorphism and SLE

Statistical analysis Allele and genotype frequencies were compared between SLE patients and controls, using Fisher’s exact probability test to calculate p values for 2×2 tables. If significant, data were expressed as p-value, odds ratios (OR) with exact 95% confidence intervals (CI). Test for Hardy-Weinberg equilibrium was done by χ2 statistics. LD analysis was performed by Haploview (Haploview Software version 3.32, Broad Institute, Cambridge, MA, USA http:// www.broad.mit.edu/mpg/haploview/) [9]. Results The two SNPs were found to be in Hardy-Weinberg Equilibrium. The observed allele and genotype frequencies of the ESR1 XbaI x/X and PvuII p/P polymorphisms among patients with SLE and healthy controls are summarized in Table 1. Table 1. Genotype and allele frequencies of ESR1 XbaI x/X and PvuII p/P polymorphisms in systemic lupus erythematosus patients and controls Genotype SLE Controls N=number of patients N=49 N=69 XbaI x/X (A/G) xx (AA) 15 (30.6%) 17 (24.6%) Xx (AG) 23 (46.9%) 42 (60.9%) XX (GG) 11 (22.5%) 10 (14.5%) x (A) 54.1% 55.1% X (G) 45.9% 44.9% PvuII p/P (T/C) pp (TT) 10 (20.4%) 17 (24.6%) Pp (TC) 26 (53.1%) 37 (53.6%) PP (CC) 13 (26.5%) 15 (21.7%) p (T) 46.9% 51.4% P (C) 53.1% 45.6% PvuII & XbaI Ppxx 10 (20.4%) 12 (17.4) Ppxx 5 (10.2%) 5 (7.2%) ppXx 0 (0.0%) 5 (7.2%) PpXx 21 (42.9%) 30 (43.5%) PPXx 2 (4.1%) 7 (10.2%) PpXX 0 (0.0%) 2 (2.9%) PPXX 11 (22.4%) 8 (11.6%)

Table 2. Comparison between the ESR1 XbaI x/X and PvuII p/P polymorphisms and ACR criteria *NS-not significant Genotype ACR criteria XbaI x/X pPvuII pP Xx (n=15) XX (n=11) pp (n=10) PP (n=13) (n=23) value (n=26) Malar rash 7(46.7%) 16(69.6%) 9)81.8%) 0.07 5(50.0%) 17(65.4%) 10(76.9%) Discoid rash 4(77.8%) 3(13.0%) 1(9.1%) NS* 2(20.0%) 5(19.2%) 1(7.7%) Photosensitivity 11(73.3%) 11(47.8%) 6(54.6%) 0.11 6(60.0%) 15(57.7%) 7(53.8%) Oral ulcer 2(13.3%) 1(4.4%) 0(0.0%) NS 1(10.0%) 2(7.7%) 0(0.0%) Arthritis 9(60.0%) 14(60.9%) 8(72.7%) 0.36 6(60.0%) 11(42.3%) 6(46.2%) Serositis 4(33.3%) 4(78.6%) 3(71.4%) NS 3(40.0%) 4(70.6%) 4(66.7%) Renal disease 15 23 11 NS 10 26 13 Neurological disease (100.0%) (100.0%) (100.0%) NS (100.0%) (100.0%) (100.0%) Hematological disease 2(13.3%) 5(21.7%) 3(27.3%) 1(10.0%) 6(23.1%) 3(23.1%) 0.035 Immunological disease 2(13.3%) 11(47.8%) 4(36.4) 0.5 0(0.0%) 9(34.4%) 8(61.5%) (Anti-dsDNA, Anti-SM, 9(60.0%) 13(56.9%) 7(63.6%) 6(60.0%) 14(53.9%) 9(69.2%) Anti-phospholipid) ANA 0.48 10(66.7%) 15(65.2%) 8(72.7%) 7(70.0%) 16(61.5%) 10(76/9%)

