Biomedical subjects

Surya V Seshan

Publications and source records attributed to Surya V Seshan.

At least 19 recordsLinked to original sources

PubMed2026

Benchmarking urinary cell transcriptomes for noninvasive differentiation of BK polyomavirus-associated nephropathy from T cell-mediated rejection.

BK polyomavirus-associated nephropathy (BKVN) adversely impacts kidney allograft survival and often mimics acute T cell-mediated rejection (TCMR), confounding diagnosis and management. To address this conundrum, we performed unbiased RNA sequencing of urinary cells matched to biopsies classified as BKVN with intragraft inflammation (BKVN-P), BKVN without inflammation (BKVN-N), TCMR, or no rejection (NR). BKVN-N displayed dominant host DNA replication, cell cycle, and repair programs, while BKVN-P samples exhibited expansive innate immune activation, antigen presentation, chemokine upregulation, and epithelial injury. Both BKVN subtypes shared signatures of T cell exhaustion and mature and tolerogenic dendritic cell activation but differed in immune orientation - Th1 predominance in BKVN-N versus Treg and CD8 enrichment in BKVN-P. Compared with TCMR samples, BKVN-P lacked robust TCR/CD28 signaling and was enriched for viral and innate modules; BKVN-N lacked alloimmune activation. B cell exhaustion characterized BKVN-N, while BKVN-P displayed robust B cell activation with metabolic downregulation. A ratiometric urinary cell biomarker, CXCL10 mRNA/CD3E mRNA, distinguished both BKVN subtypes from TCMR with diagnostic accuracy, replicated by quantitative reverse transcription PCR for clinical translation, and confirmed in an independent cohort. These findings demonstrate the utility of urinary cell transcriptomics for resolving viral injury from alloimmunity, enabling precision diagnostics and targeted immunomodulation in kidney transplantation.

Humans↗

PubMed2006

Effect of combination therapy with enalapril and the TGF-beta antagonist 1D11 in unilateral ureteral obstruction.

In unilateral ureteral obstruction (UUO), the kidney is characterized by increased fibrosis and apoptosis. Both transforming growth factor-beta (TGF-beta) and ANG II have been implicated, and ANG II may mediate its effects through TGF-beta. Previous studies demonstrated amelioration of renal damage when either TGF-beta or ANG II has been individually targeted. In this study, we sought to determine whether combining 1D11 (monoclonal antibody to TGF-beta) and an ACE inhibitor, enalapril, would be more effective in UUO than either individual treatment, as has been shown in diabetic and glomerulonephritic models. Rats underwent UUO and were given either control monoclonal antibody, 1D11 or enalapril, or 1D11/enalapril combination, for 14 days. Kidneys were harvested and examined for fibrosis [trichrome; collagen (real-time PCR, Sircol assay) and fibroblast-specific protein expression (immunohistochemistry), apoptosis (TUNEL), macrophage infiltration (immunohistochemistry), and TGF-beta expression (real-time PCR and tubular localization with immunohistochemistry)]. UUO was found to induce fibrosis, apoptosis, macrophage infiltration, and TGF-beta expression in the obstructed kidney. Administration of either 1D11 or enalapril individually significantly decreased all these changes; when 1D11 and enalapril were combined, there was little additive effect, and the combination did not provide full protection against damage. The results demonstrate that, for the most part, combination therapy is not additive in UUO. This could be due to the continued presence of a physical obstruction or to biochemical differences between UUO and other renal disease models. Furthermore, it suggests that other targets may be amenable to pharmacological manipulation in UUO.

Animals↗

PubMed2006

Mitigation of radiation nephropathy after internal alpha-particle irradiation of kidneys.

