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High Collagen I Gene Expression as an Independent Predictor of Adverse Renal Outcomes in Lupus Nephritis Patients With Preserved Renal Function.

Patients with lupus nephritis (LN) have increased risk of developing progressive renal failure requiring renal replacement therapy. Treatment of LN includes corticosteroids and immunosuppressive agents, which can lead to significantly increased risks of morbidity and mortality. (1) Lupus nephritis encompasses many forms of glomerulonephritis with inflammatory and fibrotic components. (2) A renal biopsy is typically used to obtain prognostic information and to serve as a guide to therapy. The World Health Organization (WHO) and, later, the International Society of Nephrology/Renal Pathology Society (ISN/RPS) have proposed morphologic classifications for LN. (2) While these classification systems are valuable, individual patients even within the same disease class may show large variations in long-term outcomes. Semiquantitative activity and chronicity indices have been used to augment prognostic information from the WHO classification system, but the value of these indices remains controversial. (3,4)

In patients with LN, active inflammation may be followed by progressive decline in kidney function due to the development of fibrosis. Interstitial fibrosis has been associated with poor clinical outcome in many renal diseases including LN. (5-8) The deposition of extracellular matrix (ECM) proteins such as collagen I (COL1 gene product) is a key component of fibrosis. A number of cytokines, including transforming growth factor [beta]-1 (TGFB1 gene product), may mediate fibrosis by stimulating COL1 synthesis and through other mechanisms. (5,6)

The presence of tubulointerstitial fibrosis is often predictive of poor renal outcome in the long run, but when such processes are visible by routine histology, the pathogenic processes are largely irreversible. (9) Genes involved in matrix expansion and fibrosis have been shown to correlate with fibrosis in many experimental animals including models of LN. (5,7) Inhibition of these fibrogenic pathways may be a potent therapeutic means for treating fibrosis in various kidney disease models. (10) Currently, there are limited data evaluating the relationship between the levels of fibrogenic gene expression and the response to treatment or long-term outcome in human kidney diseases. The presence of renal fibrogenic genes may predict adverse renal outcome and serve as a guide to therapy in LN. Furthermore, it could pave a way for therapeutic interventions aimed at decreasing the fibrogenic proteins.

In this study, we propose that the degree of fibrosis associated in LN is associated with increased expression of renal fibrogenic genes TGFB1 and COL1 and that increased expression of fibrogenic genes is associated with adverse renal outcomes.

MATERIALS AND METHODS

Patients' Baseline Data and Management

Patients with a formal diagnosis of systemic lupus erythematosus undergoing a kidney biopsy at Ramathibodi Hospital, Bangkok, Thailand, between 2002 and 2004 because of clinical indications were included in this study. In addition to the core of tissue sample obtained for diagnosis, an extra core of tissue was rapidly frozen and stored in liquid nitrogen for future gene expression studies. Control subjects with preserved renal function (serum creatinine < 1.2 mg/dL) were recruited from patients undergoing nephrectomy for renal cell carcinoma. A tissue core was obtained from the renal cortex of the noninvolved pole of the kidney, snap frozen, and stored in liquid nitrogen. This study was approved by the Ethical Committee of Ramathibodi Hospital, Faculty of Medicine, and all participants gave written informed consent.

Routine clinical characteristics for each patient were recorded at baseline and at each follow-up. Glomerular filtration rate (GFR) was estimated by using the Chronic Kidney Disease Epidemiology Collaboration formula. (11) Patients were managed at the physicians' discretion. In general, patients received antihypertensive therapy if their blood pressure exceeded 130/80 mm Hg and received immunosuppressive treatments according to the class and severity of glomerulonephritis.

The kidney biopsy tissues were fixed in Zenker solution (Sigma-Aldrich, St Louis, Missouri), evaluated by histology and immunofluorescence, and classified according to the ISN/RPS classification criteria (12) by a specialist nephropathologist who did not have knowledge of the clinical outcome. In addition, the activity index (AI) and chronicity index (CI) were also assessed. (12,13) The patients were also divided into categories, high or low AI (AI > 7 or AI < 7) and high or low CI (CI > 3 or CI [less than or equal to] 3), on the basis of a previous study showing these values to be predictive of adverse outcome. (14) Additionally, we determined the fibrosis score for the interstitial fibrosis component of CI as evaluated by Masson Trichrome stain. Interstitial fibrosis was scored as zero if none of the tubulointerstitial area was involved in blue/green staining, as 1 if 1% to 25% of the area was involved, and as 2 if 26% to 50% of the area was involved.

Quantification of Established Fibrosis With Picro-Sirius Red Stain

The extent of fibrosis was evaluated in renal biopsy samples by computerized quantification of Picro-Sirius-stained sections, using a protocol modified from previous studies. (15,16) Six-micrometer-thick kidney tissues were prepared on glass slides, deparaffinized, and hydrated in ethanol series. After the Zenker fixative was removed by 1% iodine in 80% ethanol and 5% sodium thiosulfate (Sigma-Aldrich), slides were stained in 0.1% Picro-Sirius Red (ProSci Tech, Queensland, Australia) overnight, placed in 0.01N hydrochloric acid, dehydrated through graded ethanol, placed in xylene, and coverslipped in Permount.

The kidney biopsy was imaged (x400 magnification) in normal light and double polarized light field with a digital camera (Nikon, Melville, New York). Ten images of the kidney cortex were randomly photographed for each subject. The microscope light source and the condenser were used at fixed settings in all images. Under normal light, ECM material is stained red, whereas under polarized light, collagen displays bright areas.

Image analysis was performed with software ImageJ (1.30; National Institutes of Health [NIH], Bethesda, Maryland). Color images were converted to gray-scale red, green, and blue stack and then differentiated into 3 gray-scale images representing red, green, and blue. For analysis, the green-gray scale images were used in brightfield picture, and the red-gray scale images were used for polarized light picture. The total area, intraluminal area, ECM area (nonpolarized), and polarized positive areas were measured by threshold graphs. The positive Sirius Red staining under regular light reflects the extent of ECM expansion, but the areas of the same stain visible under polarized light highlight the deposition of collagen I and III. Extracellular matrix index and collagen I/III index were calculated from the mean of the 10 photographs for each subject as follows:

The Total Cortical Area = Total Area - Intraluminal Area.

