Characteristics of enriched heterogeneous renal cell populations

Abstract

Characteristics of enriched heterogeneous renal cell populations useful to identify them as having therapeutic potential, to identify them as having bioactivity or durable bioactivity in a patient, or to guide dosing of a patient in need of treatment for kidney disease.

Description

CHARACTERISTICS OF ENRICHED HETEROGENEOUS RENAL CELL POPULATIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 598,467 filed on November 13, 2023, the disclosure of which is incorporated by reference herein in its entirety, including any drawings.BACKGROUND

[0002] The incidence and prevalence of chronic kidney disease (CKD) are increasing given the growing diabetes and metabolic syndrome epidemics (Thurlow JS, et al., Am J Nephrol. 2021; 52:98-107). Left untreated, CKD can progress to end-stage renal disease (ESRD) and necessitate the need for renal replacement therapy. Dialysis, associated with increased morbidity and mortality, not only remains a bridge to transplantation but also represents a significant burden on the federal health exchequer (Thurlow JS, et al., Am J Nephrol. 2021; 52:98-107; Murphy D, et al., Intern Med 2016; 65:473-481; Hill NR, et al., PLoS One 77:e0158765. doi: 10.1371 / joumal.pone.0158765; Glassock RJ, et al., Nat Rev Nephrol. 2017; 75:104-114; Migliori M, et al., J Nephrol. 2017, 50:373-383). Considering that patients often become refractory to therapeutic regimens that address disease etiology, there is an urgent need for modalities that repair renal microarchitecture, restore kidney function, and delay or avert the need for dialysis.

[0003] Autologous cell therapy employs an individual’s cells, which typically are cultured and expanded outside the body, and then re-introduced into the individual. Advantages of such an approach include reduced risks for alloimmune events, bioincompatibility, and disease transmission (Kazmi B, et al., Wounds 2009; 9:234-242). Rilparencel, which has also been described as selected renal cells (SRC) or REACT®, is an enriched heterogeneous renal cell population being advanced as autologous cell-based therapy for treatment of CKD (Stavas J, et al., A J Nephrol 53(2022):50-58.; Stavas J, et al., KI Reports 2022; 7:1619- 1629). Results from a Phase II clinical trial in a diabetic kidney disease cohort suggested that SRC administration has an acceptable safety profile and may improve renal function (Stavas J, et al., KI Reports 2022; 7:1619-1629).

[0004] There is a need in the art to identify enriched heterogeneous renal cell populations, such as rilparencel, that have therapeutic potential to treat CKD patients, preventing the CKD patients’ progression to ESRD. Further, there is a need in the art to be able to assess suchenriched heterogeneous renal cell populations, once administered to CKD patients, to ensure that cells of the populations are bioactive in the patient and remain bioactive in the patient, e.g., which may guide subsequent or re-dosing the patient with the enriched heterogeneous renal cell populations.BRIEF SUMMARY

[0005] The present disclosure describes a method of identifying a therapeutic composition as having bioactivity in a chronic kidney disease (CKD) patient in need of treatment with the therapeutic composition. In the method, level of serum bicarbonate in the CKD patient is determined at a first and a second time point. The first time point occurs prior to the second time point and the second time point occurs following administration of a first dose of the therapeutic composition. The therapeutic composition is identified as having bioactivity in the CKD patient if the level of serum bicarbonate in the patient is higher at the second time point than at the first time point. The therapeutic composition includes an enriched heterogeneous renal cell population.

[0006] The present disclosure also describes a method of improving kidney fiinction in a human CKD patient. In the method, at least a first, a second and a third dose of a therapeutic composition are administered to the human CKD patient. The second dose is administered about two to twelve months following the first dose. The third dose is administered: (i) following the second dose, and (ii) within about three months after detection of a dose trigger event. The dose trigger event is a decline from maximum serum bicarbonate level in the human CKD patient following administration of the second dose. The therapeutic composition includes an enriched heterogeneous renal cell population.

[0007] The present disclosure further describes a method of assessing durability of bioactivity of a therapeutic composition administered to a human patient in need of treatment for chronic kidney disease (CKD). In the method, the level of serum bicarbonate in the CKD patient is monitored. The monitoring includes determining level of serum bicarbonate in the CKD patient over two or more time points of a time interval. The two or more time points includes at least a first and a final time point. The first time point is, at its earliest, day of administration of a first dose of the therapeutic composition, and the final time point is, at its latest, 42 months following administration of the first dose of the therapeutic composition. Bioactivity of the therapeutic composition is assessed as being durable if the level of serum bicarbonate of the CKD patient successively increases over the two or more time points ofthe time interval. The therapeutic composition includes an enriched heterogeneous renal cell population and had been administered by injection into the renal cortex of at least one kidney of the patient.

[0008] The present disclosure further yet describes a method of identifying an enriched heterogeneous renal cell population as having a therapeutic potential. In the method: (i) expression of a sodium bicarbonate cotransporter, e.g., slc4A4, by cells of the enriched heterogeneous renal cell population is determined; and / or (ii) uptake of sodium bicarbonate by cells of the enriched heterogeneous renal cell population is determined. The enriched heterogeneous renal cell population is identified as having therapeutic potential if it is determined that: (i) greater than 60% of cells of the enriched heterogeneous renal cell population express slc4a4; and / or (ii) cells of the enriched heterogeneous renal cell population uptake bicarbonate at a level higher than cells of a control renal cell population.BRIEF DESCRIPTION OF THE FIGURES

[0009] FIG. 1: Percentage change in serum bicarbonate in stage 3a - 4 CKD patients treated with 2 doses of an enriched heterogeneous renal cell population, REACT® (rilparencel), over time. The patient’s baseline value for determining percentage change was the patient’s serum bicarbonate level 14 days prior to administration of a first REACT® (rilparencel) dose.

[0010] FIG. 2: Percentage change in serum bicarbonate in stage 3a - 4 CKD patients treated with 2 doses of an enriched heterogeneous renal cell population, REACT® (rilparencel), over time. The patient’s baseline value for determining percentage change was the patient’s serum bicarbonate level on the day of administration of a first REACT® (rilparencel) dose.

[0011] FIG. 3 : Absolute change in serum bicarbonate in stage 3a - 4 CKD patients treated with 2 doses of an enriched heterogeneous renal cell population, REACT® (rilparencel), over time. The patient’s baseline value for determining change was the patient’s serum bicarbonate level 14 days prior to administration of a first REACT® (rilparencel) dose.

[0012] FIG. 4 : Absolute change in serum bicarbonate in stage 3a - 4 CKD patients treated with 2 doses of an enriched heterogeneous renal cell population, REACT® (rilparencel), over time. The patient’s baseline value for determining change was the patient’s serum bicarbonate level on the day of administration of a first REACT® (rilparencel) dose.

[0013] FIG. 5: Percentage change in serum bicarbonate in stage 3b - 4 CKD patients treated with 2 doses of an enriched heterogeneous renal cell population, REACT® (rilparencel), overtime. The patient’s baseline value for determining percentage change was the average of the patient’s serum bicarbonate level at day of biopsy and 14 days prior to administration of a first REACT® (rilparencel) dose.

[0014] FIG. 6: Percentage change in serum bicarbonate in patients having stage 3b - 4 CKD and type 1 or 2 diabetes treated with 2 doses of an enriched heterogeneous renal cell population, REACT® (rilparencel), over time. The patient’s baseline value for determining percentage change was the average of the patient’s serum bicarbonate level at day of biopsy and on the day, but prior to administration, of a first REACT® (rilparencel) dose.

[0015] FIG. 7: Percentage change in serum bicarbonate in patients having stage 3b - 4 CKD and type 1 or 2 diabetes treated with 2 doses of an enriched heterogeneous renal cell population, REACT® (rilparencel), over time. The patient’s baseline value for determining percentage change was the patient’s serum bicarbonate level approximately 3 months after administration of a first REACT® (rilparencel) dose, but before administration of a second REACT® (rilparencel) dose.DETAILED DESCRIPTION

[0016] Described herein are methods of identifying a therapeutic composition as having bioactivity in a chronic kidney disease (CKD) patient in need of a treatment with the therapeutic composition, methods of assessing durability of bioactivity of a therapeutic composition administered to a human patient in need of treatment for CKD, methods of improving kidney function in a human CKD patient, and methods of identifying an enriched heterogeneous renal cell population as having therapeutic potential.

[0017] In the methods of identifying a therapeutic composition as having bioactivity in a CKD patient in need of treatment with the therapeutic composition, the CKD patient in need of treatment with the therapeutic composition may be in need of treatment of a kidney disease, a tubular transport deficiency, or a glomerular filtration deficiency and the patient’s CKD may originate in the kidney or result from any condition, e.g., diabetes or hypertension or a congenital anomaly. The CKD patient in need of treatment with the therapeutic composition may have the kidney disease and metabolic acidosis. The CKD patient may be at any stage kidney disease, CKD stage I, II, Illa, Illb, or IV

[0018] In the methods of identifying a therapeutic composition as having bioactivity in a CKD patient, the CKD patient’s level of serum bicarbonate may be determined, and may be determined at first and second time points. Determining levels of serum bicarbonate in theCKD patient may be by any method known in the art. In some instances, serum bicarbonate level in the patient may be determined by a photometric / enzymatic assay. Examples of reagents / kits that may be used to determine serum bicarbonate levels via a photometric / enzymatic assay include the DiaSys Diagnostic Systems GmbH, Bicarbonate FS* kit (Cat. No. 1 095099 10 021); Roche bicarbonate reagent (for use with Roche Cobase 6000 Chemistry Analyzer); Gcell Bicarbonate Assay Kit (CO2) (Cat. Nos. GB460E, GB460E / B, GB461E, GB461E / B); and Beckman Coulter Bicarbonate system reagent (Cat. Nos. OSR6137, OSR6237, OSR6537), which is for use with Beckman Coulter AU analyzers. See also Forrester, et al., Clin. Chem. 27(1975): 1093-1101. In some instances, levels of serum bicarbonate may be determined as part of an electrolyte and / or metabolite panel.

[0019] The first and second time points, at which the CKD patient’s level of serum bicarbonate may be determined may be such that the first time point occurs prior to the second time point, and the second time point occurs following administration of a first dose of the therapeutic composition. In some instances, then, the first time point may occur prior to administration of a first dose of the therapeutic composition and the second time point may occur after administration of the first dose of the therapeutic composition. In other instances, both the first and the second time point may occur after administration of the first dose of the therapeutic composition.

[0020] In instances in which the first time point occurs prior to administration of a first dose of the therapeutic composition, and the second time point occurs after administration of the first dose of the therapeutic composition, the first time point may occur between about three months to about the day of but before administration of a first dose of the therapeutic composition and the second time point may occur between about 12 months to about 24 months following administration of the first dose of the therapeutic composition. In such instances, the first time point may occur between about three months to about the day of but before, about two months to about the day of but before, or about one month to about the day of but before administration of a first dose of the therapeutic composition. In instances in which both the first and second time points occur after administration of a first dose of the therapeutic composition, the first time point may occur between about the day of and about a month, about the day of and about 3 weeks, about the day of and about 2 weeks or about the day of and about 1 week after administration of the first dose of the therapeutic composition, and the second time point may occur between about 3 months and about 24 months after administration of the first dose of the therapeutic composition. In either of these instances,the second time point may occur between about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, or about 24 months following administration of the first dose of the therapeutic composition. In any of these instances, the CKD patient may have fasted at most about 24 hours, e.g. at most about 6 hours, at most about 12 hours, at most about 18 hours or a most 24 hours, prior to administration of the first dose of the therapeutic composition.

[0021] In some instances of the methods of identifying a therapeutic composition as having bioactivity in a CKD patient, the CKD patient may have been administered at least first and second doses of the therapeutic composition. In instances in which the CKD patient had been administered at least first and second doses of the therapeutic composition, the first dose may have been administered about 2 months to about 12 months prior to the second dose. In such instances, the CKD patient may have been administered the first dose of the therapeutic composition between about 2 months to about 12 months, about 2 months to about 11 months, about 2 months to about 10 months, about 2 months to about 9 months, about 2 months to about 8 months, about 2 months to about 7 months, about 2 months to about 6 months, about 2 months to about 5 months, about 2 months to about 4 months, about 2 months to about 3 months, about 3 months to about 9 months, about 3 months to about 8 months, about 3 months to about 7 months, about 3 months to about 6 months, about 3 months to about 5 months, about 3 months to about 4 months, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months prior to the second dose of the therapeutic composition.

[0022] In the method of identifying a therapeutic composition as having bioactivity in a CKD patient, and in which the CKD patient had been administered at least first and second doses of the therapeutic composition, e.g., a first and a second dose, the first time point at which serum bicarbonate level is determined may be between about 3 months and about 2 days prior to administration of the first dose of the therapeutic composition, and the second time point at which serum bicarbonate is determined may be between about 12 months and about 24 months following administration of the second dose of the therapeutic composition. For example, in these instances: (i) the first time point may be between about 2 months and about 2 days prior, about 1 month and about 2 days prior, about 3 weeks and about 2 days prior, about 2 weeks and about 2 days prior, about 1 week and about 2 days prior, about 3 monthsand about 1 day prior, about 2 months and about 1 day prior, about 1 month and about 1 day prior, about 3 weeks and about 1 day prior, about 2 weeks and about 1 day prior, about 1 week and about 1 day prior, about 3 months, about 2 months, about 1 month, about 3 weeks, about 2 weeks, about 1 week, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days or about 1 day prior to administration of the first dose of the therapeutic; and (ii) the second time point may be between about 12 months and about 24 months, about 12 months and about 20 months, about 12 months and about 18 months, about 12 months and about 16 months, about 12 months and about 14 months, about 14 months and about 24 months, about 14 months and about 22 months, about 14 months and about 20 months, about 14 months and about 18 months, about 14 months and about 16 months, about 16 months and about 24 months, about 16 months and about 22 months, about 16 months and about 20 months, about 16 months and about 18 months, about 18 months and about 24 months, about 18 months and about 20 months, about 24 months, about 22 months, about 20 months, about 18 months, about 16 months, about 14 months, or about 12 months following administration of the second dose of the therapeutic composition.

