1 Division of Nephrology, Chang Gung Memorial Hospital, Keelung and 2 Graduate Institute of Veterinary Medicine, National Taiwan University, Taipei, Taiwan
Correspondence and offprint requests to: Mai-Szu Wu, Division of Nephrology, Chang Gung Memorial Hospital, 222, Mai-Chin Road, Keelung, Taiwan. Email: maxwu1{at}adm.cgmh.org.tw
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Abstract |
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Methods. To address these questions, we first performed detailed in vivo clearance tests in three patients with leptospirosis (Leptospira shermani) and hypokalaemia to define the tubular lesion. These tests indicated a defective Na+K+Cl co-transporter and a poor response to furosemide infusion. We performed further in vitro studies in a model of medullary thick ascending limb (mTAL) cultured cells derived from normal mouse.
Results. Outer membrane protein extract from L.shermani (0.3 µg/ml) inhibited Na+K+Cl co-transporter activity in mTAL cells (control, 10.15±0.52; L.shermani, 6.47±0.47 nmol/min/mg protein). The basolateral Na+K+ ATPase remained intact. Reverse transcriptionpolymerase chain reaction (RTPCR) further revealed that the outer membrane protein extract from L.shermani downregulated Na+K+Cl co-transporter (mNKCC2) mRNA expression. These changes were dose dependent and could be reversed by the antibody against outer membrane protein extract from L.shermani. Experiments with a less pathogenic strain of leptospira (L.bratislava) and Escherichia coli did not affect Na+K+Cl co-transporter activity.
Conclusions. We conclude that L.shermani leptospirosis downregulates mNKCC2 mRNA expression and inhibits Na+K+Cl co-transporter activity in TAL cells. These alterations may explain the observed electrolyte disorders in leptospirosis.
Keywords: kidney; leptospirosis; Na+K+ ATPase; Na+K+Cl co-transporter; thick ascending limb
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Introduction |
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Leptospirosis is an important but usually ignored cause of acute renal failure in Taiwan [2]. Renal involvement in L.shermani infection appears to be a special form of acute renal failure characterized by a higher frequency of polyuria and the presence of hypokalaemia with an elevated urinary fractional excretion of potassium [4]. Previous indirect evidence examining the relative integrity of the distal nephron in guinea pigs suggested that L.interrogans causes a dysfunctional proximal tubular lesion [5]. It is not clear whether L.shermani infection stimulates the same pathophysiological mechanisms in human. We evaluated three patients with L.shermani infection, one patient with L.bratislava infection and one patient with Escherichia coli infection. They all presented with non-oliguric acute renal failure. During the recovery phase, hypokalaemia and metabolic alkalosis developed in leptospirosis patients. A renal potassium wasting resulted in hypokalaemia. The characteristic clinical manifestations suggested a specific tubulointerstitial lesion caused by L.shermani. We hypothesized that L.shermani exerted its effects on a specific renal segment, which created the unique clinical manifestation. To address this possibility, we first defined the involved renal segments by using classic renal clearance tests. We also performed in vitro experiments to investigate the underlying cellular mechanisms involved in renal epithelial transport. In the second part of the study, we investigated the effect of the outer membrane protein extract from L.shermani on renal epithelial transport mechanisms using a model of renal epithelial cells derived from the normal mouse [6]. A better understanding of these mechanisms will enhance our knowledge about infection-related renal tubulointerstitial disease.
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Materials and methods |
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Case 3. A 46-year-old male farmer came to our ward reporting 2 weeks of fever, jaundice and anorexia. Hyperbilirubinaemia and azotaemia (Cr 4.4 mg/dl; 389 µmol/l) were found upon admission. Leptospira shermani infection was confirmed by MAT. Hypokalaemia (serum, 2.6 mEq/l; urine, 38 mEq/l) due to renal potassium wasting developed after recovery of renal function (Cr 1.6 mg/dl; 141 µmol/l). Several clearance tests were performed to define the location of the tubular lesion (Table 1).
Case 4. A 77-year-old male patient came to our ward after 1 week of fever, abdominal pain and disturbances of consciousness. The patient developed renal failure (Cr 8.3 mg/dl; 734 µmol/l) and hepatic failure. MAT showed increased titres of 1:400 to L.bratislava. Leptospira DNA was detected by polymerase chain reaction (PCR) in urine and blood samples. Renal function and hyperbilirubinaemia (Cr 1.4 mg/dl; 124 µmol/l) recovered almost completely after 1 month of penicillin therapy. Hypokalaemia (serum 2.6 mEq/l; urine, 31 mEq/l) developed during the recovery phase. Several clearance tests were performed to define the tubular lesions (Table 1).
