Non-invasive method of diagnosing renal fibrosis
Measurement of mRNAs in urinary cells offers a noninvasive means of diagnosing fibrosis in kidneys. One aspect of the invention is a method that includes: (a) measuring quantities of vimentin mRNA, NKCC2 mRNA, and E-cadherin mRNA in a test sample of cells obtained from urine; and (b) determining whether the vimentin mRNA quantity is higher, the NKCC2 mRNA quantity is lower, or the E-cadherin mRNA is higher than in healthy urinary cells; and thereby detecting that the sample is a fibrotic kidney sample. Step (a) can also include measuring the quantity of RNA expressed by a housekeeping gene (e.g., 18S rRNA). The quantities of vimentin mRNA, NKCC2 mRNA, and E-cadherin mRNA can be normalized against the quantity of housekeeping gene RNA.
1. A method comprising: treating kidney fibrosis in a subject with a kidney transplant having renal fibrosis, wherein the method comprises:
quantitative nucleic acid amplification measurement of sample RNA from urinary cells of the subject using primers specific for vimentin, NKCC2, E-cadherin, and 18S rRNA;
calculating a composite score as follows:
Composite
Score
=
36.10283
+
[
-
15.84215
*
log
10
(
18
s
RNA
)
]
+
1.56907
*
log
10
(
18
s
RNA
)
*
log
10
(
18
s
RNA
)
+
5.11698
*
max
[
0
,
log
10
(
Vimentin
)
-
5.6
]
+
[
-
1.44145
*
log
10
(
NKCC
2
)
]
+
3.31357
*
min
[
3.1
,
log
10
(
E
-
cadherin
)
]
where,
log 10 (18s RNA) is log 10 of 18S RNA quantity/10 5 in the test sample;
log 10 (Vimentin) is log 10 of a normalized vimentin mRNA quantity in the test sample;
log 10 (NKCC2)] is log 10 of a normalized NKCC2 mRNA quantity in the test sample;
log 10 (E-cadherin) is log 10 of a normalized E-cadherin mRNA quantity in the test sample;
max[0, log 10 (Vimentin)−5.6]=0 whenever log 10 (Vimentin) is ≤5.6 and =log 10 (Vimentin−5.6 whenever log 10 (Vimentin) is >5.6;
min[3.1, log 10 (E-cadherin)]=log 10 (E-cadherin) whenever log 10 (Ecadherin)<3.1 and =3.1 whenever log 10 (E-cadherin)≥3.1; and * signifies multiplication; and
removal of the kidney transplant and transplantation of a second kidney transplant, or administering one or more anti-inflammatory agent, anti-coagulant, antioxidant, blood pressure medication, angiotensin-converting enzyme inhibitor, angiotensin AT1 receptor blocker, connective tissue growth factor inhibitor, or antifibrotic agent, when the composite score is 4.7 or more to thereby treat kidney fibrosis in a subject.
2. The method of claim 1 , comprising: treating kidney fibrosis in the subject when the sample RNA of a test sample of urinary cells from the subject has:
(i) a log 10 ([vimentin]/[18S rRNA]/10 5 ) value greater than about 5.0;
(ii) a log 10 ([NKCC2]/[18S rRNA]/10 5 ) value less than about 3.0; or
(iii) a log 10 ([E-cadherin]/[18S rRNA]/10 5 ) value greater than about 3.0.
3. The method of claim 1 wherein the composite score varies from about 1 to 8, and a normal composite score is about 3.5.
4. The method of claim 1 , wherein a test sample with a composite score of more than about 4.7 indicates a subject has fibrosis.
5. The method of claim 1 , wherein performing quantitative nucleic acid amplification comprises use of a probe or primer that can stringently hybridize to a nucleic acid comprising SEQ NO:11.