[0023] In the method of identifying a therapeutic composition as having bioactivity in a CKD patient, where the CKD patient had been administered at least first and second doses of the therapeutic composition, and the first time point may be between about 3 months and about 2 days prior to administration of the first dose of the therapeutic composition, and the second time point may be between about 12 months and about 24 months following administration of the second dose of the therapeutic composition, the level of serum bicarbonate in the CKD patient may be about .1 to about 4.0 mEq / L higher at the second time point than the first time point. In some of these instances, the level of serum bicarbonate in the CKD patient may be about .1 to about 3.8, about .1 to about 3.6, about .1 to about 3.4, about .1 to about 3.2, about .1 to about 3.0, about .1 to about 2.8, about .1 to about 2.6, about .1 to about 2.4, about .1 to about 2.2, about .1 to about 2.0, about .1 to about 1.8, about .1 to about 1.6, about .1 to about 1.4, about .1 and about 1.2, about .1 to about 1.0, about .1 and about .8, about .1 and about .6, about .1 to about .4, about .1 to about .3, about .1 to about .2, about .1, about .2, about .3, about .4, about .5, about .6, about .7, about .8, about .9, about 1.0, about 1.2, about 1.4 or about 1.6, about 1.8, about 2.0, about 2.2, about 2.4, about 2.6, about 2.8, about 3.0, about 3.2, about 3.4, about 3.6, about 3.8 or about 4.0 mEq / L higher at the second time point than the first time point. Further, in some of these instances, the level of serum bicarbonate in the CKD patient may be about 2% to about 30% higher at the second time point relative to thefirst time point. In some of these other instances, the level of serum bicarbonate in the CKD patient may be about 2% to about 25%, about 2% to about 22.5%, about 2% to about 20%, about 2% to about 17.5%, about 2% to about 15%, about 2% to about 12.5%, about 2% to about 10%, about 2% to about 7.5%, about 2% to about 5%, about 2%, about 2.5%, about 5%, about 7.5%, about 10%, about 12.5%, about 15%, about 17.5%, about 20%, about 22.5%, or about 25%, at least about 2%, at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, at least about 12.5%, at least about 15%, at least about 17.5%, at least about 20%, at least about 22.5% or at least about 25% higher at the second time point relative to the first time point.

[0024] In other instances of the method of identifying a therapeutic composition as having bioactivity in a CKD patient, and in which the CKD patient had been administered at least first and second doses of the therapeutic composition, e.g., a first and a second dose, the first time point at which serum bicarbonate level is determined may be about 6 months following administration of the second dose of the therapeutic composition and the second time point at which serum bicarbonate level is determined may be between about 18 and 24 months following administration of the second dose of the therapeutic composition. For example, in these instances: (i) the first time point may be about 6 months (e.g., about 5 months, about 5.5 months, about 6 months, about 6.5 months, or about 7 months) following administration of the second dose of the therapeutic compositions; and (ii) the second time point may be between about 18 and about 24 months, about 18 months and about 23 months, about 18 months and about 22 months, about 18 months and about 21 months, about 18 months and about 20 months, about 20 months and about 24 months, about 20 months and about 23 months, about 20 months and about 22 months, about 20 months and about 21 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, or about 24 months following administration of the second dose of the therapeutic composition.

[0025] In the method of identifying a therapeutic composition as having bioactivity in a CKD patient, where the CKD patient had been administered at least first and second doses of the therapeutic composition, and the first time point may be about 6 months following administration of the second dose of the therapeutic composition, and the second time point may be between about 18 months and about 24 months following administration of the second dose of the therapeutic composition, the level of serum bicarbonate in the CKD patient may be about .1 to about 8 mEq / L higher at the second time point than the first timepoint. In some of these instances, the level of serum bicarbonate in the CKD patient may be about .1 to about 7.75, about .1 to about 7.5, about .1 to about 7.25, about .1 to about 7.0, about .1 to about 6.75, about .1 to about 6.5, about .1 to about 6.25, about .1 to about 6.0, about .1 to about 5.75, about .1 to about 5.5, about .1 to about 5.25, about .1 to about 5.0, about .1 to about 4.75, about .1 to about 4.5, about .1 to about 4.25, about .1 to about 4, about .1 to about 3.75, about .1 to about 3.5, about .1 to about 3.25, about .1 to about 3.0, about .1 to about 2.75, about .1 to about 2.5, about .1 to about 2.25, about .1 to about 2.0, about .1 to about 1.75, about .1 to about 1.5, about .1 to about 1.25, about .1 to about 1.0, about 1.0 to about 8.0, about 1.0 to about 7.5, about 1.0 to about 7.0, about 1.0 to about 6.5, about 1.0 to about 6.0, about 1.0 to about 5.5, about 1.0 to about 5.0, about 1.0 to about 4.5, about 1.0 to about 4.0, about 1.0 to about 3.5, about 1.0 to about 3.0, about 1.0 to about 2.5, about 1.0 to about 2.0, about .1, about 1.0, about 2.0, about 3.0, about 4.0, about 5.0, about 6.0, about 7.0 or about 8.0 mEq / L higher at the second time point than the first time point. Further, in some of these instances, the level of serum bicarbonate in the CKD patient may be about .5% to about 15%, about .5% to about 12.5%, about .5% to about 10%, about .5% to about 7.5%, about .5% to about 7% higher at the second time point than the first time point. In some of these other instances, the level of serum bicarbonate in the CKD patient may be about .5% to about 6.75%, about .5% to about 6.5%, about .5% to about 6.25%, about .5% to about 6.0%, about .5% to about 5.75%, about .5% to about 5.5%, about .5% to about 5.25%, about .5% to about 5.0%, about .5%, to about 4.75% about .5% to about 4.5%, about .5% to about 4.25%, about .5% to about 4.0%, about .5% to about 3.75%, about .5% to about 3.5%, about .5% to about 3.25%, about .5% to about 3.0%, about .5% to about 2.75%, about .5% to about 2.5%, about .5% to about 2.25%, about .5% to about 2.0%, about .5 to about 1.75%, about .5% to about 1.5%, about .5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5%, about 5.5%, about 6.0%, about 6.5% about 7.0%, at least about .5%, at least about 1.0%, at least about 1.5%, at least about 2.0%, at least about 2.5%, at least about 3.0%, at least about 3.5%, at least about 4.0%, at least about 4.5%, at least about 5%, at least about 5.5%, at least about 6.0%, at least about 6.5% or at least about 7.0% higher at the second time point than the first time point.

[0026] In yet other instances of the method of identifying a therapeutic composition as having bioactivity in a CKD patient, and in which the CKD patient had been administered at least first and second doses of the therapeutic composition, e.g., a first and a second dose, the first time point at which serum bicarbonate level is determined may be between the day of,and about 3 months after, administration of a first dose of the therapeutic composition and second time point at which serum bicarbonate level is determined may be between about 6 months and about 24 months following administration of a second dose of the therapeutic composition. For example, in these instances: (i) the first time point may be between the day of and about 2.5 months after, the day of and about 2 months after, the day of and about 1.5 months after, the day of and about 1 month after, the day of and about 2 weeks after, the day of and about 1 week after, about the day of, about 1 week after, about 2 weeks after, about 3 weeks after, about 1 month after, about 1.5 months after, about 2 months after, about 2.5 months after or about 3 months after administration of a first dose of the therapeutic composition; and (ii) the second time point may be between about 6 months and about 24 months, about 6 months and about 22 months, about 6 months and about 20 months, about 6 months and about 18 months, about 6 months and about 16 months, about 6 months and about 14 months, about 6 months and about 12 months, about 6 months and about 10 months, about 6 months and about 8 months, about 9 months and about 24 months, about 9 months and about 22 months, about 9 months and about 20 months, about 9 months and about 18 months, about 9 months and about 16 months, about 9 months and about 14 months, about 9 months and about 12 months, about 12 months and about 24 months, about 12 months and about 22 months, about 12 months and about 20 months, about 12 months and about 18 months, about 12 months and about 16 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months or about 24 months following administration of the second dose of the therapeutic composition. In these, yet other, instances, the CKD patient may have fasted at most 24 hours, e.g., at most about 6 hours, at most about 12 hours, at most about 18 hours or a most about 24 hours, prior to administration of the first dose of the therapeutic composition.

[0027] In the method of identifying a therapeutic composition as having bioactivity in a CKD patient, where the CKD patient had been administered at least first and second doses of the therapeutic composition, and the first time point may be between the day of, and about 1 or 3 months after, administration of the first dose of the therapeutic composition, and the second time point may be between about 6 months and about 24 months following administration of the second dose of the therapeutic composition, the level of serum bicarbonate in the CKD patient may be about .1 to about 10.0 mEq / L higher at the second time point than the firsttime point. In some of these instances, the level of serum bicarbonate in the CKD patient may be about .1 to about 9.0, about .1 to about 8.0, about .1 to about 7.0, about .1 to about 6.0, about .1 to about 5.0, about .1 to about 4.0, about .1 to about 3.0, about .1 to about 2.0, about .1 to about 1.0, about .1 to about .75, about .1 to about .5, about .1 to about .25, about 1.0 to about 10.0, about 1.0 and about 9.5, about 1.0 and about 9.0, about 1.0 and about 8.5, about 1.0 and about 8.0, about 1.0 to about 7.5, about 1.0 to about 7.0, about 1.0 to about 6.5, about 1.0 to about 6.0, about 1.0 to about 5.5, about 1.0 to about 5.0, about 1.0 to about 4.5, about 1.0 to about 4.0, about 1.0 to about 3.5, about 1.0 to about 3.0, about 1.0 to about 2.5, about .1, about .25, about .5, about .75, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5 about 8.0, about 8.5, about 9.0, about 9.5 or about 10 mEq / L higher at the second time point than the first time point. Further, in some of these instances, the level of serum bicarbonate in the CKD patient may be about .5% to about 50% higher at the second time point than the first time point. In some of these instances, the level of serum bicarbonate in the CKD patient may be about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% higher at the second time point than the first time point.

[0028] In an example of an instance of the method of identifying a therapeutic composition as having bioactivity in a CKD patient having been administered at least first and second doses of the therapeutic composition, where the first time point is between the day of, and about 1 or 3 months after, administration of a first dose of the therapeutic composition and the second time point is between about 6 months and about 24 months following administration of a second dose of the therapeutic composition, the second time point, in particular, may be at about 6 months, e.g., about 5 months, about 5.5 months, about 6 months,about 6.5 months, following administration of the second dose of the therapeutic. In this example instance, the level of serum bicarbonate in the CKD patient may be about .1 to about 5 mEq / L higher at the second time point than the first time point. For example, the level of serum bicarbonate in the CKD patient may be about .1 to about 4.75, about .1 to about 4.5, about .1 to about 4.25, about .1 to about 4.0, about .1 to about 3.75, about .1 to about 3.5, about .1 to about 3.25, about .1 to about 3.0, about .1 to about 2.75, about .1 to about 2.5, about .1 to about 2.25, about .1 to about 2.0, about .1, about .5, about 1.0, about 1.5, about2.0, about 2.25, about 2.5, about 2.75, about 3.0, about 3.25, about 3.5, about 3.75, about 4.0, about 4.25, about 4.5 or about 4.75 mEq / L higher at the second time point than the first time point. Further, in this example instance, the level of serum bicarbonate in the CKD patient may be about 1% to about 30% higher at the second time point than the first time point. In this example instance, the level of serum bicarbonate in the CKD patient may be about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30% higher at the second time point than the first time point.

[0029] In another example of an instance of the method of identifying a therapeutic composition as having bioactivity in a CKD patient having been administered at least first and second doses of the therapeutic composition, where the first time point is between the day of, and about 1 or 3 months after, administration of a first dose of the therapeutic composition and the second time point is between about 6 months and about 24 months following administration of a second dose of the therapeutic composition, the second time point, in particular, may be at about 18 months to about 24 months (e.g., about 17.5 months, about 18 months, about 18.5 months, about 19 months, about 19.5 months, about 20 months, about 20.5 months, about 21 months, about 21.5 months, about 22 months, about 22.5 months, about 23 months, about 23.5 months, about 24 months) following administration of the second dose of the therapeutic composition. In this another example instance, the level of serum bicarbonate in the CKD patient may be about 1.0 to about 10.0 mEq / L higher at the second time point than the first time point. For example, the level of serum bicarbonate inthe CKD patient may be about 1.0 to about 9.5, about 1.0 to about 9.0, about 1.0 to about 8.5, about 1.0 to about 8.0, 1.0 to about 7.5, about 1.0 to about 7.0, about 1.0 to about 6.5, about 1.0 to about 6.0, about 1.0 to about 5.5, about 1.0 to about 5.0, about 1.0 to about 4.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5 about 8.0, about 8.5, about 9.0, about 9.5 or about 10 mEq / L higher at the second time point than the first time point. Further, in this another example instance, the level of serum bicarbonate in the CKD patient may be about 1% to about 50% higher at the second time point than the first time point. In this another example instance, the level of serum bicarbonate in the CKD patient may be about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45% or at least about 50% higher at the second time point than the first time point.

