Optical Clearing in the Kidney Reveals Potassium-Mediated Tubule Remodeling

Abstract

Distal nephron remodeling contributes to the pathophysiology of many clinically relevant scenarios, including diuretic resistance and certain Mendelian disorders of blood pressure. However, constitutive genetic disruptions are likely to have substantial developmental effects in this segment, and whether tubule remodeling upon physiological stimuli is a normal homeostatic mechanism is not known. Since the distal nephron acts as a potassium sensor, we assessed proliferation and tubule length in three dimensions upon dietary or inducible genetic manipulation by using optical clearing of adult mouse kidneys, whole-mount immunolabeling, and advanced light microscopy. We show that dietary potassium restriction leads promptly to proliferation of various nephron segments, including the distal convoluted tubule, whereas disruption of the potassium sensor Kir4.1 causes atrophy, despite ambient hypokalemia. These results provide proof that kidney tubules adapt rapidly to diet and indicate the power of clearing approaches to assess cell number and tubule length in healthy and diseased kidney.

Keywords: AQP2; CLARITY; DCT; Kir4.1; NCC; ethyl cinnamate; hypokalemia; low-potassium diet; optical kidney clearing; tubule remodeling.

Figure 1.. Effects of a 3-Day Low-Potassium Diet

(A) Adult C57BL/6J mice were fed for 3 days with a low-potassium diet or normal-potassium diet and then injected with bromodeoxyuridine (BrdU) intraperitoneally before kidneys were perfusion fixed 4 hr later. (B) Blood potassium levels were lower in mice fed a low-potassium diet than in mice fed a normal-potassium diet. (C) Immunofluorescence of 4-μm-thin sections revealed that a low-potassium diet increased the number of proliferating cells (BrdU [cells in S-phase] and Ki-67 [cells in G1, S, G2, or M-phase]) in the proximal tubule (LTL⁺, arrows) and other tubules or the peritubular space (arrowhead). Scale bar, 100 μm. (D and E) A low-potassium diet induced a strong proliferative response in parvalbumin⁺ early distal convoluted tubule (DCT1) (D) and AQP2⁺ medullary collecting duct (E). (F and G) Quantification of (C). (H) Quantification of (D). (I) Quantification of (E). 12 fields at 100× magnification were quantified from each sample. Data are represented as mean ± SEM (n = 6–7 mice). p values were calculated using an unpaired two-tailed Student’s t test. ISOM, inner stripe of the outer medulla; OSOM, outer stripe of the outer medulla; PV, parvalbumin.

Figure 2.. Optical Clearing and BrdU⁺ Cells in Ethyl-Cinnamate-Cleared Kidney Slices

(A) PFA-fixed kidneys were either sliced in thick slices or not sectioned, and then two different protocols were applied. For CLARITY, tissue was immersed in hydrogel monomer solution, polymerization was induced, and then tissue was cleared. For ethyl cinnamate (ECi), antigen retrieval was performed. In both protocols, tissue was then stained with antibodies or dye and optical clearing was performed using CLARITY (easy clear index solution) or ethyl cinnamate. Confocal and light sheet microscopy were used for volume imaging. Note that different kidneys are shown here. (B and C) Three-dimensional visualization of a representative z stack shows an increased number of BrdU⁺ cells in the cortex (B) and outer medulla (C) in mice on a low-potassium diet compared to mice on a normal-potassium diet. ISOM, inner stripe of the outer medulla; OSOM, outer stripe of the outer medulla. (D and E) Quantification of (B) and (C). Two kidney slices per sample were used, and from each kidney slice, five z stacks were taken from both the cortex (D)and outer medulla (E) (covering ~50% of the outer stripe [OSOM] and 50% of the inner stripe [ISOM]). Imaging was performed with confocal microscopy using a 10× objective lens (Zeiss LSM 880 with Airyscan). Data are represented as mean ± SEM (n = 6 mice). p values were calculated using the unpaired two-tailed Student’s t test. Scale bar, 100 μm. See also Video S1 (outer medulla under low potassium diet).

Figure 3.. BrdU⁺ Cells in AQP2⁺ Tubules

(A) AQP2⁺ tubules in CLARITY-cleared control kidney. Scale bars, 1 mm. (B) AQP2⁺ tubules in control kidney. The CLARITY protocol was first applied, and then tissue was washed, dehydrated, and cleared with ethyl cinnamate. Scale bar, 200 μm. (C–D) Ethyl cinnamate-cleared kidney slices from mice on 3-day normal (C) or low (D) potassium diet were co-stained for AQP2 and BrdU. (E–G) Data analysis process to determine BrdU⁺ cells within the AQP2⁺ medullary collecting duct in mice on low potassium diet. (H) Quantification of BrdU⁺ cells within the AQP2⁺ medullary collecting duct in mice on normal (C) or low (D) normal and low-potassium diets. Two kidney slices per sample were used, and from each kidney slice, five z stacks were taken from the outer medulla (covering ~50% of the outer stripe [OSOM] and 50% of the inner stripe [ISOM]). Imaging was performed with confocal microscopy using a 10× objective lens (Zeiss LSM 880 with Airyscan). Data are represented as mean ± SEM (n = 3 mice). p values were calculated using an unpaired two-tailed Student’s t test. (I and J) A representative z stack shows an absence of nonspecific binding of secondary antibody (I, donkey anti-mouse Cy5 [pseudocolor: red]; J, donkey anti-goat Cy3 [pseudocolor: green]) without previous incubation in primary antibody. Scale bars, 100 μm. Imaging was performed with a Zeiss Light Sheet Z1 using a 5× objective lens (A) and confocal microscopy using a 10× objective lens (B–G). See also Videos S2 and S3.

Figure 4.. Quantification of DCT Length in Control and Kir4.1 Knockout Mice

(A) Visualization of pThr53-NCC⁺ DCTs and all nuclei using propidium iodide (PI) in whole optical cleared control kidney. Glomeruli are visible as bright red dots due to a higher density of PI⁺ nuclei within glomeruli than surrounding tissue. Scale bar, 1 mm. (B) One slice of a z stack showing apical staining of DCT and all nuclei using pThr53-NCC and PI. Scale bar, 100 μm. (C) Low-magnification visualization of pThr53-NCC⁺ DCTs. The z stack was taken along the cortical kidney surface. Scale bar, 300 μm. (D) High-magnification visualization shows individual DCTs. The length was measured after three dimensional rendering using measurement points in Imaris software (white lines). Scale bar, 50 μm. (E) Quantification of (D) shows that Kir4.1 deletion is associated with a shortening of the DCT compared to controls (CTRL). (F) One slice of a z stack showing all nuclei using PI and absence of nonspecific binding of secondary antibody (Ab) (donkey anti-rabbit Cy5) without previous incubation in pThr53-NCC antibody. Scale bar, 100 μm. (G) No signal was detected in secondary antibody-only experiments (donkey anti-rabbit Cy5; pseudocolor: green) in ethyl cinnamate-cleared kidneys. Scale bar, 100 μm. 7–10 z stacks were taken with a 20× objective lens from the cortex of each kidney slice, and two kidney slices per animal were used. Data are represented as mean ± SEM (n = 3 mice). p values were calculated using an unpaired two-tailed Student’s t test. Imaging was performed with Zeiss Light Sheet Z1 using 5× (A) and 20× objective lenses (B–D and F). Zeiss LSM 880 confocal microscopy was used to take the image shown in (G). See also Video S4.