Journal: International Journal of Molecular Sciences

Article Title: Renal Tubule-Specific Deletion of Nephrocystin 3 (Nphp3) Causes Infantile Nephronophthisis-like Phenotypes in Mice

doi: 10.3390/ijms27062687

Figure Lengend Snippet: Renal cysts in Cdh16-Cre ; Nphp3 flox/flox mice originate from the distal tubules and collecting ducts and can be rescued by tolvaptan and 2-(2-chloro-4-iodoanilino)-N-(cyclopropylmethoxy)-3,4-difluorobenzamide (CI-1040) treatment. ( A ) Immunofluorescence staining of kidneys from Cdh16-Cre ; Nphp3 flox/flox mice showing the staining of proximal tubules (LTL-FITC), distal convoluted tubules (SLC12A3-CY3), and collecting ducts (AQP2-CY5). These results indicate that renal cysts in these mice originate from the distal tubules and collecting ducts. White arrows indicate renal cysts. ( B ) Zonula occludens-1(ZO-1) immunofluorescence staining of the kidneys of 2-week-old mice, showing changes in epithelial cell polarity and disruption of epithelial integrity in the tubular walls, particularly the cyst walls, of Cdh16-Cre ; Nphp3 flox/flox mice, as indicated by the white arrows ( n = 3 mice/group). The blue fluorescence indicates cell nuclei stained with DAPI, while the green fluorescence shows the ZO-1 protein. ( C ) In 2-week-old Cdh16-Cre ; Nphp3 flox/flo x mice, renal tubular epithelial cells (particularly cyst-lining cells) exhibited a significant increase in Ki-67 positivity compared to control mice. ( D ) The gene expression levels of renal tubular injury markers (kidney injury molecule-1[KIM-1] and neutrophil gelatinase-associated lipocalin [NGAL]) were examined in 1 and 2-week-old mice. The results show that the expression of these tubular injury markers was significantly upregulated in Cre ; Nphp3 flox/flo x mice ( n = 3 mice/group). ( E ) The cyclic adenosine monophosphate (cAMP) content per unit weight of kidney tissue was measured in 1 and 2-week-old mice. The results show a certain degree of increase in cAMP levels in the kidneys of Cdh16-Cre ; Nphp3 flox/flo x mice at 1 week of age, and this increase became more pronounced and statistically significant at 2 weeks ( n ≥ 3 mice/group). ( F ) H&E-stained kidney sections (20×) from Cdh16-Cre ; Nphp3 flox/flox mice treated with tolvaptan or CI-1040. Compared with the vehicle control treatment, the administration of tolvaptan (5 mg/kg) or CI-1040 (10 mg/kg) significantly reduced renal cyst formation. In Cdh16-Cre ; Nphp3 flox/flox mice, treatment with either tolvaptan or CI-1040 significantly decreased the kidney KW/BW ratio, increased the number of glomeruli per maximal renal cross-section, and reduced the glomerular cross-sectional area ( n ≥ 3 mice/group). ( G ) Under untreated conditions, the p-ERK level in the kidneys of Cdh16-Cre ; Nphp3 flox/flox mice was significantly higher than that in Nphp3 flox/flox mice. The administration of CI-1040 (10 mg/kg) led to a significant reduction in renal phospho-ERK (p-ERK) levels in Cdh16-Cre ; Nphp3 flox/flox mice. The proportion of p-ERK-positive cells was significantly reduced following CI-1040 treatment ( n = 3 mice/group). Statistical significance was defined as * p < 0.05; *** p < 0.001; and p > 0.05 (NS [not significant]) using an unpaired two-tailed Student’s t -test for two-group comparisons.

Article Snippet: The primary antibodies used were as follows: NPHP3 (Proteintech, Cat No.: 22026-1-AP, dilution 1:200), p-ERK (Proteintech, Cat No.: 80031-1-RR, dilution 1:200), SLC12A3-CY3 (Affinity, Cat No.: AF5101, dilution 1:100, Changzhou, China), LTL-FITC (Vector Labs, Cat No.: B-1325, dilution 1:50, Newark, CA, USA), and AQP2-CY5 (Affinity, Cat No.: DF7560, dilution 1:100), CD11B-CY3 (abcam, ab133357, dilution 1:1000, Cambridge, UK), ZO-1 (Proteintech, Cat No.: 21773-1-AP, dilution 1:200), and Ki-67 (HuaBio, Cat No.: 28074-1-AP, dilution 1:200, Hangzhou, China).

Techniques: Immunofluorescence, Staining, Disruption, Fluorescence, Control, Gene Expression, Expressing, Two Tailed Test