[0030] In a further example of an instance of the method of identifying a therapeutic composition as having bioactivity in a CKD patient, where the CKD patient had been administered at least first and second doses of the therapeutic composition, the first time point may be between about 1 month prior to administration of the first dose of the therapeutic composition and the day of, but prior to, administration of the first dose of the therapeutic composition (e.g., about 1 month prior to, about 3 weeks prior to, about 2 weeks prior to, about 1 week prior to, about 5 days prior to, about 3 days prior to, about a day prior to, or the day of but prior to administration of the first dose of the therapeutic composition) and the second time point may be about three months after administration of the first dose of the therapeutic composition (e.g., about 2 months, about 2.5 months, about 3 months, about 3.5 months or about 4 months after administration of the first dose of the therapeutic composition). In such a method, the level of serum bicarbonate in the CKD patient may bebetween about 0.5% and about 25%, about 0.5% and about 20%, about 0.5% and about 15%, about 0.5% and about 10%, about 0.5% and about 5, about 1% and about 25%, about 1% and about 20%, about 1% and about 15%, about 1% and about 10%, about 1% and about 5%, about 0.5%, about 1%, about 5%, about 10%, at least about 0.5%, at least about 1%, at least about 5%, or at least about 10% higher at the second time point relative to the first time point.

[0031] In yet a fiirther example of an instance of the method of identifying a therapeutic composition as having bioactivity in a CKD patient, where the CKD patient had been administered at least first and second doses of the therapeutic composition, the first time point may be between about 1 month prior to administration of the first dose of the therapeutic composition and the day of, but prior to, administration of the first dose of the therapeutic composition (e.g., about 1 month prior to, about 3 weeks prior to, about 2 weeks prior to, about 1 week prior to, about 5 days prior to, about 3 days prior to, about a day prior to, or the day of but prior to administration of the first dose of the therapeutic composition) and the second time point may be about six months after administration of the first dose of the therapeutic composition (e.g., about 5 months, about 5.5 months, about 6 months, about 6.5 months, or about 7 months after administration of the first dose of the therapeutic composition). In such a method, the level of serum bicarbonate in the CKD patient may be between about 0.5% and about 25%, about 0.5% and about 20%, about 0.5% and about 15%, about 0.5% and about 10%, about 0.5% and about 5%, about 1% and about 25%, about 1% and about 20%, about 1% and about 15%, about 1% and about 10%, about 1% and about 5%, about 0.5%, about 1%, about 5%, about 10%, at least about 0.5%, at least about 1%, at least about 5%, or at least about 10% higher at the second time point relative to the first time point.

[0032] In any of the methods of identifying a therapeutic composition as having bioactivity in a CKD patient, the therapeutic composition may be identified as having bioactivity in the patient if the patient’s level of serum bicarbonate is higher at the second time point than the first time point. In some instances, the patient’s level increase in serum bicarbonate at the second time point over the first time point, which identifies the therapeutic composition as having bioactivity in the CKD patient, may be a particular increase in absolute value serum bicarbonate level, e.g., about .1 mEq / L, or a particular increase in percentage serum bicarbonate level, e.g., about .1%, as provided in the disclosure herein.

[0033] In any of the methods of identifying a therapeutic composition as having bioactivity in a CKD patient, the therapeutic composition may include an enriched heterogeneous renal cell population. Such an enriched heterogeneous renal cell population may be enriched forrenal epithelial cells and / or renal tubular cells, which may include renal proximal tubule cells. In some instances, the enriched heterogeneous renal cell population may be enriched for renal tubule cells and may further include glomerular and interstitial cells. The cells of the enriched heterogenous renal cell population, which may be included in the therapeutic composition, may be characterized by expression of certain proteins or markers in addition, or alternatively, to the types of renal cell(s) included therein. For instance, the enriched heterogeneous renal cell population that may be included in the therapeutic composition may characterized by one or more of: (i) expression of gamma-glutamyl transpeptidase (GGT)-l and / or a cytokeratin (CK); and / or (ii) secretion of vascular endothelial TGF (VEGF) and / or kidney injury molecule- 1 (KIM-1). Further, or alternatively, the cells of the enriched heterogeneous renal cell population may be characterized by expression of at least one nephrogenic marker, where the at least one nephrogenic marker is SIX Homeobox 2 (SIX2), odd-skipped-related 1 (OSR1), LIM homeobox 1 (LHX1), rearranged during transfection (RET) and / or fibroblast growth factor 8 (FGF8). In some instances, the enriched heterogeneous renal cell population may fiirther, or alternatively, be characterized as including cells that exhibit a buoyant density of greater than about 1.04 g / mL, or greater than 1.04 g / mL, or greater than or equal to 1.04 g / mL, or greater than about 1.0419 g / mL, or greater than 1.0419 g / mL, or greater than or equal to 1.0419 g / mL. Further characteristics of enriched heterogeneous renal cell populations, cells that may be included enriched heterogeneous renal cell populations, therapeutic compositions that include enriched heterogeneous renal cell populations, and doses (e.g., about 1.0 to 9.0 x 106cells per gram estimated kidney weight) of therapeutic compositions for administration to CKD patients are disclosed further herein, infra.

[0034] In any of the methods of identifying a therapeutic composition as having bioactivity in a CKD patient, the bioactivity of the therapeutic composition in the CKD patient may be, or include, survival of cells of the therapeutic composition’s enriched heterogeneous renal cell population in the CKD patient. The survival of the cells of the therapeutic composition’s enriched heterogeneous renal cell population in the CKD patient may, or may not, include survival of the cells in a kidney or kidney tissue of the CKD patient. In instances in which cells of the therapeutic composition’s enriched heterogeneous renal cell population survive in the kidney of the CKD patient, the cells of the therapeutic composition’s enriched heterogeneous renal cell population may integrate in the CKD patient’s kidney tissue. In instances in which cells of the therapeutic composition’s enriched heterogeneous renal cellpopulation integrate in the CKD’s patient’s kidney tissue, the cells may survive within the kidney tissue of the CKD patient, and they may also form structures with existing structures, e.g., tubules, of the CKD patient’s kidney tissue, form new structures such as tubules in the CKD patient’s kidney tissue, or secrete factors that decrease rate of loss (e.g., improve survival) of existing structures (or cells within existing structures) such as tubules in the CKD patient’s kidney tissue.

[0035] Thus, in instances, the bioactivity of the therapeutic composition that may be identified by the method may be or include integration of cells of the therapeutic composition’s enriched heterogeneous renal cell population in a kidney of the CKD patient. In some instances, the integration of cells of the enriched heterogeneous renal cell population in the kidney of the CKD patient may be a survival of the enriched heterogeneous renal cell population’s cells in and / or among cells of the kidney of the CKD patient. In some instances, the integration of the enriched heterogeneous renal cell population’s cells may fiirther include the enriched heterogeneous renal cell population’s cells preserving, stabilizing, and / or restoring renal tubules, or glomerular filtration, in the kidney tissue of the patient. In instances in which bioactivity of the enriched heterogeneous renal cell population’s cells is or includes preserving, stabilizing, and / or restoring renal tubules in kidney tissue of the patient, the enriched heterogeneous renal cell population’s cells may repair, or repopulate, tubules in the kidney tissue of the patient. In other instances in which bioactivity of the enriched heterogeneous renal cell population’s cells is or includes preserving, stabilizing, and / or restoring renal tubules in kidney tissue of the patient, the enriched heterogeneous renal cell population’s cells may, themselves form new tubules in the kidney tissue of the patient. In yet other instances in which bioactivity of the enriched heterogeneous renal cell population’s cells is or includes preserving, stabilizing, and / or restoring renal tubules in kidney tissue of the patient, the enriched heterogeneous renal cell population’s cells may secrete factors that promote and / or initiate repair and / or formation of new tubules by cells in the kidney of the CKD patient, with or without cells of the enriched heterogeneous renal cell population.

[0036] Furthermore, in any of the methods of identifying a therapeutic composition as having bioactivity in a CKD patient, the bioactivity of the therapeutic composition in the CKD patient may additionally include stabilizing or reducing rate of decline in estimated glomerular filtration rate (eGFR) of the patient, increasing eGFR of the patient, stabilizing or reducing rate of increase in urine albumin-creatinine ratio (uACR) of the patient, reducing uACR of the patient, restoring regulation of erythropoiesis by the kidney of the patient,restoring regulation of blood or serum potassium level by the kidney of the patient, restoring regulation of blood or serum phosphorus level by the kidney of the patient, and / or restoring regulation of blood or serum calcium of the kidney of the patient.

[0037] In any of the methods of identifying a therapeutic composition as having bioactivity in a CKD patient, the CKD patient may be identified as a responder to the therapeutic composition if the therapeutic composition is identified as having bioactivity in the CKD patient.

[0038] In any of the methods of identifying a therapeutic composition as having bioactivity in a CKD patient, the identification of the therapeutic composition as having bioactivity may identify the therapeutic composition as treating or preventing metabolic acidosis in the CKD patient. In some instances, the treating or preventing metabolic acidosis may be a stabilization of the patient’s blood pH or serum bicarbonate levels. In these instances, stabilization may be the maintenance of the patient’s blood pH or serum bicarbonate levels to within about .01%, about .025%, or about .03% of the patient’s blood pH or serum bicarbonate levels at time, e.g., day, of administration of a first dose of the therapeutic composition. In other instances, the treating or preventing metabolic acidosis may be an increasing of the patient’s blood pH or serum bicarbonate levels. In these instances, the increasing may improve the patient’s blood pH or serum bicarbonate levels such that one / both approach or reach normal blood pH or serum bicarbonate levels, which then (if one / both reach normal blood pH and / or serum bicarbonate levels) may be maintained at normal blood pH and / or serum bicarbonate levels. In yet other instances, the treating or preventing metabolic acidosis may be in a patient not suffering from metabolic acidosis, and may prevent the patient’s blood pH or serum bicarbonate levels from decreasing, or approaching levels associated with acidosis.

[0039] In other methods of the disclosure are methods of assessing durability of bioactivity of a therapeutic composition administered to a human patient in need of treatment for CKD. In any of the methods of assessing durability of bioactivity of a therapeutic composition administered to a human CKD patient the level of serum bicarbonate in the CKD patient may be (i) monitored at time points of an interval and (ii) bioactivity of the therapeutic composition may be assessed as being durable if the level of serum bicarbonate of the CKD patient successively increases over the time points of the time interval.

[0040] In some instances of any of the methods of assessing durability of bioactivity of a therapeutic composition, the monitoring may be the determining of level of serum bicarbonate in the CKD patient over two or more time points of a time interval. In some instances, the two or more time points may be or include at least a first and a final time point. In some instances, the first time point may be, at its earliest, day of administration of a first dose of the therapeutic composition. In some instances, the final time point may be, at its latest, 42 months following administration of the first dose of the therapeutic composition. In some instances, the bioactivity of the therapeutic composition may be assessed as being durable if the level of serum bicarbonate of the CKD patient successively increases over the two or more time points of the time interval.

[0041] In the methods of assessing durability of bioactivity of a therapeutic composition, the CKD patient may have been administered a single dose of the therapeutic composition. In some instances, the CKD patient may have been administered a first dose and at least a second dose of the therapeutic composition. In instances in which the CKD patient may have been administered a first and at least a second dose of the therapeutic composition, the first dose may be administered about 2 months to about 12 months prior to the second dose. In some instances, the first dose may be administered about 2 months to about 12 months, about 2 months to about 11 months, about 2 months to about 10 months, about 2 months to about 9 months, about 3 months to about 9 months, about 3 months to about 8 months, about 3 months to about 7 months, about 3 months to about 6 months, about 3 months to about 5 months, about 3 months to about 4 months, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months prior to the second dose.

[0042] In some instances of any of the methods of assessing durability of bioactivity of a therapeutic composition the first time point for determining level of serum bicarbonate in the CKD patient may be between about the day of to about 12 months following administration of a first dose of the therapeutic composition. In some instances, the first time point may be between about the day of to about 11 months following, about the day of to about 10 months following, about the day of to about 9 months following, about the day of to about 8 months following, about the day of to about 7 months following, about the day of to about 6 months following, about the day of to about 5 months following, about the day of to about 4 months following, about the day of to about 3 months following, about the day of to about 2 months following, about 3 months to about 12 months following, about 3 months to about 11 monthsfollowing, about 3 months to about 10 months following, about 3 months to about 9 months following, about 3 months to about 8 months following, about 3 months to about 7 months following, about 3 months to about 6 months following, about 3 months to about 5 months following, about 3 months to about 4 months following, about the day of, about 2 weeks following, about 1 month following, about 2 months following, about 3 months following, about 4 months following, about 5 months following, about 6 months following, about 7 months following, about 8 months following, about 9 months following, about 10 months following, about 11 months following, or 12 months following administration of the first dose of the therapeutic composition.