Extracellular Matrix Index (ECMI) = (Picro-Sirius Red-Positive Area Under Normal Light/Total Cortical Area) x 100.

Collagen I/III Index = (Positive Polarized Light Area/Total Cortical Area) x 100.

RNA Extraction and cDNA Synthesis

Total RNA was isolated from the whole kidney biopsy core by silica gel-based membrane spin technology with deoxyribonuclease I treatment (RNeasy Micro Kit, Qiagen, Chatsworth, California). Complementary DNA (cDNA) was synthesized by iScripts cDNA Synthesis Kit using Moloney murine leukemia virus-derived reverse transcriptase premixed with a ribonuclease inhibitor (BioRad, Philadelphia, Pennsylvania) with a blend of oligo(dT) and random hexamer primers.

Quantification of Renal Fibrogenic Gene Expression

Messenger RNA (mRNA) expression was quantified by real-time quantitative polymerase chain reaction (PCR) with Fast Start Universal Probe Master Kit (Applied Biosystems, Foster City, California). cDNA from kidney cortex was amplified by using iScripts cDNA Synthesis Kit in a 96-well plate. Multiplex quantitative PCR was performed with target and housekeeping genes in the same well. (17) By this method, the threshold cycle (Ct) of target genes in each sample was normalized with housekeeping genes to account for individual sample variation in RNA amount. Ct is defined as the cycle number at which the fluorescent intensity generated by the tracer dye, which is released from the probe during DNA amplification, reaches a fixed limit of detection. Ct was determined at the exponential phase and is inversely related to the initial mRNA amount.

Primer sequences for target genes TGFB1 and COL1 are shown in Table 1. The mRNA expression of target genes was calculated by using the [DELTA]Ct procedure with VIC-TAMRA-labeled cyclophilin (Applied Biosystems) as housekeeping gene for normalization among samples where [DELTA]Ct values were calculated from the following equation:

[DELTA]Ct = [Ct.sub.target gene] - [Ct.sub.housekeeping gene].

The primers and probes were tested without reverse transcriptase to ensure that they did not amplify genomic DNA. A standard curve was generated for each gene by using pooled cDNA. Conditions of the PCR reaction were optimized so that the amplification efficiency of the target genes and the endogenous reference gene was comparable across 3 log dilutions of pooled cDNA. (18) The corresponding [DELTA]Ct was plotted against the log concentrations of template cDNA. The slope of the linear regression of 0.1 indicated that the amplification efficiencies were comparable (data not shown).

The optimized conditions consisted of TGFB1 or COL1, 250 nM primers, and 250 nM probes, with 1 [micro]L cyclophilin. The PCR conditions were 50[degrees]C for 2 minutes, 95[degrees]C for 10 minutes, followed by 40 cycles at 95[degrees]C for 0.15 minute, and 60[degrees]C for 1 minute.

The mean Ct obtained in triplicate was used. Mean coefficient of variation for the Ct was 1.08% to 1.95%. The relative levels of gene expression in patients with LN were evaluated by using the comparative cycle (Ct) method. (18) The expression of target genes in LN tissues was expressed as fold change of the mean value of control subjects (n = 3), which were arbitrarily set as 1 as follows:

Relative Expression to Control Tissues = [2.sup.-[DELTA][DELTA]Ct], where

[DELTA]Ct = [Ct.sub.target gene] [Ct.sub.housekeeping gene]; [DELTA][DELTA]Ct = [DELTA][Ct.sub.LN tissues] - [DELTA][Ct.sub.normal tissues].

Outcome Definitions

The 2 outcomes were (1) remission, defined as complete remission with urine protein to creatinine ratio below 0.3g/g, or partial remission, defined as reduction of the ratio of urine protein to creatinine by 50% or below 1g/g, and (2) renal failure, defined as a reduction of GFR by 50% (GFR50) or end-stage renal disease (ESRD) (GFR < 15 mL/min/1.73 [m.sup.2] or dialysis for more than 3 months).

Statistical Analysis

Data were summarized by mean [+ or -] standard deviation and by percentages. To analyze the relationship between clinicopathologic features at the time of kidney biopsy, the expression levels of mRNA of COL1 and TGFB1 were evaluated by continuous analysis. Correlations were calculated by Spearman rank coefficient for nonparametric data and by Pearson coefficient for normally distributed data. One-way analysis of variance, Student t test, or nonparametric tests were used as appropriate to compare differences between groups for continuous data. Comparisons of categorical data were performed with the [chi square] test. To evaluate the predictive value of extremes of gene expression, the levels of mRNA were also dichotomized into high (top 20%) and low to moderate (lower 80%). Survival methods were used to evaluate the prognostic value of the relative gene expression levels in the prediction of the outcomes in the presence of censoring by log-rank statistics, and the results were presented by Kaplan-Meier plots. (19) Predictive modeling was performed with Cox proportional hazards models. (20) A multivariate model, with a stepwise backward elimination procedure, was used and based on a likelihood ratio test with P >.10 for removal and P <.05 for entry of variables. Statistical significance was defined as P <.05, 2-tailed. All statistical analysis was conducted with software package SPSS version 15.0 (SPSS, Inc, Chicago, Illinois).