Case 5. A 46-year-old female visited our emergency department complaining of intermittent fever and dyspnoea. Abnormal renal function (Cr 5.5 mg/dl; 486 µmol/l) was noted upon arrival. Blood culture revealed E.coli infection. Leptospira was not found by serology or by PCR. Acute renal failure due to E.coli sepsis was diagnosed. Renal function (Cr 1.3 mg/dl; 115 µmol/l) improved after antibiotic treatment, and renal clearance tests were performed to evaluate tubular function during recovery from acute tubular necrosis.
Diagnosis of leptospirosis
In patients with suspected leptospirosis, diagnosis was made by 4-fold changes of MAT titre in paired sera between the acute and convalescence phase, a single titre in MAT >1:400 or the demonstration of leptospira DNA in urine or in blood samples by PCR.
Clearance tests
Several in vivo clearance tests were used to define the tubular lesion as previously described [7]. Briefly, a bicarbonate test was used to test proximal tubular bicarbonate reabsorption and collecting tubular proton pump function. A furosemide test was performed to assess furosemide-sensitive sodium absorption along the thick ascending limb (TAL). A thiazide test was performed to evaluate distal tubular sodium reabsorption.
Cultured cells
The experiments were carried out on subcultured cells derived from isolated medullary TALs (mTALs) and cortical TALs (cTALs) microdissected out from the kidney of 1-month-old C57BL/6 mice as previously described [6]. Subcultured mTAL cells were routinely grown in a modified culture medium [Dulbecco's modified Eagle's medium (DMEM): Ham's F12, 1:1 (v/v); 60 nM sodium selenate; 5 µg/ml transferin; 2 mM glutamine; 5 µg/ml insulin; 50 nM dexamethasone; 1 nM triiodothyronine; 10 ng/ml epidermal growth factor; 2% fetal calf serum; 20 mM HEPES, pH 7.4] at 37°C in a 5% CO295% air atmosphere. All experiments were performed between the 6th and 15th passages on sets of confluent cells grown on Petri dishes.
RNA extraction and reverse transcriptionPCR (RTPCR)
RNA was extracted from isolated mTAL and cTAL segments microdissected from an adult mouse kidney and from confluent mTAL and cTAL cultured cells by the method of Chomczynski and Sacchi as previously described [8]. Total RNA concentration was treated with RNase-free DNase I (Boehringer Mannheim, Germany) at 37°C for 30 min and RNA concentration was evaluated by spectrophotometry. RNA (100 µg) was reverse transcribed with avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim) at 42°C for 60 min. A 150 ng aliquot of cDNA and non-reverse-transcribed RNA were amplified for 32 cycles in 100 µl total volume containing 50 mM KCl, 20 mM TrisHCl pH 8.4, 10 mM dNTP, 1.5 mM MgCl2, 1 U of Taq polymerase and 10 pmol of inducible nitric oxide sythetase (iNOS) primers. The thermal cycling program was as follows: 94°C for 1 min, 60°C for 1 min and 72°C for 3 min. The two primers from the NKCC2 gene [8] were as follows: antisense strand 5'-CTT GGC TTC GGT TTT AGA TGA CCC G-3', sense strand 5'-GCA ATG CTG GCA TTT AGA CCC TCC G-3'. Amplification products (406 bp) were run on a 4% agarose gel with ethidium bromide and photographed. As a control, the identity of the amplified products of ß-actin (460 bp) was processed at the same time.
Quantitation of PCR products: competitive PCR assay
Competitive RTPCR was performed for the measurement of NKCC2 and ß-actin mRNA. The test template for all PCRs was an aliquot of cDNA collected from mTAL cells. To quantitate the tested cDNAs, various amounts of mutant cDNA templates were added to compete with test cDNA on an equimolar basis, as previously described. For NKCC2 and ß-actin, deletion cDNA mutant templates were developed to create 105 and 103 bp deletions in the middle of the molecules, resulting in mutant cDNAs of 301 and 357 bp, respectively. Following agarose gel electrophoresis, amplification bands stained by ethidium bromide were quantitated from the film negative by scanning densitometry. The ratio of mutant to wild-type band density was calculated for each lane and plotted as a function of the amount of initial mutant template added to the reaction. The amount of cDNA was derived from linear regression analysis with duplicate or triplicate assays. The mean values for assays were expressed as a percentage change from the control.