[0043] In some instances of any of the methods of assessing durability of bioactivity of a therapeutic composition the final time point may be about 2 weeks following the first time point. In some instances, the final time point may be about 2 weeks, about 1 month, about 6 weeks, about 2 months, about 10 weeks, about 3 months, about 14 weeks, about 4 months, about 18 weeks, about 5 months, about 22 weeks, about 6 months, about 26 weeks, about 7 months, about 30 weeks, about 8 months, about 34 weeks, about 9 months, about 38 weeks, about 10 months, about 42 weeks, about 11 months, about 46 weeks, about 12 months, about 50 weeks, about 13 months, about 54 weeks or about 14 months after the first time point. In some instances, the first time point may be between the day of administration and about 3 months after administration of the first dose if the therapeutic composition and the final time point may be between about 3.5 months and about 30 months after administration of the first dose of the therapeutic composition.

[0044] In some instances of any of the methods of assessing durability of bioactivity of a therapeutic composition, the first time point may be about the day of, about 2 weeks following, about 1 month following, about 6 weeks following, about 2 months following, about 10 weeks following or about 3 months following administration of the first dose of the therapeutic composition and the final time point may be about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 25 months, about 26 months, about 27 months, about 28 months, about 29 months or about 30 months after administration of the first dose of the therapeutic composition.

[0045] In some instances of any of the methods of assessing durability of bioactivity of a therapeutic composition, the two or more time points may be at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or at least twelve time points.

[0046] The two or more time points of the time interval may occur with any frequency and need not occur at regular intervals. For example, any two of the two or more time points of the time interval may occur between about 2 weeks and about 12 months apart, between about 2 weeks and about 10 months apart, between about 2 weeks and about 8 months apart, between about 2 weeks and 6 months apart, between about 4 weeks and about 6 months apart, between about 6 weeks and about 6 months apart, between about 2 months and about 6 months apart, between about 3 months and about 6 months apart, between about 4 months and about 6 months apart, between about 1 month and about 5 months apart, between about 2 months and about 5 months apart, between about 3 months and about 5 months apart, between about 4 months and about 6 months apart, or about 2 weeks apart, about 4 weeks apart, about 2 months apart, about 3 months apart, about 4 months apart, about 5 months apart, about 6 months apart, about 7 months apart, about 8 months apart, about 9 months apart, about 10 months apart, about 11 months apart or about 12 months apart.

[0047] In the methods of assessing durability of bioactivity of the therapeutic composition, the therapeutic composition may assessed as durable over a time interval if the level of serum bicarbonate of the CKD patient successively increases over the two or more time points of the time interval. Successive increases in level of serum bicarbonate in the CKD patient may be increases in the CKD patient’s serum bicarbonate levels from a first time point to a second time point (e.g., and from a second time to a third time point, and from a third point to a fourth time point, etc.) up to the final time point of the time interval. The increases may be increases from an earlier time point to its nearest proximal later time point of at least about .01 to about 3, about .01 to about 2, .01 to about 1, about .01 to about .9, about .01 to about .8, about .01 to about .7, about .01 to about .6 about .01 to about .5, about .01 to about .4, about .01 to about .3, about .01 to about .2, about .01 to about .1, about .01 to about .09, about .01 to about .08, about .01 to about .07, about .01 to about .06, about .01 to about .05, about .01 to about .04, about .01 to about. 03 about .01 to about .02, about .1 to about 9.0, about .1 to about 8.0, about .1 to about 7.0, about .1 to about 6.0, about .1 to about 5.0, about .1 to about 4.0, about .1 to about 3.0, about .1 to about 2.0, about .1 to about 1.0, about .01, about .02, about .03, about .04, about .05, about .06, about .07, about .08, about 09, about .1 toabout .75, about .1 to about .5, about .1 to about .25, about 1.0 to about 10.0, about 1.0 and about 9.5, about 1.0 and about 9.0, about 1.0 and about 8.5, about 1.0 and about 8.0, about 1.0 to about 7.5, about 1.0 to about 7.0, about 1.0 to about 6.5, about 1.0 to about 6.0, about 1.0 to about 5.5, about 1.0 to about 5.0, about 1.0 to about 4.5, about 1.0 to about 4.0, about 1.0 to about 3.5, about 1.0 to about 3.0, about 1.0 to about 2.5, about .1, about .25, about .5, about .75, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5 about 8.0, about 8.5, about 9.0, about 9.5 or about 10 mEq / L. It will be understood that in instances in which the two or more time points includes at least three time points, amount (or level) of increase in serum bicarbonate that occurs over any two of the two or more time points need not be the same amount (or level) of increase in serum bicarbonate as that of any other two of the two or more time points.

[0048] The bioactivity of a therapeutic composition, assessed by the methods of assessing durability of bioactivity of the therapeutic composition, may be any as discussed earlier herein, e.g., in the methods of identifying a therapeutic composition as having bioactivity in a CKD patient. For example, the bioactivity may be or include integration of cells of the enriched heterogeneous renal cell population with kidney tissue of a kidney of the patient. In some instances, the integration of cells of the enriched heterogeneous renal cell population may be or include the preservation, stabilization, and / or restoration of renal tubules in the kidney tissue of the patient. In some instances, the preservation, stabilization, and / or restoration of renal tubules may be repair of, or repopulation of, tubules in the kidney tissue of the patient by cells of the enriched heterogeneous renal cell population. In some instances, the preservation, stabilization, and / or restoration of renal tubules may be formation of new tubules in the kidney tissue of the patient by cells of the enriched heterogeneous renal cell population. In some instances, the preservation, stabilization and / or restoration of renal tubules may be or include secretion of factors by cells of the heterogeneous renal cell population that promote and / or initiate repair and / or formation of new tubules in the kidney of the CKD patient. In some instances, the integration further preserves, stabilizes and / or restores glomerular function in the kidney tissue of the patient. Further, the durability of the bioactivity of the enriched heterogeneous renal cell population assessed by the methods may reference cells of the enriched heterogeneous renal cell population continuing to have bioactivity, as described herein, over any two or more time points of the time interval in which the level of serum bicarbonate of the CKD patient increases.

[0049] The therapeutic composition, whose durability of bioactivity may be assessed in the methods of assessing durability of a therapeutic composition, may be enriched for certain types of renal cells and / or include cells that may have certain characteristics as described earlier herein, e.g., in the methods of identifying a therapeutic composition as having bioactivity in a CKD patient. In addition, characteristics of enriched heterogeneous renal cell populations, cells that may be included enriched heterogeneous renal cell populations, therapeutic compositions that include enriched heterogeneous renal cell populations, doses of therapeutic compositions that may have been administered to the CKD patient, and administration of therapeutic compositions (e.g., by injection into the renal cortex of at least one kidney) to patients are disclosed further herein, infra.

[0050] The human patient in need of treatment for CKD, and who may have been administered the therapeutic composition, in the methods of assessing durability of bioactivity of therapeutic composition, may be a human patient having any kidney disease, any tubular transport deficiency, or any glomerular filtration deficiency and the patient’s CKD may originate in the kidney or result from any condition, e.g., diabetes or hypertension or a congenital anomaly. The human patient in need of treatment for CKD may also be in need of treatment for metabolic acidosis. The human patient may be at any stage kidney disease, CKD stage I, II, Illa, Illb, or IV.

[0051] In fiirther methods of the disclosure are methods of identifying an enriched heterogeneous renal cell population as having therapeutic potential. In the methods of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, (i) expression of a sodium bicarbonate cotransporter by cells of the enriched heterogeneous renal cell population may be determined and / or (ii) uptake of sodium bicarbonate by cells of the enriched heterogeneous renal cell population may be determined. In some instances, the sodium bicarbonate cotransporter may be slc4A4 (uniprot Q9Y6R1, GenBank NM 001098484.2).

[0052] In the methods of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, the therapeutic potential of the enriched heterogeneous renal cell population may be in the treatment of a kidney disease, a tubular transport deficiency, or a glomerular filtration deficiency as discussed herein. Further, the therapeutic potential may be a potential of the enriched heterogeneous renal cell population to restore kidney fimction, stabilize kidney function, improve kidney function, reduce renal fibrosis, reduce renal inflammation, induce tubulogenesis in a kidney, induce nephrogenesis in a kidney or induceglomerulogenesis in a kidney of a patient in need of treatment with the enriched heterogeneous renal cell population, e.g., patient in need of treatment for a kidney disease, a tubular transport deficiency or a glomerular filtration deficiency as discussed herein. The therapeutic potential of the heterogeneous renal cell population identified by the methods may be a restoration of mineral balance, electrolyte balance, fluid homeostasis, reabsorption of essential nutrients, cystatin C metabolism, or an alleviation of anemia in a patient in need of treatment with the enriched heterogeneous renal cell population, e.g., patient in need of treatment for a kidney disease, a tubular transport deficiency or a glomerular filtration deficiency as discussed herein.. The therapeutic potential of the enriched heterogeneous renal cell population identified by the methods may be the potential to delay or prevent the need for dialysis, or delay or prevent the need for a kidney transplant in a patient in need of a treatment for a kidney disease.

[0053] In the methods of identifying an enriched heterogeneous renal cell population as having therapeutic potential, the enriched heterogeneous renal cell population may be determined as having a therapeutic potential if it is determined that: (i) greater than about 60% of cells of the enriched heterogeneous renal cell population express slc4A4 and / or (ii) cells of the enriched heterogeneous renal cell population uptake bicarbonate at a level higher than cells of a control renal cell population. In some instances, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if it is determined that: (i) greater than about 60% of cells of the enriched heterogeneous renal cell population express slc4A4. In some instances, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if it is determined that: (i) between about 60% and about 90% of cells of the enriched heterogeneous renal cell population express slc4A4. In some instances, the enriched heterogeneous renal cell population may be identified as having therapeutic potential if between about 60% and about 85%, or between about 65% and about 85%, or greater than about 60%, about 65%, about 70%, about 75%, about 80% of cells of the enriched heterogeneous renal cell population express slc4A4.

[0054] In instances of the methods of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, where it is determined whether cells of the enriched heterogeneous renal cell population express slc4A4, detection of slc4A4 may be by detection of slc4A4 in a nucleic acid, e.g., mRNA or miRNA, or polypeptide form and may be by any suitable assay / technique. For example, if expression is determined in polypeptide form, it may be determined by assays such as Western blot, fluorescence activated cell sorting(FACS), enzyme linked immunosorbent assay (ELISA). If expression is determined in nucleic acid form, it may be determined by an assay such as, or that uses, Southern blot, polymerase chain reaction (PCR) or reverse transcriptase PCR, serial analysis of gene expression (SAGE), Mass ARRAY, or fluorescence in situ hybridization (FISH). Expression may be determined via a bulk or a single cell assay or may be determined after collection of conditioned media from a sample of cells from an enriched heterogeneous renal cell population. The assay may be one that employs a labeled detection reagent. The labeled detection reagent may include (a) a portion that complexes, directly or indirectly, with marker(s) and (b) a detection moiety. Non-limiting detection moieties include radioactive isotopes, e.g.,35S,14C,1251,3H and131I, colloidal gold particles, fluorescent labels, e.g., Texas Red, rhodamine, fluorescein, dansyl, Lissamine, phycocryterin, phycocyanin, SPECTRUM ORANGE, SPECTRUM GREEN1 and enzyme substrates, e.g., firefly luciferase, bacterial luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, or beta galactosidase.

[0055] In the methods of identifying an enriched heterogeneous renal cell population as having a therapeutic potential, alternatively, or in addition to identifying the enriched heterogeneous renal cell population as having therapeutic potential, if it is determined that (i) greater than about 60% of cells of the enriched heterogeneous renal cell population express slc4A4, the enriched heterogeneous renal cell population may be identified as having a therapeutic potential if (ii) cells of the enriched heterogeneous renal cell population uptake bicarbonate at a level higher than cells of the control renal cell population. In some instances, the control renal cell population may be a kidney tissue sample or a culture of cells prepared from the kidney tissue sample, which may be a kidney biopsy sample. In some instances, the control renal cell population, e.g., kidney tissue sample or culture of cells prepared from the kidney tissue, (e.g., biopsy), sample, may be from a CKD patient. In some instances, the CKD patient may be a patient whose kidney tissue was used a starting renal cell population for preparation of the enriched heterogeneous renal cell population.

[0056] The enriched heterogeneous renal cell population may be enriched for certain types of renal cells and / or include cells that may have certain characteristics as described earlier herein, e.g., in the methods of identifying a therapeutic composition as having bioactivity in a CKD patient. Further, the enriched heterogeneous renal cell population may be described as having, or may have cells having, characteristics disclosed further herein, and / or may be prepared according to methods disclosed further herein, infra.

[0057] In other methods of the disclosure are methods of improving kidney fimction in a human CKD patient. In the methods of improving kidney function in a human CKD patient at least a first, a second, and a third dose of a therapeutic composition may be administered to the patient. In some instances, the second dose may be administered about two to twelve months following the first dose. The third dose may be administered to the CKD patient if a dose trigger event is detected, so long as the dose trigger event is detected following the second injection. In some instances, the third dose may be administered (i) following the second dose and (ii) within about three months after detection of a dose trigger event. In some instances, the dose trigger event may be a decline from maximum serum bicarbonate level in the human CKD patient following administration of the second dose.

[0058] In the methods of improving kidney fimction in a human CKD patient the first dose may be administered about 2 months to about 12 months, about 2 months to about 11 months, about 2 months to about 10 months, about 2 months to about 9 months, about 3 months to about 9 month, about 3 months to about 8 months, about 3 months to about 7 months, about 3 months to about 6 months, about 3 months to about 5 months, about 3 months to about 4 months, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months prior to the second dose.