PURPOSE: Internal irradiation of kidneys as a consequence of radioimmunotherapy, radiation accidents, or nuclear terrorism can result in radiation nephropathy. We attempted to modify pharmacologically, the functional and morphologic changes in mouse kidneys after injection with the actinium ((225)Ac) nanogenerator, an in vivo generator of alpha- and beta-particle emitting elements. METHODS AND MATERIALS: The animals were injected with 0.35 muCi of the (225)Ac nanogenerator, which delivers a dose of 27.6 Gy to the kidneys. Then, they were randomized to receive captopril (angiotensin-converting enzyme inhibitor), L-158,809 (angiotensin II receptor-1 blocker), spironolactone (aldosterone receptor antagonist), or a placebo. RESULTS: Forty weeks after the (225)Ac injection, the placebo-control mice showed a significant increase in blood urea nitrogen (BUN) (87.6 +/- 6.9 mg/dL), dilated Bowman spaces, and tubulolysis with basement membrane thickening. Captopril treatment accentuated the functional (BUN 119.0 +/- 4.0 mg/dL; p <0.01 vs. placebo controls) and histopathologic damage. In contrast, L-158,809 offered moderate protection (BUN 66.6 +/- 3.9 mg/dL; p = 0.02 vs. placebo controls). Spironolactone treatment, however, significantly prevented the development of histopathologic and functional changes (BUN 31.2 +/- 2.5 mg/dL; p <0.001 vs. placebo controls). CONCLUSIONS: Low-dose spironolactone and, to a lesser extent, angiotensin receptor-1 blockade can offer renal protection in a mouse model of internal alpha-particle irradiation.

Actinium↗

PubMed2006

Angiotensin receptor blockade decreases fibrosis and fibroblast expression in a rat model of unilateral ureteral obstruction.

PURPOSE: Unilateral ureteral obstruction is characterized by histopathological changes including interstitial fibrosis, fibroblast specific protein expression, tubular atrophy and apoptosis, and macrophage infiltration. Angiotensin II has been implicated in some of these changes. We examined the effect of angiotensin blockade on markers of renal injury, including fibroblast specific protein expression, fibrosis, apoptosis and macrophage infiltration. We used losartan, an angiotensin II antagonist, in a unilateral ureteral obstruction model and studied animals 3 weeks after unilateral ureteral obstruction, a time at which renal damage is well established. MATERIALS AND METHODS: Rats underwent unilateral ureteral obstruction and were given either drinking water or losartan for 21 days. Kidneys were harvested and examined for fibrosis (trichrome and the Sircol assay for collagen), apoptosis (TUNEL), and fibroblast specific protein expression and macrophage infiltration (immunohistochemistry). RESULTS: Unilateral ureteral obstruction was found to induce fibrosis, apoptosis, fibroblast expression and macrophage in the obstructed kidney. Losartan significantly decreased apoptosis and macrophage infiltration in the obstructed kidney. It also decreased fibrosis, as measured by either trichrome staining assessed by a pathologist, the Sircol assay for collagen or fibroblast specific protein expression. However, approximately 50% of the changes were not affected by the current treatment, suggesting that other factors contribute to renal damage in unilateral ureteral obstruction. CONCLUSIONS: We observed the direct contribution of angiotensin II to both apoptotic and cellular transition processes (epithelial mesenchymal transition) and fibrosis in unilateral ureteral obstruction. Because these processes are active not only in unilateral ureteral obstruction, but also in other renal diseases, the value of angiotensin II blockade as an important part of the antifibrotic armamentarium has been confirmed.

Angiotensin II Type 1 Receptor Blockers↗

PubMed2005

Messenger RNA for FOXP3 in the urine of renal-allograft recipients.