pvalue 0.2 NS 0.56 NS 0.28 NS NS NS 0.008 0.3

0.3

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The ESR1 XbaI x/X and PvuII p/P polymorphisms appeared in linkage disequilibrium (D’=0.84). No statistically significant difference was found in the allele and genotype frequencies among SLE patients and controls (Table 1). Stratification by gender also showed no statistically significant differences. The predominant genotype in both groups was XxPp, followed by xxpp and XXPP without any statistically significant difference in their distribution (Table 2). The XX+Xx genotypes (p=0.035, OR 5.1, 95%CL 1-26.3) and the P allele (p=0.00003) and PP+Pp genotypes (p=0.008) were found to be strongly associated with the hematological disease in SLE patients. The XbaI X allele was found to be associated with malar rash in SLE patients (p=0.04, OR 2.4, 95%CI 1-5.7). Although statistically insignificant the XX+Xx genotypes were also found predominant among the patients with malar rash (p=0.07, OR 3.2, 95%CI 0.9-11.3).

the development of SLE clinical manifestations but they are not probably involved in the development of lupusnephritis. Acknowledgements The authors are grateful to Prof D. Monova for her support in the recruitment of the SLE patients. The genetic analysis was performed at the Molecular Medicine Center, Medical University-Sofia, supported by an infrastructure grant DUNK01-2/2009 by the National Science Fund, Ministry of Education, Youth and Science. Conflict of interest statement. None declared.

References 1.

Discussion The biological actions of estrogens are mediated through the estrogen receptors, which function as a ligand inducible transcription factor and convey signals by binding as a complex of accessory proteins to specific DNA sequences in the promoters of estrogen dependent genes. Studies in mice provide evidence that the development of signs of spontaneous renal disease involves ER-α but not ER-β and that ER-α gene polymorphisms may be associated with susceptibility to lupus nephritis [5]. However, the mechanism by which the PvuII p/P or XbaI x/X polymorphisms could affect the pathogenesis of SLE and lupus nephritis remains unclear. An association between the PvuII p/P and XbaI x/X polymorphisms with SLE and lupus nephritis has been reported in several studies involving the Asian population [1014]. We did not find any statistically significant differrence in the allele and genotype distribution among SLE patients and controls in earlier results comprising European population [15-18]. The incompatibility in the results, especially those concerning the allele and genotype distribution among patients and controls might be due to racial, ethnical or gender variation as well as illness classification. It has been proved that ethnic variation plays a major role in genetic regulation of estrogen and estrogen receptor activity [18]. ER-α is expressed in vascular endothelial cells, vascular smooth muscle cells, and cardiomyocytes [19]. It has been shown that elevated estrogen concentration interferes with stem cell differentiation and results in pancytopenia and aplastic anemia [20]. We found that the XXPP genotype is associated with hematological disease in SLE patients.

2. 3.

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9. 10.

11.

Conclusion In conclusion, an association of X allele and the XX and Xx genotypes with the development of malar rash was observed. These results are in line with previously reported findings [14,15,17]. Furthermore, the results of this pilot study indicate that XbaI and PvuII polymorphisms of the ESR1 gene represent possible disease modifying factors for

12.

13.

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BANTAO Journal 2012; 10 (2): 63-68

BJ BANTAO Journal Original article

Immunohistochemical and Morphometrical Study of the Tubulointerstitial Changes in Primary Glomerulopathies Slavica Kostadinova-Kunovska1, Rubens Jovanovic1, Ladislava Grcevska2, Jelka MasinSpasovska2, Goce Spasovski2, Momir Polenakovic3 and Gordana Petrushevska1 1

Institute of Pathology, Faculty of Medicine, University "Ss Cyril and Methodius", 2Department of Nephrology, Clinical Center, "Mother Theresa", 3Macedonian Academy of Sciences and Arts, Skopje, Republic of Macedonia