[0059] In the methods of improving kidney fimction in a human CKD patient, the administration of the third dose of the therapeutic composition to the CKD patient may be within about 10 weeks of having detected the dose trigger event. In some instances, administration of the third dose of the therapeutic composition may be within about 8 weeks, within about 6 weeks, within about 4 weeks, within about 2 weeks, within about 1 week, within about 1 week to about 8 weeks, within about 1 week to about 6 weeks, within about 1 week to about 4 weeks, within about 1 week and about 2 weeks, within about 2 weeks to about 8 weeks, within about 2 weeks to about 7 weeks, within about 2 weeks to about 6 weeks, within about 2 weeks to about 5 weeks, within about 2 weeks to about 4 weeks, within about 4 weeks to about 8 weeks, within about 4 weeks to about 6 weeks, about 3 weeks to about 6 weeks, or about 4 weeks to about 5 weeks after having detected the dose trigger event.

[0060] In some instances in any of the methods of improving kidney fimction in a human CKD patient, the detected dose trigger event may be a detected decline or decrease in the patient’s maximum serum bicarbonate level following administration of the second dose ofthe therapeutic composition. A decline or decrease in the patient’s maximum serum bicarbonate level may be a decline or decrease of about .05% to about 1% from the patient’s maximum serum bicarbonate level.

[0061] In some instances in any of the methods of improving kidney fimction in a human CKD patient the decline or decrease from maximum serum bicarbonate level in the human CKD patient may be a decline or decrease from the maximum serum bicarbonate level of about .05% to about .9%, about .05% to about .8%, about .05% to about .7%, about .05% to about .6%, about .05% to about .5%, about .05% to about .4%, about .05% to about .3%, about .05% to about .2%, about .05% to about .1%, about .05%, about .075%, about .1%, about .125%, about .15%, about .175%, about .2%, about .225%, about .25%, about .275%, about .3%, about .325%, about .35%, about .375%, about .4%, about .425%, about .45%, about .475%, about .5%, at least about .05%, at least about.1%, at least about .125%, at least about .15%, at least about .175%, at least about .2%, at least about .225% or at least about .25%, relative to the patient’s maximum serum bicarbonate level.

[0062] In some instances in any of the methods of improving kidney fimction in a human CKD patient, the dose trigger event may be a decline or decrease from maximum serum bicarbonate level that is sustained in the human CKD patient. In some instances, the decline or decrease from maximum serum bicarbonate level may be sustained for about 2 weeks to about 6 months, about 2 weeks to about 5.5 months, about 2 weeks to about 5 months, about2 weeks to about 4.5 months, about 2 weeks to about 4 months, about 2 weeks to about 3.5 months, about 2 weeks to about 3 months, about 2 weeks to about 10 weeks, about 2 weeks to about 2 months, about 2 weeks to about 6 weeks, about 2 weeks to about a month, about 1 month to about 6 months, about 1 month to about 5.5 months, about 1 month to about 5 months, about 1 month to about 4.5 months, about 1 month to about 4 months, about 1 month to about 3.5 months, about 1 month to about 3 months, about 6 weeks to about 3 months, about 2 months to about 3 months, about 2 weeks, at least about 4 weeks, at least about 1 month, at least about 6 weeks, at least about 2 months, at least about 10 weeks, at least about3 months, at least about 3.5 months, at least about 4 months, at least about 4.5 months, at least about 5 months, at least about 5.5 months or at least about 6 months. In some instances, the decline or decrease may be sustained if the maximum serum bicarbonate level in the patient decreases and does not return to the maximum serum bicarbonate level. In some instances, the decline or decrease may be sustained if the maximum serum bicarbonate level in the patient continually decreases from the maximum serum bicarbonate level.

[0063] In some instances in any of the methods of improving kidney fimction in a human CKD patient, the dose trigger event may be detected by having performed a step of determining the patient’s serum bicarbonate level at any at least two time points following administration of the second dose of the therapeutic composition, e.g., at least two time points following administration of the second dose of the therapeutic composition that may be or include time points that occur at least about one week, about two weeks, about six weeks, about eight weeks, about ten weeks, about twelve weeks, about fourteen weeks, about sixteen weeks, about eighteen weeks, about twenty weeks, about twenty two weeks, about twenty four weeks, about seven months, about eight months, about ten months, about twelve months, about fourteen months, about sixteen months, about eighteen months, about twenty months, about twenty two months or about twenty four months following the administration of the second dose of the therapeutic composition. The at least two time points may be at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten time points following administration of the second dose of the therapeutic composition. The at least two time points following administration of the second dose of the therapeutic composition may occur with any frequency, e.g., including those noted above, and need not occur at regular intervals.

[0064] In the methods of improving kidney fimction in a human CKD patient the improving kidney function may be stabilizing or reducing rate of decline in estimated glomerular filtration rate (eGFR) of the patient, increasing eGFR of the patient, stabilizing or reducing rate of increase in urine albumin-creatinine ratio (uACR) of the patient, reducing uACR of the patient, restoring regulation of erythropoiesis by the kidney of the patient, restoring regulation of blood or serum potassium level by the kidney of the patient, restoring regulation of blood or serum phosphorus level by the kidney of the patient, and / or restoring regulation of blood or serum calcium of the kidney of the patient.

[0065] In the methods of improving kidney fimction in a human CKD patient, the therapeutic compositions administered to the human CKD patient may include an enriched heterogeneous renal cell population. The enriched heterogeneous renal cell population may be enriched for certain types of renal cells and / or include cells that may have certain characteristics as described earlier herein, e.g., in the methods of identifying a therapeutic composition as having bioactivity in a CKD patient. Further, the enriched heterogeneous renal cell population may be described as having, or may have cells having, characteristics disclosed further herein, that may be administered in therapeutic compositions at particulardoses (e.g., about 1.0 to 9.0 x 106cells per gram estimated kidney weight) via example methods (e.g., by injection into the renal cortex of at least one kidney) as described farther herein, infra.

[0066] Further, in the methods of improving kidney fanction in a human CKD patient, the human CKD patient may have any kidney disease, any tubular transport deficiency, or any glomerular filtration deficiency, and the patient’s CKD may originate in the kidney or result from any condition, e.g., diabetes or hypertension or a congenital anomaly. The human patient in need of treatment for CKD may also be in need of treatment for metabolic acidosis. The human patient may be at any stage kidney disease, CKD stage I, II, Illa, Illb, or IV. Other characteristics of a human CKD patient are provided herein.

[0067] It should be understood that the use of “about” herein is an indication that any particular number, (e.g., the “5” in about 5% or about 5 days or about 5 mEq / L) need not be exactly that particular number, (e.g., need not be exactly 5% or 5 days or 5 mEq / L). Rather, it should be understood that “about” indicates that the particular number includes values within up to 10% (e.g., up to 10%, up to 5%, or up to 1%) of that particular number, (e.g., between 4.5% and 5.5% or between 4.5 days and 5.5 days or between 4.5 and 5.5 mEq / L).

[0068] In any of the methods provided herein, the enriched heterogeneous renal cell population, whether included in a therapeutic composition or identified as having therapeutic potential, may be a cell population enriched for one or more renal cell types such as renal epithelial cells, renal tubular cells, renal tubular epithelial cells, or renal proximal tubular cells. The enrichment of the enriched heterogeneous renal cell population for these one or more of renal cell types may be a reference to the enriched heterogeneous renal cell population having a greater percentage of the one or more renal cell types than does a kidney tissue of a patient, a kidney biopsy of a patient, or an in vitro culture of cells established from a kidney tissue or kidney biopsy of a patient, (which, collectively, may be referred to as a “starting renal cell population”). A starting renal cell population, if an in vitro culture of cells established from a kidney tissue of a patient or a kidney biopsy of a patient, may be a renal cell preparation comprising dissociated cells of a kidney tissue or kidney biopsy (e.g., cells dissociated from the kidney tissue or kidney biopsy via mincing and / or enzyme digestion), that may or may not have been treated to remove red blood cells and debris. The enriched heterogeneous renal cell population, in addition to being enriched for renal epithelial cells, renal tubular cells, renal tubular epithelial cells, and / or renal proximal tubular cells, may also include other renal cell types such as glomerular cells, podocytes, collecting duct cells,interstitial, and / or vascular cells. These renal cell types, in the enriched heterogeneous renal cell population, may be capable of expressing, and may express, nephrogenic markers and / or markers expressed by ureteric bud (UB), cap mesenchyme (CM), and podocyte cells, e.g, SIX2, OSR1, LHX1, RET and / or FGF8.

[0069] The enriched heterogeneous renal cell population, whether included in a therapeutic composition or identified as having therapeutic potential in the methods described herein, may be enriched for the one or more renal cell types as a result of having been prepared from a starting renal cell population, (e.g., a kidney tissue of a patient, a kidney biopsy of a patient, or an in vitro culture of cells established from a kidney tissue or kidney biopsy of a patient), via a method that includes a separation step. The separation step may be one that separates cells of the starting renal cell population that have been passaged no more than one, two, or three times, on the basis of their buoyant density. If the separation step is one that separates cells on the basis of their buoyant density, the separation step may utilize a single or multi- step continuous or discontinuous density gradient using a density gradient media such as glycerol, glucose OptiPrep, Percoll, or Ficoll-Paque. The use of such a density gradient media in this manner may result in cells of the starting renal cell population (or starting renal cell population having been passaged at most one, two or three times) separating into one or more distinguishable fractions from which cells of the enriched heterogeneous renal cell population may be distinctly identified and isolated. The distinguishable fraction(s) may be one / those in which the buoyant density of cells in the fraction(s) is greater than about 1.045 g / mL, or greater than 1.045 g / mL, or greater than or equal to 1.045 g / mL. The distinguishable fraction(s) may be one / those in which the buoyant density of cells in the fraction(s) is greater than about 1.04 g / mL, or greater than 1.04 g / mL, or greater than or equal to 1.04 g / mL, or greater than about 1.0419 g / mL, or greater than 1.0419 g / mL, or greater than or equal to 1.0419 g / mL. The distinguishable fraction(s) may be one / those in which the buoyant density is between about 1.045 g / mL and about 1.091 g / mL, or between about 1.045 g / mL and about 1.052 g / mL. Alternatively, the separation step may be one that separates cells of the starting renal cell population (or cells of the starting renal cell population that have been passaged no more than one, two or three times), on the basis of whether they express particular markers on their surface. If the separation step separates cells on the basis of their expression of particular cell surface markers, the separation step may be one that utilizes flow cytometry. The flow cytometry may sort out cells from the starting renal cell population (or starting renal cell population having been passaged at most one, two or threetimes) if they express particular surface markers, such as a cytokeratin, e.g., CK8, CK18 and / or CK19, characteristic of, e.g., renal epithelial, renal tubular, renal tubular epithelial, or renal proximal and / or distal tubular cells.

[0070] The enriched heterogeneous renal cell population, whether included in a therapeutic composition or identified as having therapeutic potential in the methods described herein, having been prepared from a starting renal cell population (or starting renal cell population having been passaged at most one, two or three times) may be cultured under hypoxic conditions prior to the separation step. If the cells are cultured under hypoxic conditions prior to the separation step, the cells may be cultured under conditions in which the oxygen levels are less than about 20%, or less than about 15%, or less than about 10%, or less than about 9%, or less than about 8%, or less than about 7%, or less than about 6%, or less than about 5%, or less than about 4%, or less than about 3% or less than about 2% oxygen. If the cells are cultured under hypoxic conditions, the cells may be cultured under the hypoxic conditions for at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, about 6 to about 48 hours, about 6 to about 36 hours, about 6 to about 24 hours, about 12 to about 48 hours, about 12 to about 36 hours, about 12 to about 24 hours, or about 12 to about 18 hours.

[0071] In general, the preparation of an enriched heterogeneous renal cell population, which may be included in a therapeutic composition or identified as having therapeutic potential in the methods provided herein, may be from any starting cell population, for example, an in vitro culture of cells established from a kidney tissue of a patient or a kidney biopsy of a patient. If the enriched heterogeneous renal cell population is prepared from the in vitro culture of cells established from the kidney tissue or kidney biopsy of the patient, the cells of the in vitro culture may be expanded by passaging at most one, or at most two or at most three times. Alternatively, if desired, cells of the in vitro culture of cells established from the kidney tissue or kidney biopsy may be cryopreserved and then expanded by passaging at most one, or at most two or at most three times. Once the cells have been expanded, the expanded cells may be cryopreserved. The expanded cells, whether or not cryopreserved, may then be subject to a separation step or may then be subject to hypoxic culture conditions followed by a separation step. The enriched heterogeneous renal cell population is isolatable by having performed the separation step. Once the enriched heterogeneous renal cell population has been isolated, it may be frozen and / or analyzed prior to use as a therapeutic.

[0072] In some of the methods provided herein, the enriched heterogeneous renal cell population may be included in a therapeutic composition. The therapeutic composition, in some instances, may be in a hydrogel composition. The therapeutic composition, in other instances, may be a liquid composition. The therapeutic composition including the enriched heterogeneous renal cell population may, or may not, include hyaluronic acid.