BACKGROUND: The outcome of renal transplantation after an episode of acute rejection is difficult to predict, even with an allograft biopsy. METHODS: We studied urine specimens from 36 subjects with acute rejection, 18 subjects with chronic allograft nephropathy, and 29 subjects with normal biopsy results. Levels of messenger RNA (mRNA) for FOXP3, a specification and functional factor for regulatory T lymphocytes, and mRNA for CD25, CD3epsilon, perforin, and 18S ribosomal RNA (rRNA) were measured with a kinetic, quantitative polymerase-chain-reaction assay. We examined associations of mRNA levels with acute rejection, rejection reversal, and graft failure. RESULTS: The log-transformed mean (+/-SE) ratio of FOXP3 mRNA copies to 18S ribosomal RNA copies was higher in urine from the group with acute rejection (3.8+/-0.5) than in the group with chronic allograft nephropathy (1.3+/-0.7) or the group with normal biopsy results (1.6+/-0.4) (P<0.001 by the Kruskal-Wallis test). FOXP3 mRNA levels were inversely correlated with serum creatinine levels measured at the time of biopsy in the acute-rejection group (Spearman's correlation coefficient = -0.38, P=0.02) but not in the group with chronic allograft nephropathy or the group with normal biopsy results. Analyses of receiver-operating-characteristic curves demonstrated that reversal of acute rejection can be predicted with 90 percent sensitivity and 73 percent specificity with use of the optimal identified cutoff for FOXP3 mRNA of 3.46 (P=0.001). FOXP3 mRNA levels identified subjects at risk for graft failure within six months after the incident episode of acute rejection (relative risk for the lowest third of FOXP3 mRNA levels, 6; P=0.02). None of the other mRNA levels were predictive of reversal of acute rejection or graft failure. CONCLUSIONS: Measurement of FOXP3 mRNA in urine may offer a noninvasive means of improving the prediction of outcome of acute rejection of renal transplants.

Acute Disease↗

PubMed2005

Renal tubulointerstitial changes after internal irradiation with alpha-particle-emitting actinium daughters.

The effect of external gamma irradiation on the kidneys is well described. However, the mechanisms of radiation nephropathy as a consequence of targeted radionuclide therapies are poorly understood. The functional and morphologic changes were studied chronologically (from 10 to 40 wk) in mouse kidneys after injection with an actinium-225 (225Ac) nanogenerator, a molecular-sized, antibody-targeted, in vivo generator of alpha-particle-emitting elements. Renal irradiation from free, radioactive daughters of 225Ac led to time-dependent reduction in renal function manifesting as increase in blood urea nitrogen. The histopathologic changes corresponded with the decline in renal function. Glomerular, tubular, and endothelial cell nuclear pleomorphism and focal tubular cell injury, lysis, and karyorrhexis were observed as early as 10 wk. Progressive thinning of the cortex as a result of widespread tubulolysis, collapsed tubules, glomerular crowding, decrease in glomerular cellularity, interstitial inflammation, and an elevated juxtaglomerular cell count were noted at 20 to 30 wk after treatment. By 35 to 40 wk, regeneration of simplified tubules with tubular atrophy and loss with focal, mild interstitial fibrosis had occurred. A lower juxtaglomerular cell count with focal cytoplasmic vacuolization, suggesting increased degranulation, was also observed in this period. A focal increase in tubular and interstitial cell TGF-beta1 expression starting at 20 wk, peaking at 25 wk, and later declining in intensity with mild increase in the extracellular matrix deposition was noticed. These findings suggest that internally delivered alpha-particle irradiation-induced loss of tubular epithelial cells triggers a chain of adaptive changes that result in progressive renal parenchymal damage accompanied by a loss of renal function. These findings are dissimilar to those seen after gamma or beta irradiation of kidneys.

Actinium↗

PubMed2005

Dietary arginine supplementation attenuates renal damage after relief of unilateral ureteral obstruction in rats.