Preparation of outer membrane protein extract of leptospira
Two commonly encountered pathogenic leptospira serovars, L.shermani and L.bratislava, and a non-pathogenic L.biflexa serovar patoc were obtained from ATCC (MD) and cultured in JohnsonHarris bovine serum albuminTween-80 medium (Difco, MI). Leptospires were counted using dark-field microscopy as previously described [9]. Protein extract from the outer membrane of leptospires was obtained as previously described [10]. Briefly, leptospira cells were cultured for 57 days at 28°C until a cell density of 108/ml was obtained. A 200 ml aliquot of cultured cells was centrifuged at 15 000 g for 30 min and resuspended in 8 ml of 1 M NaCl. The bacteria were observed under dark-field microscopy until conversion to spherical form, were centrifuged again at 15 000 g for 30 min and were resuspended in 8 ml of distilled water. Treatment of this cell suspension with 0.04% sodium dodecyl sulfate (SDS) solubilized the outer membrane. The cells were removed by centrifugation, and the supernatant was filtered through a 0.45 µm membrane filter and lyophilized. Protein concentration was measured by the Bradford method (Protein Assay, Bio-Rad Laboratories, CA). Each extraction procedure yielded 0.5 mg of protein per serovar.
Antiserum for L.shermani
Antiserum against L.shermani was prepared in New Zealand white rabbits by immunizing outer membrane protein as previously described [11]. A 400 µg aliquot of detergent-soluble protein was mixed with Freund's complete adjuvant, and injected intradermally into various sites along the back of the rabbit. A booster of the same dose was given 2 weeks later. The animals were bled 2 weeks after the last inoculation. The titre of the antiserum was determined by an MAT which revealed a titre of 1:10 000.
Transport studies
The potassium-transporting capacities of the cells were studied by radiolabelled rubidium (86Rb+), used as the tracer for potassium influx and efflux studies. Confluent cells in 24-well trays were washed with 1 ml of modified phosphate-buffered saline (PBS, in mM: NaCl 136; KCl 5; Na2HPO4 1; glucose 5; HEPES 10; pH 7.4) pre-warmed to 37°C. The medium was removed and rubidium influx was initiated by adding 1 ml of PBS containing 2 µCi/ml 86Rb+. The reaction was stopped by removing the solution and performing three rinses with 1 ml of ice-cold 100 mM MgCl2 solution. Cells were solubilized with 0.5 ml of 1 M NaOH and radioactivity was determined by scintillation counting. The K+ influx was measured by incubating cells without (total influx) or with 0.5 mM ouabain or ouabain plus 104 M furosemide in order to determine the ouabain-sensitive influx (Os) mediated by the Na+K+ ATPase pumps, and the ouabain-resistant furosemide-sensitive influx (Or-Fs) mediated by Na+K+Cl co-transport [12].
Cell viability
Cell viability was estimated by a tetrazolium-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) [13] to determine the non-specific cytotoxicity of the outer membrane protein extract of L.shermani and L.bratislava to mTAL cells. Cells were seeded in 96-well plates (Corning Co., Corning, NY). After culturing for 3 days, cells were exposed to various concentrations (0.11 µg/ml) of outer membrane protein extract. Following a 48 h incubation, 40 µl of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added to each well. After 2 h at 37°C, the cells were lysed by adding 100 µl of 20% (w/v) SDS and 50% (v/v) N,N-dimethylformamide (pH 4.7) and incubated overnight at 37°C. The absorbance at 570 nm was measured for each well using a Dynex microplate reader. The reported cell viability was the percentage of viable cells in comparison with the control wells. Duplicate measurements were made for each test on at least two separate occasions.
Statistical analysis
Results are expressed as means±SD from (n) experiments performed in duplicate or triplicate. Significant differences between groups were analysed by Student's t-test and one-way ANOVA. The statistical analysis was performed using the StatviewTM program (Macintosh). A P-value <0.05 was considered significant.