Abstract

Introduction

Introduction. The complex pathogenesis of the tubulointerstitial changes in primary glomerulopathies involves phenotypic and functional changes of the tubular epithelial cells (TECs), interstitial inflammatory infiltrate and activation of the fibroblasts, thus leading to interstitial fibrosis. The damaged TECs, besides producing various mediators, are also thought to be directly involved in the process of interstitial fibrosis through the mechanism of epithelial-mesenchymal transition. We analysed the immunohistochemical and morphometrical changes in the tubular compartment and the interstitial fibrosis in renal biopsies with glomerulonephritic lesions, as well as their correlations with the clinical parameters of the renal function. Methods. The study population consisted of 50 renal biopsies with primary glomerulopathies. Single and double immunostainings with Cytokeratin 7, Vimentin, αSMA, HLADRα, E-cadherin and Collagen IV, as well as morphometric analysis on tissue sections stained with trichrome Masson, were done. Results. Interstitial fibrosis and tubular atrophy were present in 98% of the cases. The atrophic TECs showed phenotypical changes, loss of expression of epithelial and de novo expression of mesenchymal markers, as well as enhanced expression of antigen presenting properties. The tubular basement membrane was thickened. These phenotypic changes correlated with the degree of the interstitial fibrosis and values of serum creatinine. Conclusions. The results of our study confirm the existence of phenotype variations of TECs, and suggest their association with the clinical parameters for the development and progression of the fibrotic process. These findings emphasize the value of histomorphometrical and immunohistochemical analyses of the tubular and interstitial changes in the renal biopsy.

Changes in the renal tubulointerstitial compartment are present in almost all glomerular diseases, with or without glomerular inflammatory infiltrate [1-3]. The degree of glomerular impairment corresponds to the intensity of the histological changes in the tubulointerstitial compartment [4,5]. Studies have also documented the association between the extent of the interstitial fibrosis and tubular damage on one, and various clinical parameters on the other hand [4,6-11]. The tubulointerstitial compartment makes over 80% of the total renal volume [12] and comprises of complex pattern of tubules and interstitium made of cellular elements (fibroblasts, macrophages and dendritic cells) and extracellular matrix (ECM) [13]. Besides the substances incorporated in the ECM (collagen I, III, VI, proteoglycans, fibronectin, laminin and interstitial liquid), the interstitium also includes the basal membranes composed of collagen IV. According to stereological analyses the interstitial volume varies between 7-9% in the cortical compartment and 30-40% in the inner medulla [14]. The complex pathogenesis of the tubulointerstitial changes in primary glomerulopathies involves glomerular damage, phenotypic and functional changes of the tubular epithelial cells (TECs), interstitial inflammatory infiltrate and activation of the fibroblasts, ultimately leading to interstitial fibrosis. All these events in the tubulointerstitial compartment are initiated and modulated by various proinflammatory and pro-fibrogenic mediators (cytokines, chemokines, growth factors, adhesive molecules, etc.), produced by glomerular resident and non-resident cells, TECs, and inflammatory cells. The damaged TECs, besides producing various mediators, are also thought to be directly involved in the process of interstitial fibrosis through the mechanism of epithelialmesenchymal transition (EMT). This phenomenon has been described as a process during which the mature epithelial cells lose their phenotype, acquiring a new, mesenchymal one [15]. Through this very complex and multi-

Keywords: immunohistochemistry, interstitial fibrosis, morphometry, tubular changes

___________________________________________

________________________ Correspondence to: Slavica Kostadinova-Kunovska, Institute of Pathology, Faculty of Medicine, "50 Divizija" 6, 1000 Skopje, R. Macedonia; Phone: +38923237949; E-mail: [email protected]

