| Abstract|| |
Tubulo-interstitial kidney disease is characterized by moderate proteinuria < 1 g/day of low molecular weight proteins in range of MW 10.000-50.000. Even in the physiological proteinuria of < 150 mg/day, tubulo-interstitial kidney disease may exist. Using optimized sodium dodecyl sulfate polyacrylamid gel electrophoresis (SDS-PAGE) according to the method of Melzer, even in proteinuria of less than 150 mg/day all relevant proteins for diagnosis of glomerular or tubulo-interstitial kidney disease can be detected. This study evaluates the tubulo-interstitial kidney disease due to polychemotherapy for different types of cancer in 115 children and in 16 children with pyelo-ureteral junction obstruction. Fifty-two and 63 children were followed up during and after chemotherapy, respectively. During therapy, renal damage was recorded in 43% of patients with leukemia, 56% with nephroblastoma, and 79% with other tumors. Tubular protein patterns were seen up to three years after termination of chemotherapy (25% in acute lymphoplastic leukemia, 35% in nephroblastoma and 62% in other tumors). Patients with persistent complete tubular proteinuria or mixed glomerular/tubular proteinuria were found to have a high risk for irreversible renal failure. Children with congenital pyelo-ureteral junction obstruction could also be classified according to SDS-PAGE protein patterns. Patients without parenchymal lesions did not need surgery. Most of those with pathologic findings in SDS-PAGE exhibited partial or complete remission after surgery. The highly discriminating SDS-PAGE permits a rapid, sensitive, reproducible, and reliable analysis of urine proteins for diagnosis and follow-up of all kinds of congenital or acquired renal parenchymal kidney diseases.
Keywords: Children, Polychemotherapy, Pyelo-ureteral junction obstruction, SDS-PAGE nephrotoxicity, Proteinuria, Densitogram.
|How to cite this article:|
Al-Bashir A, Rohrmann D, Mertens R, Melzer H, Mann H. Early Detection of Tubulo-Interstitial Kidney Disease in Children Using Highly Discriminating SDS-Gel Electrophoresis. Saudi J Kidney Dis Transpl 2001;12:503-10
|How to cite this URL:|
Al-Bashir A, Rohrmann D, Mertens R, Melzer H, Mann H. Early Detection of Tubulo-Interstitial Kidney Disease in Children Using Highly Discriminating SDS-Gel Electrophoresis. Saudi J Kidney Dis Transpl [serial online] 2001 [cited 2019 Dec 10];12:503-10. Available from: http://www.sjkdt.org/text.asp?2001/12/4/503/33548
| Introduction|| |
Nephrotoxicity is a common side effect of chemotherapy for solid tumors and systemic disease in childhood and adolescence. SDS polyacrylamide gel electrophoresis is a suitable method to analyze pathologic urine proteins. SDS with densitometric quantification permits a rapid and reliable analysis of urinary proteins and is suitable for followup tests of renal function during and after therapy. There are many parameters to evaluate the renal damage such as serum creatinine, urea, uric acid and creatinine clearance. But in many instances there is no pathologic change until damage is already advanced. With SDS-PAGE as a noninvasive diagnostic method, renal damage can be detected at an early stage. Analysis of the protein excreted in the urine discriminates the prerenal, glomerular, tubulo-interstitial and postrenal lesions. ,, Glomerular disease is characterized by the presence of high molecular weight, while proximal tubular dysfunction is characterized by low molecular weight proteins.  This study evaluates the applicability of the method of SDS-PAGE to improve the detection and differentiation of protein bands, which may permit identifying patients at the risk of future deterioration of renal function.
| Patients and Methods|| |
One hundred and fifteen children with lymphoblastic leukemia and solid tumors during and after chemotherapy, 16 children with pyelo-ureteral junction obstruction before and after operation were included in this study. The patients' details are shown in [Table - 1].
Urine samples were collected from all patients before, during and after therapy. In very young children urine was collected in urine collecting bags.
None of the patients underwent abdominal irradiation. No patient received aminoglycoside antibiotics. Initial SDS-PAGE test, prior to initiation of therapy, was used to exclude any existing lesions.
The chemotherapeutic agents (cisplatin, carboplatin, cyclphosphamide, vincristine, daunorubicin, cytarabine, methotrexat, doxorubicin, and ifosfamide) were used in different protocols.