[0073] If the therapeutic composition is formulated as a hydrogel composition, cells of the enriched heterogeneous renal cell composition may be combined with a temperature-sensitive cell-stabilizing biomaterial. The temperature-sensitive cell-stabilizing biomaterial may be a biomaterial in a gel state at certain temperatures and a liquid state at others. For example, if the biomaterial is temperature-sensitive, the biomaterial may be in a gel state at about 8°C or below, a substantially liquid state at about ambient temperature or above, and a solid-to-liquid transitional state between about 8°C and about ambient temperature; or a gel state at about 4°C or below, a liquid state at about 37°C or above, and a solid-to-liquid transitional state between about 8°C and about 18°C; or a gel state at about 2°C or below, a liquid state at about 37°C or above, and a solid-to-liquid transitional state between about 8°C and about 18°C; or a gel state at about 2°C or below and a liquid state at about 37°C or above; or a gel state at about 4°C or below and a liquid state at about 34°C or above; or a gel state at about 6°C or below and a liquid state at about 32°C or above. The temperature-sensitive cellstabilizing the biomaterial may include, or be made up of, one or more naturally sourced or recombinant proteins or peptides. The naturally sourced or recombinant proteins or peptides may be extracellular matrix proteins of recombinant origin, or extracellular matrix sourced from kidney or another tissue or organ, or gelatin. If the temperature-sensitive cellstabilizing biomaterial is, or includes, gelatin, the gelatin may be derived from a Type I, alpha I collagen such as porcine Type I, alpha I collagen or recombinant human Type I, alpha I collagen. If the temperature-sensitive cell-stabilizing biomaterial is, or includes, gelatin, the gelatin may present in the therapeutic composition at about 0.5% to about 1% weight per volume (w / v), or about 0.8% to about 0.9% (w / v), or about 0.75% (w / v) or about 0.88% (w / v). Cells of the enriched heterogeneous renal cell population may be dispersed throughout the biomaterial, or substantially uniformly distributed throughout the biomaterial. Cells of the enriched heterogeneous renal cell population may be formulated in the biomaterial, e.g., gelatin, such that the number of cells per mL biomaterial is about 20 x 106cells per mL, about 40 x 106cells per mL, about 60 x 106cells per mL, about 100 x 106cells per mL, about120 x 106cells per mL, about 140 x 106cells per mL, about 160 x 106cells per mL, about 180 x 106cells per mL, or about 200 x 106cells per mL.

[0074] If the therapeutic composition is formulated as a liquid composition, the enriched heterogeneous renal cell population may be combined with any suitable liquid, e.g., appropriate cell storage or culture medium, a saline, or combinations thereof, for immediate use or for cryopreservation up until the timing of its use. If the therapeutic composition is a liquid composition, the cells of the enriched heterogeneous renal cell population may be suspended in a pharmaceutically acceptable carrier or excipient, such as saline, buffered saline, dextrose, water, polyethyleneglycol, and / or any combinations thereof. Cells of the enriched heterogeneous renal cell population may be combined with the suitable liquid, e.g., cell storage or culture medium, such that the number of cells per mL liquid is about 20 x 106cells per mL, about 40 x 106cells per mL, about 60 x 106cells per mL, about 100 x 106cells per mL, about 120 x 106cells per mL, about 140 x 106cells per mL, about 160 x 106cells per mL, about 180 x 106cells per mL, or about 200 x 106cells per mL.

[0075] In some of the methods provided herein, the therapeutic composition is, or had been, administered to a CKD patient. In these methods, administration of the therapeutic composition may be, or have been, administered to a CKD patient by any suitable administration route known in the art. For instance, the therapeutic composition may be or had been systemically administered to the CKD patient or at, e.g., into, the kidney (s) of the CKD patient. If the therapeutic composition is or had been administered at or into the kidney(s) of the CKD patient, it may be or had been administered over a single or over multiple injection(s). It may be or had been administered via direct laparotomy, via direct laparoscopy, transabdominally, or percutaneously. The therapeutic composition may be or had been administered by percutaneous injection into the renal cortex of a kidney, or may be or had been administered by inserting a guiding cannula percutaneously to puncture the kidney capsule and then injecting the therapeutic composition into the kidney.

[0076] In the methods provided herein where the therapeutic composition is, or had been, administered to a CKD patient, the therapeutic composition may have been administered at a particular, e.g., therapeutic, dose. A dose, e.g., therapeutically effective dose, for administration to the CKD patient by any of the suitable routes described herein may include about 1-9 x 106enriched heterogeneous renal cell population cells per gram estimated kidney weight of the patient. A therapeutically effective amount of the therapeutic composition may be a dose of about 1.0 x 106, about 2.0 x 106, about 3.0 x 106, about 4.0 x 106, about 5.0 x 106,about 6.0 x 106, about 7.0 x 106, about 8.0 x 106, about 9.0 x 106, about 2.0 - 7.0 x 106, between about 4.0 - 7.0 x 106, or between about 5.0 x 106- 7.0 x 106cells of an enriched heterogeneous renal cell population per gram estimated kidney weight of the CKD patient.

[0077] The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, up to ± 5%, or up to ± 1%.

[0078] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the appended claims.

[0079] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference in their entirety.EXAMPLESExample 1: Detection of bioactivity of an enriched heterogeneous renal cell population, rilparencel, following injection in CKD patients

[0080] Rilparencel, a heterogeneous renal cell population including renal tubular cells, is an investigational therapeutic prepared from a patient’s own biopsy kidney cells, in clinical trials for treatment of CKD. In a clinical trial study, REGEN-002 (NCT02836574), stage 3 / 4 CKD diabetic patients received two injections of rilparencel in the same kidney between two and ten months apart. Over the course of the study, serum bicarbonate, among other, assessments were made during routine clinical visits on an on-going basis up to 24 months from administration of a second of the two rilparencel injections.

[0081] Patient’s assessments, e.g., serum bicarbonate levels / values, were made at the following times: 14 days before administration of a first rilparencel injection, day of administration of the first injection, day of administration of a second rilparencel injection, and every 3 months after administration of the second injection. If a patient’s second rilparencel injection was administered more than three months after the first injection, serum bicarbonate levels of the patient were additionally determined every three months after administration of the first injection until administration of the second injection. Patients fasted overnight, up to 24 hours, before administration of each of their first and second injections.

[0082] It was discovered that patients’ serum bicarbonate levels continuously rose over time from the day of administration of a first rilparencel injection up to at least about 24 months after administration of a second rilparencel injection. Figures 2 and 4. When patients’ serum bicarbonate levels 14 days prior to first rilparencel injection were used as their baseline serum bicarbonate levels, patients’ serum bicarbonate levels began increasing over baseline no later than about 12 months after administration of the second rilparencel injection, and continuously increased up to at least about 24 months after administration of the second rilparencel injection. Figures 1 and 3. For patients’ early, first rilparencel injection to 6 months post-first rilparencel injection, serum bicarbonate levels see Figure 5; baseline levels were an average of patients’ serum bicarbonate levels at day of biopsy and 14 days prior to first rilparencel injection. Notably, serum bicarbonate levels decreased in CKD patients treated with standard of care, e.g., non-cell based, therapeutics (Figures 1-4).

[0083] As rilparencel predominantly includes renal tubule cells and renal tubule cells, inter alia, reabsorb bicarbonate, an indicator that administered rilparencel cells are biologicallyactive, e.g., have integrated in and may be stabilizing kidney structure / function, is an increase in serum bicarbonate at increasing levels in patients over time. Surprisingly, these increasing bicarbonate levels were able to be detected in patients’ serum.

[0084] Furthermore, falling serum bicarbonate levels is common in patients with kidney disease and is an indicator of metabolic acidosis. Patients with advanced kidney disease typically have serum bicarbonate levels below normal, e.g., 22-29 mEq / L. The discovery that administration of rilparencel to CKD patients measurably increases their serum bicarbonate levels provides evidence not only that rilparencel cells are bioactive in the patients, but also that they may also be usefill to ameliorate or prevent metabolic acidosis, e.g., associated with CKD.Example 2: Consistent with their observed bioactivity in CKD patients, cells in the enriched heterogeneous renal cell population express solute carrier family 4 member 4 (slc4A4)

[0085] As CKD patients treated with rilparencel were found to exhibit continually increasing serum bicarbonate levels over time, expression of slc4A4 (uniprot Q9Y6R1), a sodium bicarbonate cotransporter gene, by rilparencel cells was investigated.

[0086] To assess whether the cells in rilparencel expressed slc4A4, they were subjected to scRNA-seq. To perform scRNAseq, aliquots of the cells were stained with acridine orange and propidium iodide and cells were assessed for viability, concentration, and singleness using the LUNA-FX7 Dual Fluorescence Cell Counter (Logos Biosystems). Cells were then processed using the lOx Genomics Chromium Controller and the Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3.1 Dual Index Kit (PN-1000268) following the manufacturer’s user guide (https: / / tinyurl . com / 4855859x). Briefly, approximately 5,000 cells per sample were loaded onto the Chromium Chip G with a target recovery of 3,000 cells per sample for library preparation. Single cells, reverse transcription reagents and gel beads coated with barcoded oligos were encapsulated together in an oil droplet to produce gel beads in emulsion (GEMs). Reverse transcription was performed using a Cl 000 thermal cycler (Bio-Rad) to generate complementary DNA (cDNA) libraries tagged with a cell barcode and unique molecular index (UMI). GEMs were then broken and cDNA was purified using Dynabeads MyOne SILANE beads (Invitrogen) prior to 12 cDNA amplification cycles. Amplified cDNA libraries were purified with SPRIselect magnetic beads (Beckman Coulter) and were quantified using an Agilent Bioanalyzer High Sensitivity DNA chip (AgilentTechnologies). Fragmentation, end repair, A-tailing and double-sided size selection using SPRIselect beads were then performed. Illumina-compatible adapters were ligated onto the size-selected cDNA fragments. Adapter-ligated cDNA was then purified using SPRIselect beads. Uniquely identifiable indexes were added to this cDNA during 12 amplification cycles. The completed sequencing libraries were then purified using SPRIselect beads, visualized using the Bioanalyzer High Sensitivity DNA chip, and pooled in an equimolar ratio. Pooled libraries were sequenced on a NextSeq 2000 machine (Illumina) at the UNC High Throughput Sequencing Facility. Libraries were denatured and diluted following standard Illumina protocol, spiked with 1% PhiX sequencing control (Illumina), and sequenced on one P3 flow cell in paired-end format (Read 1 : 28 cycles, i7 index: 10 cycles, i5 Index: 10 cycles, Read 2: 90 cycles) to a total depth of 1.2 billion read pairs passing quality filters. Demultiplexing and preliminary analysis were performed using Cell Ranger 7.1.0 with default settings, and analysis was performed using 10X Genomics’ Loupe Browser.

[0087] scRNAseq revealed that cells found in rilparencel express slc4A4. Over 50%, e.g., 69% to 80%, of cells in rilparencel were found to express slc4A4. See Table 1.Table 1: slc4A4 expression by cells of an enriched heterogeneous renal cell population

[0088] slc4A4 expression by cells in additional rilparencel samples, e.g., samples manufactured for patient administration in phase 2 clinical study REGEN-007 (clinical trials ID NCT05018416), were also determined. Consistent with earlier studies, slc4A4 was found to be expressed by approximately 74.8% (average of 17 samples) of cells in the REGEN-007 patient rilparencel samples.Example 3: Confirmation of detection of bioactivity of an enriched heterogeneous renal cell population, rilparencel, following injection in CKD patients in a second clinical trial

[0089] In a second clinical trial study, REGEN-007 (NCT05018416), patients having CKD (eGFR between 20 and 50 mL / min / 1.73m2) and type 1 or 2 diabetes were treated with 2 doses, one into each kidney, of enriched heterogeneous renal cell population rilparencel.

[0090] Similar to Example 1, over the course of the study, serum bicarbonate, among other, assessments were made during patients’ routine clinical visits.

[0091] Patient serum bicarbonate levels, as with the REGEN-002 study described in Example 1, continually increased over time from the day of first rilparencel injection to six months following first rilparencel injection. See Figure 6. While patient serum bicarbonate levels also tended to increase following administration of a second rilparencel injection, e.g., from pre-second injection to 3 months post-second injection and from 9 months post-second injection to 15 months post-second injection, they did less so those described in Example 1. See Figure 7.

Claims

CLAIMS1. A method of identifying a therapeutic composition as having bioactivity in a chronic kidney disease (CKD) patient in need of treatment with the therapeutic composition, comprising: determining level of serum bicarbonate in the CKD patient at a first and a second time point, wherein the first time point occurs prior to the second time point, and the second time point occurs following administration of a first dose of the therapeutic composition; and identifying the therapeutic composition as having bioactivity in the CKD patient if the level of serum bicarbonate in the patient is higher at the second time point than at the first time point; wherein the therapeutic composition comprises an enriched heterogeneous renal cell population.

2. The method of claim 1, wherein the enriched heterogeneous renal cell population is enriched for renal tubular cells.

3. The method of claim 2, wherein the renal tubule cells comprise proximal tubule cells.

4. The method of any of claim 2 or 3, wherein the enriched heterogeneous renal cell population further comprises glomerular and interstitial cells.

5. The method of any preceding claim, wherein cells of the enriched heterogeneous renal cell population are characterized by: (i) expression of gamma-glutamyl transpeptidase (GGT)-l and a cytokeratin (CK); and (ii) secretion of vascular endothelial growth factor (VEGF) and kidney injury molecule- 1 (KIM-l).

6. The method of any preceding claim, wherein cells of the enriched heterogeneous renal cell population are characterized by expression of at least one nephrogenic marker, wherein the at least one nephrogenic marker is SIX Homeobox 2 (SIX2), odd-skipped-related 1 (OSR1),LIM homeobox 1 (LHX1), rearranged during transfection (RET) and / or fibroblast growth factor 8 (FGF8).

