Low nephron endowment increases susceptibility to renal stress and chronic kidney disease

Preterm birth results in low nephron endowment and increased risk of acute kidney injury (AKI) and chronic kidney disease (CKD). To understand the pathogenesis of AKI and CKD in preterm humans, we generated potentially novel mouse models with a 30%–70% reduction in nephron number by inhibiting or deleting Ret tyrosine kinase in the developing ureteric bud. These mice developed glomerular and tubular hypertrophy, followed by the transition to CKD, recapitulating the renal pathological changes seen in humans born preterm. We injected neonatal mice with gentamicin, a ubiquitous nephrotoxic exposure in preterm infants, and detected more severe proximal tubular injury in mice with low nephron number compared with controls with normal nephron number. Mice with low nephron number had reduced proliferative repair with more rapid development of CKD. Furthermore, mice had more profound inflammation with highly elevated levels of MCP-1 and CXCL10, produced in part by damaged proximal tubules. Our study directly links low nephron endowment with postnatal renal hypertrophy, which in this model is maladaptive and results in CKD. Underdeveloped kidneys are more susceptible to gentamicin-induced AKI, suggesting that AKI in the setting of low nephron number is more severe and further increases the risk of CKD in this vulnerable population.


Supplemental Methods:
Immunohistochemistry, RNA scope, and antibody information: For immunohistochemistry, kidney sections were fixed with 4% PFA in PBS for 4 hours for adults or 24 hours for mice < 2 weeks of age, permeabilized with 0.1% Triton X-100 in PBS for 30 min, then blocked with 10% goat serum and 0.5% bovine serum albumin (BSA) in PBS. Incubations with primary antibodies were carried out at 4 °C overnight. After washing, the sections were further incubated for 1 h at room temperature with the appropriate secondary antibodies. Nuclei were counterstained with DAPI (Vector, H-1200). Sections were visualized with a Zeiss AxioObserver.Z1 inverted fluorescence microscope or Zeiss Axio Imager M2 photographed with a digital camera, and analyzed with Axiovision software or FIJI software.
The following primary antibodies were used: Quantification of phospho-histone 3 (p-H3): Kidney sections were immunostained with antibodies to phospho-histone 3 and co-labeled with LTA conjugated to FITC and entire kidney sections were traced at 5x and imaged at 200x magnification as described above. The number of p-H3 positive cells in the cortex and outer strip of the outer medulla were counted and normalized to surface area using FIJI software.

Antibody Vendor Catalogue Number Dilution
Quantification of Lamp-1 expression: Immunohistochemistry of kidney sections was performed with antibodies to Lamp1 and NK-ATPase and co-labeled with LTA to identify proximal tubules. Fifty random images per kidney were taken at 63x in the cortical region. Proximal tubules were identified as regions of interest. Lamp1 integrated fluorescent density was measured using FIJI software, and corrected for the number of nuclei in identified regions of interest (proximal tubules).
Whole mount immunostaining and image analysis: P1 kidneys were harvested, fixed in 4% PFA at 4 o C for 24 hours, and then transferred to PBS. Immunostaining was performed with primary antibody to calbindin or Six2

Tubular injury score and quantification of tubular injury and renal inflammation: Ten random images
from the cortex to the outer stripe of the outer medulla for each kidney were PAS-stained and scanned at 100x magnification using Aperio ImageScope from Leica Biosystems and FIJI software (NIH) was used to obtain tubular injury scores. A graticule grid was randomly placed on each picture to generate 96 grids.
Histology of tubular profiles of each grid was carefully examined, and a score 0 or 1 was given for each grid: 0=normal histology; 1=tubular vacuolization, tubular atrophy, casts in the tubular lumen, and interstitial inflammatory cells infiltration. The injury score was calculated by adding all 96 grids from each image and all genotypes and treatment.