Electrophoretic separation of urine proteins was performed by means of ready-to-use gels in the horizontal PhastSystem (Pharmacia, Freiburg, Germany). The continuous polyacrylamid gradient in separating gel was 825%, and the degree of cross netting was 2%. The samples used for protein analysis were taken from single urine specimens supplemented with sodium acid (final concentration 0.1 g/l). A 10 ml aliquot was centrifuged for 10 min at 3000 rpm. Trichloroacetic (TAC) precipitation was performed on the supernatant, and protein concentrations were determined in the precipitate by the Biuret method. Depending on the protein concentration, electrophoretic protein analysis was performed either in the native urine (protein concentration < 0.6 g/l) or in urine samples diluted to a protein concentration of 0.6 g/l.
Thereafter, 0.1 ml of a 5% SDS buffer solution was added to 0.4 ml urine and incubated for 60 min at 37°C. The final protein concentration of the sample was 0.48 g/l. Then, 1.0 gl of the SDS urine sample was applied to the Phast gel. Electrophoretic protein separation was performed in a cooled separating chamber of the Phast System with a current intensity of 10 mA. 
The protein fraction then underwent silver staining in the phast gel according to a modified procedure by Heuckeshoven and Dernik. ,, Modified silver staining permits high-resolution separation of the urine proteins even at low total protein concentration of < 50 mg/l. Due to the absence of background staining, these stained bands can be transferred into visually recognizable protein patterns by means of laser densitometry (Ultrascan XL Pharmacia, Freiburg, Germany). Proteins are evaluated in a molecular weight range from 10 to 200,000 Dalton. At the same time these patterns can be electronically stored and reproduced at any time, e.g., during long-term observation. The method has been standardized using known concentrations of proteins such as albumin, α-1-microglobulin (α-1-M), β-2 microglobulin (β-2-M), phosphorylase b, carbonic anhydrase, and IgG. 5 Single proteins are detectable at concentrations of 5-10 g/µl corresponding to the sensitivity of immunological methods.
[Figure - 1] shows typical SDS-gel patterns of the urine. Normal urine has an albumin band, which is detectable even at a concentration of 8 ng/ml. With tubular proteins, the occurrence of further protein bands in 30,000-50,000 Dalton range is referred to as partial tubular proteinuria, with α-1-M particularly being detectable. With complete tubular proteinuria numerous protein bands, especially β-2-M, are found in the 8.000-50.000 Dalton range. Such proteinuria corresponds to tubulointerstitial nephritis or interstitial fibrosis in most cases and in general is nonreversible. Glomerular patterns were also observed. In cases of selective glomerular pattern proteinuria, protein excretion is virtually confined to albumin and transferrin. Selective glomerular proteinuria in cases of minimal change glomerulonephritis is fully reversible. In nonselective proteinuria, which occurs in most forms of glomerulonephritis or glomerulosclerosis, the additional detection of various immunglobulin fractions indicates structural damage to the glomerular basal membrane. No typical patterns of specific renal biopsy findings can currently be derived from the nonselective glomerular patterns. If glomerulonephritis is complicated by interstitial fibrosis, a mixed glomerulartubular pattern is detected.
The different types of proteinuria were defined as follows:
1) Glomerular proteinuria: proteins of molecular mass 60.000 Dalton or greater: albumin (Alb) transferrin (Tr), monomeric and dimeric immunoglobulin (Di IgG).
2) Tubular proteinuria: proteins of less than 50.000 Dalton: α-1-microglobulin, retinol binding protein (RBP), β-2-microglobulin, apolipoprotein A1.
3) Mixed proteinuria: a combination of both glomerular and tubular patterns.
| Results|| |
Patients undergoing polychemotherapy
All patients had normal findings before therapy. The severity of kidney injury is different in different kinds of tumors.
Twenty one percent of patients with acute lymphoblastic leukemia (ALL), had mixed tubulo-glomerular, 11% tubular, 11% glomerular and 57% normal patterns six weeks after initiation (during) therapy. Six to eight weeks after completing therapy, 10% of the ALL patients showed mixed patterns of proteinuria, 5% showed glomerular, 10% partial tubular and 75% normal patterns [Figure - 2].
During therapy, 22% of patients with Wilms tumor showed mixed glomerular and tubular, 12% tubular, 22% selective glomerular patterns and 44% normal patterns. After completing therapy, 14% of these patients showed mixed glomerular and tubular, 14% glomerular, 7% partial tubular and 65% normal patterns [Figure - 3] .
During therapy, 50% of patients with solid tumor showed mixed glomerular and tubular, 16.5% tubular, 12.5% glomerular and 21% normal patterns. After completing therapy, 34% of these patients showed mixed glomerular and tubular, 7% glomerular, 21% partial tubular and 38% normal pattern [Figure - 4].
Comparing the different types of tumors and chemotherapy, it appears that with more aggressive chemotherapy there are more pronounced and persistent renal parenchymal lesions.