BACKGROUND: Progression of renal injury after relief of unilateral ureteral obstruction (UUO) has been demonstrated. Nitric oxide (NO) may be an effective intervention due to its vasodilatory, antifibrotic, and anti-apoptotic effects. Herein, we used dietary L-arginine (ARG) supplementation in a UUO relief model. METHODS: This study comprised group 1, control (no treatment). All other rats were subject to 3-day UUO, which was then relieved, and the rats maintained for 7 additional days. Group 2, no additional treatment; group 3, L-ARG; group 4, L-NAME, NO synthase inhibitor; group 5, ARG and L-NAME. Urinary NO(2/3) was quantified. GFR and ERPF were measured at day 10. Interstitial fibrosis and fibroblast expression, macrophage infiltration, tubular apoptosis, and proliferation, NOS expression, and the levels of tissue TGF-beta were evaluated. RESULTS: Urinary NO(2/3) was significantly increased by ARG treatment and decreased by L-NAME. GFR and ERPF measured 7 days following relief were not significantly different in the previously obstructed kidneys (POK) of groups 2 and 3. L-NAME significantly reduced GFR and ERPF in the POK. ARG significantly reduced apoptosis, macrophage infiltration, and fibroblast expression in the POK. L-NAME exacerbated the effects on apoptosis and fibroblasts. Fibrosis was minimal in groups 1 through 3, but was significantly increased by L-NAME. ARG did not affect renal NOS expression and tissue TGF-beta1 levels. CONCLUSION: Dietary ARG supplementation during UUO relief did not improve ERPF or GFR. However, renal damage, including fibrosis, apoptosis, and macrophage infiltration was significantly improved by ARG treatment. This suggests that increasing NO availability could be beneficial in the setting of UUO relief.

Animals↗

PubMed2005

Seasonal variation of lupus nephritis: high prevalence of class V lupus nephritis during the winter and spring.

OBJECTIVE: Systemic lupus erythematosus is a multisystem disease with many clinical variations, including renal involvement. Our aim was to determine whether lupus nephritis (LN) has a specific seasonality. METHODS: Reports of renal biopsies performed from 1990 to 2002 were reviewed. Three hundred and seventy-three patients with class II, III, IV, and V LN were identified. Using the modified WHO classification of LN, diagnoses were tabulated and the seasonality (season of diagnosis) of LN was statistically analyzed. RESULTS: Class IV LN was detected in 179 patients (48%), class II in 63 patients (16.9%), class III in 73 patients (19.57%), and class V in 74 patients (19.9%). No difference could be detected in the number of patients diagnosed in each season when all 373 patients were analyzed as one group. The number of patients with class IV LN was higher during summer and fall than during the winter and spring. In contrast, a higher number of patients with class V LN were observed during the winter and spring seasons than during the summer and fall seasons. The percentage of patients with class V LN was significantly higher during winter and spring than during summer and fall. A similar, though non-significant, trend was seen for class III LN. A striking parallelism was found between the month of occurrence of class III and class V LN. The monthly distribution of the percentage of patients in each month with class III and V LN showed a significant correlation. The monthly distribution of patients with class IV LN was different from those with either class III or V LN. CONCLUSION: We found that the prevalence of class V LN was significantly higher and that of class III LN non-significantly higher in winter and spring. Parallelism between the monthly occurrences of class III and class V may suggest a common trigger. Analysis of the seasonality of LN may contribute to the understanding of the pathogenesis of LN, which may be multifactorial, as the different classes of LN represent different types of glomerular injury. Further studies are needed to clarify this potentially important observation.

Adolescent↗

PubMed2004

Renal damage progresses despite improvement of renal function after relief of unilateral ureteral obstruction in adult rats.

Progression of renal damage after relief of unilateral ureteral obstruction (UUO) has been demonstrated, especially in neonatal rats. We evaluated renal function and renal damage after relief of 3-day UUO in five groups of adult rats: group 1, no treatment; group 2, 3-day UUO; groups 3-5, 3-day UUO followed by relief; group 3, 7-day relief; group 4, 14-day relief; and group 5, 28-day relief. Glomerular filtration rate (GFR), renal blood flow (RBF), tissue transforming growth factor-beta (TGF-beta), interstitial fibrosis and fibroblast expression, tubular apoptosis, macrophage infiltration, expression of nitric oxide synthases (NOS), and urinary nitrate/nitrite (NO(2)/NO(3)) were evaluated. RBF and GFR were decreased to <10% of baseline by 3 days of UUO. GFR and RBF in a previously obstructed kidney (POK) returned to baseline by 14 days after relief. Both tissue TGF-beta(1) and interstitial fibrosis were significantly higher in POK of groups 3-5 compared with groups 1 and 2 . In group 5, the numbers of infiltrating macrophages, fibroblasts, and apoptotic tubular cells were higher in POK compared with group 1. Urinary NO(2)/NO(3) was significantly higher than baseline from 3 to 27 days after relief of UUO. Expression of NOS isoforms was increased in tubules. As interstitial fibrosis contributes to decreased renal function, these results suggest that the acute recovery in function may be compromised in the long term by the progressive renal fibrosis which was found. Furthermore, pharmacological intervention at the time of relief of UUO, targeted to fibrotic processes, may contribute to long-term recovery of renal function.

