Soluble klotho as a marker of renal fibrosis and podocyte injuries in human kidneys

Nam-Jun Cho, Dong-Jae Han, Ji-Hye Lee, Si-Hyong Jang, Jeong Suk Kang, Hyo-Wook Gil, Samel Park, Eun Young Lee, Nam-Jun Cho, Dong-Jae Han, Ji-Hye Lee, Si-Hyong Jang, Jeong Suk Kang, Hyo-Wook Gil, Samel Park, Eun Young Lee

Abstract

Klotho deficiency is relevant to renal fibrosis and podocyte injury in vivo and in vitro. We examined whether histological findings of renal biopsy specimens were associated with the levels of soluble klotho in humans. We investigated renal biopsy specimens of 67 patients and detailed microscopic findings were reviewed. Soluble serum/urinary klotho and urinary angiotensinogen were assessed by enzyme-linked immunosorbent assays, and tissue klotho expression was assessed by immunohistochemical staining. The median age of the study participants was 35.6 years. High serum klotho levels (≥14 pg/mL) were associated with decreased odds ratios (ORs) of interstitial fibrosis (OR = 0.019, P = 0.003) and segmental sclerosis (OR = 0.190, P = 0.022) in multivariable logistic regression analysis. Patients with a lower urinary klotho-to-creatinine ratio (UKCR) were significantly more likely to have diffuse foot process effacement (OR = 0.450, P = 0.010). The area under the receiver-operating characteristic curve (AUC) of serum klotho for predicting interstitial fibrosis was 0.920 (95% CI, 0.844-0.996), and the best cut-off value of serum klotho was 138.1 pg/mL. The AUC of UKCR for predicting diffuse foot process effacement was 0.754 (95% CI, 0.636-0.872), and the best cut-off value of UKCR was 96.7 pg/mgCr. Urinary angiotensinogen-to-creatinine ratio was not associated with serum klotho, UKCR, or any pathological finding. Our data suggested that soluble serum and urinary klotho levels represent a potential biomarker to predict renal fibrosis and podocyte injury in humans.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Comparison of serum klotho levels…
Fig 1. Comparison of serum klotho levels in different groups for each pathology.
Boxplots for serum klotho levels are presented according to the severity of each pathologic finding: (A) Interstitial fibrosis, (B) tubular atrophy, (C) segmental sclerosis of glomeruli, and (D) intimal thickening of arterial wall. *P < 0.01; **P < 0.001.
Fig 2. Representative images of Masson’s trichrome-stained…
Fig 2. Representative images of Masson’s trichrome-stained kidney sections according to serum klotho levels.
(A) The specimen from 68-year-old male patient with IgA nephropathy and serum klotho level of 790.9 pg/mL showed nearly intact interstitium and glomerulus. (B) The specimen from a 38-year-old male patient with IgA nephropathy and serum klotho level of 6.4 pg/mL showed marked interstitial fibrosis and tubular atrophy (white arrow), and sclerosing glomerulus (black arrow). Scale bar = 200 μm.
Fig 3. Association of log-transformed urinary klotho-to-creatinine…
Fig 3. Association of log-transformed urinary klotho-to-creatinine ratio with global sclerosis and foot process effacement.
Boxplots for log-transformed urinary klotho-to-creatinine ratio are shown according to the severity of each pathological finding: (A) Global glomerular sclerosis, (B) foot process effacement of podocyte. *P < 0.05; **P < 0.01. UKCR: urinary klotho-to-creatinine ratio.
Fig 4. Representative transmission electron microscopy images…
Fig 4. Representative transmission electron microscopy images according to urinary klotho-to-creatinine ratio.
(A) The specimen obtained from a 55-year-old male patient with IgA neprhopathy and a urinary klotho-to-creatinine ratio of 30.83 pg/mgCr showed preserved foot process of podocyte. (B) The specimen obtained from a 58-year-old male patient with IgA nephropathy and a urinary klotho-to-creatinine ratio of 0.44 pg/mgCr showed diffuse effacement of foot process (arrows). Scale bar = 5 μm.
Fig 5. Receiver-operating characteristic (ROC) analyses for…
Fig 5. Receiver-operating characteristic (ROC) analyses for predicting renal pathology.
ROC curves are shown according to the predictors and outcomes: (A) ROC curve for predicting interstitial fibrosis based on serum klotho, (B) ROC curve for predicting segmental sclerosis based on serum klotho, and (C) ROC curve for predicting foot process effacement based on the urinary klotho-to-creatinine ratio. Best cut-off values were presented as black circles and certain values (with specificity and sensitivity). AUC, area under the ROC curve; UKCR, urinary klotho-to-creatinine ratio.
Fig 6. Comparison of the klotho immunohistochemistry…
Fig 6. Comparison of the klotho immunohistochemistry (IHC) staining score based on each pathologic finding.
Boxplots for klotho IHC staining score are shown according to the severity of each pathologic finding: (A) Interstitial fibrosis and (B) tubular atrophy. IHC staining score for distal tubule were calculated as: percentage of strongly stained distal tubule × 2 + percentage of weakly stained distal tubule.
Fig 7. Representative images of immunohistochemistry (IHC)…
Fig 7. Representative images of immunohistochemistry (IHC) staining for klotho based on adjacent pathological features.
(A) Distal tubules surrounded by normal tubuloinsterstitium showed strong IHC staining for klotho (white arrows). (B) Distal tubules surrounded by inflamed interstitium and atrophic tubules showed absent or weak IHC staining for klotho (black arrows). Scale bar = 100 μm.

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