Supplemental Figures:
Supplemental Figure 1: A. Western blot of protein homogenates from vehicle exposed and NA-PP1 exposed kidneys harvested on E17.5. Pregnant Ret flox-V805A mice were injected daily with vehicle or a small molecule Ret tyrosine kinase inhibitor, NA-PP1, beginning E16.5 daily for 2 days (dose of 62.5 mg/kg) and kidneys were harvested 4 hours after 2 nd injection on E 17.5. Immunoblots were probed with antibodies to Ret or actin as a loading control. Ret antibody recognized both Ret (lower band corresponding to 155 kd) and phopho-Ret (upper band corresponding to 175 kd) and immunoblot revealed kidneys exposed to NA-PP1 had reduced phospho-Ret as well as Ret proteins. Line marks where the membrane was cut for incubation with anti-Ret or anti-actin. B. Distal colon innervation in Ret flox-V805A mice exposed to vehicle and NA-PP1. Pregnant Ret flox-V805A mice were injected daily with vehicle or a small molecule Ret tyrosine kinase inhibitor, NA-PP1, beginning E15.5 daily for 4 days (top panel, dose of 62.5 mg/kg) or beginning E16.5 for 3 days (bottom panel, dose 50 mg/kg). Colonic innervation pattern was assessed in whole mount distal colons immunostained with neurofilament heavy chain (NFH) to label axonal projections and HuC/HuD to label neuronal cell bodies followed by optical clearing. The pattern of enteric nervous system in the distal colon in mice with exposure to NA-PP1 E15.5 was similar to vehicle-exposed mice (top panel). Medium dose (50 mg/kg) NA-PP1 exposure beginning E16.5 resulted in no difference in enteric neuron cell number in the distal colons compared to vehicle exposure (mean ± SD, vehicle 220 ± 30.7 vs. NA-PP1 205 ± 17.5, Welch's t-test, p=0.4 vehicle n=5, NA-PP1 n=4).
Supplemental Figure 2: Ret UB del mice have tubules, glomeruli and capillaries that are similar to controls.
Kidneys from mice exposed to Dox beginning E16.5 were analyzed at 2 weeks of age. Kidneys were stained with proximal tubule marker, LTA (white), and distal tubule marker, TSC (red), shown in top panel. Thick ascending limb is labeled with THP (red) and collecting ducts with DBA (green) shown in middle panel. Renal capillaries and small veins are labeled with endomucin (green) in 6 week old mice after injection of high molecular weight dextran (70 kD). High molecular weight dextran is retained in the glomerular capillary loops and peritubular capillaries. While glomeruli appear larger at this age, dextran retention and peritubular vascular density was qualitatively normal in Ret UB del mice. Figure 3: Adult Ret UB del mice have obsolescent glomeruli and develop pathological changes of CKD. Top, PAS stained kidneys reveal focal obsolescent glomeruli in Ret UB del mice, characterized by small, shrunken and partially solidified capillary tufts (arrow) at 12 weeks of age (imaged at 600x). By 12 weeks Ret UB del mice develop focal tubular atrophy (arrows) with surrounding areas of interstitial fibrosis and chronic inflammatory cell infiltrates (imaged at 400x).

Supplemental
Supplemental Figure 4: Uninephrectomy in adult mice does not cause glomerular hypertrophy but mild proximal tubular enlargement. Eight week old mice with normal nephron endowment (Ret flox-V805A background) underwent sham operation or uninephrectomy. A. PAS stained kidneys 4 weeks after sham or uninephrectomy reveal normal appearing glomeruli and tubules (top panel, imaged at 400x), with no pathologic changes of chronic kidney disease. B. Quantification of glomerular surface area and tubular diameter. Glomerular surface area is not significantly increased after nephrectomy (4.3% larger in nephrectomy compared to controls, CI =-4.1, 13.5 p=0.51, n=4 mice per group). In contrast, tubular diameter is 10.8% larger than controls (CI = 0.12, 17.9) or an average of 3.14 µm larger (CI = 1.09, 5.18), p=0.02, n=4 mice per group. Data was analyzed by mixed-effects regression.
Supplemental Figure 5: Body weight in control and Ret UB del mice exposed to gentamicin from P3-9. Pups were weighed on P10, after 7 days of exposure to 100 mg/kg of gentamicin. Ret UB del mice had significantly lower body weight compared to controls (mean body weight of 3.5g in Ret UB del n=16 compared to 4.3g in controls n=17), *p<0.01, Welch's t-test.
Supplemental Figure 6: Electron micrograph reveal mild focal glomerular injury after gentamicininduced AKI. While the majority of glomeruli appear intact with minimal pathologic changes, there are areas of short-segment, mild foot process effacement in mice examined 1 day after completion of 7 days of gentamicin injection in a Ret UB del mouse.