Measurement of glomerular filtration rate reveals that subcapsular injection of shear‐thinning hyaluronic acid hydrogels does not impair kidney function in mice

Abstract The continued development of minimally invasive therapeutic implants, such as injectable hydrogels, necessitates the concurrent advancement of methods to best assess their biocompatibility via functional outcomes in vivo. Biomaterial implants have been studied to treat kidney disease; however, assessment of biocompatibility has been limited to biomarker and histological assessments. Techniques now exist to measure kidney function serially in vivo in murine studies via transcutaneous measurements of glomerular filtration rate (tGFR). In this study, adult male and female wild‐type BalbC mice underwent right unilateral nephrectomy. The remaining solitary left kidney was allowed 4 weeks to recover via compensatory hypertrophy, after which subcapsular injection of either saline or shear‐thinning hyaluronic acid hydrogel was performed. Serial tGFR measurements before and after treatment were used to assess the effect of hydrogel injection on kidney filtration. Urine and serum biomarkers of kidney function, and kidney histology were also quantified. Hydrogel injection did not affect kidney function, as assessed by tGFR. Results were in agreement with standard metrics of serum and urine biomarkers of injury as well as histological assessment of inflammation. The model developed provides a direct functional assessment of implant compatibility for the treatment of kidney disease and impact on kidney function.


| INTRODUCTION
Injectable hydrogels have been widely investigated for biomaterial applications for their facile delivery of growth factors and cell-based therapies. [1][2][3][4][5][6][7][8][9] Numerous studies have been published investigating injectable hydrogel systems for the treatment of kidney disease in preclinical models. Various hydrogel systems have been utilized, including chitosan, collagen, polyethylene-glycol, gelatin, and hyaluronic acid (HA)-based gels to deliver cytokines, growth factors, and cell-based therapies in models of acute and chronic kidney disease. [10][11][12][13][14][15][16][17][18][19][20] As with any biomaterial application, the safety and biocompatibility of the system are of paramount importance for translational investigation. When utilized in the treatment of kidney disease, preclinical studies to-date have based determinants of hydrogel biocompatibility on histological outcomes and biomarkers of kidney function. Histology has been used to determine the inflammatory response to the biomaterial.
Serum biomarkers of kidney function, such as blood urea nitrogen (BUN) and serum creatinine (SCr) have been used to assess the effect of the hydrogel on kidney function. Kidneys filter BUN and creatinine, excreting these solutes into the urine, so a rise in either of these biomarkers can indicate a decline in kidney function. BUN and SCr are clinically relevant and commonly utilized, however, they have significant limitations when it comes to assessing kidney function. Both BUN and SCr are poor predictors of actual glomerular filtration rate (GFR) and their accuracy is dependent upon the assay utilized. 21,22 GFR measures the rate of plasma filtered by the kidneys per minute. In animal models with two kidneys, the GFR reflects the filtration of both kidneys combined. In a solitary kidney model, the GFR reflects the filtration rate of the solitary kidney. A decrease in GFR is used to define acute kidney injury and chronic kidney disease. While serum biomarkers such as BUN and SCr are commonly utilized to estimate changes in GFR, their ability to detect kidney injury is not sensitive. [23][24][25] Measurements of GFR remain the gold standard for assessment of kidney function, however, such measurements are timely, invasive, and costly. 26 The National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) published recommendations on improving the translatability of animal models of kidney disease and highlighted the importance of accurate measurement of kidney function. 27 There is now a commercially available method to measure GFR in rats and mice via minimally invasive transcutaneous measurement. 28,29 We have previously demonstrated the ability to measure the GFR serially for 1 year in a murine model of bilateral ischemia-reperfusion acute kidney injury. 30 Specifically germane to unilateral hydrogel therapy, we have measured the GFR of one kidney of interest by performing a contralateral nephrectomy and allowing the kidney of interest to recover for 4 weeks. Following the unilateral contralateral nephrectomy, the isolated kidney of interest begins to hyperfilter in order to compensate for the increased workload. We have shown that 4 weeks of recovery provides adequate time for the solitary kidney to provide normal GFR following ischemia-reperfusion injury. 31 To-date, measured GFR has not been included in the assessment of the biocompatibility of biomaterial applications to treat kidney disease. We have previously utilized our injectable HA hydrogels to mitigate kidney disease progression in both acute and chronic kidney disease models in mice. 18,19 We have demonstrated that HA does not negatively affect either biomarkers of kidney function (BUN and SCr) nor renal histology. Indeed, HA hydrogels have mitigated the progression of kidney fibrosis in both ischemic and obstructive murine models of kidney disease. The purpose of this study was to demonstrate a method by which kidney function may be directly measured after treatment with our injectable HA hydrogel system in preclinical murine models. We hypothesized that delivery of HA hydrogel under the kid-

| Measurement of transcutaneous glomerular filtration rate
Serial transcutaneous glomerular filtration rates (tGFR) were measured as previously described. 31 Briefly, FITC-sinistrin was administered intravenously via retro-orbital injection and the renal clearance

| Unilateral nephrectomy
On study Day 0, a right unilateral nephrectomy was performed as previously described. 31 The animals were given 4 weeks to recover postoperatively in order to allow for compensatory hypertrophy of the remaining left solitary kidney.