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Results |
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Characteristics of cultured TAL cells
We examined whether L.shermani specifically inhibits Na+K+Cl co-transporter activity in TAL cells and explored the cellular mechanisms of this effect. We performed in vitro studies to explore these possibilities. We microdissected the mTAL from normal mice and put the segments into culture (Figure 1A). Confluent mouse mTAL cells grown on collagen-coated Petri dishes formed a monolayer of cuboid shaped cells and formed small domes (Figure 1A) [6]. Cultured cTAL cells were also cultivated using similar methods. RTPCR was used to detect NKCC2, the kidney-specific Na+K+Cl co-transporter mRNA. Substantial amounts of NKCC2 transcripts were detected in both microdissected mTAL and cultured mTAL cells (Figure 1B). cTAL segment and cultured cTAL cells also expressed substantial amounts of NKCC2 (data not shown). A single 406 bp band of the expected size was amplified in both microdissected mTALs and cultured cells. As a negative control, samples lacking amplified products were obtained by using non-reversed-transcribed RNA from cultured TAL cells or by omitting cDNA (Figure 1B). The different components of 86Rb+ influx measured in the absence or presence of ouabain and furosemide allowed us to distinguish between the ouabain-sensitive (Os) component of 86Rb+ influx mediated by the Na+K+ ATPase pumps and the ouabain-resistant furosemide-sensitive (Or-Fs) component of 86Rb+ influx reflecting Na+K+Cl co-transport activity (Figure 1C). In all cases, the influx of 86Rb+ increased linearly with time for up to 10 min. These results indicate that cultured TAL cells is an appropriate model to test the effect of L.shermani outer membrane extract on Na+K+Cl co-transport.
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Discussion |
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The clinical clearance tests in the present study suggested that there were defective responses to furosemide in L.shermani leptospirosis patients. These results indicated that the tubular lesion is mainly located in TAL. The renal tubular target of L.shermani leptospirosis may be the Na+K+Cl co-transporter, which is uniquely found in TAL cells. Because of this finding, we further evaluated the effect of L.shermani outer membrane extract on the Na+K+Cl co-transporter. We found that the L.shermani outer membrane protein extract reduced Na+K+Cl co-transporter activity and mRNA expression. Neutralizing antibody reversed the effect of outer membrane extract on Na+K+Cl co-transporter. These results suggest that the outer membrane protein extract exerts a specific effect on TAL cells. These delicate transport processes are well regulated by many intra-renal hormones, and keep our internal milieu in homeostasis. The inhibitory effect of the L.shermani outer membrane on Na+K+Cl co-transporter may provide pathogenic clues regarding the frequent appearance of renal potassium wasting [16] and concentration defects [17] in patients with leptospirosis. The inhibition of the Na+K+Cl co-transporter results in more distal sodium delivery and subsequently induces renal potassium loss. A reduction in Na+K+Cl co-transport activity also compromises medullary hypertonicity and may be responsible for the lack of vasopressin responsiveness.
Ionic transport additionally acts as a cellular signal in many cellular functions [18]. Cell volume regulation, apoptosis, cytokine release and many other cellular functions are regulated by the ionic transport process. Thus, alterations in ionic transport may affect many cellular functions and lead to acute or chronic renal dysfunction [19]. Our recent studies examining leptospirosis effects on cytokine and chemokine expression further supported this view by indicating that altered transport activity may be the first event in acute and chronic tubulointerstitial renal disease during leptospirosis [9]. Together, these findings point to a role for infection in renal disease.
Importantly in the present study, the inhibitory effect was limited to the medullary segment but not the cortical region. Recent studies indicated that mouse TAL expresses a total of six NKCC2 isoforms generated by the combinatorial association of three 5' exon cassettes (A, B and F) with two alternative 3' ends [20]. The two 3' ends predict C-terminal cytoplasmic domains of 129 amino acids (the C4 C-terminus) and 457 amino acids (the C9 terminus). The three C9 isoforms (A9/F9/B9) all express Na+K+2Cl co-transport activity, whereas C4 isoforms (A4/F4/B4) are non-functional in Xenopus oocytes. Activation or inhibition of protein kinase A does not affect the activity of the F9 isoforms [20]. This finding suggests that the outer membrane protein extract may have a specific isoform of the NKCC2 as a target. The affected isoform was limited to the medullary segment. Our previous studies also indicated that the affected NKCC2 was cAMP-dependent, potassium-dependent and bumetamide-sensitive [6]. The characteristics of NKCC2 suggested that it may be isoform A, which appeared mainly in the apical membrane of mTAL cells. These results indirectly suggest that specific isoforms were affected by the outer membrane protein extract. Further experiments will be necessary to specify the affected isoforms.
The current study provides a good example of the fact that clinical findings and laboratory investigations can be linked. Potential mechanisms can be verified in the laboratory, and laboratory findings can be applied towards clinical problems. Using this method, further investigations and clinical observations can be developed.
In conclusion, the outer membrane protein extract from L.shermani is able to reduce the expression of mNKCC2 mRNA and Na+K+Cl co-transporter activity. This effect may represent the initial event in tubulointerstitial injury in L.shermani leptospirosis renal injury.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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