RESULTS

Baseline Clinicopathologic Characteristics

Thirty-nine patients had adequate kidney cortex tissues (>10 glomeruli) with optimal quality RNA (ratio of the absorbance at 260 and 280 nm ranging from 1.9-2.1). Nearly all (n = 38) were women (Tables 2 and 3). The age of the patients at the time of biopsy was 31.4 [+ or -] 10.6 years. Time of disease onset to kidney biopsy was 4.6 [+ or -] 4.9 years. Most patients (89.7%) had received prior treatment with either prednisolone or immunosuppressive agents. Systolic blood pressure (BP) was 130.3 [+ or -] 13.5 mm Hg, and diastolic BP was 80.5 [+ or -] 10.2 mm Hg. Hypertension (BP > 140/90 mm Hg) was observed in 53.9% of patients, and serum creatinine level was 1.14 [+ or -] 0.93 mg/dL. Glomerular filtration rate at the time of biopsy was 85.0 [+ or -] 33.8 mL/ min. Eight patients (20.5%) had low GFR (GFR < 60 mL/ min/1.73 [m.sup.2]), and 31 patients (79.5%) had high GFR (GFR [greater than or equal to] 60 mL/min/1.73 [m.sup.2]). Proteinuria was 2.78 [+ or -] 1.87 g/24 h, and 36% had nephrotic range proteinuria at the time of biopsy. Urine red blood cell count was 4.7 [+ or -] 2.8 per high-power field and 12.8% had urine red blood cell count greater than 10 per high-power field.

One patient had mixed class V + II kidney disease, and the patient was designated as having class V disease. Seven patients (18.0%) had class II, 3 (7.7%) had class III, 20 (51.3%) had class IV, and 9 (23.1%) had class V disease. Activity index was 4.4 [+ or -] 5.2 and CI was 2.0 [+ or -] 1.7. In all, 33% of patients had high AI (AI > 7) and 33% had high CI (CI > 3). Interstitial fibrosis scores were 0 (n = 29), 1 (n = 9), and 2 (n = 1). Mean ECMI was 7.4 [+ or -] 3.8, while mean collagen I/III index was 2.05 [+ or -] 1.71.

Treatment and Outcomes

The median follow-up was 43.9 months (interquartile range, 14.4-66.0 months). Most patients received intravenous cyclophosphamide according to NIH regimen (n = 26). Other treatment protocols consisted of corticosteroid alone (n = 6), corticosteroids + azathioprine (n = 5), and corticosteroids + mycophenolate mofetil (n = 2). Twenty-seven patients (69.2%) were treated with angiotensin-converting enzyme inhibitors. Two (5.1%) were treated with angiotensin receptor blockers. Ten (25.6%) did not receive angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, or other antihypertensive drugs.

Twenty patients (51.28%) had remission, of whom 9 had complete remission and 14 had partial remission. Relapse was observed in 15 cases (38.46%). A total of 13 patients (25.64%) had renal failure (10 had GfR50, 4 reached ESRD).

Relative Gene Expression Levels of COL1 and TGFB1

The expression levels of COL1 and TGFB1 in LN were expressed as fold change of the controls. The mean fold increase for COL1 was higher than for TGFB1 (22.7 [+ or -] 35.8 versus 10.1 [+ or -] 16.1, P <.001.) There was no correlation between COL1 and TGFB1 (R = -.02, P =.91).

Gene Expression and Baseline Clinicopathologic Characteristics

Representative cases are shown in Figure 1, A through D, and Figure 2, A and B. There were significant correlations between baseline clinicopathologic characteristics and renal fibrosis gene expression, but these were not consistent (Table 4). COL1 was positively correlated with proteinuria and collagen I/III index, whereas TGFB1 did not correlate with these parameters.

The levels of gene expression were compared between patients in different categories according to clinicopathologic characteristics (Table 5). There were no significant differences in the TGFB1 gene expression between any of these categories. COL1 expression was significantly higher for patients with the highest ECMI or collagen I/III index (top 20%) than for those with lower (0%-79%) scores.

We analyzed the gene expression levels according to the ISN/RPS classification of renal histopathology. Because of low sample numbers, we combined patients with class III or IV disease together. Although class III or IV disease appeared to have higher relative expression levels than the other classes, these differences were not significant by analysis of variance (TGFB1: class II, 2.1 [+ or -] 1.5; class III or IV, 14.2 [+ or -] 19.5; class V, 5.9 [+ or -] 7.6; P =.21; COL1: class II, 5.1 [+ or -] 2.2; class III or IV, 32.1 [+ or -] 42.9; class V, 12.3 [+ or -] 19.5; p =.22). To explore the possibility that classes III or IV had higher levels of gene expression, we also combined patients with either class II or V disease. In these combinations, patients with class III, IV disease had significantly higher COL1 expression and tended to have higher TGFB1 expression than those with class II, V disease (Table 5).

Fibrosis Gene and Remission

The levels of TGFB1 or COL1 were not different between those patients who achieved remission and those who did not (Table 4). We analyzed the predictive capability of high gene expression (top 20%) on achieving remission, compared to lower gene expression (lower 80%), by using survival analysis. Neither high levels of TGFB1 nor of COL1 predicted remission or complete remission as compared to low gene expression values (P =.51).

Fibrosis Genes and Renal Failure

We evaluated the independent prognostic value of high gene expression (top 20%) compared to lower gene expression (lower 80%) in predicting renal failure by using multivariate analysis in addition to traditional clinicopathologic predictors (Table 6). High expression of COL1 ([greater than or equal to] 8-fold) was compared to low to moderate COL1 expression (<8-fold) and high TGFB1 expression ([greater than or equal to] 7-fold) was compared to low to moderate TGFB1 expression (< 7-fold). Glomerular filtration rate lower than 60 mL/min/1.73 [m.sup.2] was used to define moderate chronic kidney disease according to the Kidney Disease Improving Global Outcomes initiative. (21)

When all patients were considered (n = 39), high COL1 expression, high TGFB1 expression, high ECMI, high collagen I/III index, and low GFR tended to be predictive of renal failure (P =.10) by univariate analysis. By multivariate analysis, high ECMI (hazard ratio [HR], 4.3; P =.04) and low GFR (HR, 5.4, P =.01) were independent predictors of renal failure.