In [Figure - 5], three typical cases of kidney impairment are shown: The left side, shows in the findings in a child with ALL. After the first cycle of chemotherapy, a tubular protein pattern (yellow part of the curve) appeared, which was still present one month later. After about six months, this tubular lesion of the kidney completely reversed. The total protein concentration in these protein patterns was always below 50 mg/l.
The middle of [Figure - 5], reveals the findings in a girl with stage IV neuroblastoma, where severe tubular (yellow part of the curve) and glomerular (red part of the curve) injury of the kidney could be detected. Even after nine months, the combined tubular and glomerular protein patterns were detectable. The right side of [Figure - 5], reflects urine protein patterns in a 15 year old patient with rhabdomyosarcoma stage IV with similar combined tubular (yellow part of the curve) and glomerular (red part of the curve) lesion of the kidney, both of which persisted for six months. In this patient there was also a decrease of creatinine clearance to 50 ml/min.
Patients with pyelo-ureteral junction obstruction
There were two groups of patients. Eight patients with signs of obstruction by sonographic imaging but without signs of any tubular or glomerular lesion in SDSPAGE. These patients did not undergo surgery. After five months of observation the signs of pyelo-ureteral obstruction disappeared and SDS-PAGE in all of them continued to be normal. In the other group, there were eight patients who underwent surgery; two of them had normal SDSPAGE before and after treatment, three patients with severe tubular or glomerular lesions normalized their proteinuria patterns after surgery, and three other patients showed amelioration of tubular lesions after surgical correction.
[Figure - 6], shows one child with obstruction of the pyelo-ureteral junction before and after the first and second surgery. Before the first surgery, the patient showed severe tubular and glomerular damage. After the first operation, the signs of tubular injury on the SDS-PAGE disappeared, but the signs of glomerular damage persisted. After the second operation was performed because of recurrent obstruction, the signs of glomerular lesion of the kidney disappeared.
| Discussion|| |
Highly discriminating SDS-PAGE with silver staining, and modification of Melzer including Laser densitometry is a method, which is able to detect tubular and glomerular abnormalities in proteinuria < 200 mg/l that is generally considered to be a normal value.  This method has been applied to detect damage of the kidney in children treated by chemotherapy or had urinary obstructive disease.
Cytotoxic agents can cause renal damage. The synergistic toxicity of cisplatin or carboplatin and ifosfamide is well known. ,,,,, Other risk factors in the development of renal toxicity are the patients age, pervious damage of the renal tissue, additional antibiotic therapy and radiotherapy. Young patients and unilaterally nephrectomized patients run a high risk of developing non reversible nephrotoxicity. ,, Renal damage does not necessarily occur during or immediately after cytotoxic therapy, but may manifest itself several years after therapy and lead to chronic renal insufficiency. This calls for long-term follow-up of such patients.
Chemotherapy can lead to tubular or glomerular damage. In our study, pathologic protein excretion in urine in most cases was apparent after the first cycle of chemotherapy. In many cases it persisted after 6-8 weeks of the first cycle. In some patients, the drug induced renal parenchymal damage was irreversible even after two years of termination of chemotherapy. Signs of renal toxicity were more pronounced following the more aggressive therapeutic protocols that included ifosfamide, cisplatin and daunorubicin, as used in treating tumors such as ALL and Wilms-Tumor. ,,, In such patients, not only lesions of the tubules could be detected but also of the glomeruli.
In any case, the renal parenchymal lesions could be detected much earlier by defining the protein patterns than by the measurement of creatinine clearance or serum creatinine concentration. Most of the patients exhibited a complete tubular protein pattern corresponding to an acquired Fanconi syndrome. The protein amount and pattern could be used to decide whether chemotherapy should be modified or discontinued. In our study the patients with acute lymphatic leukemia had slight but reversible nephropathy. The situation is different in patients with Wilms tumor and various solid tumors, since they had severe nephrotoxicity in a large percentage.
Pyelo-ureteral junction obstruction in which surgical treatment is still controversial is usually diagnosed antenatally or in the early postnatal period. ,, In our study, most of the patients who exhibited no pathologic protein pattern in the urine, surgery was not necessary and all signs of obstruction disappeared during the period of observation. On the other hand, in those patients who showed pathologic findings on the SDSPAGE there was complete or partial recovery after surgical treatment. This shows that SDS-PAGE can be used as a diagnostic tool to discriminate between those patients who require surgical treatment and those who do can be avoided when the kidneys show no signs of tissue injury.  Obviously, SDS PAGE seems to be more sensitive in the diagnosis of renal injury than creatinine clearance and renal imaging.