Animals↗

PubMed2004

The classification of glomerulonephritis in systemic lupus erythematosus revisited.

The currently used classification reflects our understanding of the pathogenesis of the various forms of lupus nephritis, but clinicopathologic studies have revealed the need for improved categorization and terminology. Based on the 1982 classification published under the auspices of the World Health Organization (WHO) and subsequent clinicopathologic data, we propose that class I and II be used for purely mesangial involvement (I, mesangial immune deposits without mesangial hypercellularity; II, mesangial immune deposits with mesangial hypercellularity); class III for focal glomerulonephritis (involving <50% of total number of glomeruli) with subdivisions for active and sclerotic lesions; class IV for diffuse glomerulonephritis (involving > or = 50% of total number of glomeruli) either with segmental (class IV-S) or global (class IV-G) involvement, and also with subdivisions for active and sclerotic lesions; class V for membranous lupus nephritis; and class VI for advanced sclerosing lesions]. Combinations of membranous and proliferative glomerulonephritis (i.e., class III and V or class IV and V) should be reported individually in the diagnostic line. The diagnosis should also include entries for any concomitant vascular or tubulointerstitial lesions. One of the main advantages of the current revised classification is that it provides a clear and unequivocal description of the various lesions and classes of lupus nephritis, allowing a better standardization and lending a basis for further clinicopathologic studies. We hope that this revision, which evolved under the auspices of the International Society of Nephrology and the Renal Pathology Society, will contribute to further advancement of the WHO classification.

Biopsy↗

PubMed2004

Dietary L-arginine supplementation improves the glomerular filtration rate and renal blood flow after 24 hours of unilateral ureteral obstruction in rats.

PURPOSE: Unilateral ureteral obstruction (UUO) results in a significant change in renal blood flow (RBF) and glomerular filtration rate (GFR) by 24 hours. The intake of L-arginine, a substrate of nitric oxide (NO) synthase (NOS), can augment NO production. NO can maintain renal function through its vasodilatory action. Therefore, we examined the effect of dietary arginine supplementation on renal function in UUO. MATERIALS AND METHODS: GFR and RBF were measured by inulin and para-aminohippurate clearance, respectively, in control rats and in rats 24 hours after UUO. Rats were given arginine with or without the concomitant administration of N-nitro-L-arginine methyl ester. Urinary nitrate/nitrite (NO2/NO3) was measured by the Griess reaction and urinary cyclic guanosine monophosphate was determined by enzyme-linked immunosorbent assay. The expression of renal inducible NOS was determined by immunohistochemistry. RESULTS: Urinary NO2/NO3 was significantly increased after 2 weeks of arginine, confirming increased NO production. In control rats GFR and RBF were not significantly different in untreated vs arginine treated groups. In contrast, arginine treatment significantly increased GFR in the obstructed kidney (0.06 +/- 0.01 to 0.14 +/- 0.02 ml per minute per 100 gm) and the contralateral kidney compared with control UUO. RBF was also significantly increased by arginine. The increases in renal function with arginine were blunted by a NOS inhibitor in obstructed and contralateral kidneys. Inducible NOS expression was increased in obstructed and contralateral kidneys. CONCLUSIONS: This study demonstrates that L-arginine supplementation can improve renal function in acute UUO. This finding suggests that NO system may be a future site of pharmacological intervention for UUO.