Because of the strong impact of low baseline GFR on the risks of developing renal failure, we evaluated the predictive roles of fibrogenic genes in the subgroup of patients (n = 31) with preserved renal function at baseline (GFR [greater than or equal to] 60 mL/ min/1.73 [m.sup.2]). Among this group, 8 patients had GFR50 and none had ESRD. Figure 3, A, shows that the cumulative risk of renal failure is significantly higher in patients with high COL1 expression ([greater than or equal to] 8-fold) than those with low to moderate COL1 expression (<8-fold) and in those with high TGFB1 expression ([greater than or equal to] 7-fold) compared to low to moderate TGFB1 expression (< 7-fold) (Figure 3, B). By univariate analysis, high COL1 (HR 6.5, P =.01) and high TGFB1 (HR 5.8, P = .02) expression were predictive of renal failure. By multivariate analysis, only COL1 was an independent (HR, 4.4; P =.04) predictor for renal failure. Other clinicopathologic parameters were not predictive of renal failure in this subgroup. Neither gene was significantly predictive of renal failure when they were analyzed as continuous parameters.

COMMENT

This study found that the expression of fibrogenic genes was increased in LN, with COL1 showing higher relative increase than TGFB1. The expression of COL1 correlated with the levels of proteinuria and collagen I/III index. Low baseline GFR and high ECMI were independent predictors of adverse renal outcome when all patients were considered. However, high renal COL1 gene expression was the most important independent predictor of adverse renal outcome in patients with preserved renal function.

A number of investigators have evaluated the predictive values of different clinicopathologic variables as markers of outcome in LN. The WHO classification is useful for guiding immunosuppression when applied to a large group as a whole, but, because of the large variations within these groups, it does not consistently identify which patients will develop progressive renal disease. (22) The activity and chronicity indices, derived from composite scores of various histologic parameters, (13) were found to be predictive in some studies (23,24) but not in others. (9,25) The role of tubulointerstitial damage in predicting outcome has been noted previously for many types of kidney diseases including LN. (5,7) Esdaile et al (26) showed that an index for tubular atrophy, tubulointerstitial fibrosis, was the best predictor of outcome. On the other hand, similar to our semiquantitative analysis of interstitial fibrosis, Hill et al (9) did not find that interstitial changes in the initial biopsy specimen was predictive of outcome, demonstrating that routine histopathologic studies cannot consistently identify those likely to have adverse outcome. (27)

In our study, high levels of ECM deposition and decreased GFR at baseline were the strongest predictors of adverse outcomes when all patients were considered. Low GFR has been shown to be an important predictor of renal failure in LN in many studies. (1) Indeed in our study, only patients with GFR below 60 mL/min/1.73 [m.sup.2] developed ESRD. This is perhaps not surprising as these patients already have substantial renal injury and nephron loss. Other investigators have found that quantitative morphometry evaluating ECM deposition by Picro-Sirius Red staining was useful in predicting adverse outcomes in LN and other nephropathies. (15,16) Severe renal fibrosis is pathogenically linked to nephron loss and has been shown to be an important determinant of progressive decline of GFR. (5-8)

In kidney diseases, the amount of ECM deposition depends on the balance between factors promoting accumulation and factors affecting ECM degradation. (5,7) The findings of high expression of TGFB1 and COL1 in this study support the role of increased matrix synthesis in LN. Collagen I is normally present in relatively small amounts in the adult kidney. (28) Accumulation of this molecule has been reported in a variety of chronic human kidney diseases (29) and chronic allograft nephropathy. (30) Collagen I mRNA was overexpressed in MRL/lpr lupus mice compared to controls. (27) In our study, collagen I mRNA expression correlated with the degree of proteinuria and Sirius Red staining, which is known to stain many types of collagens including collagen I. A recent PCR array analysis of archival kidney biopsy specimens of patients with LN has identified COL1 as the gene that has one of the highest correlations with chronicity scores in a cross-sectional analysis. (31) We extended these findings and found that very high levels of COL1 mRNA tended to predict renal failure, although the prognostic value of the gene was lower than established ECM deposition when all patients were included in the analysis. On the other hand, when only patients with preserved renal function were analyzed, high COL1 mRNA appeared to be a more important predictor of renal failure than ECM deposition. In this setting, high COL1 mRNA levels may have identified patients who were primed to synthesize collagen before ECM accumulation was fully established and before significant nephron loss has occurred. As such, high COL1 mRNA could emerge as a useful early marker in LN, capable of identifying those at risk of renal disease progression while the kidney function is still close to normal. The survival analysis pointed to a predictive value of COL1 mRNA only when patient groups were dichotomized to compare those with markedly increased expression with groups with milder increases. This suggests that the relationship between COL1 gene expression at baseline and long-term outcome may not be linear, and only those individuals with the largest increases in collagen gene expression might be expected to have poorer outcome.

Numerous experimental studies support the role of TGFfl as an important mediator of collagen synthesis and fibrosis in response to renal injury. (5-7) Upregulated expression of TGFB1 is a feature of virtually all human and experimental models of renal fibrosis including murine lupus models. (27) In a previous study, increased TGF[beta]1 immunostaining was associated with adverse outcome in human LN. (32) In our study, patients with LN and high TGFBlgene expression tended to have poorer long-term survival than those with lower expression, although this was not significant by multivariate analysis. Larger numbers of subjects would probably have demonstrated significantly adverse renal outcomes among those with high TGFB1 gene expression. The interactions between collagen, TGF[beta]1, and fibrosis are complex. (5,6) In this study, the expression levels of the TGFB1 and COL1 genes were not correlated. Factors besides TGF[beta]1 can affect collagen synthesis. (5,7) Furthermore, the biological action of TGF[beta]1 is dependent on a number of other proteins, which regulate its ability to stimulate ECM synthesis. Other factors, such as prior therapy, could affect the transcription levels of genes to different extents in each individual. Such factors could explain the inconsistent correlations between TGFB1 and COL1, and between the levels of gene expression and clinical parameters.