7. The method of any preceding claim, wherein cells of the enriched heterogeneous renal cell population exhibit a buoyant density of greater than about 1.04 g / mL.

8. The method of any preceding claim, wherein the enriched heterogeneous renal population is administered by injection into the renal cortex of at least one kidney of the patient.

9. The method of any preceding claim, wherein the therapeutic composition is administered at a dose of about 1.0 to 9.0 x 106cells per gram estimated kidney weight.

10. The method of any preceding claim, wherein the bioactivity comprises integration of cells of the enriched heterogeneous renal cell population with kidney tissue of a kidney of the patient.

11. The method of claim 10, wherein the integration of cells of the enriched heterogeneous renal cell population comprises preservation, stabilization and / or restoration renal tubules in the kidney tissue of the patient.

12. The method of claim 11, wherein the preservation, stabilization and / or restoration renal tubules comprises repair of, or repopulation of, tubules in the kidney tissue of the patient by cells of the enriched heterogeneous renal cell population.

13. The method of claim 11, wherein the preservation, stabilization and / or restoration renal tubules comprises formation of new tubules in the kidney tissue of the patient by cells of the enriched heterogeneous renal cell population.

14. The method of claim 10, wherein the integration further preserves, stabilizes and / or restores glomerular function in the kidney tissue of the patient.

15. The method of any preceding claim, wherein CKD patient is administered the first dose and at least a second dose of the therapeutic composition.

16. The method of claim 15, wherein the first dose is administered about 2 months to about 12 months prior to the second dose.

17. The method of claim 16, wherein the first dose is administered about 2 months to about 12 months, about 2 months to about 11 months, about 2 months to about 10 months, about 2 months to about 9 months, about 3 months to about 9 month, about 3 months to about 8 months, about 3 months to about 7 months, about 3 months to about 6 months, about 3 months to about 5 months, about 3 months to about 4 months, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months prior to the second dose.

18. The method of any preceding claim, wherein:(i) the first time point is between the day of, and about a month after, administration of the first dose of the therapeutic composition; and(ii) the second time point is between about 3 months and about 24 months following administration of the first dose of the therapeutic composition; and wherein the CKD patient fasts at most about 24 hours prior to administration of the first dose of the therapeutic composition.

19. The method of any of claims 15-17, wherein:(i) the first time point is between about 3 months and about 2 days prior to administration a first dose of the therapeutic composition; and(ii) the second time point is between about 12 months and about 24 months, following administration of the second dose of the therapeutic composition.

20. The method of claim 19, wherein the level of serum bicarbonate in the CKD patient is about .1 to about 4.0 mEq / L higher at the second time point than the first time point.

21. The method of claim 20, wherein the level of serum bicarbonate in the CKD patient is about .1 to about 3.8, about .1 to about 3.6, about .1 to about 3.4, about .1 to about 3.2, about .1 to about 3.0, about .1 to about 2.8, about .1 to about 2.6, about .1 to about 2.4, about .1 to about 2.2, about .1 to about 2.0, about .1 to about 1.8, about .1 to about 1.6, .1 to about 1.4, about .1 and about 1.2, about .1 to about 1.0, about .1 and about .8, about .1 and about .6,about .1 to about .4, about .1 to about .3, about .1 to about .2, about .1, about .2, about .3, about .4, about .5, about .6, about .7, about .8, about .9, about 1.0, about 1.2, about 1.4 or about 1.6, about 1.8, about 2.0, about 2.2, about 2.4, about 2.6, about 2.8, about 3.0, about 3.2, about 3.4, about 3.6, about 3.8 or about 4.0 mEq / L higher at the second time point than the first time point.

22. The method of claim 19, wherein the level of serum bicarbonate in the CKD patient is about 2% to about 30% higher at the second time point relative to the first time point.

23. The method of claim 22, wherein the level of serum bicarbonate in the CKD patient is about 2% to about 25%, about 2% to about 22.5%, about 2% to about 20%, about 2% to about 17.5%, about 2% to about 15%, about 2% to about 12.5%, about 2% to about 10%, about 2% to about 7.5%, about 2% to about 5, about 2%, about 2%, about 2.5%, about 5%, about 7.5%, about 10%, about 12.5%, about 15%, about 17.5%, about 20%, about 22.5% or about 25% higher at the second time point relative to the first time point.

24. The method of any of claims 15-17, wherein:(i) the first time point is about 6 months following administration the second dose of the therapeutic composition; and(ii) the second time point is between about 18 to about 24 months following administration of the second dose of the therapeutic composition.

25. The method of claim 24, wherein the level of serum bicarbonate in the CKD patient is about .1 to about 8 mEq / L higher at the second time point than the first time point.

26. The method of claim 25, wherein the level of serum bicarbonate in the CKD patient is about .1 to about 7.75, about .1 to about 7.5, about .1 to about 7.25, about .1 to about 7.0, about .1 to about 6.75, about .1 to about 6.5, about .1 to about 6.25, about .1 to about 6.0, about .1 to about 5.75, about .1 to about 5.5, about .1 to about 5.25, about .1 to about 5.0, about .1 to about 4.75, about .1 to about 4.5, about .1 to about 4.25, about .1 to about 4, about.1 to about 3.75, about .1 to about 3.5, about .1 to about 3.25, about .1 to about 3.0, about .1 to about 2.75, about .1 to about 2.5, about .1 to about 2.25, about .1 to about 2.0, about .1 to about 1.75, about .1 to about 1.5, about .1 to about 1.25, about .1 to about 1.0, about 1.0 to about 8.0, about 1.0 to about 7.5, about 1.0 to about 7.0, about 1.0 to about 6.5, about 1.0 toabout 6.0, about 1.0 to about 5.5, about 1.0 to about 5.0, about 1.0 to about 4.5, about 1.0 to about 4.0, about 1.0 to about 3.5, about 1.0 to about 3.0, about 1.0 to about 2.5, about 1.0 to about 2.0, about .1, about 1.0, about 2.0, about 3.0, about 4.0, about 5.0, about 6.0, about 7.0 or about 8.0 mEq / L higher at the second time point than the first time point.

27. The method of claim 24, wherein the level of serum bicarbonate in the CKD patient is about .5% to about 15% higher at the second time point than the first time point.

28. The method of claim 27, wherein the level of serum bicarbonate in the CKD patient is about .5% to about 15%, about .5% to about 12.5%, about .5% to about 10%, about .5% to about 7.5%, .5% to about 6.75%, about .5% to about 6.5%, about .5% to about 6.25%, about .5% to about 6.0%, about .5% to about 5.75%, about .5% to about 5.5%, about .5% to about 5.25%, about .5% to about 5.0%, about .5%, to about 4.75% about .5% to about 4.5%, about .5% to about 4.25%, about .5% to about 4.0%, about .5% to about 3.75%, about .5% to about 3.5%, about .5% to about 3.25%, about .5% to about 3.0%, about .5% to about 2.75%, about .5% to about 2.5%, about .5% to about 2.25%, about .5% to about 2.0%, about .5 to about 1.75%, about .5% to about 1.5%, about .5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5%, about 5.5%, about 6.0%, about 6.5% or about 7.0% higher at the second time point than the first time point.

29. The method of any of claims 15-17, wherein:(i) the first time point is between the day of, and about a month after, administration of a first dose of the therapeutic composition; and(ii) the second time point is at least about 6 months and at most about 24 months, following administration of the second dose of the therapeutic composition; and wherein the CKD patient fasts at most 24 hours prior to the administration of the first dose of the therapeutic composition.

30. The method of claim 29, wherein the level of serum bicarbonate in the CKD patient is about .1 to about 10.0 mEq / L higher at the second time point than the first time point.

31. The method of claim 30, wherein the level of serum bicarbonate in the CKD patient is about .1 to about 9.0, about .1 to about 8.0, about .1 to about 7.0, about .1 to about 6.0, about .1 to about 5.0, about .1 to about 4.0, about .1 to about 3.0, about .1 to about 2.0, about .1 toabout 1.0, about .1 to about .75, about .1 to about .5, about .1 to about .25, about 1.0 to about 10.0, about 1.0 and about 9.5, about 1.0 and about 9.0, about 1.0 and about 8.5, about 1.0 and about 8.0, about 1.0 to about 7.5, about 1.0 to about 7.0, about 1.0 to about 6.5, about 1.0 to about 6.0, about 1.0 to about 5.5, about 1.0 to about 5.0, about 1.0 to about 4.5, about 1.0 to about 4.0, about 1.0 to about 3.5, about 1.0 to about 3.0, about 1.0 to about 2.5, about .1, about .25, about .5, about .75, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5 about 8.0, about 8.5, about 9.0, about 9.5 or about 10 mEq / L higher at the second time point than the first time point.

32. The method of claim 29, wherein the level of serum bicarbonate in the CKD patient is about .5% to about 50% higher at the second time point than the first time point.

33. The method of claim 32, wherein the level of serum bicarbonate in the CKD patient is about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50% higher at the second time point than the first time point.

34. The method of claim 29, wherein the second time point is about 6 months following administration of the second dose of the therapeutic composition.

35. The method of claim 34, wherein the level of serum bicarbonate in the CKD patient is about .1 to about 5 mEq / L higher at the second time point than the first time point.

36. The method of claim 35, wherein the level of serum bicarbonate in the CKD patient is about .1 to about 4.75, about .1 to about 4.5, about .1 to about 4.25, about .1 to about 4.0, about .1 to about 3.75, about .1 to about 3.5, about .1 to about 3.25, about .1 to about 3.0,about .1 to about 2.75, about .1 to about 2.5, about .1 to about 2.25, about .1 to about 2.0, about .1, about .5, about 1.0, about 1.5, about 2.0, about 2.25, about 2.5, about 2.75, about 3.0, about 3.25, about 3.5, about 3.75, about 4.0, about 4.25, about 4.5 or about 4.75 mEq / L higher at the second time point than the first time point.

37. The method of claim 34, wherein the level of serum bicarbonate in the CKD patient is about 1% to about 30% higher at the second time point than the first time point.

38. The method of claim 37, wherein the level of serum bicarbonate in the CKD patient is about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25% or about 30% higher at the second time point than the first time point.

39. The method of claim 29, wherein the second time point is about 18 to about 24 months following administration of the second dose of the therapeutic composition.

40. The method of claim 39, wherein the level of serum bicarbonate in the CKD patient is about 1.0 to about 10.0 mEq / L higher at the second time point than the first time point.

41. The method of claim 40, wherein the level of serum bicarbonate in the CKD patient is about 1.0 to about 9.5, about 1.0 to about 9.0, about 1.0 to about 8.5, about 1.0 to about 8.0, 1.0 to about 7.5, about 1.0 to about 7.0, about 1.0 to about 6.5, about 1.0 to about 6.0, about 1.0 to about 5.5, about 1.0 to about 5.0, about 1.0 to about 4.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5 about 8.0, about 8.5, about 9.0, about 9.5 or about 10 mEq / L higher at the second time point than the first time point.

42. The method of claim 39, wherein the level of serum bicarbonate in the CKD patient is about 1% to about 50% higher at the second time point than the first time point.

43. The method of claim 42, wherein the level of serum bicarbonate in the CKD patient is about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50% higher at the second time point than the first time point.

44. The method of any of claims 15-17, wherein:(i) the first time point is between about one month prior to administration of the first dose of the therapeutic composition and the day of, but prior to, administration of the first dose of the therapeutic composition; and(ii) the second time point is about three months after administration of the first dose of the therapeutic composition.

45. The method of claim 44, wherein the level of serum bicarbonate in the CKD patient is about 0.5% to about 25% higher at the second time point than the first time point.

46. The method of claim 45, wherein the level of serum bicarbonate in the CKD patient is about 0.5% to about 10% higher at the second time point than the first time point.

47. The method of claim 44, wherein the level of serum bicarbonate in the CKD patient is at least about 0.5% higher at the second time point than the first time point.

48. The method of claim 47, wherein the level of serum bicarbonate in the CKD patient is at least about 1.0% higher at the second time point than the first time point.

49. The method of any of claims 15-17, wherein:(i) the first time point is between about one month prior to administration of the first dose of the therapeutic composition and the day of, but prior to, administration of the first dose of the therapeutic composition; and(ii) the second time point is about six months after administration of the first dose of the therapeutic composition.

50. The method of claim 49, wherein the level of serum bicarbonate in the CKD patient is about 0.5% to about 25% higher at the second time point than the first time point.

51. The method of claim 50, wherein the level of serum bicarbonate in the CKD patient is about 1.0% to about 12% higher at the second time point than the first time point.

52. The method of claim 49, wherein the level of serum bicarbonate in the CKD patient is at least about 0.5% higher at the second time point than the first time point.

53. The method of claim 52, wherein the level of serum bicarbonate in the CKD patient is at least about 1.0% at the second time point than the first time point.

54. The method of any preceding claim, wherein the bioactivity further comprises stabilizing or: reducing rate of decline in estimated glomerular filtration rate (eGFR) of the patient, reducing rate of increase in urine albumin-creatinine ratio (uACR) of the patient, restoring regulation of erythropoiesis by the kidney of the patient, restoring regulation of blood or serum potassium level by the kidney of the patient, restoring regulation of blood or serum phosphorus level by the kidney of the patient, and / or restoring regulation of blood or serum calcium of the kidney of the patient.