Animals↗

PubMed2004

Proliferative glomerulonephritis with monoclonal IgG deposits: a distinct entity mimicking immune-complex glomerulonephritis.

BACKGROUND: Renal disease related to the deposition of monoclonal immunoglobulins containing both heavy and light chains can occur in type 1 cryoglobulinemia, Randall type light and heavy chain deposition disease (LHCDD), and immunotactoid glomerulonephritis. We report a novel phenotype of glomerular injury that does not conform to any of the previously described patterns of glomerular involvement by monoclonal gammopathy. METHODS: Ten cases of unclassifiable proliferative glomerulonephritis manifesting glomerular monoclonal immunoglobulin G (IgG) deposits were identified retrospectively from the archives of the Renal Pathology Laboratory of Columbia University over the past 3 years (biopsy incidence 0.21%). RESULTS: The monoclonal immunoglobulins formed granular electron dense deposits in mesangial, subendothelial, and subepithelial sites, mimicking ordinary immune complex-mediated glomerulonephritis and producing a diffuse endocapillary proliferative or membranoproliferative glomerulonephritis. However, by immunofluorescence, the deposits were monoclonal, staining for a single light chain isotype and a single gamma subclass (including two IgG1kappa, one IgG1lambda, one IgG2lambda, four IgG3kappa, and one IgG3lambda). All cases stained for the three constant domains of the gamma heavy chain (CH1, CH2, and CH3), suggesting deposition of a nondeleted immunoglobulin molecule. Tissue fixation of complement was observed in 90% of cases, and 40% of patients had hypocomplementemia. Clinical presentations included renal insufficiency in 80% (mean serum creatinine 2.8 mg/dL, range 0.9 to 8.0), proteinuria in 100% (mean urine protein 5.8 g/day; range 1.9 to 13.0), nephrotic syndrome in 44%, and microhematuria in 60%. A monoclonal serum protein with the same heavy and light chain isotype as that of the glomerular deposits was identified in 50% of cases (including three IgGkappa and two IgGlambda); however, no patient had clinical or laboratory features of type 1 cryoglobulinemia. No patient had overt myeloma or lymphoma at presentation or over the course of follow-up (mean 12 months). CONCLUSION: Glomerular deposition of monoclonal IgG can produce a proliferative glomerulonephritis that mimics immune-complex glomerulonephritis by light and electron microscopy. Proper recognition of this entity requires confirmation of monoclonality by staining for the gamma heavy chain subclasses.

Adult↗

PubMed2004

The classification of glomerulonephritis in systemic lupus erythematosus revisited.

The currently used classification reflects our understanding of the pathogenesis of the various forms of lupus nephritis, but clinicopathologic studies have revealed the need for improved categorization and terminology. Based on the 1982 classification published under the auspices of the World Health Organization (WHO) and subsequent clinicopathologic data, we propose that class I and II be used for purely mesangial involvement (I, mesangial immune deposits without mesangial hypercellularity; II, mesangial immune deposits with mesangial hypercellularity); class III for focal glomerulonephritis (involving <50% of total number of glomeruli) with subdivisions for active and sclerotic lesions; class IV for diffuse glomerulonephritis (involving > or =50% of total number of glomeruli) either with segmental (class IV-S) or global (class IV-G) involvement, and also with subdivisions for active and sclerotic lesions; class V for membranous lupus nephritis; and class VI for advanced sclerosing lesions. Combinations of membranous and proliferative glomerulonephritis (i.e., class III and V or class IV and V) should be reported individually in the diagnostic line. The diagnosis should also include entries for any concomitant vascular or tubulointerstitial lesions. One of the main advantages of the current revised classification is that it provides a clear and unequivocal description of the various lesions and classes of lupus nephritis, allowing a better standardization and lending a basis for further clinicopathologic studies. We hope that this revision, which evolved under the auspices of the International Society of Nephrology and the Renal Pathology Society, will contribute to further advancement of the WHO classification.

Humans↗

PubMed2004

Noninvasive detection of renal allograft inflammation by measurements of mRNA for IP-10 and CXCR3 in urine.