The major limitations to this study include the rather small sample size. Nonetheless, to our knowledge this is the largest study to evaluate the long-term effects of collagen I in patients with LN. This is a mixed group of subjects with LN. A large proportion of patients was already receiving prior therapy. Most patients received cyclophosphamide according to the NIH regimen, but dose adjustments of medication were individualized. Some patients also received other treatments. Multivariate analysis did not show any effects of immunosuppression on outcome. Nonetheless, it remains likely that differences in therapeutic regimen could influence long-term results. During the course of disease, transformation of one lupus class into another may occur either spontaneously or as a consequence of therapy and could impact long-term renal outcome. Unfortunately, we did not perform serial biopsies on the patients to fully evaluate the impact of these events. Additionally, we did not perform microdissection to evaluate the interstitium separately from the glomeruli. Renal fibrogenic processes likely involve both glomeruli and tubulointerstitium compartments, but differences in tissue sampling may account for some of the observed variations in gene expression. Given these constraints, however, it is perhaps more striking that high COL1 mRNA expression is the strongest independent predictor of long-term outcome in patients with preserved GFR. COL1 synthesis therefore may be a critical determinant of renal fibrosis in LN in real-world clinical settings even among those who receive prior immunosuppressive therapy.

CONCLUSIONS

Overall, low GFR and severe ECM deposition were found to be risk factors for renal disease progression in patients with LN. In individuals with preserved GFR, high COL1 mRNA expression was an independent predictor of renal outcome, such that patients with COL1 gene expression 8-fold above normal had greater than 4-fold increased risk of progressive renal failure. If these findings can be confirmed in larger studies, COL1 gene expression may emerge as a valuable index of poor disease outcome, capable of detecting patients who have a strong shift toward renal fibrosis even before the onset of GFR reduction or advanced ECM deposition. Additional studies will be necessary to evaluate if renal fibrogenic gene expression could be used to guide immunosuppressive or specific antifibrotic therapies in the future.

Please Note: Illustration(s) are not available due to copyright restrictions.

Dr Tachaudomdach was supported by grants from the Strategic Scholarships for Frontier Research Network for the Join Ph.D. Program Thai doctoral degree, and the Office of the Higher Education Commission. Dr Kitiyakara was supported by grants from the Office of the Higher Education Commission, and Thailand Research Fund (RMU4880048), Mahidol University, and Ramathibodi Hospital. Dr Kantachuvesiri was supported by the Research Fund for "Genetic Studies in Transgenic Animal Models of SLE" (No. 02011854-0005), Mahidol University.

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Chiraporn Tachaudomdach, PhD; Surasak Kantachuvesiri, MD; Suwikran Wongpraphairot, MD; Suchin Worawichawong, MD; Pleumjit Tankee, MD; Suda Riengrojpitak, PhD; Chagriya Kitiyakara, MD

Accepted for publication April 1, 2014.

From the Molecular Medicine Graduate Program, Faculty of Science, Mahidol University (Dr Tachaudomdach), the Department of Medicine, Faculty of Medicine, Ramathibodi Hospital (Drs Kantachuvesiri and Kitiyakara), the Department of Pathology, Faculty of Medicine, Ramathibodi Hospital (Dr Worawichawong), and the Department of Pathobiology, Faculty of Science (Dr Riengrojpitak), Mahidol University, Bangkok, Thailand; the Department of Medicine, Prince of Songkla University, Songkla, Thailand (Dr Wongpraphairot); and the Department of Medicine, Vachira Phuket Hospital, Phuket, Thailand (Dr Tankee).

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Chagriya Kitiyakara, MD, Department of Medicine, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand (e-mail: kitiyakc@yahoo.com).

Caption: Figure 1. Picro-Sirius Red staining in representative cases. Case No. 45 with low extracellular matrix index and low collagen I/III index (A) under normal light and (B) under polarized light. Case No. 69 with higher extracellular matrix index and higher collagen I/III index (C) under normal light and (D) under polarized light (original magnifications x400 [A through D]).

Caption: Figure 3. Cumulative risk of renal failure in subgroup with preserved renal function at baseline (n = 31). A, Patients with high ([greater than or equal to] 8-fold) COL1 gene expression versus low to moderate (<8-fold) COL1 gene expression; P = .01. B, Patients with high ([greater than or equal to] 7-fold) TGFB1 gene expression versus patients with low to moderate (<7-fold) TGFB1 gene expression; P = .02.
Table 1. Primers and Probe Sequences

Genes   Accession No.           Primer (Forward/Reverse)

TGFB1   NM 000660.4     F: 5'-CCAGCATCTGCAAAGCTC-3'
                        R: 5'-GTCAATGTACAGCTGCCGCA-3'
                        Probe: (FAM)-50-
                        ACACCAACTATTGCTTCAGCTCCACGGA-3'-(TAMRA)
COL1    NM 000088.3     F: 5-CCTCAA GGCTCCAACGAG-3'
                        R: 5'-TCAATCACTGTCTTGCCCCA-3'
                        Probe: (FAM)-50-
                        ATGGCTGCACGAGTCACACCGGA-3'-(TAMRA)

Genes   Product Length, bp

TGFB1          100

COL1           117

Abbreviations: F, forward; FAM, carboxyfluorescein;R, reverse.

Table 2. Individual Patient Data (n = 39)