In conclusion, SDS-PAGE with the consecutive laser-densitometric documentation may be a sensitive, non-invasive and reproducible method for early detection and follow-up of renal tubular and glomerular injury even with minimal proteinuria, especially in children on cancer chemotherapy or having pyelo-ureteral junction obstruction.
| References|| |
|1.||Rossi R, Godde A, Kleinebrand A, et al. Unilateral nephrectomy and cisplatin as risk factor of ifosfamide-induced nephrotoxicity: analysis of 120 patients. J Clin Oncol 1994;12:159-65. |
|2.||Brocklebank T, Cooper EH, Richmond K. Sodium dodecyl sulphate polyacrylamide gel electrophoresis patterns of proteinuria in various renal diseases of childhood. Pediatr Nephrol 1991;5:371-5. [PUBMED] |
|3.||Boesken OH. SDS-PAGE: Gel Electrophoresis of Urinary proteins. New York: de Gruyter 1981. |
|4.||Weber WH. Urinary proteins analysis. J Chromatogr 1988;429:315-44. |
|5.||Kierdorf H, Melzer H, Mann H, Sieberth HG, Differentiation of proteins in polyacrylamide gels by a modification of silver staining for the phast system and a laser densitometer. Electrophoresis 1993;14:820-2. |
|6.||Ashraf MS, Brady J, Breatnach F, Deasy PF, O'Meara A. Ifosfamide nephrotoxicity in paediatric cancer patients. Eur J Pediatr 1994;153:90-4. [PUBMED] |
|7.||Boddy AV, English M, Pearson AD, Idle JR, Skinner R. Ifosfamide nephrotoxicity: limited influence of metabolism and mode paediatrics. Eur J Cancer 1996;7:1179-84. |
|8.||Berns JS, Haghighat A, Staddon A, et al. Severe, irreversible renal failure after ifosfamide treatment: a clinicopathologic report of two patients. Cancer 1995;76:497-500. [PUBMED] |
|9.||Loebstein R, Koren G. Ifosfamide-induced nephrotoxicity in children: critical review of predictive risk factors. Pediatrics 1998; 101:E8. |
|10.||Skinner R, Sharkey IM, Pearson AD, Graft AW. Ifosfamide, mesna, and nephrotoxicity in children. J Clin Oncol 1993;11:173-90. |
|11.||Skinner R, Pearson AD, English MW, et al. Cisplatin dose rate as a risk factor for nephrotoxicity in children. Br J Cancer 1998;77:1677-82. [PUBMED] |
|12.||Shore R, Greenberg M, Geary D, Koren G. Iphosphamide-induced nephrotoxicity in children. Pediatr Nephrol 1992;6:162-5. [PUBMED] |
|13.||Skinner R, Pearson AD, Price L, Coulthard MG, Craft AW. The influence of age on nephrotoxicity following chemotherapy in children. Br J Cancer Suppl 1992;18:S30-35. [PUBMED] |
|14.||Rossi R, Danzebrink S, Hillebrand D, Linnenburger K, Ullrich K, Jurgens H. Ifosfamide-induced sub-clinical nephrotoxicity and its potentiation by cisplatinum. Med Pediatr Oncol 1994;22:27-32. |
|15.||Friedlaender MM, Haviv YS, Rosenmann E, Peylan-Ramu N. End-stage renal interstitial fibrosis in an adult ten years after ifosfamide therapy. Am J Nephrol 1998;18: 131-3. [PUBMED] [FULLTEXT]|
|16.||Ashraf MS, Skinner R, English MW, Craft AW, Pearson AD. Late reversibility of chronic ifosfamide-associated nephrotoxicity in a child. Med Pediatr Oncol 1997;28:62-4. [PUBMED] [FULLTEXT]|
|17.||Brock PR, Koliouskas DE, Barratt TM, Yeomans E, Pritchard J. Partial reversibility of cisplatin nephrotoxicity in children. J Pediatr 1991;118:531-4. [PUBMED] |
|18.||Takla NV, Hamilton BD, Cartwright PC, Snow BW. Apparent unilateral ureteropelvic junction obstruction in the newborn: expectations for resolution. J Urol 1998; 160:2175-8. [PUBMED] |
|19.||Ulman I, Jayanthi VR, Koff SA. The longterm follow-up of new-borns with severe unilateral hydronephrosis initially treated non-operatively. J Urol 2000;164:1101-5. [PUBMED] |
|20.||Koff SA. The prenatal diagnosis of hydronephrosis: when and why not to operate? Arsh Esp Urol 1998;51:569-74. |
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[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6]
[Table - 1]