BACKGROUND: We explored the hypothesis that measurements of mRNA encoding interferon-inducible protein-10 (IP-10) or the chemokine receptor CXCR3 in urinary cells offer a noninvasive means of elucidating cellular traffic causing acute rejection of human renal allografts. METHODS: We obtained 63 urine specimens from 58 renal allograft recipients who underwent 63 allograft biopsies to resolve the basis for graft dysfunction, and 27 additional urine samples from 24 other patients with stable allograft function. Twenty-seven of the 63 biopsies were classified as acute rejection, 20 as other, and 16 as chronic allograft nephropathy. We measured the levels of transcripts for IP-10 and CXCR3, and a constitutively expressed gene 18S rRNA in the urine specimens and correlated transcript levels with renal allograft diagnosis. RESULTS: mRNA levels of IP-10 (P < 0.0001) or CXCR3 (P < 0.0001) but not the levels of 18S rRNA (P= 0.56) predicted intragraft cellular traffic causing acute rejection. Receiver-operating characteristic curve analysis demonstrated that acute rejection can be predicted with a sensitivity of 100% and a specificity of 78% using the (log-transformed) cutoff value of 9.11 copies of IP-10, and with a sensitivity of 63% and a specificity of 83% using the cutoff value of 11.59 copies of CXCR3. Immunohistologic analysis of allograft biopsies showed exuberant expression of IP-10 and CXCR3 during acute rejection whereas both were absent in grafts with stable function. CONCLUSION: Our investigation demonstrates that intragraft cellular events associated with acute rejection of human renal allografts can be noninvasively identified by measurements of mRNA for IP-10 and CXCR3 in urinary cells.

Adult↗

PubMed2004

Liposome-mediated transfer of nitric oxide synthase gene improves renal function in ureteral obstruction in rats.

BACKGROUND: The protective effect of nitric oxide has been demonstrated in several renal disease models. We augmented renal nitric oxide production by transfer of the inducible nitric oxide synthase (iNOS) gene into rat kidney in controls and in unilateral ureteral obstruction (UUO). METHODS: The human iNOS gene was inserted into a pcDNA 3.1-backbone plasmid with the FLAG epitope (FLAG-iNOS). In vitro, transduction of FLAG-iNOS was confirmed by Western blot and Griess reaction. In vivo, we transfected either FLAG-iNOS or control plasmid (CMV-LacZ), using cationic liposomes. Urinary nitric oxide metabolites and immunohistochemistry confirmed iNOS transduction. Renal function was also assessed. RESULTS: In vitro, increased iNOS expression was demonstrated in human embryonic kidney (HEK293) cells, along with increased release of nitric oxide metabolites, NO(2)/NO(3). In vivo, FLAG-iNOS was detected by polymerase chain reaction (PCR) up to 35 days after the transfection. Urine collection documented increased urinary NO(2)/NO(3). Immunohistochemistry localized iNOS to collecting ducts, distal tubules, and glomerulus of the injected kidney. Renal function measured up to 21 days after transfection in control animals was not significantly different between the two groups. In contrast, renal function after 24 hours of UUO was significantly improved in FLAG-iNOS-treated animals. CONCLUSION: This study demonstrates the feasibility of liposome-mediated iNOS gene transfer into the kidney. Furthermore, the improvement of renal function in UUO demonstrates that the transfected iNOS gene is active and suggests that decreased iNOS activity contributes to the decreased renal function in UUO. This iNOS construct may have therapeutic utility in the pathophysiologic sequelae of UUO and other renal diseases.

Cells, Cultured↗

PubMed2003

Molecular signatures of urinary cells distinguish acute rejection of renal allografts from urinary tract infection.