| Delivery of injectable hydrogel under the left kidney capsule
On study Day 28, the animals received one of the three following were formulated and subcapsular injections were performed as previously described. 18,19 In brief, HA (74 kDa) was pendently modified by either adamantane (Ad-HA) or β-cyclodextrin (CD-HA), with each component having an average of 20%-25% of disaccharides modified by the pendant group. 32 For optical imaging studies, Ad-HA was further labeled by the near-IR dye Cy7.5 via methacrylation of CD-HA and subsequent Michael-addition with a fluorophoreterminated peptide containing a cysteine residue (GCKKG-Cy7.5). 33 Hydrogels were prepared by dissolution of the polymers at 3.5 wt % in sterile saline and mixing to afford a shear-thinning and injectable hydrogel.

| Serial in vivo optical imaging
Hydrogel localization under the kidney capsule was verified by dorsal and lateral images, while hydrogel quantification was performed on the lateral images. Hydrogel degradation was tracked via optical imaging utilizing the Pearl (LI-COR) small animal imaging machine, as previously described. 16,18 Signal intensity was measured in photons/pixel/second and normalized for maximum intensity per animal. Animals treated with HA hydrogel underwent serial optical imaging daily for 3 days, then again on Day 33 and prior to sacrifice on study Day 35.

| Histological quantification of kidney fibrosis
Kidney tissue was formalin-fixed, paraffin embedded, and sectioned (4 μm thickness) for staining. Kidney fibrosis was assessed by staining and quantifying via picrosirius red as previously described 31 ; hydroxyproline content was measured as previously described. 31 Only the left treated kidneys were analyzed; the right nephrectomized kidneys were flash frozen.

| Statistics
Inference of the means assuming normal distribution was used to determine the number of mice required per cohort in order to detect a 15% change in measured tGFR with 85% power, p < .05; each experimental cohort contains n = 7 males and n = 7 females. T-tests were utilized to compare measures between two groups, assuming  Figure 2 shows the sustained degradation of HA after injection, with no difference between male and female cohorts.
One day after the right nephrectomy (Day 1), there were no statistically significant differences in serum biomarkers of kidney function (Cystatin C, SCr, and BUN) ( Figure S1). There was no difference in cystatin C ( Figure 3A) or SCr ( Figure 3B Urine was collected serially when available. Earlier time points were underpowered, however, the available data are shown in Figure S2. At sacrifice on Day 35 urine KIM-1 levels were increased in Saline F compared to both Saline M and HA F groups ( Figure 4A). There was no difference in urine NGAL levels amongst the cohorts ( Figure 4B).  Figure 5B), nor hydroxyproline content ( Figure 5C).

| DISCUSSION
We have previously utilized our injectable HA hydrogel to provide unilateral therapy in murine models of acute and chronic kidney disease and have shown histological improvement in groups treated with HA hydrogel alone. 18,19 These effects may be due in part to the cytoprotective, antifibrotic, and immunomodulatory effects of HA itself, observed by ourselves and others. 35 Our study has numerous strengths. The addition of tGFR measurements to standard serum and urine biomarker and histology assessment aligns with the NIDDK's recommendation on overcoming barriers in translational research for kidney disease. 27 Likewise, by including both male and female mice and presenting the data unpooled by sex, our study aligns with current recommendations to improve scientific rigor and transparency in translational research. 34 Sex hormones have been demonstrated to effect outcomes in ischemia-reperfusion acute kidney injury, nephrotoxic-mediated kidney injury, and the development of chronic kidney disease. 11,[38][39][40] Overall, our data suggest that sex does not affect renal outcomes after HA delivery under the kidney capsule in healthy mice.

| CONCLUSION
In conclusion, we have described a quantitative way to measure the effect that injectable hydrogels have on GFR in murine models. Unilateral treatment of hydrogel to one kidney can be assessed after performing a contralateral unilateral nephrectomy. Assessment of measured kidney function is an important marker of biocompatibility for biomaterial systems used to treat kidney disease and should be incorporated into the assessment of preclinical treatment models in addition to standard biomarkers and histology. Direct in vivo assessments of kidney function can ensure safety and efficacy beyond that of biomarkers and histological outcome.