Case No.   Age, y/Sex   Cr    UProt   URbc   Class   AI   CI   ECMI

01            24/F      0.6   0.4       3      2     0    3     5.6
07            23/F      0.7   1.7       5      5     0    0     8.1
14            29/F      1.1   6.7       5      4     7    2     5.3
16            18/F      3.0   3.2       5      4     13   1     5.9
19            22/F      0.6   5.4      15      4     7    0     3.6
20            27/F      0.8   3.3       5      4     6    4     8.3
21            40/F      0.8   1.5       5      2     0    1     5.3
23            34/F      1.9   1.4       3      4     3    7     7.8
25            25/F      0.9   2.8       2      4     7    2     7.7
26            45/F      0.8   2.8       3      5     0    3     8.4
35            23/F      3.0   2.8       3      4     1    5    15
37            47/F      0.7   6.2       2      5     0    3     6.6
40            38/F      1.6   1.5       5      4     12   5     4.5
42            17/F      0.8   7.2       3      4     11   2    12.5
45            51/F      0.7   2.0       5      2     0    1     4.2
47            28/F      0.7   1.3       2      2     0    1     4.3
48            29/F      0.8   2.6      10      4     10   3     8.7
50            24/F      2.8   8.6       5      4     6    4    12.5
51            26/F      0.7   1.7      10      5     0    2     5.3
52            24/F      0.7   0.5       5      2     0    0     5.7
54            37/F      0.6   2.8       5      4     9    1    12.8
56            17/F      0.7   3.2       3      4     5    1    11.6
57            24/F      0.7   2.1       5      4     8    1     6.9
58            41/F      0.7   3.4       2      5     0    1     4.7
60            59/F      0.8   0.8       3      2     0    2     4.3
63            47/F      0.8   0.5       2      2     0    2     9.3
65            18/F      0.8   3.5       5      4     14   3     5.6
66            21/F      1.5   2.5      10      4     7    0     9.4
67            40/F      0.8   0.8       5      5     0    0     9.4
68            21/F      2.7   2.0       2      4     18   5     6
69            33/F      0.8   2.4       2      4     3    3     7.4
70            42/F      0.7   2.5      10      5     0    0     6.8
75            41/F      0.6   3.7       5      5     0    0     4.9
78            35/F      5     3.7       5      4     15   2     3.8
79            23/F      0.8   2.5       2      2     0    3     8.4
80            34/F      0.7   1.1       5      5     6    2     5.8
81            30/F      0.6   3.5       5      5     0    1     5.6
83            45/F      0.9   1.0       5      2     0    4     5.6
84            22/M      0.7   3.2       2      4     4    0     5.5

Case No.   Col I/III   COL1    TGFB1   Rx      Outcome

01           3.1        6.5     1.0    IM    GFR50
07           3.2        4.4     2.3    P
14           2.9        6.1     5.0    CY
16           2.7        1.1     3.4    CY    ESRD
19           3.2       99.2     7.3    CY    GFR50
20           1.3       19.0     8.7    CY
21           2.9        7.9     2.7    IM
23           2.6       20.0     0.5    CY    GFR50
25           4.5        4.2     2.7    CY
26           3.5        7.1     5.4    CY
35           4.6       99.2    12.1    CY
37           2.7       64.2     4.1    P     GFR 50
40           1.9        1.3     5.0    CY    GFR50, ESRD
42           4.5       64.0     2.8    CY
45           1.9        5.2     3.2    CY
47           2          1.2     4.7    CY
48           7.6        4.4    10.2    CY
50           4.1       99.0     6.4    CY    ESRD
51           3.9        8.2     3.2    IM
52           4.4        6.4     1.6    P
54           4.5       120.4    8.8    CY
56           4.8        4.4     4.1    CY
57           3.3        4.3    96.7    CY    GFR50
58           3.4        4.7     2.9    CY
60           2.4        4.3     0.50   CY
63           4.9        4.3     0.50   MMF
65           2.5        6.6    18.5    CY    GFR50
66           5.9        5.2     1.2    MMF
67           4.4        5.9     1.1    P
68           2.5        9.2    18.0    CY    ESRD
69           2.2       11.0    22.8    CY    GFR50
70           4.1        4.1     8.2    P
75           3          5.0     0.50   CY
78           3.2       19.9     7.9    CY
79           4.2       120.4   18.6    CY
80           2.7        6.7    27.1    P     GFR50
81           3.1        7.1    25.3    IM    GFR50
83           2.2        7.4    18.5    IM
84           3.4        4.9    20.0    CY

Abbreviations: AI, activity index; CI, chronicity index; Col I/III,
collagen I/III index; COL1, collagen type I gene; Cr, serum
creatinine (mg/dL); CY, intravenous cyclophosphamide + P; ECMI,
extracellular matrix index; ESRD, end-stage renal disease (dialysis
or GFR < 15 mL/min/1.73 [m.sup.2]); GFR50, 50% decrease in glomerular
filtration rate; IM, azathioprine + P; MMF, mycophenolate mofetil +
P; P, prednisolone alone; Rx, therapy; TGFB1, transforming growth
factor [beta]-1 gene; UProt, urine protein (g/24 h or protein to
creatinine ratio [g/g]); URbc, urine red blood cells per high-power
field.

Table 3. Summary of Baseline Clinical Data (n = 39)

                                    Mean [+ or -] SD
                                      or Percentage

Clinical characteristics
  Age at initial biopsy, y        31.4 [+ or -] 10.6
  Time from disease onset
    to biopsy, y                  4.6 [+ or -] 4.9
  Women, %                        97.4
  Prior immunosuppressive
    agent, %                      87.2
  Systolic BP, mm Hg              130.3 [+ or -] 13.5
  Diastolic BP, mm Hg             80.5 [+ or -] 10.2
  Serum albumin, g/L              29.7 [+ or -] 7.4
  Creatinine, mg/dL               1.1 [+ or -] 0.93
  GFR, mL/min/1.73 [m.sup.2]      84.9 [+ or -] 33.8
  Urine protein, g/d              2.8 [+ or -] 1.8
  Hypertensive, BP > 140/90
    mm Hg, %                      53.9
  Low GFR, GFR < 60 mL/
    min/1.73 [m.sup.2], %         20.5
  Nephrotic range proteinuria,
    >3 g, %                       35.8
  Urine red blood cells,
    > 10/HPF, %                   12.8
Kidney biopsy
  RPS/ISN class: M/III/IV/V,      7/3/20/9 (17.9/7.7/
    No. (%)                            51.3/23.1)
  Activity index                  4.4 [+ or -] 5.2
  Chronicity index                2.1 [+ or -] 1.7
  Extracellular matrix index      3.4 [+ or -] 1.2
  Collagen I/III index            7.2 [+ or -] 2.7
Outcome, No. (%)
  All remission: complete/        20:14/6 (51.28:35.89/
    partial                              15.38)
  Decreased 50% GFR               10 (25.64)
  Dialysis                        4 (10.25)

Abbreviations: BP, blood pressure; GFR, glomerular filtration rate; HPF,
high-power field; ISN/RPS, International Society of Nephrology/Renal
Pathology Society; SD, standard deviation.