Acute rejection (AR) and urinary tract infection (UTI) continue to plague renal transplantation. We tested the hypotheses that UTI does not increase granzyme B mRNA levels in urinary cells, and that the levels distinguish AR from UTI. We measured the levels of granzyme B mRNA in 15 urine specimens from renal allograft recipients with UTI, 29 specimens from patients with AR but without UTI, and 14 specimens from patients without AR and without UTI. We also measured transcript levels in urine specimens from 41 nontransplant individuals, 11 with UTI and 30 without UTI. UTI did not increase granzyme B mRNA levels. Granzyme B mRNA levels were lower in renal allograft recipients with UTI compared with those with AR (P<0.0001). We conclude that bacterial UTI is unlikely to confound AR diagnosis made by measurement of granzyme B mRNA levels in urinary cells.

Acute Disease↗

PubMed2003

Serine proteinase inhibitor-9, an endogenous blocker of granzyme B/perforin lytic pathway, is hyperexpressed during acute rejection of renal allografts.

BACKGROUND: Serine proteinase inhibitor (PI)-9 with a reactive center P1 (Glu)-P1' is a natural antagonist of granzyme B and is expressed in high levels in cytotoxic T lymphocytes (CTL). In view of the role of CTL in acute rejection, we explored the hypothesis that PI-9 would be hyperexpressed during acute rejection. Because PI-9 can protect CTL from its own fatal arsenal and potentially enhance the vitality of CTL, we examined whether PI-9 levels correlate with the severity of rejection as well as predict subsequent graft function. METHODS: We obtained 95 urine specimens from 87 renal allograft recipients. RNA was isolated from the urinary cells and mRNA encoding PI-9, granzyme B, or perforin and a constitutively expressed 18S rRNA was measured with the use of real-time quantitative polymerase chain reaction assay, and the level of expression was correlated with allograft status. RESULTS: The levels of PI-9 (P=0.001), granzyme B (P<0.0001), and perforin mRNAs (P<0.0001), but not the levels of 18S rRNA (P=0.54), were higher in the urinary cells from the 29 patients with a biopsy-confirmed acute rejection than in the 58 recipients without acute rejection. PI-9 levels were significantly higher in patients with type II or higher acute rejection changes compared with those with less than type II changes (P=0.01). Furthermore, PI-9 levels predicted subsequent graft function (r=0.43, P=0.01). CONCLUSIONS: PI-9 mRNA levels in urinary cells are diagnostic of acute rejection, predict renal allograft histology grade, and predict functional outcome following an acute rejection episode.

Acute Disease↗

PubMed2003

CD103 mRNA levels in urinary cells predict acute rejection of renal allografts.

BACKGROUND: CD103 is displayed on the cell surface of alloreactive CD8 cytotoxic T lymphocytes (CTLs) and is a critical component for the intraepithelial homing of T cells. Because intratubular localization of mononuclear cells is a feature of acute cellular rejection of renal allografts, we explored the hypothesis that CD103 messenger (m)RNA levels in urinary cells predict acute rejection. METHODS: We collected 89 urine specimens from 79 recipients of renal allografts. RNA was isolated from the urinary cells, and we measured CD103 mRNA levels and a constitutively expressed 18S ribosomal (r)RNA with the use of real-time quantitative polymerase chain reaction assay. RESULTS: CD103 mRNA levels, but not 18S rRNA levels, were higher in urinary cells from 30 patients with an episode of acute rejection (32 biopsies and 32 urine samples) compared with the levels in 12 patients with other findings on allograft biopsy (12 biopsies and 12 urine samples), 12 patients with biopsy evidence of chronic allograft nephropathy (12 biopsies and 12 urine samples), and 25 patients with stable graft function after renal transplantation (0 biopsies and 33 urine samples) (P = 0.001; one-way analysis of variance). Acute rejection was predicted with a sensitivity of 59% and a specificity of 75% using natural log-transformed value 8.16 CD103 copies per microgram as the cutoff value (P = 0.001). CONCLUSION: CD103 mRNA levels in urinary cells are diagnostic of acute rejection of renal allografts. Because CD103 is a cell surface marker of intratubular CD8 CTLs, a noninvasive assessment of cellular traffic into the allograft may be feasible by the measurement of CD103 mRNA levels in urinary cells.

Acute Disease↗