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step process, the atrophic TECs lose the epithelial and manifest de novo mesenchymal properties. The fibroblasts, both resident and the newly formed ones through the process of EMT, deposit excessive amounts of ECM [16], resulting in destruction of the renal interstitium, obliteration and atrophy of the tubules and peritubular capillaries and formation of atubular glomeruli (missing the distal nephron segment). The final result is a consequent reduction of the glomerular filtration rate (GFR). The aim of this study was to analyze: i) changes in the tubular segments, both in epithelial cells and in basement membrane, and ii) the extent of interstitial fibrosis in renal biopsies affected by glomerulonephritic lesions, as well to correlate them with various clinical parameters related to the evaluation of renal function. Study Population We analyzed archival renal biopsies from fifty patients, 35 male and 15 female, diagnosed as primary glomerulopathies with standard histological and immunofluorescent analyses. Their mean age at the time of biopsy was 41.8±14.4 (range 15-80). The control group consisted of 20 samples from nephrectomised kidneys due to renal carcinoma, from 13 male and 7 female patients, at mean age of 55.2±14.41 (range 27-76), with regular renal histomorphology. Upon receipt, the biopsy samples were divided into two parts: a part for standard paraffin-embedding and a part for embedding in an epoxy resin. Clinical data The clinical data were derived from the clinical histories of patients. Urine and blood samples collected at the time of biopsy were analysed for the concentration of the serum creatinine (µmol/L) and 24-hour total protein excretion (g/dU).

Tubulointerstitial Changes in Primary Glomerulopathies

OV-TL 12/30), Vimentin (clone V9), αSMA (clone 1A4), HLA-DRα (clone TAL.1B5), E-cadherin (clone NCH-38) and Collagen IV (clone CIV22), followed by counterstaining with hematoxylin (Merck, Darmstadt). The slides were evaluated with a NIKON Eclipse 1000 light microscope. The intensity of staining with HLA-DRα was assessed in a semi-quantitative manner and the results were expressed as absent (-), weak (+), moderate (++) and intensive (+++) signal. Tubular staining with Vimentin and αSMA (smooth muscle actin) was also assessed in semi-quantitiative fashion as follows: 0-no signal; 1-less than 1% tubules stained; 2-1-5% tubules stained; 3-5-10 % tubules stained. Double immunostaining Double immunostaining method was applied in order to examine the co-localization of Cytokeratin 7 and Vimentin or αSMA. We used the DAKO EnVision Doublestaining kit. The deparaffined tissue sections were firstly incubated with Cytokeratin 7 as first primary antibody, followed by secondary antibody conjugated with horseradish peroxidase and the DAB chromogen. The next series of steps included incubation with the second primary antibody (Vimentin or αSMA), followed by secondary antibody conjugated with alkaline phosphatase and the Fast Red chromogen. The tissue samples were then counterstained with hematoxylin (Merck, Darmstadt) and covered with buffered glycerol. Morphometric analysis The tissue samples from each case stained with trichrome Masson were used for software color extraction of the interstitial fibrous tissue with the Image Analysing System Lucia M-Nikon. Sequentially taken images on 10 consecutive high power fields (HPF), avoiding glomeruli and large vessels, were analysed with the integrated software, expressing the interstitial fibrosis as a percentage of the total analysed area.

Tissue preparation for light microscopy Statistical analysis The renal samples underwent the standard procedure of formalin fixation and the paraffin embedded tissue sections were stained with hematoxylin and eosin, periodic acid-Schiff, trichrome Masson and Silvermethenamine Jones. Sections for immunostainings were also obtained. Tissue preparation for electron microscopy A portion of each tissue sample was fixed in 2.5% phosphate-buffered glutaraldehyde, postfixed in OsO4 and embedded in epoxy resin. Semi-thin sections (1µ) were stained with Toluidine blue and periodic acid-Schiff silvermethenamine (PASM) and analysed on a light microscope. Immunoperoxidase staining The EnVision method (DAKO, Denmark) was applied on paraffin sections using the following monoclonal antibodies (all from DAKO, Denmark): Cytokeratin 7 (clone

The results of statistical analyses were expressed as mean values ± standard deviation (SD) and range. The MannWhitney test, Kruskal-Wallis test, chi square, Spearman`s coefficient of correlation (R) and modified Student`s ttest were used, as needed. Values of p-equal or lower than 0.01 and 0.05 were considered as statistically significant. Results Interstitial fibrosis and tubular atrophy Interstitial fibrosis and tubular atrophy with variable extent were present in almost all but one of the analyzed cases, unlike the cases from the control group. The extent of the interstitial fibrosis in the analyzed group of biopsies varied between 8.6 and 32%, with significantly higher mean value of 18.75%±5.04% as compared to the control group (5.32%±1.03; p