55. The method of any preceding claim, further comprising identifying the CKD patient as a responder to the therapeutic composition if the therapeutic composition is detected as having bioactivity in the patient.

56. The method of any of claims 1-53, wherein the identifying the therapeutic composition as having bioactivity identifies the therapeutic composition as treating or preventing metabolic acidosis in the patient.

57. The method of claim 56, wherein the treating or preventing metabolic acidosis in the patient comprises stabilizing blood pH of the patient.

58. The method of claim 56, wherein the treating or preventing metabolic acidosis in the patient comprises increasing blood pH of the patient.

59. A method of improving kidney fimction in a human CKD patient, comprising: administering at least a first, a second and a third dose of a therapeutic composition to the human CKD patient, wherein the second dose is administered about two to twelve months following the first dose, and wherein the third dose is administered:(i) following the second dose, and(ii) within about three months after detection of a dose trigger event, wherein the dose trigger event is a decline from maximum serum bicarbonate level in the human CKD patient following administration of the second dose, wherein the therapeutic composition comprises an enriched heterogeneous renal cell population.

60. The method of claim 59, wherein the enriched heterogeneous renal cell population is enriched for renal tubular cells.

61. The method of claim 60, wherein the renal tubule cells comprise proximal tubule cells.

62. The method of any of claim 60 or 61, wherein the enriched heterogeneous renal cell population further comprises glomerular and interstitial cells.

63. The method of any of claims 59-62, wherein cells of the enriched heterogeneous renal cell population are characterized by: (i) expression of gamma-glutamyl transpeptidase (GGT)-l and a cytokeratin (CK); and (ii) secretion of vascular endothelial growth factor (VEGF) and kidney injury molecule- 1 (KIM-1).

64. The method of any of claims 59-63, wherein cells of the enriched heterogeneous renal cell population are characterized by expression of at least one nephrogenic marker, whereinthe at least one nephrogenic marker is SIX Homeobox 2 (SIX2), odd-skipped-related 1 (OSR1), LIM homeobox 1 (LHX1), rearranged during transfection (RET) and / or fibroblast growth factor 8 (FGF8).

65. The method of any of claims 59-64, wherein cells of the enriched heterogeneous renal cell population exhibit a buoyant density of greater than about 1.04 g / mL.

66. The method of any of claims 59-65, wherein the therapeutic composition is administered by injection into the renal cortex of at least one kidney of the patient.

67. The method of any of claims 59-66, wherein the therapeutic composition is administered at a dose of about 1.0 to 9.0 x 106cells per gram estimated kidney weight.

68. The method of claim 59, wherein the first dose is administered about 2 months to about 12 months, about 2 months to about 11 months, about 2 months to about 10 months, about 2 months to about 9 months, about 3 months to about 9 month, about 3 months to about 8 months, about 3 months to about 7 months, about 3 months to about 6 months, about 3 months to about 5 months, about 3 months to about 4 months, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months prior to the second dose.

69. The method of any of claims 59-68, wherein the third dose is administered within about 10 weeks after detection of the dose trigger event.

70. The method of claim 69, wherein the third dose is administered within about 8 weeks, within about 6 weeks, within about 4 weeks, within about 2 weeks, within about 1 week, within about 1 week to about 8 weeks, within about 1 week to about 6 weeks, within about 1 week to about 4 weeks, within about 1 week and about 2 weeks, within about 2 weeks to about 8 weeks, within about 2 weeks to about 6 weeks or within about 2 weeks to about 4 weeks of the dose trigger event.

71. The method of any of claims 59-70, wherein the decline from maximum serum bicarbonate level in the human CKD patient is a decrease from the maximum serum bicarbonate level of about .05% to about 1%.

72. The method of claim 71, wherein the decline from maximum serum bicarbonate level in the human CKD patient is a decrease from the maximum serum bicarbonate level of about .05% to about .9%, about .05% to about .8%, about .05% to about .7%, about .05% to about .6%, about .05% to about .5%, about .05% to about .4%, about .05% to about .3%, about .05% to about .2%, about .05% to about .1%, about .05%, about .075%, about .1%, about .125%, about .15%, about .175%, about .2%, about .225%, about .25%, about .275%, about .3%, about .325%, about .35%, about .375%, about .4%, about .425%, about .45%, about .475% or about .5%.

73. The method of any of claims 59-72, wherein the decline from maximum serum bicarbonate level is sustained.

74. The method of claim 73, wherein the decline is sustained for about 1 week to about 4 months.

75. The method of claim 74, wherein the decline is sustained for about 2 weeks to about 3 months, about 2 weeks to about 10 weeks, about 2 weeks to about 2 months, about 2 weeks to about 6 weeks, about 2 weeks to about a month, about 1 month to about 3 months, about 6 weeks to about 3 months, about 2 months to about 3 months, about 2 weeks, at least about 4 weeks, at least about 1 month, at least about 6 weeks, at least about 2 months, at least about 10 weeks or at least about 3 months.

76. The method of any of claims 73-75, wherein the decline is sustained if the maximum serum bicarbonate level in the patient decreases and does not return to the maximum serum bicarbonate level.

77. The method of claim 76, wherein the decline is sustained if the maximum serum bicarbonate level in the patient continually decreases from the maximum serum bicarbonate level.

78. The method of any of claims 59-77, wherein the improving kidney fimction comprises stabilizing or: reducing rate of decline in estimated glomerular filtration rate (eGFR) of the patient, reducing rate of increase in urine albumin-creatinine ratio (uACR) of the patient, restoring regulation of erythropoiesis by the kidney of the patient, restoring regulation of blood or serum potassium level by the kidney of the patient, restoring regulation of blood or serum phosphorus level by the kidney of the patient, and / or restoring regulation of blood or serum calcium of the kidney of the patient.

79. A method of assessing durability of bioactivity of a therapeutic composition administered to a human patient in need of treatment for chronic kidney disease (CKD), comprising: monitoring level of serum bicarbonate in the CKD patient, wherein the monitoring comprises determining level of serum bicarbonate in the CKD patient over two or more time points of a time interval, wherein the two or more time points comprises at least a first and a final time point, wherein the first time point is, at its earliest, day of administration of a first dose of the therapeutic composition, and wherein the final time point is, at its latest, 42 months following administration of the first dose of the therapeutic composition; and assessing bioactivity of the therapeutic composition as being durable if the level of serum bicarbonate of the CKD patient successively increases over the two or more time points of the time interval, wherein the therapeutic composition comprises an enriched heterogeneous renal cell population, and wherein the therapeutic composition had been administered by injection into the renal cortex of at least one kidney of the patient.

80. The method of claim 79, wherein the enriched heterogeneous renal cell population is enriched for renal tubular cells.

81. The method of claim 80, wherein the renal tubule cells comprise proximal tubule cells.

82. The method of any of claim 80 or 81, wherein the enriched heterogeneous renal cell population further comprises glomerular and interstitial cells.

83. The method of any of claims 79-82, wherein cells of the enriched heterogeneous renal cell population are characterized by: (i) expression of gamma-glutamyl transpeptidase (GGT)-l and a cytokeratin (CK); and (ii) secretion of vascular endothelial growth factor (VEGF) and kidney injury molecule- 1 (KIM-1).

84. The method of any of claims 79-83, wherein cells of the enriched heterogeneous renal cell population are characterized by expression of at least one nephrogenic marker, wherein the at least one nephrogenic marker is SIX Homeobox 2 (SIX2), odd-skipped-related 1 (OSR1), LIM homeobox 1 (LHX1), rearranged during transfection (RET) and / or fibroblast growth factor 8 (FGF8).

85. The method of any of claims 79-84, wherein cells of the enriched heterogeneous renal cell population exhibit a buoyant density of greater than about 1.04 g / mL.

86. The method of any of claims 79-85, wherein the therapeutic composition is administered at a dose of about 1.0 to 9.0 x 106cells per gram estimated kidney weight.

87. The method of any of claims 79-86, wherein the bioactivity comprises integration of cells of the enriched heterogeneous renal cell population with kidney tissue of a kidney of the patient.

88. The method of claim 87, wherein the integration of cells of the enriched heterogeneous renal cell population comprises preservation, stabilization and / or restoration of renal tubules in the kidney tissue of the patient.

89. The method of claim 88, wherein the preservation, stabilization and / or restoration of renal tubules comprises repair of, or repopulation of, tubules in the kidney tissue of the patient by cells of the enriched heterogeneous renal cell population.

90. The method of claim 88, wherein the preservation, stabilization and / or restoration of renal tubules comprises formation of new tubules in the kidney tissue of the patient by cells of the enriched heterogeneous renal cell population.

91. The method of claim 87, wherein the integration farther preserves, stabilizes and / or restores glomerular function in the kidney tissue of the patient.

92. The method of any of claims 79-91, wherein the CKD patient is administered the first dose and at least a second dose of the therapeutic composition.

93. The method of claim 92, wherein the first dose is administered about 2 months to about 12 months prior to the second dose.

94. The method of claim 93, wherein the first dose is administered about 2 months to about 12 months, about 2 months to about 11 months, about 2 months to about 10 months, about 2 months to about 9 months, about 3 months to about 9 month, about 3 months to about 8 months, about 3 months to about 7 months, about 3 months to about 6 months, about 3 months to about 5 months, about 3 months to about 4 months, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months prior to the second dose.

95. The method of any of claims 79-94, wherein the first time point is about the day of to about 12 months following administration of a first dose of the therapeutic composition.

96. The method of claim 95, wherein the first time point is about the day of to about 11 months following, about the day of to about 10 months following, about the day of to about 9 months following, about the day of to about 8 months following, about the day of to about 7 months following, about the day of to about 6 months following, about the day of to about 5 months following, about the day of to about 4 months following, about the day of to about 3 months following, about the day of to about 2 months following, about 3 months to about 12 months following, about 3 months to about 11 months following, about 3 months to about 10 months following, about 3 months to about 9 months following, about 3 months to about 8 months following, about 3 months to about 7 months following, about 3 months to about 6 months following, about 3 months to about 5 months following, about 3 months to about 4 months following, about the day of, about 2 weeks following, about 1 month following, about 2 months following, about 3 months following, about 4 months following, about 5 months following, about 6 months following, about 7 months following, about 8 months following,about 9 months following, about 10 months following, about 11 months following or 12 months following administration of the first dose of the therapeutic composition.

97. The method of any of claims 79-96, wherein the final time point is about 2 weeks following the first time point.

98. The method of any of claims 79-96, wherein the final time point is about 2 weeks, about 1 month, about 6 weeks, about 2 months, about 10 weeks, about 3 months, about 14 weeks, about 4 months, about 18 weeks, about 5 months, about 22 weeks, about 6 months, about 26 weeks, about 7 months, about 30 weeks, about 8 months, about 34 weeks, about 9 months, about 38 weeks, about 10 months, about 42 weeks, about 11 months, about 46 weeks, about 12 months, about 50 weeks, about 13 months, about 54 weeks or about 14 months after the first time point.

99. The method of any of claims 79-94, wherein the first time point is between the day of administration and about 3 months after administration of the first dose of the therapeutic composition and the final time point is between about 3.5 months and about 30 months after administration of the first dose of the therapeutic composition.

100. The method of claim 99, wherein the first time point is about the day of, about 2 weeks following, about 1 month following, about 6 weeks following, about 2 months following, about 10 weeks following or about 3 months following administration of the first dose of the therapeutic composition and wherein the final time point is about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 25 months, about 26 months, about 27 months, about 28 months, about 29 months or about 30 months after administration of the first dose of the therapeutic composition.

101. The method of any of claims 79-100, wherein the two or more time points comprises at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or at least twelve time points.

102. A method of identifying an enriched heterogeneous renal cell population as having therapeutic potential, comprising: determining:(i) expression of a sodium bicarbonate cotransporter by cells of the enriched heterogeneous renal cell population, wherein the sodium bicarbonate cotransporter is slc4A4; and / or(ii) uptake of sodium bicarbonate by cells of the enriched heterogeneous renal cell population; and identifying the enriched heterogeneous renal cell population as having therapeutic potential if it is determined that:(i) greater than about 60% of cells of the enriched heterogeneous renal cell population express slc4a4; and / or(ii) cells of the enriched heterogeneous renal cell population uptake bicarbonate at a level higher than cells of a control renal cell population.

103. The method of claim 102, wherein the enriched heterogeneous renal cell population is identified as having therapeutic potential if it is determined that: (i) greater than about 60% of cells of the enriched heterogeneous renal cell population express slc4A4.

104. The method of claim 103, wherein the enriched heterogeneous renal cell population is identified as having therapeutic potential if it is determined that: (i) between about 60% and about 80% of cells of the enriched heterogeneous renal cell population express slc4A4.

105. The method of claim 102, wherein the enriched heterogeneous renal cell population is identified as having therapeutic potential if it is determined that: (ii) cells of the enriched heterogeneous renal cell population uptake bicarbonate at a level higher than cells of the control renal cell population.

106. The method of claim 103 or 104, wherein the enriched heterogeneous renal cell population is identified as having therapeutic potential if it is farther determined that (ii) cellsof the enriched heterogeneous renal cell population uptake bicarbonate at a higher level than cells of the control renal cell population.

107. The method of any of claims 102, 105 or 106, wherein the control renal cell population comprises a kidney tissue sample or a culture of cells prepared from the kidney tissue sample.

108. The method of claim 107, wherein the kidney tissue sample is a kidney biopsy sample.

109. The method of claim 107 or 108, wherein the kidney tissue sample is from a CKD patient.

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