Table 4. Relationship Between Gene Expression and Baseline
Clinicopathologic Parameters

Genes      AI         CI      Extracellular Matrix Index

COL1    R = .05     R = .18         R = .195
        P = .76     P = .26         P = .23
TGFB1   R = -0.18   R = .00         R = -.09
        P = .27     P = .99         P = .55

Genes      AI       Collagen I/III Index     GFR       Proteinuria

COL1    R = .05       R = .52#             R = -.08   R = .48#
        P = .76       P = .001# (a)        P = .62    P = .002# (a)
TGFB1   R = -0.18     R = -.03             R = .15    R = -.05
        P = .27       P = .82              P = .37    P = .75

Genes      AI       Hematuria

COL1    R = .05     R = .07
        P = .76     P = .68
TGFB1   R = -0.18   R = -.14
        P = .27     P = .38

Abbreviations: AI, activity index; CI, chronicity index; COLZ,
collagen type I gene; GFR, glomerular filtration rate; TGFB1,
transforming growth factor [beta]-1 gene.

(a) Bolded values: P < .05.

# Bolded values: P < .05.

Table 5. Relative Gene Expression by Baseline Clinicopathologic
Categories (a)

Categories                               TGFB1                P Value

GFR, mL/min/1.73
[m.sup.2]
  Low, [less than or                Low: 6.8 [+ or -] 5.8       .26
    equal to] 60 (n = 8)
  High, > 60 (n = 31)             High: 11.0 [+ or -] 7.8

Proteinuria
  Nephrotic (n = 14)               Neph: 8.4 [+ or -] 7.7       .31
    versus
  Subnephrotic (n = 26)            Sub: 11.0 [+ or -] 19.0

Activity index
  High, [greater than or          High: 14.4 [+ or -] 25.3      .11
    equal to] 7 (n = 13)
  Low, <7 (n = 26)                  Low: 8.0 [+ or -] 8.5

Chronicity index
  High, [greater than or          High: 24.2 [+ or -] 36.8      .33
    equal to] 3 (n = 13)
  Low, <3 (n = 26)                 Low: 16.9 [+ or -] 33.3

Interstitial fibrosis
  High, [greater than or           High: 9.7 [+ or -] 7.8       .45
    equal to] 1 (n = 10)
  Low, 0 (n = 29)                  Low: 10.4 [+ or -] 18.5

Extracellular matrix
index
  Highest, [greater             Highest: 6.3 [+ or -] 5.2       .08
    than or equal to] 8.0
     (n = 8)
  Lower, <8.0 (n = 31)           Lower: 12.0 [+ or -] 19.2

Collagen I/III index
  Highest, [greater             Highest: 6.0 [+ or -] 5.4       .10
    than or equal to] 4.0
    (n = 8)
  Lower, <4.0 (n = 31)           Lower: 12.2 [+ or -] 19.2

ISN/RPS class
  III, IV (n = 23)              III, IV: 4.2 [+ or -] 19.5      .07
  V, II (n = 16)                  II, V: 4.3 [+ or -] 6.0

Remission
  No remission (n = 19)     No remission 7.8 [+ or -] 8.3       .22
  Remission (n = 20)         Remission: 12.3 [+ or -] 21.0

Categories                                COL1                 P Value

GFR, mL/min/1.73
[m.sup.2]
  Low, [less than or                Low: 31.9 [+ or -] 42.2      .31
    equal to] 60 (n = 8)
  High, > 60 (n = 31)              High: 20.3 [+ or -] 34.4

Proteinuria
  Nephrotic (n = 14)               Neph: 29.6 [+ or -] 37.7      .23
    versus
  Subnephrotic (n = 26)             Sub: 19.2 [+ or -] 35.1

Activity index
  High, [greater than or           High: 26.6 [+ or -] 40.7      .32
    equal to] 7 (n = 13)
  Low, <7 (n = 26)                  Low: 20.7 [+ or -] 33.8

Chronicity index
  High, [greater than or           High: 24.2 [+ or -] 36.8      .49
    equal to] 3 (n = 13)
  Low, <3 (n = 26)                  Low: 16.9 [+ or -] 33.3

Interstitial fibrosis
  High, [greater than or           High: 18.7 [+ or -] 28.9      .21
    equal to] 1 (n = 10)
  Low, 0 (n = 29)                   Low: 21.4 [+ or -] 36.2

Extracellular matrix
index
  Highest, [greater             Highest: 42.9 [+ or -] 49.4     <.001
    than or equal to] 8.0                                        (b)
     (n = 8)
  Lower, <8.0 (n = 31)            Lower: 12.5 [+ or -] 21.4

Collagen I/III index
  Highest, [greater             Highest: 41.7 [+ or -] 50.3     <.001
    than or equal to] 4.0                                        (b)
    (n = 8)
  Lower, <4.0 (n = 31)           Lower: 13.16 [+ or -] 21.4

ISN/RPS class
  III, IV (n = 23)              III, IV: 32.1 [+ or -] 42.9     <.001
  V, II (n = 16)                   II, V: 9.2 [+ or -] 14.8      (b)

Remission
  No remission (n = 19)     No remission 20.1 [+ or -] 34.6      .55
  Remission (n = 20)          Remission: 25.1 [+ or -] 37.7

Abbreviations: COL1, collagen type I gene; GFR, glomerular filtration
rate; ISN-RPS, International Society of Nephrology/Renal Pathology
Society; Neph, Nephrotic; Sub, Subnephrotic; TGFB1, transforming
growth factor [beta]-1 gene.

(a) Data shown as mean [+ or -] standard deviation.

(b) Bolded values: P < .05.

