Discovery and validation of a molecular signature for the noninvasive diagnosis of human renal allograft fibrosis.

BACKGROUND: Tubulointerstitial fibrosis (fibrosis), a histologic feature associated with a failing kidney allograft, is diagnosed using the invasive allograft biopsy. A noninvasive diagnostic test for fibrosis may help improve allograft outcome. METHODS: We obtained 114 urine specimens from 114 renal allograft recipients: 48 from 48 recipients with fibrosis in their biopsy results and 66 from 66 recipients with normal biopsy results. Levels of messenger RNAs (mRNAs) in urinary cells were measured using kinetic, quantitative polymerase chain reaction assays, and the levels were related to allograft diagnosis. A discovery set of 76 recipients (32 with allograft fibrosis and 44 with normal biopsy results) was used to develop a diagnostic signature, and an independent validation set of 38 recipients (16 with allograft fibrosis and 22 with normal biopsy results) was used to validate the signature. RESULTS: In the discovery set, urinary cell levels of the following mRNAs were significantly associated with the presence of allograft fibrosis: vimentin (P<0.0001, logistic regression model), hepatocyte growth factor (P<0.0001), α-smooth muscle actin (P<0.0001), fibronectin 1 (P<0.0001), perforin (P=0.0002), plasminogen activator inhibitor 1 (P=0.0002), transforming growth factor ß1 (P=0.0004), tissue inhibitor of metalloproteinase 1 (P=0.0009), granzyme B (P=0.0009), fibroblast-specific protein 1 (P=0.006), CD103 (P=0.02), and collagen 1A1 (P=0.04). A four-gene model composed of the levels of mRNA for vimentin, NKCC2, and E-cadherin and of 18S ribosomal RNA provided the most accurate, parsimonious diagnostic model of allograft fibrosis with a sensitivity of 93.8% and a specificity of 84.1% (P<0.0001). In the independent validation set, this same model predicted the presence of allograft fibrosis with a sensitivity of 77.3% and a specificity of 87.5% (P<0.0001). CONCLUSIONS: Measurement of mRNAs in urinary cells may offer a noninvasive means of diagnosing fibrosis in human renal allografts.

Renal tubular dysfunction in patients with American cutaneous leishmaniasis.

Renal dysfunction seen in patients with American cutaneous leishmaniasis (ACL) has been attributed to the use of antimonials for treatment. To determine whether ACL itself causes tubular dysfunction, we measured renal function in 37 patients with ACL prior to their treatment and compared results to that in 10 healthy volunteers of similar mean age. None of the patients presented with glomerular dysfunction; however, 27 had a urinary concentrating defect. There was no statistical difference between groups in the pre- and post-desmopressin test of urine osmolality, but the post-test urine osmolality of the controls was significantly higher. Urinary AQP2 levels, determined by western blot of isolated exosomes, were found to be significantly lower in patients than in controls, whereas that of the cotransporter (NKCC2) was significantly higher. A urinary acidification defect (post-test pH greater than 5.50 following calcium chloride) was found in 15 patients. Pretest plasma bicarbonate was below normal in 12 patients as was the pretest plasma pH in 14. Expression of the Na/H exchanger (NHE3), H(+)-ATPase, and pendrin were all significantly higher in patients with ACL than in controls. A combined urinary concentration and acidification defect was found in 12 patients. Thus, the urinary concentrating defect of ACL may be caused by decreased AQP2, with increased NKCC2 compensatory. Pendrin upregulation may be related to the urinary acidification defect with increased NHE3 and H(+)-ATPase also compensatory. Hence, ACL can cause asymptomatic renal tubular dysfunction.

Reduced urinary excretion of thiazide-sensitive Na-Cl cotransporter in Gitelman syndrome: preliminary data.

BACKGROUND: The relationship between SLC12A3 mutations and actual sodium-chloride (Na-Cl) cotransporter (NCC) expression in patients with Gitelman syndrome (GS) was rarely evaluated. Detection of urinary thiazide-sensitive NCC was not tried in patients with GS. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: 6 patients with GS and 1 patient with surreptitious vomiting. OUTCOMES & MEASUREMENTS: Renal clearance study, mutation analysis using reverse-transcription polymerase chain reaction and direct sequencing for the SLC12A3 gene, and immunohistochemical staining for NCC, Na-K-2Cl-cotransporter, alpha1-subunit of Na(+),K(+)-ATPase, and calbindin-D(28K) of the renal biopsy specimens were performed. Membrane fractions of urine were obtained by using differential centrifugation and probed with antibodies against human NCC and aquaporin 2. RESULTS: Results of clearance studies were consistent with GS, showing decreased distal fractional chloride reabsorption with only furosemide. SLC12A3 gene mutations were found in all patients with GS. Immunohistochemistry showed markedly decreased NCC expression in the distal convoluted tubule, whereas expression of other transporters remained intact. Urinary NCC excretion was markedly decreased in patients with GS, but not in the patient with surreptitious vomiting. LIMITATIONS: Small number of patients and lack of mutation analysis of CLCNKB. CONCLUSIONS: There were no relations between NCC expression and types of mutations. Detection of urinary NCC might be helpful for the differential diagnosis of GS.