Table 6. Predictors of Renal Failure in All Patients and in the
Subgroup With Preserved Renal Function (a, b)

Parameters                                      Univariate

                                      Hazard     95% CI      P Value
                                      Ratio

All patients (n = 39)

  COL1, [greater than or equal         3.77                    .05
    to] 8-fold versus <8-fold
  TGFB1, [greater than or equal        3.72                    .05
    to] 7-fold versus <7-fold
  ISN/RPS class, II + V versus         0.91                    .31
    III + IV
  Activity index, [greater than        0.97                    .33
    or equal to] 7 versus <7
  Chronicity index, [greater           1.67                    .19
    than or equal to] 3 versus <3
  Interstitial fibrosis,               0.53                    .81
    [greater than or equal to] 1
    versus 0
  Extracellular matrix index,          6.25    1.03-6.74#     .01#
    [greater than or equal to] 8
    versus <8
  Collagen I/III index, [greater       3.19                    .07
    than or equal to] 4 versus <4
  Age at nephritis onset,              1.44                    .23
    [greater than or equal to] 25
    versus <25 y
  Time to biopsy, [greater than        1.41                    .24
    or equal to] 1 versus <1 y
  Systolic BP, [greater than or        0.61                    .22
    equal to] 140 versus <140 mm
    Hg
  Diastolic BP, 90 versus <90 mm       0.99                    .31
    Hg
  Glomerular filtration rate,          5.35    1.56-18.15#    .02#
    <60 versus [greater than or
    equal to] 60 mL/min/1.73
    [m.sup.2]
  Proteinuria, nephrotic versus        0.37                    .54
    subnephrotic
  Hematuria, [greater than or          0.05                    .83
    equal to] 10 versus <10
    RBCs/HPF
  Immunosuppression versus             0.28                    .59
    steroid alone
  ACEIs or ARBs, Yes versus No         0.80                    .65

Subgroup: patients with GFR
[greater than or equal to] 60
mL/min/1.73 [m.sup.2] (n = 31)

  COL1, [greater than or equal         6.52    1.30-6.61#     .01#
    to] 8-fold versus <8-fold
  TGFB1, [greater than or equal        5.86     1.18-6.36     .02#
    to] 7-fold versus <7-fold
  ISN/RPS class, II + V versus         0.59                    .40
    III + IV
  Activity index, [greater than       0.81                    .37
    or equal to] 7 versus <7
  Chronicity index, [greater           0.63                    .42
    than or equal to] 3 versus <3
  Interstitial fibrosis,               2.71                    .10
    [greater than or equal to] 1
    versus 0
  Extracellular matrix index,          2.29                    .13
    [greater than or equal to] 8.0
    versus <8.0
  Collagen I/III index, [greater       1.81                    .18
    than or equal to] 4.0 versus
    <4.0
  All other clinical parameters
    P > .1 by univariate analysis

Parameters                                    Multivariate

                                      Hazard     95% CI     P Value
                                      Ratio

All patients (n = 39)

  COL1, [greater than or equal
    to] 8-fold versus <8-fold
  TGFB1, [greater than or equal
    to] 7-fold versus <7-fold
  ISN/RPS class, II + V versus
    III + IV
  Activity index, [greater than
    or equal to] 7 versus <7
  Chronicity index, [greater
    than or equal to] 3 versus <3
  Interstitial fibrosis,
    [greater than or equal to] 1
    versus 0
  Extracellular matrix index,         8.74#    1.13-9.43#    .04#
    [greater than or equal to] 8
    versus <8
  Collagen I/III index, [greater
    than or equal to] 4 versus <4
  Age at nephritis onset,
    [greater than or equal to] 25
    versus <25 y
  Time to biopsy, [greater than
    or equal to] 1 versus <1 y
  Systolic BP, [greater than or
    equal to] 140 versus <140 mm
    Hg
  Diastolic BP, 90 versus <90 mm
    Hg
  Glomerular filtration rate,         4.95#    1.45-16.8#    .01#
    <60 versus [greater than or
    equal to] 60 mL/min/1.73
    [m.sup.2]
  Proteinuria, nephrotic versus
    subnephrotic
  Hematuria, [greater than or
    equal to] 10 versus <10
    RBCs/HPF
  Immunosuppression versus
    steroid alone
  ACEIs or ARBs, Yes versus No

Subgroup: patients with GFR
[greater than or equal to] 60
mL/min/1.73 [m.sup.2] (n = 31)

  COL1, [greater than or equal        4.38#    1.05-4.55#    .04#
    to] 8-fold versus <8-fold
  TGFB1, [greater than or equal       2.694                   .10
    to] 7-fold versus <7-fold
  ISN/RPS class, II + V versus
    III + IV
  Activity index, [greater than
    or equal to] 7 versus <7
  Chronicity index, [greater
    than or equal to] 3 versus <3
  Interstitial fibrosis,
    [greater than or equal to] 1
    versus 0
  Extracellular matrix index,
    [greater than or equal to] 8.0
    versus <8.0
  Collagen I/III index, [greater
    than or equal to] 4.0 versus
    <4.0
  All other clinical parameters
    P > .1 by univariate analysis

Abbreviations: ACEIs, angiotensin-converting enzyme inhibitors;
ARBs, angiotensin receptor blockers; BP, blood pressure; CI,
confidence interval; COL1, collagen type I gene; GFR, glomerular
filtration rate; HPF, high-power field; ISN/RPS, International
Society of Nephrology/Renal Pathology Society; RBCs, red blood
cells; TGFB1, transforming growth factor [beta]-1 gene. Ninety-five
percent CI shown only for significant parameters.

(a) Parameters with P < .1 by univariate analysis were included in
the multivariate analysis.

(b) Bolded values show significant parameters by multivariate
analysis.

# indicates bold values.

Figure 2. Representative cases No. 45 and No. 69. A, Extracellular
matrix index (dark bar) and collagen I/III index (white bar). B,
Relative gene expression levels with COL1 (dark bar) and TGFB1
(white bar).

A

                %

No. 45   4.3 **    2.0 *
No. 69   15.0 **   4.6 *

B        Relative Expression

No. 45   5.2 **    3.2 *
No. 69   11.0 **   22.7 *

* represents the dark bar
** represents the white bar

Note: Table made from bar graph.
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Author:Tachaudomdach, Chiraporn; Kantachuvesiri, Surasak; Wongpraphairot, Suwikran; Worawichawong, Suchin;
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Clinical report
Date:Mar 1, 2015
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